ENGINEERED PLANT MICROORGANISMS FOR RNAi DELIVERY AND USES THEREOF

Abstract

Provided herein are RNAi delivery compositions and methods for use in targeting organisms found in a plant environment. Features include modification of a chassis microorganism to include exogenous nucleic acids encoding for an siRNA. The compositions and methods provided herein can be used for delivery to a wide variety of crops and for targeting one or more plant-associated pathogens.

Description

ENGINEERED PLANT MICROORGANISMS FOR RNAi DELIVERY AND USES THEREOFCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 716,511, filed November 5, 2024, and U.S. Provisional Application No. 63 / 769,208, filed March 10, 2025, the entire contents of each of which are incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on XXX, is named XXX and is XXX bytes in size.BACKGROUND

[0003] Crop protection is a major concern across all venues of agricultural production. From seed to harvest, farmers face numerous challenges to producing a safe and profitable harvest. Fungi, insects, invasive weeds, nematodes, and other detrimental organisms are a significant source of crop loss and revenue around the world. In the U.S., fungal plant pathogens lead to crop loss ranging between 10- 20% and worth $100-200 billion annually, insect pests lead to crop loss worth approximately $70 billion annually, nematodes may lead to crop loss in soy and com of up to 80% or approximately $125 billion annually, and weeds lead to crop losses of 20-25% or approximately $40 billion annually. Current solutions include the use of non-specific treatments such as broad-spectrum antimicrobials and heavy metals. These non-specific treatments lead to the disruption of the native plant phyto- and rhizo- microbiomes as well as the overall soil microbiome. Currently there is a need for effective and species-specific targeted control of pathogenic organisms, pests, and weeds.BRIEF SUMMARY

[0004] Provided herein are engineered microorganisms comprising at least one dsRNA expression cassette, wherein the dsRNA expression cassette is episomal or integrated in a genome of the engineered microorganism, and wherein the at least one dsRNA expression cassette comprises: an expression region comprising a complementary pair of sequences encoding for a dsRNA about 20- 5000 base pairs in length and comprising a plurality of small interfering RNAs (siRNAs), each 18-31 nucleotides in length, wherein one of the sequences of the complementary pair of sequences comprises a sequence of at least one region of at least one target gene in at least one target organism, and whereinthe siRNAs suppress expression of the at least one target gene; and two antisense promoters flanking the sequences encoding the dsRNA, wherein the promoters are in opposite orientation to each other.

[0005] Provided herein are engineered microorganisms comprising at least one dsRNA expression cassette encoding for a hairpin dsRNA comprising a plurality of small interfering RNAs (siRNAs) each 18-31 nucleotides in length, wherein the dsRNA expression cassette is integrated in a genome of the engineered microorganism and wherein the at least one dsRNA expression cassette comprises a sequence comprising: a promoter; a first coding sequence about 20-5000 nucleotides in length comprising a sequence of at least one region of at least one target gene in at least one target organism; a second coding sequence, wherein the second sequence is the reverse complement of the first sequence; and an intron region separating the first coding region and the second coding region about 50-400 nucleotides in length; wherein the siRNAs suppress expression of the at least one target gene.

[0006] Provided herein are engineered microorganisms, wherein the engineered microorganism comprises a plurality of dsRNA expression cassettes integrated in a genome of the engineered microorganism, wherein each dsRNA expression cassette comprises: a complementary pair of sequences encoding a small interfering RNA (siRNA) 18-31 nucleotides in length, wherein the siRNA comprises a sequence complementary to a region of a target gene in a target organism, and wherein the siRNA suppresses expression of the target gene; and two antisense promoters flanking the sequences encoding the siRNA, wherein the promoters are in opposite orientation to each other.

[0007] Provided herein are compositions comprising the engineered microorganisms described herein or a spore, mycelium, or vegetative cell thereof, and a plant.

[0008] Provided herein are methods of treating a plant, the methods comprising introducing the engineered microorganisms described herein or a spore, mycelium, or vegetative cell thereof, to an ecosystem of a plant.

[0009] Provided herein are methods of treating a plant, the methods comprising introducing the engineered microorganisms described herein or a spore, mycelium, or vegetative cell thereof, to an ecosystem of a plant.

[0010] Provided herein are formulations, wherein the formulations comprise: the engineered microorganisms described herein, and an adjuvant.

[0011] Provided herein are methods of manufacture, wherein the methods comprise: generating the engineered microorganism described herein; growing the engineered microorganism; and formulating a composition comprising the engineered microorganism or a spore, mycelium, or vegetative cell thereof, and an adjuvant.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0013] FIGURES 1A - IB illustrate development and application of an engineered microorganism in a plant environment. FIG. 1A illustrates a general workflow starting with identification of a target organism and chassis microorganism selection, and modification and transformation of the chassis microorganism, expression and delivery of RNAi payload and uptake by the target organism, silencing of target genes in the target organism, and resulting effects of (1) cell death, lowered viability, attenuated pathogenicity, or other modification to viability or pathogenicity in the target organism, or (2) plant growth promotion, improved disease resistance, trait improvement if the target organism is a plant. FIG. IB is a figure showing mechanism of action after the engineered microorganism is introduced to an ecosystem of a plant, from applying spores of an engineered microorganism as described herein, application through seed treatment, colonization of the rhizosphere, production of dsRNA, uptake of dsRNA by target Fusarium, DICER processing of the dsRNA within the Fusarium cell, and RISC degradation of target mRNA.

[0014] FIGURES. 2A-2G are schematics illustrating the different types of genetic constructs integrated into the chromosome of the chassis microorganism as well as the different types of dsRNA constructs transcribed from those constructs FIG. 2A shows complementary sense and antisense strands with two promoters in opposite orientation (referred to as antisense promoters) are transcribed to generate a long double-stranded RNA molecule (dsRNA). FIG. 2B shows two inversely complementary regions of a coding sequence separated by an intron region. Transcription is driven by a single promoter, producing a hairpin-like dsRNA molecule. The intron is excised to generate the dsRNA. FIG. 2C shows complementary sense and antisense strands with two promoters in opposite orientation are transcribed to produce a short siRNA-like molecule. FIG. 2D illustrates a long dsRNA processed by DICER protein generating siRNA 18-31 nucleotides in length. FIG. 2E illustrates a synthetic concatemer dsRNA array comprising exon regions of the target transcript predicted to generate the most effective siRNAs through DICER activity expressed as one continuous dsRNA, or synthetic concatemer dsRNA array, and cleaved by DICER to generate siRNA 18-31 nucleotides in length. FIG. 2F illustrates a synthetic siRNA system containing 2-to-160 siRNA-mimicking (18-3 Int long) coding sequences spaced by DICER modulating spacer regions (DMSR) cleaved by DICER togenerate siRNA 18-31 nucleotides in length. FIG. 2G shows two inversely complementary regions of a coding sequence separated by a hairpin region. Transcription is driven by a single promoter, producing a hairpin-like dsRNA molecule.

[0015] FIGURES 3A and 3B are histograms demonstrating the size distributions of small RNAs derived from dsRNAs expressed in Fusarium oxysporum f. sp. lactucae alongside hph-derived small RNAs expected to be from sheared mRNAs. Small RNAs from Fusarium oxysporum f. sp. lactucae expressing a randomized dsRNA (Fol random) is shown in FIG. 3A and small RNAs from Fusarium oxysporum f. sp. lactucae expressing an anti-mScarlet dsRNA (Fol anti -m Scarlet) is shown in FIG. 3B alongside size distributions for a simultaneously expressed mRNA not expected to form siRNAs (Fol hph).

[0016] FIGURES 4A and 4B are bar graphs illustrating Fusarium oxysporum f. sp. lycopersici exhibiting spray-induced gene silencing. FIG. 4A is a bar plot showing the mean fluorescence in wildtype Fusarium oxysporum f. sp. lycopersici or engineered by AMT to express mScarlet. FIG. 4B is a bar graph representing the average relative concentration of mScarlet mRNA transcript in Fusarium oxysporum f. sp. lycopersici expressing mScarlet reporter gene co-incubated with purified dsRNA targeting mScarlet transcript (SEQ ID NO: 16, “mScarlet-targeting dsRNA”) or a control dsRNA (ct- ), showing reduced expression in mScarlet dsRNA samples.

[0017] FIGURE 5A and 5B are images and bar graph illustrating Fusarium oxysporum f. sp. lycopersici exhibiting self-induced gene silencing. FIG. 5A are photos of Fusarium oxysporum f. sp. lycopersici wildtype (upper panel) and engineered (lower panel) to express dsRNA complementary to Fmkl, as demonstrated by droplet dispersion. The black bars illustrate the dispersion diameter of the droplets. FIG. 5B is a bar graph representing the average relative level of Fmkl mRNA transcript in Fusarium oxysporum f. sp. lycopersici WT and Fusarium oxysporum f. sp. lycopersici expressing a dsRNA targeting the Fmkl transcript (Fmkl dsRNA).

[0018] FIGURES 6A-6D are images showing Trichoderma harzianum-wiAucQA mycoparasitism against Fusarium oxysporum f. sp. lycopersici and a bar graph illustrating growth inhibition of Fusarium oxysporum f. sp. lycopersici by Trichoderma spp.. FIG. 6A is an image of a culture plate co-inoculated with F. oxysporum f. sp. lycopersici (left) and a non-mycoparasitic strain of Trichoderma reseei (right), showing a standoff situation in which neither fungal colony overgrows the other FIG. 6B is an image of a culture plate co-inoculated with F. oxysporum f. sp. lycopersici (left) and a mycoparasitic strain of Trichoderma harzianum (right), showing overgrowth of the F. oxysporum by the Trichoderma. FIG. 6C is a microscope image showing coiling of the thinner hyphaeof Trichoderma harzianum around the thicker hyphae of Fusarium oxysporum (an indication of my coparasitism) and what appears to be an appressorium (black caret). FIG. 6D is a bar graph representing the average relative level of radial growth of Fusarium oxysporum f. sp. lycopersici WT and Fusarium oxysporum f. sp. lycopersici co-cultured with Trichoderma harzianum, Trichoderma virens, Trichoderma asperellum, and Trichoderma reesei after 4 and 7 days post inoculation (dpi).

[0019] FIGURE 7 is a line graph illustrating inhibition of fluorescence of T. harzianum engineered to constitutively express fluorescent protein mScarlet (TH mScarlet) and by co-incubation with wildtype (TH WT) or engineered Trichoderma harzianum expressing dsRNA targeting the following: P- glucuronidase (TH anti-GUS) and mScarlet (TH anti mScarlet).

[0020] FIGURES 8A - 8C are an image of a gel analyzing RT-PCR on RNA extracted from engineered Bacillus subtilis, gels confirming presence of dsRNA by enzymatic digestion, and dot blots confirming dsRNA by dsRNA-specific antibodies. RNA from engineered Bacillus subtilis expressing dsRNA targeting PMT2 (D3), mScarlet (D4), P-glucuronidase (D5), and CYP51 (D7 - D10) were amplified with primers specific to each strain’s dsRNA. FIG. 8A shows RT-PCR performed with reverse transcriptase (RT) in the reaction (left), and without RT in the reaction (right). The absence of bands in the reaction without RT confirms that there is no genomic DNA contaminating the RNA samples. FIG. 8B shows total RNA treated with RNase If, an enzyme that degrades single-stranded RNA. Left: 3 uL of total RNA purified from a strain that does not have the dsRNA expression cassette ((-) dsRNA) and a strain that does express dsRNA ((+)dsRNA). Total RNA from both strains looks similar. Right: 25 uL of total RNA from each strain treated with ssRNA specific RNase If. Only the (+) dsRNA sample retains a band, indicating that the strain is producing double stranded RNA. FIG. 8C shows dsRNA-specific dot blots using a dsRNA-binding antibody. A dilution series of in vitro transcribed (IVT) dsRNA (positive control) and total RNA purified from five engineered Bacillus subtilis strains expressing dsRNA and one Bacillus subtilis strain without a dsRNA expression cassette (negative control) were dotted on a membrane and probed with a dsRNA-specific antibody. Signal from IVT dsRNA dots suggests the antibody specifically detects dsRNA. The six dots corresponding to the RNA from dsRNA expressing strains have clear signal, while the dot corresponding to RNA from a non dsRNA expressing strain shows no signal. This confirms that the RNA our strains are expressing is double stranded.

[0021] FIGURES 9A and 9B are photos of an electrophoresis gels showing single-stranded binding protein (ssb) and dsRNA recovered from cell-free supernatant from cultures of wild-type (WT) or engineered Bacillus subtilis expressing pan-specific targeting dsRNA (SEQ ID NO: 24). FIG. 9Ashows samples after RT-PCR with reverse transcriptase. FIG. 9B shows samples after RT-PCR without reverse transcriptase.

[0022] FIGURE 10 is an image of an electrophoresis gel with samples from engineered Trichoderma harzianum engineered to express a dsRNA cassette after cassette-specific PCR (upper panel) or RT- PCR of the dsRNA constructs, validating cassette integration and expression of inserted sequences.

[0023] FIGURES 11A - 11C are graphs illustrating the growth rates of wild-type and engineered Bacillus subtilis, Trichoderma asperellum, and Trichoderma harzianum grown in liquid culture. FIG 11A shows the growth rate of Bacillus subtilis engineered to express dsRNA4 targeting pan-specific targets, dsRNA31 targeting ERG3 / ERG5, and dsRNA38 targeting PMT1-PMT2-PMT4 were compared to wild-type Bacillus subtilis when grown in 2L DASGIP bioreactors. FIG. 11B shows the growth rate of Trichoderma asperellum engineered to express dsRNA5 targeting [3-glucuronidase (GUS) compared to wild-type Trichoderma asperellum. FIG. 11C shows the growth rate of Trichoderma harzianum engineered to express dsRNA3 targeting PMT2 compared to wild-type Trichoderma harzianum. All engineered strains show comparable colonization compared to their wild-type parent strains.

[0024] FIGURES 12A-12C are line graphs demonstrating viability of engineered organism spores over time compared to wild-type organisms. FIG. 12A shows viable cells recovered from stored engineered vs. wild-type Bacillus subtilis spores over 12 weeks. FIG. 12B shows viable cells recovered from stored engineered vs. wild-type Trichoderma asperellum spores over 8 weeks. FIG. 12C shows viable cells recovered from stored engineered vs. wild-type Trichoderma harzianum spores over 12 weeks.

[0025] FIGURES 13A-13C are graphs illustrating microbial abundance over time in lettuce root tissue when inoculated with wildtype or engineered microbes applied as a soil drench. FIG. 13A shows abundance of Trichoderma asperellum, FIG. 13B shows abundance of Trichoderma harzianum, and FIG. 13C shows abundance of Bacillus subtilis. All engineered strains show comparable colonization compared to their wild-type parent strains.

[0026] FIGURE 14 is a line graph illustrating inhibition of fluorescence of Fusarium oxysporum f. sp. lactucae engineered to constitutively express fluorescent protein mScarlet (Fol*) and coincubation with wild-type (WT B. subtilis) or engineered Bacillus subtilis expressing dsRNA targeting the following: P-glucuronidase (GUS, GEBs-GUS), mScarlet (GEBs-m Scarlet), and CYP51 (GEBs- CYP51).

[0027] FIGURES 15A-15C are graphs illustrating recovery of soybean biomass when inoculated with Fusarium virguliforme (Fv) and treated with engineered Bacillus subtilis. FIG. 15A and FIG. 15B are graphs representing leaf area in untreated plants (“Healthy plant”), inoculated with Fv (“Pathogen only”), and treated with wild-type or engineered Bacillus subtilis expressing non-targeting randomized dsRNA (D41) or dsRNA targeting: panspecific targets (D4), fks 1 (D28), rhol-rhoA-rho2- rho3 (D29), ERG1 (D30), ERG3 / ERG5 (D31), ERG11 / CYP51 (D32), ERG4 (D33), ERG24 (D34), SGE1 (D35), FvToxl (D36), Myosin-5 (D37), PMT1-PMT2-PMT4 (D38), Luc7 (D39), Tom40 (D40), and rhol-rhoA-rho2-rho3-fksl (D43), demonstrating improved growth in Fv-infected plants treated with Bacillus subtilis engineered to express dsRNA targeting ERG3 / ERG5, ERG11 / CYP51, ERG4, ERG24, Myosin-1, PMT1-PMT2-PMT4, Luc7, Tom40, and rhol-rhoA-rho2-rho3-fksl compared to controls. FIG. 15C is a graph showing measure leaf sizes in replicated test groups including ERG3 / ERG5 (D31), ERG11 / CYP51 (D32), ERG24 (D34), Tom40 (D40), and rhol-rhoA- rho2-rho3-fksl (D43) compared to commercial fungicide Saltro®.

[0028] FIGURE 16 is a graph showing measured leaf sizes in replicated test groups evaluating two application methods; an in-furrow root drench using a liquid spore suspension applied at four different dosages (1E6, 1E7, 1E8, 1E9 CFU / mL I seed), and a seed treatment using a liquid formulation applied at a dosage of 1E7 CFU / seed, indicating equivalent area in root drench treated with 1E9 CFU / ml / seed and seeds treated with 1E7 CFU / seed.

[0029] FIGURES 17A-17E are graphs illustrating recovery of soybean biomass when inoculated with Fusarium virguliforme (Fv) isolates and treated with engineered Bacillus subtilis applied as a seed treatment on three elite commercial soybean cultivars. FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D are graphs representing leaf area in untreated plants (“Healthy plant”), inoculated with a mixture of two Fv isolates, and treated with wild-type or engineered Bacillus subtilis expressing dsRNA targeting: ERG3 / ERG5 (D31), PMT1-PMT2-PMT4 (D38), Tom40 (D40), and rhol-rhoA-rho2-rho3- fksl (D43), and compared to commercial fungicide Saltro®. All four dsRNA-expressing Bacillus subtilis showed improved growth in Fv-infected plants compared to controls in all soybean cultivars. FIG. 17E are images of the first true leaves of soybean plants (an elite cultivar) 10 days post planting, comparing untreated (“Healthy”), inoculated with Fv (“Pathogen Only”), engineered Bacillus subtilis expressing dsRNA43 targeting rhol-rhoA-rho2-rho3-fksl, or Saltro®.

[0030] FIGURES 18A-18B are graphs illustrating dsRNA optimization to improve biocontrol efficacy while reducing off-target activity against non-target organisms. FIG. 18A is a graph representing the number of on-target hits and the number of off-target hits with generated dsRNAsequences. FIG. 18B is a graph representing the leaf area in soybean plants when untreated (“Healthy plant”), inoculated with Fusarium virguliforme (Fv) (“Pathogen only”), and treated with engineered Bacillus subtilis expressing dsRNA targeting CYP51 (“DIO”) and (“D32”)

[0031] FIGURES 19A-19B are graphs illustrating improved recovery of plant biomass when inoculated with fungal pathogen and treated with engineered Bacillus subtilis expressing dsRNA designed to target multiple genes. FIG. 19A is a graph representing increased leaf area in Fv-infected Williams 82 soybean plants when treated with engineered Bacillus subtilis expressing dsRNA targeting rhol-rhoA-rho2-rho3 (D29), fksl (D28), and rhol-rhoA-rho2-rho3-fksl (D43) compared to plants inoculated with Fv alone (Pathogen only). FIG. 19B is a graph representing the increased leaf area of Fol-infected Salanova lettuce when treated with engineered Bacillus subtilis expressing dsRNA targeting rhol-rho2-rho3 (DI 7), fksl (DI 6), and rhol-rho2-rho3-fksl (D42) compared to plants inoculated Fol alone (Pathogen only).

[0032] FIGURES 20A-20E are images showing image processing to measure lettuce leaf area and graphs illustrating recovery of lettuce biomass when inoculated with Fusarium oxysporum f. sp. lactucae (Fol) and treated with engineered Bacillus subtilis and Trichoderma harzianum. FIG. 20A is an image of a lettuce plant (Salanova® Green Oakleaf) before and after image processing that isolates and quantifies green pixels to measure leaf area as a proxy for biomass. FIG. 20B is a dot plot representing the leaf area of Salanova® lettuce when untreated (NaCl), inoculated with Fol (Fol), and treated with wild-type (WT) or engineered Bacillus subtilis expressing dsRNA targeting PMT2 (SEQ ID NO: 8, GEBs-dsRNA3, “3”), multiple genes (SEQ ID NO: 16, GEBs-dsRNA4, “4”), and CYP51 (SEQ ID NO: 10 GEBs-dsRNA7, “7” and SEQ ID NO: 18 GEBs-dsRNA8, “8”) (n = 11 plants / condition). FIG. 20C is a dot plot representing a pixel count in NaCl, Fol, WT / A, and GEBs-dsRNA4 replicated from FIG. 20B (n = 12 plants / condition). FIG. 20D are images of lettuce plants (Salanova® Green Oakleaf) 21 days post planting, comparing untreated, inoculated with Fol (“Pathogen Only”), wild-type or engineered Bacillus subtilis expressing dsRNA4 targeting panspecific targets. FIG. 20E is a dot plot representing the leaf area of Salanova® lettuce when untreated (NaCl), inoculated with Fol (Fol), and treated with wild-type (WT77?) or engineered Trichoderma harzianum expressing dsRNA targeting the following: P-glucuronidase (SEQ ID NO: 17 GUS, GETh- dsRNA5, “5”) and PMT2 (SEQ ID NO: 20 GETh-dsRNAl l “11,” SEQ ID NO: 21 GETh-dsRNA12 “12,” and SEQ ID NO: 22 GETh-dsRNA13 “13”) (n = 12 plants / condition).

[0033] FIGURES 21A-21D are graphs illustrating recovery of lettuce biomass when inoculated with Fusarium oxysporum f. sp. lactucae (Fol) and treated with engineered Bacillus subtilis. FIG. 21 A andFIG. 21B are graphs representing the leaf area of Salanova lettuce when untreated (“Healthy plant”), inoculated with Fol (“Pathogen only”), and treated with wild-type or engineered Bacillus subtilis expressing non-targeting randomized dsRNA (D41) or dsRNA targeting the following: panspecific (D4), fksl (DI 6), rhol-rhoA-rho2-rho3 (DI 7), ERG1 (DI 8), ERG3 / ERG5 (DI 9), CYP51 (D20), ERG4 (D21), ERG24 (D22), SGE1 (D23), Myosin-1 (D24), PMT1-PMT2-PMT4 (D25), Luc7 (D26), Tom40 (D27), and rhol-rhoA-rho2-rho3-fksl (D42) Engineered strains are compared to two commercial biological controls Asparello® T34 (Biobest Group) and Rootshield® (BioWorks) applied as root drenches at commercial rates. Results show increased leaf area in Fol-infected lettuce treated with Bacillus subtilis engineered to express dsRNA targeting panspecific targets, fksl, ERG1, ERG3 / ERG5, ERG4, PMT1-PMT2-PMT4, SGE1, Luc7, Tom40, and rhol-rhoA-rho2-rho3-fksl, relative to Pathogen only. FIG. 21C is a graph representing growth in Fol-infected lettuce treated with Bacillus subtilis engineered to express dsRNA targeting panspecific targets compared to controls, showing a significant increase in growth compared to both groups treated with pathogen alone or infected plants treated with wild-type Bacillus. FIG. 21D is a graph representing the leaf area of Salanova lettuce when untreated (nontreated), inoculated with two different field isolates of Fol (Fol- Ci 1 and F ol-02), and treated with wild-type or engineered Bacillus subtilis expressing dsRNA targeting panspecific targets (D4) compared to commercial biological control Rootshield® (BioWorks), indicating growth in plants treated with engineered Bacillus subtilis at least equal to or greater than growth in plants treated with commercially available product.DETAILED DESCRIPTION OF THE INVENTION

[0034] Provided herein are compositions of engineered microorganisms for microbe-induced gene silencing (MIGS). Further provided are methods of use and manufacture of said engineered microorganisms. Described engineered microorganisms, including both prokaryotes and eukaryotes, deliver siRNA targeting genes in plant-associated organisms, including fungi, oomycetes, nematodes, plants (mono- & dicots), insects, bacteria, or viruses. Methods described herein include treatment of a plant or plant environment to reduce or prevent the growth of a detrimental organism, or promote expression of a beneficial feature in a plant. This system provides several advantages over current treatments, including but not limited to the breadth of targets and niches targeted, the choice of niche- associated chassis microorganism to deliver the payload in a site-directed manner, the combination of payloading of active ingredients with the intrinsic bioactivity or behavior of the chassis microorganism (e.g, my coparasitism in Trichoderma spp., swarming, production of antimicrobial peptides, andbiofilm formation in Bacillus spp.) to enhance potency, the use of natural vesicles (e.g., clathrin- mediated EV uptake) to mediate transfer of payloads directly into target organism cells, the ability to design high quality RNAi payloads via bioinformatics and assemble them in a singular construct for efficient and controlled activity, and the ability to enhance production, targeting, and delivery of dsRNA and / or siRNA payloads to the target pathogen through construct design, chassis microorganism choice, vesicle payloading, and multiplexing of multiple targets in a single host. Further, a single RNAi delivery cassette can target a single gene, multiple genes, or multiple organisms. Thus, this RNAi approach produces a greater quantity of siRNA molecules with an enhanced likelihood of targeting one or more genes in one or more organisms when compared to methods described previously. Further described are (1) chassis microorganisms engineered to generate dsRNA and / or siRNA; (2) RNA expression cassettes for insertion into the genome of a chassis microorganism; (3) methods of delivery of payload dsRNAs and siRNAs into target organisms; and (4) target organisms; (5) methods of production; and (6) outcomes as benchmarks of measurement of a successful treatment.

[0035] A general workflow describing development of an engineered microorganism and delivery of an RNAi payload is shown in FIG. 1A. Briefly, a target organism or multiple target organisms are is identified for treatment. Identification includes determination and optionally optimization of one or more target gene sequences on the target organism genome to effect gene silencing. Concurrently, a chassis microorganism is selected. The chassis microorganism is engineered to comprise an expression cassette comprising one or more target gene sequences. Expression and processing of the one or more target gene sequences generates a multiplicity of RNA fragments 19 to 23 nucleotides in length, including RNA fragments with gene silencing activity, or siRNA. Gene silencing is evidenced by cell death, lowered viability, attenuated pathogenicity, or other modifications to viability or pathogenicity in the target organism.

[0036] Exemplary fungal treatment using an engineered microorganism as described herein is illustrated in FIG. IB. Briefly, spores of an engineered microorganism are collected and formulated for application to the environment comprising a target organism. In the illustrated embodiment, application comprises a seed treatment or irrigation application. Following germination, engineered microorganisms colonize the local environment, for example the rhizosphere. The engineered microorganisms generate dsRNA from expression cassettes inserted in the genome. The produced dsRNA strands are taken up by a pathogenic Fusarium cohabiting the environment with the engineered microorganism. In cases in which the dsRNA is over 19 bp, DICER ribonuclease in theFusarium cell cleaves the dsRNA into siRNA fragments 18-31 nucleotides long. The siRNA fragments are taken up by the RNA-induced silencing complex (RISC) and used as a template to recognize a complementary mRNA. Argonaute protein in RISC cleaves the mRNA, thereby silencing the target gene.Definitions

[0037] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” can include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “contains,” “containing,” “including”, “includes,” “having,” “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0038] Unless specifically stated, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / — 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0039] Throughout this disclosure, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subj ect to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention, unless the context clearly dictates otherwise.

[0040] Provided herein are compositions which include bacteria having a percent identity based on 16S rRNA bacterial genetic sequence, a hypervariable region of the 16S rRNA, or whole genome comparison to a reference strain. Typically, comparison of the 16S rRNA bacterial genetic sequence allows a strain to be identified as within the same species as another strain by comparing sequences with known bacterial DNA sequences using NCBI BLAST search. The level of identity in relation toa nucleotide sequence may be determined for at least 20 contiguous nucleotides, for at least 30 contiguous nucleotides, for at least at least 40 contiguous nucleotides, for at least 50 contiguous nucleotides, for at least 60 contiguous nucleotides, or for at least 100 contiguous nucleotides. A level of identity in relation to a nucleotide sequence can be determined for the entire sequence searched. Percent identity can be at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to a reference bacterial 16S rRNA sequence, 16S rRNA V4 region sequence, or whole genome sequence. Percent identity can be at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to a reference bacteria 16S rRNA: VI region, V2 region, V3 region, V5 region, V6 region, V7 region, V8 region or V9 region sequence.

[0041] Provided herein are compositions which include fungi having a percent identity based on 18S, ITS, 28S, RPB1, RPB2, tub2 / BenA, partial calmodulin (CaM) sequence or whole genome comparison to a reference strain. Typically, comparison of the 18S, ITS, 28S, RPB1, RPB2, tub2 / BenA, and partial calmodulin (CaM) DNA sequences allows a strain to be identified as within the same species as another strain by using NCBI BLAST search. A level of identity in relation to one or more reference gene sequences can be determined. Percent identity can be at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to a reference fungal whole genome sequence. Percent identity can be at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to reference fungal gene sequences.

[0042] Reference to a population of bacteria, a population of fungi, or a purified population refers to a plurality of specified microorganisms. A purified microorganism can be enriched from a source sample. A population of the microorganism can comprise about: 10%, 20%, 30%, 40%, 50%, 60%, 70% or more of a single strain of the microorganism.

[0043] As used herein, a substance is “pure” or “substantially pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified”, when applied to a microorganism, can refer to a microorganism that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A microorganism or a microorganism population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microorganism or microorganism population, or by passage through culture, and a purified microorganism or microorganism population may contain other materials up to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” Purifiedmicroorganism and microorganism populations can be more than at least about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than at least about 99% pure by weight (w / w). In the instance of microbial compositions provided herein, the one or more microorganism types, species, or strains present in the composition can be independently purified from one or more other microorganisms produced and / or present in the material or environment containing the microorganism type. Microbial compositions and the microorganism components thereof are generally purified from residual habitat products.

[0044] As used herein, “microorganism” or “microbe” refers to an organism of microscopic size that is too small to be seen with the naked eye. Microorganisms include, but are not limited to bacteria, fungi, archaea, algae, and unicellular eukaryote (e.g. protozoa). For the purposes of this disclosure, a microorganism also refers to any active or dormant forms thereof. Reference to microorganisms comprises their corresponding spores, endospores, mycelia, hyphae, vegetative cells or any other form.

[0045] As used herein, “plant-associated microorganisms” or “plant-associated pathogens” refers to any microorganism found in the phytosphere or soil in the region of a plant. Plant-associated microorganisms, as used herein, include microorganisms found in any interior or exterior region of the plant. In some embodiments, plant-associated microorganisms are found in the phyllosphere, the rhizosphere, the soil, the root system, the shoot system, the flowers, the leaves, the fruit, the stem, or the roots. Plant-associated microorganisms, as used herein, include microorganisms found in any layer of soil that will affect the growth or productivity of a plant. In some embodiments, a plant-associated microorganism is found in the humus layer, the topsoil layer, the eluviation layer, or the subsoil layer. In some embodiments, a plant-associated pathogen is a fungus, an oomycete, a nematode, a virus, an insect, or a weed.

[0046] As used herein, “ecosystem” or “ecosystem of a plant” refers to the phytosphere of a plant, the organisms and conditions affecting the plant, and any other aspect of the physical environment affecting the plant.

[0047] An isolated microbe or microorganism may have been (1) separated from at least some of the components with which it was associated when initially obtained (whether in nature or in an experimental setting), and / or (2) produced, prepared, purified, and / or manufactured by the hand of man, e.g. using artificial culture conditions such as (but not limited to) culturing on a plate, solid substrate, and / or in a fermentor. Isolated microbes can include those microbes that are cultured, even if such cultures are not monocultures. Isolated microbes can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, ormore of the other components with which they were initially associated. Isolated microbes can be more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. A microbe population of a biological sample provided herein can comprise one or more microbes, which may then be isolated from such sample. Isolated microbes may be provided in a form that is not naturally occurring.I. Engineered Chassis Microorganisms

[0048] Provided herein are chassis microorganisms engineered to release RNA in the rhizosphere of a plant. For the purposes of this disclosure, such microorganisms are referred to as “chassis microorganisms” prior to genetic modification, and “engineered microorganisms” following genetic modification. Chassis microorganisms are selected based on native traits of the microorganism, delivery environment, and potential modifications in view of the intended target. Microbe-induced gene silencing (MIGS) utilizing live microorganisms in their natural niches, circumvents many of the delivery and dosage challenges faced by spray-induced gene silencing. In some embodiments, soil-, rhizosphere-, and phyllosphere-associated microbes deliver RNA payloads directly to the site of action of important plant pests, including fungi, oomycetes, bacteria, nematodes, weeds, insects, and viruses or to provide nutrients or growth advantage via Plant Growth Promotion (PGP) pathways. In some embodiments, an RNA payload delivered by engineered microorganisms at one location, such as the roots, translocates inside the plant to another location, such as the soil, stem, leaf, or flower, before delivery to a target organism.

[0049] In some embodiments, chassis microorganisms are identified and selected for preferential colonization of any plant and soil niche, including the phyllosphere, the rhizosphere, soil, roots, phloem, xylem, stems, leaves, flowers, and fruit bodies, to effect a variety of outcomes in a site directed manner. In some embodiments, a chassis microorganism is selected for intrinsic bioactivity to further enhance delivered potency of the product concept. In some embodiments, a chassis microorganism is engineered to exhibit a bioactivity not intrinsic to the species. In some embodiments, the bioactivity comprises my coparasitism, biofilm formation, swarming, secretion, production of extracellular vesicles, or natural antifungal or antibacterial defense toxic compounds or peptides.Mycoparasitism and mycophagy

[0050] In some embodiments, a chassis microorganism used for engineering MIGS are mycotrophic or mycoparasites. In some embodiments, a chassis microorganism is biotrophic or saprophytic. In anexemplary embodiment, an engineered mycoparasitic fungi in genus Trichoderma is capable of secreting fungal cell wall degrading enzymes. The Trichoderma hyphae are capable of coiling around the hyphae of their host / prey fungus. The Trichoderma hyphae then penetrate the target, digesting and absorbing their cytoplasmic components. In another exemplary embodiment, Trichoderma produces gliotoxin, an epipolythio-dioxopiperazine compound whose disulfide bridge reacts with thiol groups on proteins, resulting in varied detrimental effects including apoptosis, inhibition of the catalytic activities of the proteasome and angiogenesis.

[0051] Differential gene expression among species of Trichoderma controls the different ways in which they antagonize other fungi. In an exemplary embodiment, T. atroviridae produces fungicidal secondary metabolites and small proteins, and cell wall degrading beta-glucanases. In another exemplary embodiment, T. virens upregulates genes encoding cellulases and hemicellulases for cell wall degradation, and genes involved in solute transport.

[0052] Microbial mycophagy is not restricted to fungi. There have been several genera of bacteria described as such, including some species in the genus Bacillus. In some embodiments, my coparasitism and mycophagy in the microbial chassis used for engineering contribute to effective delivery of the engineered payload to target organisms.Biofilm formation

[0053] In some embodiments, a chassis microorganism described herein forms a biofilm. Representative microbes in the genus Bacillus form biofilms, a process in which bacterial cells form collectives by aggregating and embedding in a self-produced extracellular matrix composed mostly of exopolysaccharides. Biofilms formed on the surface of roots, and hyphae of fungi associated with plants, facilitate multiple interactions between members of the rhizosphere community. In some embodiments, biofilm formation in a bacterial chassis used for engineering contributes to effective delivery of engineered cargo to target organisms.Swarming behavior

[0054] In some embodiments, a chassis microorganism described herein exhibits swarming behavior. Representative bacteria in the genus Bacillus possess swarming behavior. The leading edge of swarms is characterized by highly flagellated cells that produce surfactants. Swarming behavior can be elicited by root secretions and eliciting factors produced by roots and pathogenic fungi. Moreover, swarming motility and biofilm formation are among the traits believed to be responsible for Bacillus biocontrolabilities. In some embodiments, swarming behavior of the bacterial chassis used for engineering contributes to effective delivery of engineered cargo to target pathogenic fungi.Production of defensive molecules by chassis microorganisms

[0055] In some embodiments, a chassis microorganism described herein exhibits intrinsic beneficial activity in the form of expression of defensive molecules including but not limited to bioactive peptides, enzymes, RNA, DNA, small molecules, and natural products. Such bioactive molecules can suppress viability of target organisms, induce cell death of target organisms, or induce plant defense response in the target crop. In some embodiments, overexpression through constitutive or highly expressed promoters of genes with intrinsic beneficial activity in the chassis microorganism can be leveraged to increase the efficacy of the MIGS system. In an exemplary embodiment, overexpression of Sml in Trichoderma virens Tv29-8 enhances induced plant defense response and systemic resistance in the associated treated crop. In another exemplary embodiment, overexpression of plantazolicin (RBAM_007470) by Bacillus amyloliquefaciens FZB42 enhances nematicidal activity of the bacteria.Extracellular Vesicles

[0056] In some embodiments, an engineered microorganism described herein produces extracellular vesicles (EVs). EVs are lipid bilayer particles ranging in size from about 20 nanometers to about 10 microns or more. In some embodiments, EVs carry proteins, lipids, nucleic acids, metabolites, organelles, or any combination thereof.

[0057] In some embodiments, an engineered microorganism described herein expresses VNp, a 38- amino acid peptide. VNp induces curvature in some bacterial membranes which can increase the rate of extracellular vesiculation.

[0058] Microorganisms described herein occur naturally or are introduced in a plant environment. In some embodiments, the plant environment comprises the phyllosphere, the rhizosphere, the soil, the root system, the shoot system, the flowers, the leaves, the fruit, the stem, or any combination thereof.Reduction in disease

[0059] In some embodiments, application of an engineered microorganism described herein induces attenuation of growth or death of a target organism. In some embodiments, treatment with an engineered microorganism described herein induces a suppression of a target organism. In some embodiments, the suppression is preventative. In some embodiments, the suppression is to inhibitcolonization of the target organism within a plant tissue. In some embodiments, the suppression is to inhibit colonization of the target organism on the surface of a plant, within a plant, within or on the roots of a plant, or in the soil surrounding a plant. In some embodiments, the suppression is to inhibit colonization of the target organism in soil. In some embodiments, the suppression is to inhibit colonization in roots. In some embodiments, the suppression prevents an increase in the abundance of a target organism across many plants. In some embodiments, the suppression prevents an increase of a target organism in an individual plant. In some embodiments, the suppression restricts spread of a target organism within a population of plants of the same species. In some embodiments, the suppression prevents spread of a target organism from one species to a different species. In some embodiments, the suppression is a challenge to one or more existing target organisms. In some embodiments, the suppression is to an established colonization within a plant tissue. In some embodiments, the suppression is to an established colonization on the surface of a plant, within a plant, within or on the roots of a plant, or in the soil surrounding a plant. In some embodiments, the suppression is to an established colonization in soil. In some embodiments, the suppression is to an established colonization in roots. In some embodiments, the suppression comprises a reduction in growth of the plant-associated organisms. In some embodiments, the suppression comprises a reduction in total abundance of the target plant-associated organisms across many plants. In some embodiments, the suppression comprises a reduction in abundance of the plant-associated organisms in an individual plant. In some embodiments, the suppression comprises a reduction in microorganism concentration as measured by colony forming units (CFU) per gram of plant tissue. In some embodiments, the suppression promotes the growth of one or more beneficial microbes. Beneficial microbes include, but are not limited to bacteria or fungi. In some embodiments, beneficial microbes include saprophytes. In some embodiments, beneficial microbes include mycorrhizae. In some embodiments, beneficial microbes comprise Rhizobia spp., Bacillus spp., Pseudomonas spp., Enterobacter cloacae, Arthrobacter spp., Burkholderia spp., or Citrobacter freundii .Improvement to plant

[0060] In some embodiments, application of an engineered microorganism described herein provides for an improvement in a condition or production of a plant. In some embodiments, suppression of a target organism impedes or disrupts plant disease progression. Common symptoms of plant disease caused by pathogenic organisms in plants include, but are not limited to, black rot, blight, necrotic lesions, defoliation, stunted growth, blossom drop, and citrus greening. In some embodiments,suppression of pathogenic organisms causes increased leaf integrity. In some embodiments, suppression of pathogenic organisms causes increased height. In some embodiments, suppression of pathogenic organisms causes increased growth rate. In some embodiments, suppression of pathogenic organisms causes increased yield of a product of the plant. In some embodiments, suppression of pathogenic organisms causes increased biomass. In some embodiments, suppression of pathogenic organisms causes increased crop quantity. In some embodiments, suppression of pathogenic organisms causes increased vegetable quantity. In some embodiments, suppression of pathogenic organisms causes increased fruit quantity. In some embodiments, suppression of pathogenic organisms causes increased fruit mass. In some embodiments, suppression of pathogenic organisms causes improved plant quality. In some embodiments, suppression of pathogenic organisms causes improved fruit quality. In some embodiments, suppression of pathogenic organisms causes improved vegetable quality. In some embodiments, suppression of pathogenic organisms causes increased longevity. In some embodiments, suppression of pathogenic organisms causes increased tolerance to environmental stress. In some embodiments, suppression of pathogenic organisms causes increased salinity tolerance. In some embodiments, suppression of pathogenic organisms causes increased drought tolerance.

[0061] Provided herein are microorganisms engineered to deliver dsRNA and / or siRNA to a plant environment. In some embodiments, the engineered microorganism is found in the plant microbiome. In some embodiments, the engineered microorganism is found in a plant phyllomicrobiome or rhizomicrobiome. In some embodiments, the engineered microorganism provides a beneficial mechanism of action to plant growth. In some embodiments, the engineered microorganism itself does not affect the growth or production of the plant. In some embodiments, the engineered microorganism is not native to the plant microbiome. Selected genera and exemplary species of microorganism considered for modification according to methods described herein are listed in Table 1. Any named species is understood to additionally include any variant or strain thereof.Table 1. Exemplary Chassis Microorganisms.II. Expression Cassette

[0062] In some embodiments, an engineered microorganism provided herein comprises an expression cassette. A “cassette”, as used herein, describes a nucleic acid sequence comprising at least one promoter sequence and an expression region. In some embodiments, an expression cassette comprises an RNAi cassette. In some embodiments, an expression region described herein comprises an siRNA coding sequence. In some embodiments, an RNAi cassette comprises a dsRNA coding sequence. In some embodiments, an expression cassette described herein encodes for one siRNA. In some embodiments, an expression cassette described herein encodes for more than one siRNA. siRNA

[0063] In some embodiments, an expression region in an expression cassette described herein encodes double stranded siRNA. In some embodiments, siRNA is used to induce mRNA degradation and suppress target gene expression. Sequences of siRNA are complementary to a target exonic region of a gene in a target organism. In some embodiments, the siRNA is complementary to a native target gene sequence. In some embodiments, the siRNA is a synthetic design. In some embodiments, a synthetic sequence provides for increased specificity and efficacy of the siRNA relative to an siRNA derived from a native sequence.

[0064] In some embodiments, an siRNA described herein suppresses from about 1% to about 100% expression of a target gene. In some embodiments, an siRNA described herein suppresses about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% expression of a target gene. In some embodiments, an siRNA described herein suppresses from about 1% to about 10%, from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 70% to about 80%, from about 80% to about 90%, from about 90% to about 100% expression of a target gene.Expression Region

[0065] In some embodiments, a synthetic dsRNA sequence is designed against a consensus sequence of a target gene. An exemplary method of synthetic dsRNA sequence design is described in Example 1. In some embodiments, a consensus sequence comprises from 1 to about 1000 variants of a target gene. In some embodiments, a consensus sequence comprises sequences from target genes from 1 to about 1000 subspecies of a species. In some embodiments, a consensus sequence comprises sequences from target genes from 1 to about 100 species. In some embodiments, a consensus sequence comprises sequences from target genes from 1 to about 100 genera. In some embodiments, a consensus sequence comprises sequences from target genes from 1 to about 100 families. In some embodiments, a consensus sequence comprises sequences from target genes from 1 to about 100 orders. In some embodiments, a consensus sequence comprises sequences from target genes from 1 to about 100 classes.

[0066] In some embodiments, one or more consensus exon sequences are selected from the consensus sequence. In some embodiments, more than one exon sequence is selected. In some embodiments from 1 to 100 exon sequences are selected. In some embodiments, about 2, about 3, about 4, about 5,about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 exon sequences are selected.

[0067] In some embodiments, a consensus exon sequence is selected based on analysis of predicted siRNA binding activity. In some embodiments, a consensus exon sequence is selected based on analysis of probability of a specific length of RNA (siRNA k-mer) to be hybridized with potential splice variant mRNA sequences, DICER activity, and siRNA binding to mRNA.

[0068] In some embodiments, an expression region of an expression cassette comprises a native exon sequence. In some embodiments, an expression region comprises a consensus exon sequence. In some embodiments, an expression region comprises a single exon sequence. In some embodiments, an expression region comprises selected sequences from a native or consensus exon sequence. In some embodiments, an expression region comprises more than one exon sequence. In some embodiments, an expression region comprises a combination of native and consensus exon sequences. In some embodiments, an expression region comprises from 1 to about 50 exon sequences. In some embodiments, an expression region comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more exon regions. In some embodiments, an expression region is expressed as a single continuous dsRNA.Promoters

[0069] In some embodiments, an engineered microorganism described herein comprises a promoter. Promoters drive transcription of an expression cassette. In some embodiments, the promoter is a fungal promoter. In some embodiments, the promoter is a bacterial promoter. In some embodiments, expression of a coding gene is driven by a constitutive promoter. In some embodiments, expression of a coding gene is driven by an inducible promoter. In some embodiments, expression of a coding gene is driven by a single promoter. In some embodiments, expression of a coding gene is driven by a pair of promoters in opposite orientation on complementary strands (antisense). In some embodiments, expression regions on complementary strands of nucleic acid are arranged in a “head to head” configuration, and opposite strands are transcribed divergently. In some embodiments, the promoters are head to head promoters. In some embodiments, expression regions are transcribed using bidirectional promoters. In some embodiments, the promoter comprises Pural, Pcbhl, PceI7, Ppgll, Pamyl, Ptefl, PcDNAl, PgpdA, Prp2, U6, Peel, Pxyn, Ptcul, Pyatl, Pactin, PoliC, PtetR, Pxyl, or any combination thereof. In some embodiments, the promoter comprises PphoA, P43, P45, PT7, P34,PxylA, Physpank, Pspac, Pveg, PamyE, PgrD, PbacA, PcotC, Pspank, PnrpE, PamyP, PespsA, PSP6, PrrnB 1+2 WT, PrrnB 1+2 A starts, or any combination thereof. Sequences of representative promoters are listed in Table 2.Table 2. Exemplary Promoter sequences.Construct Design

[0070] Expression cassettes described herein comprise a genetic construct for transcription of a double-stranded RNA. In some embodiments, a genetic construct comprises a plurality of sequences encoding for one or more siRNA as described herein. In some embodiments, an engineeredmicroorganism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 25, about 30, about 40, or more expression cassettes.

[0071] In some embodiments, siRNA sequences encoded by a genetic construct target one gene in a target organism. In some embodiments, siRNA sequences encoded by a genetic construct target 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 25, about 30, about 40, or more genes in a target organism. In some embodiments, siRNA sequences encoded by a genetic construct target one or more genes in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 25, about 30, about 40, or more target organisms. In some embodiments, siRNA sequences encoded by a genetic construct target genes of a single species. In some embodiments, siRNA sequences encoded by a genetic construct target genes of different species. In some embodiments, siRNA sequences encoded by a genetic construct target genes of two species, three species, four species, five species, six species, seven species, eight species, nine species, ten species, or more.Long dsRNA

[0072] In some embodiments, an RNAi expression cassette comprises complementary sense and antisense strands encoding multiple siRNA fragments in a single long double-stranded RNA (dsRNA) molecule. An illustration of an exemplary cassette encoding a long dsRNA is shown in FIG. 2A. In some embodiments, two promoters as described herein with opposite orientations (antisense promoters) drive transcription of the genetic construct. In some embodiments, the antisense promoters comprise two promoters that are the same. In some embodiments, the antisense promoters comprise two promoters that are different. Transcription of the complementary strands generates a long dsRNA molecule.

[0073] In some embodiments, a long dsRNA described herein is from about 20 to about 5000 base pairs in length. In some embodiments, a long dsRNA described herein is about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2200, about 2400, about 2600, about 2800, about 3000, about 3400, about 3800, about 4000, about 4400, about 4600, about 4800, or about 5000 base pairs in length.Hairpin dsRNA

[0074] In some embodiments, the expression cassette encodes for a hairpin dsRNA. In some embodiments, the hairpin expression cassette comprises two complementary sequences in reverse orientation on a strand and a single promoter as illustrated in FIG. 2B and FIG. 2G, wherein the two complementary sequences hybridize to form a hairpin structure. In some embodiments, the twocomplementary sequences are separated by an intron region. In some embodiments, the transcribed intron region is cleaved after transcription. In some embodiments, the intron region is not cleaved after transcription. In some embodiments, the two complementary sequences are separated by a hairpin region. In some embodiments, each complementary sequence is about 20 to about 5000 nucleotides in length. In some embodiments, each complementary sequence is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900 about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2200, about 2400, about 2600, about 2800, about 3000, about 3400, about 3800, about 4000, about 4400, about 4600, about 4800, or about 5000 nucleotides in length. In some embodiments, the intron region is from about 50 nucleotides to about 400 nucleotides in length. In some embodiments, the intron region is about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length. Exemplary intron and hairpin loop sequences are provided in Table 3.Table 3: Exemplary intron and hairpin loop sequencessiRNA

[0075] In some embodiments, an expression cassette comprises complementary sense and antisense strands encoding a single siRNA 18-31 nucleotides in length as described herein and illustrated in FIG. 2C. In some embodiments, two promoters as described herein with opposite orientations (antisense promoters) drive transcription of the genetic construct. In some embodiments, the antisense promoters comprise two promoters that are the same. In some embodiments, the antisense promoters comprise two promoters that are different. Transcription of the complementary strands generates an siRNA molecule. In some embodiments, an engineered microorganism comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 25, about 30, about 40, or more expression cassettes, each comprising complementary sense and antisense strands encoding a single siRNA 18-31 nucleotides in length.DICER processing

[0076] DICER, also known as endoribonuclease Dicer or helicase with RNase motif, is an endonuclease involved in miRNA and siRNA production. DICER works with the RNA-induced silencing complex (RISC) to degrade messenger RNA (mRNA) and suppress, inhibit, or silence, gene expression. For the purposes of this disclosure, “DICER” refers to any ribonuclease found in a plant, animal, insect, fungal, or oomycete cell, which exhibits DICER-like activity, including RNAse III and other RNA-cleaving enzymes.

[0077] DICER processing cleaves measured lengths of dsRNA from the 5’ and 3’ ends to generate RNA fragments 18-31 nucleotides in length. Modulation of factors to affect the initial DICER cleavage from the 5’ and 3’ strand ends and determined fragment length provides for one or more cleavage motifs. In some embodiments, a dsRNA comprises from 1 to about 5000 different cleavage motifs. In some embodiments, a dsRNA comprises from 1 to about 10, from about 10 to about 20, from about 20 to about 30, from about 30 to about 40, from about 40 to about 50, from about 50 to about 60, from about 60 to about 70, from about 70 to about 80, from about 80 to about 90, from about 90 to about 100, from about 100 to about 120, from about 120 to about 140, from about 140 to about160, from about 160 to about 180, from about 180 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 300, from about 300 to about 350, from about 350 to about 400, from about 400 to about 450, from about 450 to about 500, from about 500 to about 550, from about 550 to about 600, from about 600 to about 650, from about 650 to about 700, from about 700 to about 750, from about 750 to about 800, from about 800 to about 850, from about 850 to about 900, from about 900 to about 1000, from about 1000 to about 1200, from about 1200 to about 1400, from about 1400 to about 1800, from about 1800 to about 2000, from about 2000 to about 2500, from about 2500 to about 3000, from about 3000 to about 3500, from about 3500 to about 4000, from about 4000 to about 4500, from about 4500 to about 5000 different cleavage motifs. In some embodiments, the different cleavage motifs vary by cleavage start site. In some embodiments, the different cleavage motifs vary by RNA fragment length. In some embodiments, DICER cleavage generates RNA fragments 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length. In some embodiments, a distribution of DICER cleavage sites provides for different cleavage motifs. In some embodiments, DICER cleavage generates RNA fragments starting at the 5’ end, the 3’ end, or both. In some embodiments, DICER cleavage generates RNA fragments from 1 to about 31 nucleotides from the 5 ’ end, the 3 ’ end, or both. In some embodiments, DICER cleavage generates RNA fragments 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides from the 5’ end, the 3’ end, or both. In some embodiments, the different cleavage motifs vary by both cleavage start site and RNA fragment length.

[0078] In some embodiments, DICER cleavage according to different cleavage motifs generates a multiplicity of RNA fragments from a particular dsRNA. In some embodiments, a dsRNA comprises from about 1 to about 100 different cleavage motifs.

[0079] In some embodiments, a multiplexed RNAi is generated from multiple single RNAi expression cassettes. In some embodiments, DICER processing of a long dsRNA strand generates siRNA fragments as illustrated in FIG. 2D. In some embodiments, multiple RNAi expression cassettes are generated from a single operon. In some embodiments, RNAi expression cassettes are separated by 3’ UTR sequences. In some embodiments, DICER processing of a synthetic dsRNA strand generates siRNA fragments as illustrated in FIG. 2E. In some embodiments, a long dsRNA or synthetic dsRNA strand comprises one or more DICER cleavage sites. In some embodiments, a long dsRNA or syntheticdsRNA strand comprises more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 20, more than 30, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, more than 100, more than 150, 200, more than 250, more than 300, more than 350, more than 400, more than 450, more than 500, more than 550, more than 600, more than 650, more than 700, more than 750, more than 800, more than 850, more than 900, more than 950, more than 1000, more than 1500, more than 2000, more than 2500, more than 3000, more than 3500, more than 4000, more than 45000, or up to 5000 DICER cleavage sites.

[0080] In some embodiments, a dsRNA or synthetic RNA strand comprises about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, or about 5000 DICER cleavage sites.

[0081] In some embodiments, DICER processing provides for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 3500, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, 65,000 distinct RNA sequences 18-31 nucleotides in length from a dsRNA strand. In some embodiments, DICER processing provides for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 3500, about 4000, about 4500, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 15,000, about 20,000, about 25,000, about 30,000, about 35,000, about 40,000, about 45,000, about 50,000, about 55,000, about 60,000, about 65,000, about 70,000 or more distinct RNA sequences 18-31 nucleotides in length from a dsRNA strand.

[0082] In some embodiments, one or more distinct RNA sequences generated from DICER processing exhibit RNA interference (RNAi) activity. In some embodiments, RNAi activity comprises suppression of expression of a target gene. In some embodiments, distinct RNA sequences with RNAi activity are small interfering RNA (siRNA). In some embodiments, a plurality of distinct RNAsequences are siRNAs. In some embodiments, from about 0.1% to about 100% of generated RNA sequences are siRNAs. In some embodiments, about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of generated RNA sequences are siRNAs. In some embodiments, from about 0.1% to about 1%, from about 1% to about 10%, from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 70% to about 80%, from about 80% to about 90%, from about 90% to about 100% of generated RNA sequences are siRNAs. In some embodiments, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of generated RNA sequences are siRNAs.

[0083] In some embodiments, gene silencing activity is optimized by generating a multiplicity siRNA fragments from an exon region assessed for increased predicted siRNA activity. In some embodiments, each predicted siRNA k-mer is analyzed for off-target binding. In some embodiments, analysis is performed by aligning each k-mer against genomes and transcriptomes from the host, pathogen, rhizosphere microbiome, plant, human, other organisms, or any combination thereof to determine potential off-target binding.

[0084] In some embodiments, DICER processing occurs in the engineered microorganism. In some embodiments, a dsRNA is cleaved by DICER prior to release from the engineered microorganism comprising the expression cassette. In some embodiments, DICER processing occurs in a target organism cell. In some embodiments, DICER processing occurs in an environment external to the engineered microorganism or the target organism.

[0085] In some embodiments, the expression cassette encodes for one or more DICER modulating spacer regions (DMSR). DMSR are synthetic spacers that are poorly processed by DICER proteins while promoting the processing of a dsRNA. In some embodiments, an expression cassette encodes for at least one DMSR inserted between two siRNA regions. And exemplary schema illustrating alternating siRNA and DMSR regions is provided in FIG. 2F.III. Delivery Methods

[0086] Transcribed RNAs are released into the environment local to the engineered microorganism and passively or actively taken up by the target organism or host plant. In some embodiments, the expression product is released by secretion, cell lysis, or by incorporation in extracellular vesicles. Insome embodiments, an engineered microorganism natively produces extracellular vesicles. In some embodiments, an engineered microorganism is further engineered to produce extracellular vesicles. In some embodiments, an engineered microorganism expresses, or is engineered to express, vesicle nucleating peptide (VNp). In some embodiments, an engineered microorganism transfers transcribed RNAs directly into the target organism through parasitism or anastomosis, which may include hyphal fusion, appresorium formation, or a combination thereof.

[0087] Anastomosis is a type of connection between cells or organisms whereby materials can be shared between they cytoplasm of the two cells or organisms. In fungi, anastomosis is the fusion between two hyphae. In some embodiments, the hyphae are from the same organism. In some embodiments, the hyphae are from distinct organisms.

[0088] Transcribed RNA described herein is incorporated into the cell of a target organism or host plant by passive or active uptake. In some embodiments, passive uptake comprises free diffusion, facilitated diffusion, diffusion channels, or any combination thereof. In some embodiments, active uptake comprises receptor-mediated endocytosis, direct feeding, or any combination thereof.

[0089] In some embodiments, dsRNA is processed to siRNA after uptake into an organism. In some embodiments, siRNA is amplified by RNA-dependent RNA polymerases. In some embodiments, siRNA processed and / or amplified in a plant becomes systemically distributed throughout the plant.

[0090] Target organisms encounter siRNAs through active or passive uptake. In some embodiments, uptake is effected by plant-derived vesicles. In some embodiments, siRNAs delivered to the soil are taken up by other organisms in the local environment.IV. Target Organisms

[0091] In some embodiments, an siRNA and / or dsRNA generated by an engineered microorganism described herein targets one or more genes in one or more target organisms. In some embodiments, a target organism affects the growth or condition of a plant. In some embodiments, a target organism is a fungus, an oomycete, a nematode, a plant, an insect, a bacteria, or a virus. In some embodiments, the plant target organism is a monocot or a dicot.Fungi and Oomycetes

[0092] In some embodiments, a target organism described herein comprises a fungus or oomycete. A fungal or oomycete infection on a plant can inhibit normal growth (hypotrophy) or induce excessive abnormal growth (hypertrophy or hyperplasia). In some embodiments, hyperplasia appears as clubroot, galls, warts, or leaf curls. A fungal or oomycete infection can induce necrosis, locally orextensively. In some embodiments, necrosis in a plant appears as leaf spots, blight, scab, rots, damping-off, anthracnose, dieback, and canker. In some embodiments, a fungus or oomycete produces spores (conidia, sporangiospores, blastospores, arthrospores, chlamydospores, zoospores, ascospores, basidiospores, zygospores, oospores) or mycelia. In some embodiments, the target organism comprises a fungus or oomycete of a genus or species listed in Table 4.Table 4. Exemplary Fungal and Oomycete genera and species.

[0093] In exemplary embodiments, an engineered microorganism described herein generates siRNA targeting a fungal gene listed in Table 5. Genes listed in Table 5 are exemplary and not exclusive of genes targetable by the compositions and methods described herein.Table 5. Exemplary Fungal Target Genes.

[0094] In some embodiments, a generated dsRNA or siRNA targeting a fungal gene comprises one or more of the sequences listed in Table 6. Sequences listed in Table 6 are exemplary and not exclusive.Table 6. Exemplary Sequences Used for Designing dsRNAs Targeting Fungal Genes.Nematodes

[0095] In some embodiments, a target organism described herein comprises nematodes. Nematodes are thread-like roundworms that feed on fungi, bacteria, protozoans, other nematodes, and plants. A nematode infection in a plant can appear as yellowing, stunting, and wilting, along with yield decline. In some embodiments, the target organism comprises a nematode of a genus or species listed in Table 7.Table 7. Exemplary Nematode genera and species.

[0096] In exemplary embodiments, an engineered microorganism described herein generates siRNA targeting a nematode gene listed in Table 8. Genes listed in Table 8 are exemplary and not exclusive of genes targetable by the compositions and methods described herein.Table 8. Exemplary Nematode Target Genes.

[0097] In exemplary embodiments, sequences listed in Table 9 are used to design dsRNA used compositions and methods described herein. Sequences listed in Table 9 are exemplary and not exclusive of sequences targetable by the compositions and methods described herein.Table 9: Exemplary Sequences Used for Designing dsRNAs Targeting Nematode GenesPlants

[0098] Embodiments described herein are designed to affect a trait of a plant. In some embodiments, application of a composition described herein affects the growth, productivity, appearance, or another feature of a plant. In some embodiments, the affect is an herbicidal effect. In some embodiments, the plant is a weed, an invasive species, or another undesirable organism. In some embodiments, application of a composition described herein causes limited, reduced, prevented, or stopped growth of a plant. In some embodiments, application of a composition described herein causes death of a plant. In some embodiments, the plant is a crop plant, an ornamental plant, or another desirable plant. In embodiments, application of a composition described herein increases or improves an appearance, a productivity, or another aspect of a plant.

[0099] In some embodiments, a plant is an intermediary in delivery to a target organism. In some embodiments, a plant uptakes a dsRNA or siRNA that targets a sequence in another organism. In some embodiments, dsRNA is processed to siRNA after uptake in a plant.

[0100] RNAi systems described herein provide for siRNA delivery to a plant to provide for silencing of life-critical or trait-affecting genes. In some embodiments, the plant is a monocot or a dicot. In some embodiments, an RNAi system described herein provides for attenuation or killing of a weed or invasive plant. In some embodiments, an herbicidal siRNA targets a gene provided in Table 10.Table 10. Exemplary Herbicidal Target Genes.

[0101] In some embodiments, an RNAi system described herein provides for modulation of a gene affecting a trait of a crop plant In some embodiments, the crop is a food crop, a feed or forage crop, a fiber crop, an oil crop an ornamental crop, an industrial crop, or any combination thereof. In some embodiments, the target is a gene listed in Table 11.Table 11. Exemplary crop plant target genes.Insects

[0102] Insects can cause detriment to plants by direct damage, for example feeding on plant tissue or sap, or injecting a toxin when feeding. They also foster conditions promoting growth of other plant pathogens such as bacteria or fungi. Additionally, insects can carry diseases, transmitting pathogenic organisms throughout a population of plants. In some embodiments, siRNA described herein target genes in insect species listed in Table 12.Table 12. Exemplary Insect species

[0103] In exemplary embodiments, an engineered microorganism described herein generates siRNA targeting an insect gene listed in Table 13. Genes listed in Table 13 are exemplary and not exclusive of genes targetable by the RNAi systems described herein.Table 13. Exemplary Insect Target Genes.Bacteria

[0104] In some embodiments, a target organism described herein comprises a bacteria. Bacterial infections in plants can cause symptoms similar to fungal infections. In some embodiments, symptoms of a bacterial infection include leaf spots, wilts, scabs, cankers and rots of roots and fruit. In some embodiments, the target organism comprises a bacteria of a genus or species listed in Table 14.Table 14. Exemplary Bacterial species

[0105] In exemplary embodiments, an engineered microorganism described herein generates siRNA targeting a bacterial gene listed in Table 15. Genes listed in Table 15 are exemplary and not exclusive of genes targetable by the compositions and methods described herein.Table 15. Exemplary Bacterial Target Genes.Virus

[0106] In some embodiments, a target organism described herein comprises a virus. In some embodiments, the virus is a virus that infects plants (“plant virus”). Plant viruses are less studied than animal viruses. However, plant viruses, such as tobacco mosaic virus, cause about US$60 billion loss in crop yields worldwide per year. In some embodiments, the target organism comprises a virus of a genus listed in Table 16.Table 16. Exemplary Viral generaV. Methods of Use

[0107] Engineered microorganisms described herein are applied, in some embodiments, to a plant, to a soil, or to a seed. In some embodiments, a spore from an engineered microorganism is applied. In some embodiments, the methods of application include a foliar spray, irrigation, a seed treatment, a wipe, a root drench, a needle inoculation, a syringe infiltration, a mist, a fog, a spray, or an injection. Methods of application to a soil include a root drench or a soil amendment. Methods of application to a seed include a seed coating. In some embodiments, the application is pre-emergent to the plant growth. In some embodiments, the application is post-emergent to the plant growth.

[0108] Compositions and methods described herein can be used to improve production or growth of a plant. In some embodiments, compositions or methods described herein treat an established disease in a plant. In some embodiments, compositions or methods described herein prevent a disease in a plant. In some embodiments, compositions or methods described herein kill a target organism. In some embodiments, compositions or methods described herein, prevent growth of a target organism. In some embodiments, compositions or methods described herein provide for increased or comparable killing or growth prevention of a target organism relative to other treatment modalities. In some embodiments, the other treatment modalities comprise Actigard®, copper, Kocide® 3000, Contans®, Saltro®, Tymirium®, Ilevo®, Acceleron®, CruiserMaxx®, Avicta Complete®, N-Hibit®, Votivo®, Clariva Complete®, ILevo®, Nemastrike Technology®, Aveo®, BioWorks RootShield®, Serenade®, TerraClean 5®, Metam CLR®, Pic-Clor 60 EC®, Paladin Pic-21®, Captan®, Rally®, Thiram®, Merivon®, MOVENTO®, Transform WG®, Delegate WG®, ROUNDUP POWERMAX 3®, Enlist Duo®, Trivence®, antibiotics, a plant immune activator, or natural microbes. In some embodiments, compositions or methods described herein are used in conjunction with other treatment modalities. In some embodiments, use of compositions and methods described herein provides forreduced use of other treatments, such as pesticides, insecticides, herbicides, fungicides, bactericides, or any other treatment. In some embodiments, compositions and methods described herein can be used as a biostimulant, providing for increased growth or production of a plant. In some embodiments, compositions and methods described herein provide factors for increased tolerance to environmental stress in a plant. In some embodiments, compositions and methods described herein provide factors for increased drought tolerance in plants. In some embodiments, compositions and methods described herein provide factors for increased heat tolerance in plants. In some embodiments, compositions and methods described herein provide factors for increased cold tolerance in plants. In some embodiments, compositions and methods described herein provide factors for increased flood tolerance in plants. In some embodiments, compositions and methods described herein provide factors for increased salinity tolerance in plants. In some embodiments, a composition provides for root signal recognition, stomatai closure, signal transport, osmotic adjustment, increased root growth, increased absorption area, or reduced transpiration area.VI. Production

[0109] In some embodiments, expression cassettes are generated comprising one or more promoters and expression regions as described herein. Cassettes are inserted either episomally or into the genome of the engineered microorganisms leveraging either random chromosomal integration or site-specific recombination.

[0110] Engineered microorganisms as described herein can be produced in liquid culture or solid state culture. In some embodiments, a liquid culture is grown in a fermentor. A fermentor is also called a bioreactor. In some embodiments, a fermentor is an airlift fermentor, a continuous stirred tank fermentor, a packed bed fermentor, a fluidized bed fermentor, a continuous flow fermentor, or a bubble column fermentor. In some embodiments, the engineered microorganism is grown in solid state culture. In some embodiments, a solid state culture is grown in bags, temperature and humidity controlled trays, a rotating drum fermenter, a packed bed fermenter, or a solid state fermenter. The substrate for solid state growth includes but is not limited to wheat, wheat bran, rice, rice husk, rice bran, buckwheat seeds, oats, refused potatoes, soybean flour, cassava flour, sesame seeds, linseed meal, cottonseed meal, sorghum seeds, corn husks, and soybean hulls. In some embodiments, the engineered microorganism culture is further processed. In some embodiments, the engineered microorganism is washed, lyophilized, spray dried, dried, concentrated, frozen, milled, granulated, thickened, or gelled.Formulation[OHl] Formulations of compositions described herein are provided in forms for improved delivery, accuracy of delivery, or reduced off-target delivery. In some embodiments, spores, endospores, mycelia, hyphae, vegetative cells or any other form of an engineered microorganism are generated for formulation in a composition. In some embodiments, the formulations of compositions described herein are provided in forms for improved microbial stabilization. In some embodiments, the formulations of compositions described herein are provided in forms for improved cell viability. In some embodiments, the formulations of compositions described herein are provided in forms for improved wettability. In some embodiments, the formulations of compositions described herein are provided in forms for improved flowability of seeds. In some embodiments, the formulations of compositions described herein are provided in forms for improved engineered microorganism colonization. In some embodiments, a composition described herein can be provided as an emulsion, a colloid, a dust, a granule, a pellet, a powder, a liquid, a spray, a mist, a gel, a paste, a fog, or a solution.

[0112] In some embodiments, formulations of compositions described herein provide for maintenance of the engineered microorganism in the plant environment. In some embodiments, the engineered microorganism is maintained for a week, a month, 6 months, or a year after inoculation. In some embodiments, modifications to the engineered microorganisms and compositions described herein provide for increased duration of colonization compared to wild type microorganisms. In some embodiments, a composition comprises a formulation for increased duration of colonization. In some embodiments, modifications to the engineered microorganisms and compositions described herein provide for increased colonization abundance compared to wild type microorganisms. In some embodiments, a composition comprises a formulation for increased colonization abundance.

[0113] In some embodiments, compositions described herein further comprise an adjuvant. An adjuvant is a substance added to enhance the performance and / or physical properties of the composition. In some embodiments, an adjuvant comprises a surfactant, an oil, a compatibility agent, a stability agent, a buffering agent, a conditioning agent, a defoaming agent, a deposition agent, a drift control agent, a binding agent, a flowability agent, a granulation agent, a UV protectant, an osmotic protectant, nutrients, or a thickener. In some embodiments, an adjuvant comprises Tween 20, Triton X-100, Silwet L77, sucrose, dextrose, lactose, mannitol, maltodextrin, magnesium chloride, magnesium stearate, sodium benzoate, sodium bicarbonate, sodium phosphate, sodium ascorbate, seed coating polymer, agar, glycerol, sodium alginate, calcium chloride, ammonium sulfate, ammoniumphosphate, polyacrylic acid, citric acid, polyvinyl alcohol, lecithin, cellulose, microcrystalline cellulose, carboxymethylcellulose, chitosan, starch, titanium dioxide, talcum powder, soybean oil, xanthan gum, molasses, peptone, clay, or a combination thereof. In some embodiments, the composition comprises from about 0.01% to about 10% sodium alginate by volume. In some embodiments, the composition comprises about 0.01%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 5%, or about 10% sodium alginate by volume. In some embodiments, the composition comprises magnesium chloride. In some embodiments, the composition comprises from about 0.01% to about 50% seed coating polymer by volume. In some embodiments, the composition comprises about 0.01%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% seed coating polymer by volume. In some embodiments, the seed coating polymer is Flo Rite® Pro 02 Plantability Polymer. In some embodiments, the composition comprises from about ImM to about IM magnesium chloride. In some embodiments, the composition comprises about ImM, about lOmM, about 20mM, about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about lOOmM, about 200mM, about 300mM, about 400mM, about 500mM, about 600mM, about 700mM, about 800mM, about 900mM, or about IM magnesium chloride. In some embodiments, the magnesium chloride is in a solution before drying the composition. In some embodiments, the composition comprises Tween 20. In some embodiments, the composition comprises from about 0.01% to about 10% Tween 20 by volume. In some embodiments, the composition comprises about 0.01%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 5%, or about 10% Tween 20 by volume. In some embodiments, the composition comprises Triton X-100. In some embodiments, the composition comprises from about 0.01% to about 10% Triton X-100 by volume. In some embodiments, the composition comprises about 0.01%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 5%, or about 10 % Triton X-100 by volume. In some embodiments, the composition comprises SilwetL77. In some embodiments, the composition comprises from about 0.002% to about 2% Silwet L77 by volume. In some embodiments, the composition comprises about 0.002%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.1%, about 0.5%, about 1%, or about 2% Silwet L77.Dose

[0114] Compositions described herein are applied at a dose effective to suppress or reduce growth of a target organism. In some embodiments, the engineered microorganism is applied at a ratio of CFU engineered microorganism vs CFU target organism. In some embodiments, the application comprises 1000-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 100-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises50-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 40-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 30-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 20-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 10-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 5-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 1-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 0.5-fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 0.1- fold more engineered microorganisms than target organisms in the plant or plant environment. In some embodiments, the application comprises 0.01 -fold more engineered microorganisms than target organisms in the plant or plant environment.

[0115] In some embodiments, compositions of engineered microorganisms described herein are applied in an amount of total CFU per mL liquid media. In some embodiments, engineered microorganisms are applied at 10A2 CFU / mL liquid media. In some embodiments, engineered microorganisms are applied at 10A3 CFU / mL liquid media. In some embodiments, engineered microorganisms are applied at 10A4 CFU / mL liquid media. In some embodiments, engineered microorganisms are applied at 10A5 CFU / mL liquid media. In some embodiments, engineered microorganisms are applied at 10A6 CFU / mL liquid media. In some embodiments, engineered microorganisms are applied at 10A7 CFU / mL liquid media. In some embodiments, engineered microorganisms are applied at 10A8 CFU / mL liquid media. In some embodiments, engineeredmicroorganisms are applied at 10A9 CFU / mL liquid media. In some embodiments, engineered microorganisms are applied at 10Al 0 CFU / mL liquid media.

[0116] In some embodiments, compositions of engineered microorganisms described herein are applied in an amount of total CFU per unit of soil area. In some embodiments, engineered microorganisms are applied at 10A2 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A3 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A4 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A5 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A6 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A7 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A8 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A9 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A10 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10Al 1 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A12 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10Al 3 CFU per 1,000 square feet of soil area. In some embodiments, engineered microorganisms are applied at 10A14 CFU per 1,000 square feet of soil area.

[0117] In some embodiments, compositions of engineered microorganisms described herein are in an amount of total CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A2 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A3 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A4 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are prepared at 10A5 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are prepared at 10A6 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A7 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A8 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A9 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10Al 0 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10Al 1 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A12 CFU per 100 gallons of water applied. In someembodiments, engineered microorganisms are at 10Al 3 CFU per 100 gallons of water applied. In some embodiments, engineered microorganisms are at 10A14 CFU per 100 gallons of water applied.

[0118] In some embodiments, compositions of engineered microorganisms described herein are applied in an amount of total CFU per unit of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A2 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A3 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A4 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A5 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A6 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A7 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A8 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A9 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A10 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10Al l CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A12 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A13 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces. In some embodiments, engineered microorganisms are applied at 10A14 CFU per 100 pounds of bulbs, seeds, seed tubers or cut seed pieces.EXEMPLARY EMBODIMENTS

[0119] Provided herein are engineered microorganisms comprising at least one dsRNA expression cassette, wherein the dsRNA expression cassette is integrated in a genome of the engineered microorganism, and wherein the at least one dsRNA expression cassette comprises: an expression region comprising a complementary pair of sequences encoding for a dsRNA about 250-700 basepairs in length and comprising a plurality of small interfering RNAs (siRNAs), each 19-23 nucleotides in length, wherein one of the sequences of the complementary pair of sequences comprises a sequence of at least one region of at least one target gene in at least one target organism, and wherein the siRNAs suppress expression of the at least one target gene; and two antisense promoters flanking the sequencesencoding the dsRNA, wherein the promoters are in opposite orientation to each other. Provided herein are engineered microorganisms, wherein the genome of the engineered microorganism comprises from 1 to 10 dsRNA expression cassettes. Provided herein are engineered microorganisms, wherein the region of the at least one target gene is an exon region. Provided herein are engineered microorganisms, wherein the siRNA suppresses expression of 2 to 20 target genes. Provided herein are engineered microorganisms, wherein the target genes are in more than one target organism. Provided herein are engineered microorganisms, wherein the target genes are in 2 to 20 target organisms. Provided herein are engineered microorganisms, wherein the siRNA comprises a synthesized sequence. Provided herein are engineered microorganisms, wherein the synthesized sequence is generated based on factors comprising consensus splice variant target sequence, DICER activity sites, thermodynamics of siRNA binding to mRNA, and any combination thereof. Provided herein are engineered microorganisms, wherein the dsRNA comprises a plurality of DICER cleavage sites. Provided herein are engineered microorganisms, wherein the dsRNA comprises from 1 to 700 DICER cleavage sites. Provided herein are engineered microorganisms, wherein a distribution of DICER cleavage sites provides for more than one cleavage motif to generate the plurality of siRNAs 19-23 nucleotides in length. Provided herein are engineered microorganisms, wherein the more than one cleavage motif comprises from 1 to 40 cleavage motifs. Provided herein are engineered microorganisms, wherein the from 1 to 40 cleavage motifs provide for from 1 to 4000 distinct RNA sequences 19-23 nucleotides in length. Provided herein are engineered microorganisms, wherein a plurality of distinct RNA sequences are siRNAs. Provided herein are engineered microorganisms, wherein from about 1% to about 100% of the distinct RNA sequences are siRNAs. Provided herein are engineered microorganisms, wherein the siRNAs suppress from about 1% to about 100% expression of a target gene. Provided herein are engineered microorganisms, wherein the dsRNA comprises a plurality of DICER modulating spacer regions (DMSR). Provided herein are engineered microorganisms, wherein the sequences encoding the dsRNA further comprise high-affinity DICER cut sites distinct from the DMSR. Provided herein are engineered microorganisms, wherein the engineered microorganism exhibits a characteristic comprising my coparasitism, biofilm formation, swarming, secretion, production of extracellular vesicles, or expression of natural antifungal or antibacterial defense toxic compounds or peptides. Provided herein are engineered microorganisms, wherein the engineered microorganism is further modified to exhibit the characteristic. Provided herein are engineered microorganisms, wherein the engineered microorganism further expresses Vesicle Nucleating peptide (NVp). Provided herein are engineered microorganisms, wherein the targetorganism is a fungus, an oomycete, a nematode, a plant, an insect, a bacteria, or a virus. Provided herein are engineered microorganisms, wherein the target organism is the fungus or the oomycete. Provided herein are engineered microorganisms, wherein the fungus or the oomycete is a Blumeria spp., Botrytis spp., Colletotrichum spp., Fusarium spp., Macrophomina spp., Mycosphaerella spp., Phakopsora spp., Phytophthora spp., Puccinia spp., Pyricularia spp., Pythium spp., Rhizoctonia spp., Sclerotinia spp., Sclerotium spp., or Verticillium spp. Provided herein are engineered microorganisms, wherein the at least one target gene comprises CDC2, CDC28, SIR2, RAD52, POL3, SEC14, H0G1, GAPDH, RPL, FMK1, PMT2, CYP51, HSP90, SOD1, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is the nematode. Provided herein are engineered microorganisms, wherein the at least one target gene comprises Gr-EXPB2, Mi-SXPl, Mi- CRT, Hg30C02, Hsl0A06, Mj-col-45, Mj-col-13, Hg-cpa-6, CHS 1 & 2, Mj-ndk-1 (nucleoside diphosphate kinase), Hg-acs-1 (acyl-CoA synthetase), Mj-egl-30 (Ras-like GTPase), Hg-kin-1 (serine / threonine-protein kinase), Mj-crt, Mj-ral-2, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is the plant. Provided herein are engineered microorganisms, wherein the at least one target gene comprises a gene encoding 5- enolpyruvylshikimate-3 -phosphate synthase (EPSPS), Acetolactate synthase (ALS), PPO (Protoporphyrinogen oxidase), ACCase (Acetyl-CoA carboxylase), Photosystem II (PSII) reaction center proteins (DI, D2), ALS (Alpha- and beta-tubulin), HPPD (4-hydroxyphenylpyruvate dioxygenase), HPPD (4-hydroxyphenylpyruvate dioxygenase), GS (Glutamine synthetase), ALS (ALS1 and ALS2, Arabidopsis thaliana homologs), AHAS (Acetohydroxyacid synthase), GAT (Glutamate- 1 -semialdehyde aminotransferase), RUBISCO (Ribulose-l,5-bisphosphate carboxylase / oxygenase), PPGPP (Guanosine pentaphosphate synthetase), ATPase (Adenosine triphosphatase), MVA pathway genes (Mevalonate pathway), ADC (Arginine decarboxylase), DHPS (Dihydropteroate synthase), GTPCH (GTP cyclohydrolase I), GST (Glutathione S-transferase), ABC transporters (ATP -binding cassette transporters), Cytochrome P450 monooxygenases, CYP81A (Cytochrome P450 81 A subfamily), or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is the insect. Provided herein are engineered microorganisms, wherein the at least one target gene comprises a gene encoding Chitin Synthase (CHS), Vitellogenin (Vg), Hexamerin, Juvenile Hormone Esterase (JHE), Acetylcholinesterase (AChE), Arginine Kinase, Cathepsin L, Trehalose-6-phosphate synthase (TPS), Snf7, Cullin-1, or Rabl l, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is found in a phyllosphere, a rhizosphere, soil, a root system, a shoot system, a flowers,a leaf, a fruit, a stem, a root, or any combination thereof, of a plant. Provided herein are engineered microorganisms, wherein the engineered microorganism is natively co-located with the target organism in the phyllosphere, the rhizosphere, the root system, soil, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant. Provided herein are engineered microorganisms, wherein the engineered microorganism is further modified to grow in the phyllosphere, the rhizosphere, the root system, soil, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant, and wherein the engineered microorganism is co-located with the target organism. Provided herein are engineered microorganisms, wherein the engineered microorganism is a spore-forming microorganism. Provided herein are engineered microorganisms, wherein the engineered microorganism is a fungus. Provided herein are engineered microorganisms, wherein the fungus is a Trichoderma spp. Provided herein are engineered microorganisms, wherein the antisense promoters each comprise a strong constitutive or inducible promoter. Provided herein are engineered microorganisms, wherein the antisense promoters each comprise independently Pural, Pcbhl, PceI7, Ppgll, Pamyl, Ptefl, PcDNAl, PgpdA, Prp2, U6, Peel, Pxyn, Ptcul, Pyatl, Pactin, or any combination thereof. Provided herein are engineered microorganisms, wherein the engineered microorganism is a bacteria. Provided herein are engineered microorganisms, wherein the bacteria is a Bacillus spp. Provided herein are engineered microorganisms, wherein the antisense promoters each comprise a strong constitutive or inducible promoter. Provided herein are engineered microorganisms, wherein the antisense promoters each comprise independently PphoA, P43, P45, PT7, P34, PxylA, Phy spank, Pspac, Pveg, PamyE, PphoA, P43, P45, PgrD, PbacA, PcotC, Pspank, PnrpE, PamyP, PespsA, or any combination thereof. Provided herein are engineered microorganisms, wherein the at least one target gene comprises a gene that affects a plant trait. Provided herein are engineered microorganisms, wherein the at least one target gene comprises GmFAD3, El, ZmMADSl, GmMIPSl, ZLKR, SDH, ZmBCH2, ghSAD-1, ghFAD2- 1, ANR, FaMYBl, TFL1, plC, FaPGl, FaWRKY29, FaWRKY64, AGPase, LsNCED4, or any combination thereof.

[0120] Provided herein are engineered microorganisms comprising at least one dsRNA expression cassette encoding for a hairpin dsRNA comprising a plurality of small interfering RNAs (siRNAs) each 19-23 nucleotides in length, wherein the dsRNA expression cassette is integrated in a genome of the engineered microorganism and wherein the at least one dsRNA expression cassette comprises a sequence comprising: a promoter; a first coding sequence about 250-700 nucleotides in length comprising a sequence of at least one region of at least one target gene in at least one target organism;a second coding sequence, wherein the second sequence is the reverse complement of the first sequence; and an intron region separating the first coding region and the second coding region about 50-400 nucleotides in length; wherein the siRNAs suppress expression of the at least one target gene. Provided herein are engineered microorganisms, wherein the genome of the engineered microorganism comprises from 1 to 10 dsRNA expression cassettes. Provided herein are engineered microorganisms, wherein the region of the at least one target gene is an exon region. Provided herein are engineered microorganisms, wherein the siRNA suppresses expression of 2 to 20 target genes. Provided herein are engineered microorganisms, wherein the target genes are in more than one target organism. Provided herein are engineered microorganisms, wherein the target genes are in 2 to 20 target organisms. Provided herein are engineered microorganisms, wherein the siRNA comprises a synthesized sequence. Provided herein are engineered microorganisms, wherein the synthesized sequence is generated based on factors comprising consensus splice variant target sequence, DICER activity sites, thermodynamics of siRNA binding to mRNA, and any combination thereof. Provided herein are engineered microorganisms, wherein the dsRNA comprises a plurality of DICER cleavage sites. Provided herein are engineered microorganisms, wherein the dsRNA comprises from 1 to 700 DICER cleavage sites. Provided herein are engineered microorganisms, wherein a distribution of DICER cleavage sites provides more than one cleavage motif to generate the plurality of siRNAs 19- 23 nucleotides in length. Provided herein are engineered microorganisms, wherein the more than one cleavage motif comprises from 1 to 40 cleavage motifs. Provided herein are engineered microorganisms, wherein the from 1 to 40 cleavage motifs provide for from 1 to 4000 distinct RNA sequences 19-23 nucleotides in length. Provided herein are engineered microorganisms, wherein a plurality of distinct RNA sequences are siRNAs. Provided herein are engineered microorganisms, wherein from about 1% to about 100% of the distinct RNA sequences are siRNAs. Provided herein are engineered microorganisms, wherein the siRNAs suppress from about 1% to about 100% expression of a target gene. Provided herein are engineered microorganisms, wherein the dsRNA comprises a plurality of DICER modulating spacer regions (DMSR). Provided herein are engineered microorganisms, wherein the sequence encoding the dsRNA further comprises high-affinity DICER cut sites distinct from the DMSR. Provided herein are engineered microorganisms, wherein the engineered microorganism exhibits a characteristic comprising my coparasitism, biofilm formation, swarming, secretion, production of extracellular vesicles, or expression of natural antifungal or antibacterial defense toxic compounds or peptides. Provided herein are engineered microorganisms, wherein the engineered microorganism is further modified to exhibit the characteristic. Providedherein are engineered microorganisms, wherein the engineered microorganism further expresses Vesicle Nucleating peptide (NVp). Provided herein are engineered microorganisms, wherein the target organism is a fungus, an oomycete, a nematode, a plant, an insect, a bacteria, or a virus. Provided herein are engineered microorganisms, wherein the target organism is the fungus or the oomycete. Provided herein are engineered microorganisms, wherein the fungus or the oomycete is a Blumeria spp., Botrytis spp., Colletotrichum spp., Fusarium spp., Macrophomina spp., Mycosphaerella spp., Phakopsora spp., Phytophthora spp., Puccinia spp., Pyricularia spp., Pythium spp., Rhizoctonia spp., Sclerotinia spp., Sclerotium spp., or Verticillium spp. Provided herein are engineered microorganisms, wherein the at least one target gene comprises CDC2, CDC28, SIR2, RAD52, POL3, SEC14, H0G1, GAPDH, RPL, FMK1, PMT2, CYP51, HSP90, SOD1, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is the nematode. Provided herein are engineered microorganisms, wherein the at least one target gene comprises Gr-EXPB2, Mi-SXPl, Mi- CRT, Hg30C02, Hsl0A06, Mj-col-45, Mj-col-13, Hg-cpa-6, CHS 1 & 2, Mj-ndk-1 (nucleoside diphosphate kinase), Hg-acs-1 (acyl-CoA synthetase), Mj-egl-30 (Ras-like GTPase), Hg-kin-1 (serine / threonine-protein kinase), Mj-crt, Mj-ral-2 or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is the plant. Provided herein are engineered microorganisms, wherein the at least one target gene comprises a gene encoding 5- enolpyruvylshikimate-3 -phosphate synthase (EPSPS), Acetolactate synthase (ALS), PPO (Protoporphyrinogen oxidase), ACCase (Acetyl-CoA carboxylase), Photosystem II (PSII) reaction center proteins (DI, D2), ALS (Alpha- and beta-tubulin), HPPD (4-hydroxyphenylpyruvate dioxygenase), HPPD (4-hydroxyphenylpyruvate dioxygenase), GS (Glutamine synthetase), ALS (ALS1 and ALS2, Arabidopsis thaliana homologs), AHAS (Acetohydroxyacid synthase), GAT (Glutamate- 1 -semialdehyde aminotransferase), RUBISCO (Ribulose-l,5-bisphosphate carboxylase / oxygenase), PPGPP (Guanosine pentaphosphate synthetase), ATPase (Adenosine triphosphatase), MVA pathway genes (Mevalonate pathway), ADC (Arginine decarboxylase), DHPS (Dihydropteroate synthase), GTPCH (GTP cyclohydrolase I), GST (Glutathione S-transferase), ABC transporters (ATP -binding cassette transporters), Cytochrome P450 monooxygenases, CYP81A (Cytochrome P450 81 A subfamily), or any combination thereof Provided herein are engineered microorganisms, wherein the target organism is the insect. Provided herein are engineered microorganisms, wherein the at least one target gene comprises a gene encoding Chitin Synthase (CHS), Vitellogenin (Vg), Hexamerin, Juvenile Hormone Esterase (JHE), Acetylcholinesterase (AChE), Arginine Kinase, Cathepsin L, Trehalose-6-phosphate synthase (TPS), Snf7, Cullin-1, orRabl l, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is found in a phyllosphere, a rhizosphere, a root system, a shoot system, a flowers, a leaf, a fruit, a stem, a root, or any combination thereof, of a plant. Provided herein are engineered microorganisms, wherein the engineered microorganism is natively co-located with the target organism in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant. Provided herein are engineered microorganisms, wherein the engineered microorganism is further modified to grow in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant, wherein the engineered microorganism is colocated with the target organism. Provided herein are engineered microorganisms, wherein the engineered microorganism is a spore-forming microorganism. Provided herein are engineered microorganisms, wherein the engineered microorganism is a fungus. Provided herein are engineered microorganisms, wherein the fungus is a Trichoderma spp. Provided herein are engineered microorganisms, wherein the promoter comprises a strong constitutive or inducible promoter. Provided herein are engineered microorganisms, wherein the promoter comprises Pural, Pcbhl, PceI7, Ppgll, Pamyl, Ptefl, PcDNAl, PgpdA, Prp2, U6, Peel, Pxyn, Ptcul, Pyatl, Pactin, or any combination thereof. Provided herein are engineered microorganisms, wherein the engineered microorganism is a bacteria. Provided herein are engineered microorganisms, wherein the bacteria is a Bacillus spp. Provided herein are engineered microorganisms, wherein the promoter comprises a strong constitutive or inducible promoter. Provided herein are engineered microorganisms, wherein the promoter comprises independently PphoA, P43, P45, PT7, P34, PxylA, Physpank, Pspac, Pveg, PamyE, PphoA, P43, P45, PgrD, PbacA, PcotC, Pspank, PnrpE, PamyP, PespsA, or any combination thereof. Provided herein are engineered microorganisms, wherein the at least one target gene is a gene that affects a plant trait. Provided herein are engineered microorganisms, wherein the at least one target gene comprises GmFAD3, El, ZmMADSl, GmMIPSl, ZLKR, SDH, ZmBCH2, ghSAD-1, ghFAD2-l, ANR, FaMYBl, TFL1, plC, FaPGl, FaWRKY29, FaWRKY64, AGPase, LsNCED4, or any combination thereof.

[0121] Provided herein are engineered microorganisms, wherein the engineered microorganism comprises a plurality of dsRNA expression cassettes integrated in a genome of the engineered microorganism, wherein each dsRNA expression cassette comprises: a complementary pair of sequences encoding a small interfering RNA (siRNA) 19-23 nucleotides in length, wherein the siRNA comprises a sequence complementary to a region of a target gene in a target organism, and whereinthe siRNA suppresses expression of the target gene; and two antisense promoters flanking the sequences encoding the siRNA, wherein the promoters are in opposite orientation to each other. Provided herein are engineered microorganisms, wherein the engineered microorganism comprises 2 to 20 genomic insertions. Provided herein are engineered microorganisms, wherein the region of the target gene is an exon region. Provided herein are engineered microorganisms, wherein the siRNA suppresses expression of 2 to 20 target genes. Provided herein are engineered microorganisms, wherein the siRNAs target genes in more than one target organism. Provided herein are engineered microorganisms, wherein the target genes are in 2 to 20 target organisms. Provided herein are engineered microorganisms, wherein the siRNA comprises a synthesized sequence. Provided herein are engineered microorganisms, wherein the synthesized sequence is generated based on factors comprising consensus splice variant target sequence, thermodynamics of siRNA binding to mRNA, and any combination thereof. Provided herein are engineered microorganisms, wherein the engineered microorganism exhibits a characteristic comprising my coparasitism, biofilm formation, swarming, secretion, production of extracellular vesicles, or expression of natural antifungal or antibacterial defense toxic compounds or peptides. Provided herein are engineered microorganisms, wherein the engineered microorganism is further modified to exhibit the characteristic. Provided herein are engineered microorganisms, wherein the engineered microorganism further expresses Vesicle Nucleating peptide (NVp). Provided herein are engineered microorganisms, wherein the target organism is a fungus, an oomycete, a nematode, a plant, an insect, a bacteria, or a virus. Provided herein are engineered microorganisms, wherein the target organism is the fungus or the oomycete. Provided herein are engineered microorganisms, wherein the fungus or the oomycete is a Bhimerici spp., Botrytis spp., Colletotrichum spp., Fusarium spp., Macrophomina spp., Mycosphaerella spp., Phakopsora spp., Phytophthora spp., Puccinia spp., Pyricularia spp., Pythium spp., Rhizoctonia spp., Sclerotinia spp., Sclerotium spp., or Verticillium spp. Provided herein are engineered microorganisms, wherein the at least one target gene comprises CDC2, CDC28, SIR2, RAD52, POL3, SEC14, HOG1, GAPDH, RPL, FMK1, PMT2, CYP51, HSP90, SOD1, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is the nematode. Provided herein are engineered microorganisms, wherein the at least one target gene comprises Gr-EXPB2, Mi-SXPl, Mi-CRT, Hg30C02, Hsl0A06, Mj-col-45, Mj-col-13, Hg-cpa-6, CHS 1 & 2, Mj-ndk-1 (nucleoside diphosphate kinase), Hg-acs-1 (acyl-CoA synthetase), Mj-egl-30 (Ras-like GTPase), Hg-kin-1 (serine / threonine-protein kinase), Mj-crt, Mj-ral-2, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is the plant. Provided herein are engineeredmicroorganisms, wherein the at least one target gene comprises a gene encoding 5- enolpyruvylshikimate-3 -phosphate synthase (EPSPS), Acetolactate synthase (ALS), PPO (Protoporphyrinogen oxidase), ACCase (Acetyl-CoA carboxylase), Photosystem II (PSII) reaction center proteins (DI, D2), ALS (Alpha- and beta-tubulin), HPPD (4-hydroxyphenylpyruvate dioxygenase), HPPD (4-hydroxyphenylpyruvate dioxygenase), GS (Glutamine synthetase), ALS (ALS1 and ALS2, Arabidopsis thaliana homologs), AHAS (Acetohydroxyacid synthase), GAT (Glutamate- 1 -semialdehyde aminotransferase), RUBISCO (Ribulose-l,5-bisphosphate carboxylase / oxygenase), PPGPP (Guanosine pentaphosphate synthetase), ATPase (Adenosine triphosphatase), MVA pathway genes (Mevalonate pathway), ADC (Arginine decarboxylase), DHPS (Dihydropteroate synthase), GTPCH (GTP cyclohydrolase I), GST (Glutathione S-transferase), ABC transporters (ATP -binding cassette transporters), Cytochrome P450 monooxygenases, CYP81A (Cytochrome P450 81 A subfamily), or any combination thereof Provided herein are engineered microorganisms, wherein the target organism is the insect. Provided herein are engineered microorganisms, wherein the at least one target gene comprises a gene encoding Chitin Synthase (CHS), Vitellogenin (Vg), Hexamerin, Juvenile Hormone Esterase (JHE), Acetylcholinesterase (AChE), Arginine Kinase, Cathepsin L, Trehalose-6-phosphate synthase (TPS), Snf7, Cullin-1, or Rabl l, or any combination thereof. Provided herein are engineered microorganisms, wherein the target organism is found in a phyllosphere, a rhizosphere, a root system, a shoot system, a flowers, a leaf, a fruit, a stem, a root, or any combination thereof, of a plant. Provided herein are engineered microorganisms, wherein the engineered microorganism is natively co-located with the target organism in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant. Provided herein are engineered microorganisms, wherein the engineered microorganism is further modified to grow in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant, wherein the modified microorganism is colocated with the target organism. Provided herein are engineered microorganisms, wherein the engineered microorganism is a spore-forming microorganism. Provided herein are engineered microorganisms, wherein the engineered microorganism is a fungus. Provided herein are engineered microorganisms, wherein the fungus is a Trichoderma spp. Provided herein are engineered microorganisms, wherein the antisense promoters each comprise a strong constitutive or inducible promoter. Provided herein are engineered microorganisms, wherein the antisense promoters each comprise independently Pural, Pcbhl, PceI7, Ppgll, Pamyl, Ptefl, PcDNAl, PgpdA, Prp2, U6, Peel,Pxyn, Ptcul, Pyatl, Pactin, or any combination thereof. Provided herein are engineered microorganisms, wherein the engineered microorganism is a bacteria. Provided herein are engineered microorganisms, wherein the bacteria is a Bacillus spp. Provided herein are engineered microorganisms, wherein the antisense promoters each comprise a strong constitutive or inducible promoter. Provided herein are engineered microorganisms, in the antisense promoters each comprise independently PphoA, P43, P45, PT7, P34, PxylA, Physpank, Pspac, Pveg, PamyE, PphoA, P43, P45, PgrD, PbacA, PcotC, Pspank, PnrpE, PamyP, PespsA, or any combination thereof. Provided herein are engineered microorganisms, wherein the target gene is a gene that affects a plant trait. Provided herein are engineered microorganisms, wherein the target gene comprises GmFAD3, El, ZmMADSl, GmMIPSl, ZLKR, SDH, ZmBCH2, ghSAD-1, ghFAD2-l, ANR, FaMYBl, TFL1, plC, FaPGl, FaWRKY29, FaWRKY64, AGPase, LsNCED4, or any combination thereof.

[0122] Provided herein are compositions comprising the engineered microorganisms described herein or a spore, mycelium, or vegetative cell thereof, and a plant. Provided herein are compositions wherein the plant is of the genus Brassica, Solarium, Malus, Citrus, Vitis, Saccharum, Zea, Oryza, Triticum, or any combination, strain, or variant thereof.

[0123] Provided herein are methods of treating a plant, the methods comprising introducing the engineered microorganisms described herein or a spore, mycelium, or vegetative cell thereof, to an ecosystem of a plant. Provided herein are methods, wherein the engineered microorganism generates one or more copies of the dsRNA comprising the siRNAs. Provided herein are methods, wherein the engineered microorganism further comprises a DICER enzyme, and wherein the DICER enzyme cleaves the dsRNA to generate the plurality of siRNAs. Provided herein are methods, wherein the methods further comprise transferring the siRNA to a cell of the target organism by passive transfer. Provided herein are methods, wherein the passive transfer is effected by diffusion, extracellular vesicles, and / or cell lysis. Provided herein are methods, further comprising transferring the dsRNA to a cell of the target organism, wherein the transfer is a passive transfer. Provided herein are methods, wherein the transfer is effected by diffusion, extracellular vesicles, and / or cell lysis. Provided herein are methods, wherein the cell of the target organism comprises a DICER enzyme, and wherein the DICER enzyme cleaves the dsRNA to generate siRNA.

[0124] Provided herein are methods of treating a plant, the methods comprising introducing the engineered microorganisms described herein or a spore, mycelium, or vegetative cell thereof, to an ecosystem of a plant. Provided herein are methods, wherein the engineered microorganism generates one or more copies of a plurality of siRNAs from the plurality of dsRNA expression cassettes.Provided herein are methods, wherein the methods further comprise transferring the siRNA to a target organism by passive transfer. Provided herein are methods, wherein the transfer is effected by diffusion, extracellular vesicles, and / or cell lysis.

[0125] Provided herein are formulations, wherein the formulations comprise: the engineered microorganisms described herein, and an adjuvant. Provided herein are formulations, wherein the adjuvant comprises, alone or in combination, a surfactant, an oil, a compatibility agent, a buffering agent, a conditioning agent, a defoaming agent, a deposition agent, a flowability agent, a granulation agent, a drift control agent, a UV protectant, or a thickener. Provided herein are formulations, wherein the adjuvant comprises Tween 20, Triton X-100, Silwet L77, magnesium chloride, seed coating polymer, sodium alginate, cellulose, microcrystalline cellulose, starch, titanium dioxide, talcum powder, or clay. Provided herein are formulations, wherein the formulation is an emulsion, a colloid, a dust, a granule, a pellet, a powder, a liquid, a spray, a mist, a gel, a paste, a fog or a solution.

[0126] Provided herein are methods of manufacture, wherein the methods comprise: generating the engineered microorganism described herein; growing the engineered microorganism; and formulating a composition comprising the engineered microorganism or a spore, mycelium, or vegetative cell thereof, and an adjuvant. Provided herein are methods, wherein the adjuvant comprises, alone or in combination, a surfactant, an oil, a compatibility agent, a buffering agent, a conditioning agent, a defoaming agent, a deposition agent, a drift control agent, a flowability agent, a granulation agent, a UV protectant, or a thickener. Provided herein are methods, wherein the adjuvant comprises phosphate buffered saline. Provided herein are methods, wherein the adjuvant comprises Tween 20, Triton X- 100, Silwet L77, magnesium chloride, a seed coating polymer, sodium alginate, cellulose, microcrystalline cellulose, starch, titanium dioxide, talcum powder, or clay. Provided herein are methods, wherein the composition is an emulsion, a colloid, a dust, a granule, a pellet, a powder, a liquid, a spray, a mist, a gel, a paste, a fog or a solution.EXAMPLESExample 1: dsRNA Sequence Selection, Design, and Off-target analysis

[0127] A target gene sequence is checked for any transcription information from the literature, and alternative splicing variant sequences are compared on EnsemblFungi database. Exon sequences common to all variants are then selected for dsRNA design. Selected exon sequence is used as an input sequence in Oligowalk. Oligowalk determines the probability of a specific length of RNA (siRNA k- mer) to be hybridized with potential mRNA sequences with the probability of hybridization and free energy for each sequence. The exon regions demonstrating the most effective siRNA production arearrayed to be expressed as one continuous dsRNA (synthetic concatemer dsRNA array). This design of dsRNA allows for a greater quantity of siRNA molecules with an enhanced probability of targeting one or more genes or organisms. Off-target analysis for each of the predicted siRNA k-mers is performed by aligning each k-mer against the host, pathogen, rhizosphere microbiome, plant, human, other organism genomes and transcriptomes that could interact with an engineered microbe described herein.Example 2. Design of pan-specific targeting dsRNA

[0128] An mScarlet CDS (SEQ ID NO: 147) codon-optimized for Fusarium oxysporum f. sp. lactucae was designed using Optipyzer. In order to test gene silencing of the mScarlet construct, a dsRNA cassette (SEQ ID NO: 24) was designed to target the first 300 base pairs of mScarlet. Given that the original mScarlet sequence was codon-optimized for Fusarium oxysporum f. sp. lactucae, kmers of this dsRNA targeting the genome were determined. Targeting kmers were identified by splitting the mScarlet dsRNA into kmers of lengths 19-23 bp. Each kmer was aligned to the genome using BBMap with a required minimum identity of 0.89 (in this case, a Hamming distance of 2). Matches of kmers to coding sequences in the genome were determined. The full list of genes with at least one matching kmer that can potentially serve as siRNAs for gene silencing in Fusarium oxysporum f. sp. lactucae are listed in Table 17.Table 17: Genomic targets in Fusarium oxysporum of kmers of the panspecific dsRNA targeting mScarletExample 3: Fusarium spp. reporter construct

[0129] Fungal species are engineered to incorporate a reporter protein using random chromosomal integration. Fusarium oxysporum fragiae, Fusarium oxysporum lactucae and Fusarium virgulifarmeare engineered to express the fluorescent proteins mNeonGreen, mScarlet, mVenus, and mCerulean, or the p -glucuronidase protein (GUS). The reporter is integrated in the chromosome of the Fusarium genome based on random chromosomal integration . Sequences of the inserted reporter genes are shown in Table 18. The expression of the reporter genes is driven by the constitutive Ptefl promoter (SEQ ID NO: 1). Transformed Fusarium are sequenced to identify the integration location and assessed for growth effects and fluorescence or P-glucuronidase activity.Table 18. Reporter Gene sequencesExample 4. siRNA production in F. oxysporum f. sp. lactucae

[0130] Fusarium oxysporum was engineered to express a dsRNA cassette integrated into the chromosome. Transformants carrying the dsRNA cassette are validated via cassette-specific PCR and sequencing of the two halves of the dsRNA hairpin using genomic DNA as a template. Following PCR confirmation of the dsRNA sequence, expression of the dsRNA construct is validated via one step RT-PCR. To confirm that F. oxysporum sp. lactucae further processes dsRNAs to siRNAs, the production of these siRNAs is verified via small RNA sequencing.

[0131] Small RNA sequencing reads from dsRNA-expressing strains of F. oxysporum are aligned to the F. oxysporum genome and dsRNA target sequences using a short-read aligner accepting only perfectly matching sequences. The size-frequency distribution of the resultant mapped reads arecompared to the distribution of reads mapping to an introduced marker gene sequence (hph ) not expected to produce siRNAs. True siRNAs are expected to predominantly fall between 19-23 bp in length, with a peak at 21 bp, whereas sheared RNAs not derived from dsRNAs are expected to have a uniform size distribution. Size distributions of small RNAs derived from Fusarium oxysporum expressing a randomized control dsRNA (Fol random, SEQ ID NO: 58) are shown alongside an introduced marker gene sequence (Fol hph, SEQ ID NO: 155) in histograms in FIG 3A. Size distributions of small RNAs derived from Fusarium oxysporum expressing anti-mScarlet (Fol anti- mScarlet, SEQ ID NO: 24) are shown alongside an introduced marker gene sequence (Fol hph, SEQ ID NO: 155) in histograms in FIG 3B.Example 5: Spray-induced gene silencing assay (SIGS) in Fusarium oxysporum f. sp. lycopersici

[0132] Effectiveness of gene silencing in Fusarium was demonstrated using a purified dsRNA designed to target a heterologously-expressed fluorescent reporter gene, mScarlet. First, agrobacterium-mediated transformation (AMT) was used to introduce the mScarlet genetic sequence into the Fusarium oxysporum f. sp. lycopersici genome. Fusarium oxysporum f. sp. lycopersici were germinated in Potato Dextrose Broth (PDB) growth media for 48 hours at 25 degrees Celsius prior to imaging. Fluorescent signal was acquired using an EVOS M7000 microscope fitted with an RFP LED filter cube (Thermo Fisher). Images were analyzed using ImageJ software. A bar plot showing mean fluorescence of wild-type (“WT”) and engineered (“mScarlet”) F. oxysporum f. sp. lycopersici strains is provided in FIG. 4A Results indicate AMT provided insertion of the mScarlet genetic sequence into the F. oxysporum lycopersici genome and the robust production of mScarlet protein. Then, about 3 x 102spores of Fusarium oxysporum f. sp. lycopersici expressing the mScarlet reporter gene were incubated for 48 hours at 25 degrees Celsius in 300 pL liquid Potato Dextrose Broth (PDB) with purified dsRNA with panspecific targets including the mScarlet transcript (SEQ ID NO: 24) or a control non-targeting dsRNA (100 pg / ml final). The resulting cultures were subjected to RNA extraction followed by qRT-PCR. FIG. 4B is a bar plot showing the relative levels of mScarlet mRNA transcripts in Fusarium oxysporum f. sp. lycopersicico incubated with dsRNA targeting mScarlet transcript (“targeting dsRNA”) or a non-targeting control dsRNA (“ct-“). mScarlet expression in the negative control is normalized to 1 and the gpda housekeeping gene is used for relative comparison across samples. Fusarium co-incubated with purified targeting dsRNA showed about 20% reduction of mScarlet mRNA transcripts relative to Fusarium co-incubated with the control dsRNA, indicating suppression of mScarlet expression.Example 6: Engineered hairpin dsRNA causes silencing of the target gene in Fusarium oxysporum f. sp. lycopersici

[0133] Gene silencing by a hairpin dsRNA construct expressing dsRNA targeting a gene encoding a mitogen-activated protein kinase, Fmkl, was demonstrated. Fusarium oxysporum f. sp. lycopersici was engineered to express a hairpin-forming dsRNA (SEQ ID NO: 19 and SEQ ID NO: 12) under the control of the constitutive promoter Ptefl (SEQ ID NO: 1). The intron allowing the formation of the hairpin-forming dsRNA is excised and the resulting dsRNA targets the native fmkl gene from Fusarium oxysporum f. sp. lycopersici. Gene silencing of the Fmkl transcript results in a decrease in surface hydrophobicity of Fusarium. Silencing is experimentally demonstrated by adding droplets of an aqueous dye solution on top of the mycelium and spores of a colony of Fusarium oxysporum f. sp. lycopersici and observing dispersion of the droplets, as opposed to beading of the aqueous solution. FIG. 5A shows Fusarium oxysporum f. sp. lycopersici growth on agar plate expressing no dsRNA (top row) or a dsRNA targeting the fmkl transcript (bottom row) (n=3 biological replicates). Fusarium oxysporum f. sp. lycopersici grown on agar plate exhibits self-induced gene silencing. Black bars illustrate the dispersion diameter of the droplets. Gene silencing of the Fmkl transcript results in a decrease in surface hydrophobicity of the Fmkl dsRNA-expressing Fusarium. FIG. 5B is a bar plot showing the relative levels of Fmkl mRNA transcripts in wild-type (“WT”) and engineered (“Fmkl dsRNA”) Fusarium oxysporum f. sp. lycopersici^ indicating suppression of fmkl expression in the engineered fungal (n=2 biological replicates).Example 7: In vitro demonstration of silencing efficacy of engineered dsRNA [analogous to spray-induced gene silencing assay (SIGS)] in Fusarium oxysporum and Fusarium virguliforme

[0134] Fusarium spores (Fusarium oxysporum f. sp. fragiae - Fof, Fusarium oxysporum f. sp. lactucae - FoL, and Fusarium virguliforme) (1 x 103spores / ml) are incubated at 25 degrees Celsius in 200pL liquid Potato Dextrose Broth (PDB) with 1-10 pg of purified engineered dsRNA targeting ERG11 / CYP51 or non-targeting control dsRNA for 72 hours. Cultures are measured for optical density (OD600) every hour. After 72 hours, the resulting cultures are pelleted and stored at minus 80 degrees Celsius until RNA extraction. Silencing of ERG11 / CYP5 lis indicated by reduced or impaired growth (measured by OD600) relative to spores co-incubated with non-targeting control dsRNA. Gene silencing can be confirmed using qRT-PCR of ERG11 and CYP51 in RNA extracted from Fusarium co-incubated with ERG11 / CYP51 targeting dsRNA and control dsRNA. Alternatively, spores (1 x 103spores) from reporter strains expressing a heterologous mScarlet fluorescence protein can be incubatedin 200pL PDB at 25 degrees Celsius with 1-1 Ogg purified dsRNA with panspecific targets including mScarlet for 72 hours. Cultures are measured for RFP fluorescence every hour. mScarlet silencing is indicated by reduced fluorescence when compared with spores incubated with non-targeting control.Example 8: Mycoparasitism activity of Trichoderma spp.

[0135] The suppressive effect of mycoparasitism was demonstrated. Fusarium oxysporum f. sp. lycopersici mycelia were plug-inoculated onto the left side of fresh PDA plates. Mycelia of mycoparasitic strains of the species Trichoderma harzianum and T. asperellum or non-mycoparasitic strains of the species Trichoderma reesei and T. virens were inoculated onto the right side of the plate five days later. Plates were incubated at room temperature for up to 10 days. At regular intervals, the plates were imaged and the diameter of the mycelia recorded at 4 and 7 days post inoculation of Trichoderma (dpi). Growth diameter was measured compared to plates inoculated with only Fusarium oxysporum f. sp. lycopersici WT. Images were acquired using an EVOS M7000 microscope (Thermo Fisher) and analyzed using ImageJ software.

[0136] FIG. 6A is a photo of a non-mycoparasitic strain of T. virens, inoculated on the right side of the plate, showing no inhibition of growth of the F. oxysporum f. sp. lycopersici, inoculated on the left side of the plate. FIG. 6B is a photo of the mycoparasitic strain of T. harzianum (right), showing a diminished growth radius of the F. oxysporum f. sp. lycopersici (left) relative to the growth radius when co-cultured with T. virens. FIG. 6C is a microscope image of a T. harzianum appressorium (indicated by black caret) forming on a Fusarium hyphae during mycoparasitism killing. An appressorium is the flattened thickened tip of a hyphal branch by which Trichoderma spp. penetrate the hyphae of their host or prey fungi.

[0137] FIG. 6D is a bar graph representing the average relative level of radial growth of Fusarium oxysporum f. sp. lycopersici WT compared io Fusarium oxysporum f. sp. lycopersici co-cultured with Trichoderma harzianum, Trichoderma virens, Trichoderma asperellum, and Trichoderma reesei after 4 and 7 days post inoculation (dpi). All species of Trichoderma showed suppression of Fusarium growth by 7 dpi. T. harzianum suppressed growth of the co-cultured Fusarium oxysporum f. sp. lycopersici by about 30% compared to the growth diameter of Fusarium _cultured without Trichoderma. Radial growth of the Fusarium culture was suppressed by about 26% at 7 dpi when co- cultured with T. asperellum. Radial growth of the Fusarium culture was suppressed by about 16% at 7 dpi when co-cultured with T. reesei. Radial growth of the Fusarium culture was suppressed by about 12% at 7dpi when co-cultured with T. virens.Example 9: Self-silencing using in vivo microbe-induced gene silencing (MIGS) assay for engineered Trichoderma harzianum

[0138] Gene silencing in fungal species using Trichoderma harzianum engineered to express a dsRNA complementary to an introduced mScarlet gene was demonstrated. Trichoderma harzianum was engineered to constitutively express fluorescent protein mScarlet (TH mScarlet). 5 x 103spores / ml Trichoderma harzianum spores were co-incubated for 72 hours at 25 degrees Celsius in liquid Potato Dextrose Broth (PDB) with or without the addition of wild-type (WT) or engineered Trichoderma harzianum expressing dsRNA targeting the following: P-glucuronidase (GUS, TH anti- GUS, SEQ ID NO: 25) and mScarlet (TH anti-mS carl et, SEQ ID NO: 24). Fluorescence at 594 nm was measured at 40 minute intervals.

[0139] Fluorescence measurements are shown in line graphs in FIG. 7. Suppression of fluorescence indicates MIGS from dsRNA expressed by the engineered T. harzianum. TH mScarlet co-incubated with TH anti-mScarlet showed about 80% reduction in fluorescence after 72 hours relative to cocultures with TH anti-GUS or TH WT.Example 10: Confirmation of dsRNA production in Bacillus

[0140] For the following vNO Bacillus subtilis 3610 cells were transformed via natural competence. Engineered sequences were confirmed via nanopore sequencing of colony PCR products. To engineer Bacillus subtilis 3610 to express dsRNA, multiple alterations to the genome were made. rncS encodes the enzyme RNase III, which cleaves dsRNA. In some cases, where the full length dsRNA is desirable, rncS was knocked out (ArncS). Two genes, yonT and txpA, are toxic in the absence of rncS. Thus, genome knockouts of yonT and txpA ( yonT txpA) were engineered before knocking out rncS via homologous recombination with an antibiotic resistance marker construct, followed by cre / lox mediated removal of the marker, leaving a scar. In strains with dsRNA expression driven by T7 RNA polymerase, the coding region for T7 RNA polymerase, driven by the P43 promoter, was inserted into the putP gene loci using homologous recombination. The antibiotic resistance cassette used to select for transformants was removed via cre / lox recombination. Next, dsRNA expression cassettes consisting of a dsRNA coding sequence under the control of two head-to-head promoters with corresponding terminators were transformed into the amyE gene loci using homologous recombination. The chloramphenicol antibiotic resistance cassette used to select for transformants is floxed, but has not yet been removed via cre / lox recombination. Finally, in strains where full length dsRNA is desired, rncS was knocked out via homologous recombination with an erythromycinresistance marker construct. The erythromycin resistance marker used to select for transformants is floxed, but has not yet been removed by cre / lox recombination. Sequences for terminators described herein are listed in Table 19.Table 19: Bacterial terminators and antibiotic resistance sequences

[0141] To extract total RNA from Bacillus, 1 x 108cells were pelleted and resuspended in 200 uL of 0.5 M sucrose, 50 mM EDTA, 2.5 mg / mL lysozyme solution. Resuspended pellets were incubated at 37 C for 30 min, then mixed with 200 uL Trizol and frozen at -80 C for at least 10 min. Samples were thawed and then purified using Zymo direct-zol RNA miniprep kits following manufacturer instructions, eluting in RNase free H2O. DNA was removed from samples by addition of DNase I enzyme, followed by incubation for 60 min at 37 deg C. RNA from DNase treated reactions was repurified using NEB monarch RNA cleanup columns and eluted in RNase free water. Quality of RNA was determined by running samples on agarose gels to confirm intact rRNA bands, and concentration of RNA was determined by Nanodrop.

[0142] To confirm RNA of the correct sequence was being expressed, RT-PCR was performed on extracted RNA using the SuperScript IV UniPrime One-Step RT-PCR system (Invitrogen) according to the manufacturer instructions, in 10 uL reactions using -20-40 ng of total RNA as template with 30 PCR cycles. Primers specific to each strain’s dsRNA were used. Two reactions were run for each strain: (1) with reverse transcriptase (RT) in the reaction to detect RNA, and (2) without RT to detect any genomic DNA that would cause a false positive result. FIG. 8A shows strong bands in reactions containing RT, and no bands in reactions without RT, indicating that RNA of the correct sequence is being expressed by the engineered Bacillus, and that no genomic DNA contamination was in the samples.

[0143] Two methods were used to confirm the engineered Bacillus subtilis were producing double stranded RNA (dsRNA). In the first method, total RNA purified from engineered Bacillus subtilis with the dsRNA expression cassette and total RNA purified from wild-type Bacillus subtilis were digested with RNAse If (New England Biolabs (NEB), Ipswich, MA), an enzyme that specifically cleaves single stranded RNA. 3 uL of total RNA from wildtype and engineered Bacillus subtilis were run on an agarose gel to confirm total RNA had not been degraded (FIG. 8B, left). The remaining 25 uL of total RNA was digested with 4 uL of RNase If (NEB). The entire digested samples were run on an agarose gel (FIG. 8B, right). All RNA from wildtype Bacillus subtilis was degraded, while theengineered Bacillus subtilis RNA showed an undigested band that is the expected size for its dsRNA, confirming dsRNA is being expressed in the engineered Bacillus subtilis.

[0144] In the second method, dsRNA-specific dot blots were performed. RNA was purified from 5 different engineered Bacillus subtilis strains expressing dsRNA targeting a non-targeting randomized control (D41), PMT2 (D14), fksl (D28), pan-specific targets (D4), and ERG3 / ERG5 (D31), and compared to RNA purified from wild-type Bacillus subtilis as a negative control. 2 uL of total RNA purified from each strain was spotted onto a super charged Nytran membrane. Additionally, a 10-fold dilution series of in vitro transcribed (IVT) dsRNA from 100 ng / uL to 0.00001 ng / uL was made as a positive control. 2 uL of each dilution was dotted onto the membrane. Blots were probed with the dsRNA-specific antibody J2 then incubated with a secondary antibody conjugated with horseradish peroxidase (HRP). Finally, blots were incubated with Femto ECL reagent and imaged on a chemiluminescent detector. FIG. 8C shows the dot blot with a strong signal from the IVT dsRNA positive control which decreases with every serial dilution, suggesting the antibody is functional and specifically detects dsRNA. The six dots corresponding to the RNA from five of the engineered dsRNA-expressing strains (D28 was dotted twice) all show a clear signal, while the dot corresponding to RNA from wild-type Bacillus subtilis shows no signal, confirming that the engineered Bacillus subtilis express dsRNA.

[0145] Sequences used for generating engineered Bacillus described in this example are provided in Table 23Table 23: Engineered Bacillus sequencesExample 11: Confirmation of dsRNA release in Bacillus culture

[0146] 50 mL of Luria Broth (LB) was inoculated with wild-type or engineered Bacillus subtilis expressing a pan-specific targeting dsRNA (SEQ ID NO: 24) and incubated overnight at 37 C with 250 rpm shaking. To harvest only cell-free supernatant (CFS) from the cultures, cultures were centrifuged for 5 minutes at 10,000 x g at 4 deg C. The supernatant was then filtered through a 0.2 pM aPES filtration unit before being transferred to a 3 kDa centrifugal filter unit (Millipore Sigma, Amicon, St. Louis, MO) for centrifugation at 4,000 x g for 1 hour at 4 deg C. Roughly 500 pL of CFS was recovered from the filter units and added to 1 mL Trizol. Samples were frozen at -80 C for at least 10 minutes and thawed at room temperature. Samples were then mixed at a 1 : 1 ratio with 100% ethanol before being used as input for Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA), following the manufacturer’s protocol. Following RNA purification, samples were treated with DNAse I (Thermo Fischer, Carlsbad, CA) according to the manufacturer’s protocol.

[0147] To confirm that RNA of the correct sequence was present in the CFS, RT-PCR was performed on extracted RNA using the SuperScript IV UniPrime One- Step RT-PCR system (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Two sets of reactions were run for each strain, one with reverse transcriptase in the reaction (RT+) (FIG. 9A), and one without (RT-) (FIG. 9B). In each reaction, two primer sets were used, one specific to the RNA for single-stranded binding protein (ssb), which serves as a control present in both wild-type (WT) and engineered Bacillus subtilis (GEBs), and one specific to the pan-specific targeting dsRNA (dsRNA). The reaction without RT is included to detect any genomic DNA that would cause a false positive result. There is a strong band in the RT(+) reaction for GEBs with dsRNA specific primers which is much brighter than that in theRT(-) reaction, indicating minor contaminating DNA but a higher amount of dsRNA in the CFS of GEBs. Meanwhile, this band is not present in the wild-type samples as expected. Small amounts of positive control ssb RNA were detected in both WT and GEBs samples. These data confirm that RNA of the expected sequence is being released from engineered Bacillus in liquid culture.Example 12. Confirming dsRNA production in Trichoderma

[0148] Trichoderma spp. were engineered to express a dsRNA cassette integrated into the chromosome. Transformants carrying the dsRNA cassette were validated via cassette-specific PCR and sequencing of the two halves of the dsRNA hairpin using genomic DNA as a template. Following PCR confirmation of the dsRNA sequence, expression of the dsRNA constructs were validated via one step RT-PCR. Confirmation of the PCR and RT-PCR by gel electrophoresis is shown for two clones of Trichoderma harzianum expressing dsRNAl l (PMT2, SEQ ID NO: 28) and dsRNA12 (PMT2, SEQ ID NO: 29) in FIG. 10. As Trichoderma spp. further process dsRNAs to siRNAs, in some instances the production of these siRNAs is verified via small RNA sequencing.Example 13: Comparison of growth rates of wild-type and engineered bacteriaBacillus subtilis

[0149] The growth rates of engineered Bacillus subtilis expressing dsRNA4 targeting pan-specific targets, dsRNA31 targeting ERG3 / ERG5, and dsRNA38 targeting PMT1-PMT2-PMT4 were compared to wild-type Bacillus subtilis. The Bacillus substilis strain expressing dsRNA4 also contained the BrncS mutation knocking out RNAse III. Each Bacillus subtilis strain was cultured in a 2L DASGIP bioreactor (Eppendorf), using the same fermentation protocol: 2x DSM medium, 30% dissolved oxygen (DO) cascade, 30°C, pH 7.0. Samples were taken at harvest at approximately 40 hours after inoculation. Bioreactors were supplemented with 5 g / L glucose. Samples were plated for percent sporulation by plating heat treated (80°C, 20 min) versus non-heat treated. Recovered spore titers were approximate 2 x 109CFU / mL culture for engineered Bacillus subtilis expressing dsRNA4 and 8 x 108CFU / mL for all other strains. FIG. 11A shows the growth rates were comparable across wild-type and engineered strains containing rncS, while the mcS strain expressing dsRNA4 grew to a higher cell density.Trichoderma

[0150] Spores of Trichoderma asperellum and Trichoderma harzianum were collected from sporulating fungi grown on PDA plates. 5-10 ml of 0.1% Tween 80 were added to each plate and the spores were scraped up using a sterile L-spreader. The collected liquid was passed through a sterilegauze pad into a 15 ml conical tube. The concentration of spores in solution was determined using a Countess 3 hemocytometer following the manufacturer’s recommendation (Thermo Fisher). The concentration of the spore solutions were normalized 2 x 104spores / mL in Potato Dextrose Broth (PDB). 200 uL of spore suspension, or 200 uL of a 1 : 1 mixture of wild-type and engineered spore suspension, were distributed in a 96-well plate with a transparent lid. The lid was sealed with parafilm. The 96-well plate was incubated 72 hours at 25°C in a Varioskan Plate Reader (Thermo Fisher) without shaking, set to measure OD630 every hour. FIG. 11B shows the growth rates of engineered Trichoderma asperellum expressing dsRNA5 targeting P-glucuronidase (GUS) compared to wild-type Trichoderma asperellum. FIG. 11C shows the growth rates of engineered Trichoderma harzianum expressing dsRNA3 targeting PMT2 compared to wild-type Trichoderma harzianum. No difference in growth rates was observed between engineered and wild-type strains.Example 14: Stability of engineered microbial spores over time

[0151] Wild-type and engineered Bacillus subtilis spores were prepared using shake flasks batch fermentation method. Briefly, strains were inoculated in IxDSM medium, incubated at 37°C shaking incubator set at 250 rpm. Strains were incubated for 27 to 30 hours before harvesting by centrifugation at 9500xg for 10 minutes. After harvest by centrifugation, spores were resuspended in spent fermentation media and stored for up to three months at 4°C. The spore suspension was sampled at regular intervals and spore viability was enumerated by plating on LB agar. FIG. 12A shows the viability of wild-type and engineered Bacillus subtilis over two months. Wild-type and engineered Trichoderma asperellum and Trichoderma harzianum spores were cultured on sorghum for 10-14 days as described in Example 10 (“( / ) Producing infested sorghum seed inoculum”)' . Spores were harvested from the sorghum by washing the sorghum with 300 mL saline solution (0.9% sodium chloride), then centrifuged, and resuspended in fresh saline solution to a concentration of ~1 x 109CFU / mL and stored for up to three months at 4°C. The spore suspension was sampled monthly and spore viability was enumerated by plating on PDA with 0.1% triton using a spread plate method (100 puL of serially diluted suspension per plate). FIG. 12B shows the viability of wild-type and engineered Trichoderma asperellum over two months. FIG. 12C shows the viability of wild-type and engineered Trichoderma harzianum over three months. Engineered microbes showed comparable stability to the wild-type microbes, maintaining >95% viability over two to three months.Example 15: Microbial abundance in root tissue of engineered microbes compared to wild-type

[0152] Salanova Green Oakleaf lettuce seeds were planted into soil and maintained in a growth chamber for 21 days with 12 hours photoperiod and 20 degrees Celsius light / 16 degrees Celsius dark temperature cycles and 60% air humidity. Plants were allocated into 3 plants / treatment group. Immediately after planting, the soil above the seed was inoculated with 1ml ~1 x 109CFU / ml wild type or engineered microbe spore suspension with Trichoderma asperellum, Trichoderma harzianum, and Bacillus subtilis each evaluated. Treatment groups of the engineered microbes included: Trichoderma asperellum engineered to express dsRNA4 targeting pan-specific targets (FIG. 13A), Trichoderma harzianum engineered to express dsRNA3 targeting PMT2 (FIG. 13B), and Bacillus subtilis engineered with all genomic edits (coml Q12L AyonT AtxpA AmcS::chlor putP: :T7 RNA Pol erm) but not expressing any dsRNA (FIG. 13C). At 1, 2, and 3 months post planting, roots were collected from 3 biological plant replicates per treatment. Root material was washed with distilled water to remove soil and then surface sterilized in a 10% bleach solution for 10 minutes before homogenization by bead beating and total DNA extraction using the MP Bio Fast DNA soil kit. Total DNA from roots was analyzed by qPCR to quantify the abundance of applied microbes in the soil. Extrapolation of CT values to CFU per gram of soil was achieved by developing a standard curve (limit of detection ~ 102CFU / g). All engineered strains show comparable colonization compared to their wild-type parent strains.Example 16: In vivo microbe-induced gene silencing (MIGS) assay for engineered Bacillus spp.

[0153] Gene silencing in fungal species using a Bacillus bacterium engineered to express a dsRNA complementary to a Fusarium gene was demonstrated. Fusarium oxysporum f. sp. lactucae was engineered to constitutively express fluorescent protein mScarlet (Fol*). 5 x 103spores / ml Fusarium spores were co-incubated for 65 hours at 25 degrees Celsius in 200pL liquid PDB with or without the addition of 5 x 104 spores / mL wild-type (WT) or engineered Bacillus subtilis expressing dsRNA targeting the following: 0 -glucuronidase (GUS, GEBs-GUS, SEQ ID NO: 25), panspecific targets (GEBs-mScarlet, SEQ ID NO: 24), and CYP51 (GEBs-CYP51, SEQ ID NO: 26), an essential gene for cell wall synthesis. Fluorescence (excitation: 520 nm, emission: 580-640 nm) was measured at 45 minute intervals.

[0154] Fluorescence measurements are shown in line graphs in FIG. 14. Suppression of fluorescence indicates MIGS from dsRNA expressed by the engineered B. subtilis. All Fol* samples co-incubated with B. subtilis showed a reduction in fluorescence. Incubation with B. subtilis expressing dsRNA targeting CYP51 (GEBs_CYP51) showed almost complete suppression of fluorescence at 65 hours.Incubation of Fol* with B. subtilis expressing dsRNA targeting panspecific targets including mScarlet (GEBs m Scarlet) showed about 90% suppression of fluorescence at 65 hours. Incubation of Fol* with either wildtype B. subtilis (WT B. subtilis) or B. subtilis expressing dsRNA targeting (3-glucuronidase (GEBs GUS) showed similar levels of suppression, about 50% less than Fol* alone.Example 17: Additional characterization of gene silencing by engineered Bacillus subtilis

[0155] MIGS on solid medium is demonstrated. Fusarium spores (Fusarium f. sp. oxysporum fragariae, Fusarium oxysporum f. sp. lactucae and Fusarium virgulforme 2 x 105spores / ml) are inoculated into fresh PDAplate by resuspending the spores directly into melted PDA. Cultures of wild-type or engineered Bacillus expressing the specified dsRNA constructs are spot-inoculated onto the plate. Plates are incubated at 25 degrees Celsius for up to 5 days. At regular intervals, the plates are imaged and the diameter of areas in which Fusarium's sporulation is inhibited is recorded. MIGS is indicated by increase in inhibition area around engineered Bacillus spots relative to wild-type Bacillus. Alternatively, 1 x 104 - 1 x 106 spores / mL of wild-type or engineered Bacillus are inoculated into a fresh PDA plate by resuspending the spores directly into melted PDA. Cultures of Fusarium are plug-inoculated onto the plate and incubated at 25 degrees Celsius for up to 10 days. At regular intervals, the plates are imaged and the diameter of Fusarium growth is measured. MIGS is indicated by decrease in Fusarium growth on plates embedded with engineered Bacillus spores relative to growth on wild-type Bacillus spore-embedded plates.

[0156] The efficacy of numerous dsRNA constructs including, but not limited to, long dsRNA (FIG. 2D), synthetic concatemer dsRNA arrays (FIG. 2E), synthetic siRNAs interspaced with DICER modulating spacer regions (DMSR) (FIG. 2F), and multiplexed siRNA (FIG. 2C) are evaluated leveraging in vivo MIGS assay with engineered Bacillus. In addition to the phenotypic readout, the pool of siRNAs generated by these different genetic construct designs is assessed using small RNA sequencing (sRNA-seq).Example 18: In vivo microbe-induced gene silencing (MIGS) assay for engineered Trichoderma spp.

[0157] Gene silencing in fungal species using a Trichoderma engineered to express a dsRNA complementary to a Fusarium gene is demonstrated.

[0158] MIGS in liquid culture'. Fusarium spores (Fusarium oxysporum f. sp. fragiae, Fusarium oxysporum f. sp. lactucae and Fusarium virguliforme) (5 x 103spores / ml) are incubated at 25 degrees Celsius and 250 rpm in liquid PDB for 6h-12h to obtain germinating spores. Spores of wildtype orengineered Trichoderma (1- 3 x IO3spores / ml) expressing the specified dsRNA constructs are incubated at 25 degree Celsius and 250 rpm in liquid PDB for 6h-8h to obtain germinating spores. Fungal cultures are mixed at the indicated ratios (1000: 1, 100:1, 10: 1, 1;1, 1 :10, 1: 100, 1: 1000; Trichoderma: Fusarium) and cultivated for 18 hours at 25 degrees Celsius. Aliquots of the resulting cultures are pelleted and stored at minus 80 degrees Celsius until DNA and / or RNA extraction. Serial dilutions of the cultures are plated on PDA plates for phenotypic observation.

[0159] A growth reduction or growth impairment of Fusarium colonies generated by co-incubation with engineered Trichoderma relative to ones generated by co-incubation with wild-type Trichoderma is indicative of MIGS. Additionally, relative levels of gene expression of Fusarium genes targeted by siRNAs generated by the expressed dsRNA (e.g., PMT2) is estimated by reverse transcription real time PCR (RT-qPCR). Gene expression is compared between cultures co-incubated with wild-type Trichoderma and engineered Trichoderma. MIGS is indicated by reduction in relative gene expression.

[0160] MIGS on solid medium / z / / e.s of inhibition): Fusarium spores (Fusarium oxysporum f. sp. fragariae, Fusarium oxysporum f. sp. lactucae and Fusarium virguliforme; 2 x 105spores / ml) are inoculated into a fresh PD agar plate by resuspending the spores directly into melted PDA. Mycelia of wild-type or engineered Trichoderma expressing the specified dsRNA constructs are plug- inoculated onto the plate. Plates are incubated at 25 degrees Celsius for up to 5 days. At regular intervals, the plates are imaged and the diameter of areas in which Fusarium's sporulation is inhibited is recorded. MIGS is indicated by increase in inhibition area around engineered Trichoderma plugs relative to wild-type Trichoderma plugs.

[0161] MIGS on solid medium (dual culture): Fusarium (Fusarium oxysporum f. sp. fragariae, Fusarium oxysporum f. sp. lactucae, Fusarium virguliforme) is plug inoculated onto 1 side of a fresh PDA plate, approximately 1cm from the edge of the plate, and incubated at 25 degrees Celsius for 5 days. Subsequently, engineered Trichoderma or wild-type Trichoderma is plug-inoculated on the opposite side of the plate, approximately 1cm from the edge of the plate. Dual culture plate is incubated at 25 degrees Celsius for up to 10 days. Radius of Fusarium growth (towards Trichoderma plug) is measured at regular intervals. MIGS is indicated by decrease in Fusarium growth radius in dual culture plates with engineered Trichoderma relative to dual culture plates with wild-type Trichoderma.

[0162] The efficacy of numerous dsRNA constructs including, but not limited to, long dsRNA, synthetic concatemer dsRNA arrays, synthetic siRNAs interspaced with DICER modulating spacerregions (DMSR), and multiplexed siRNA is evaluated leveraging in vivo MIGS assay with engineered Trichoderma spp. (see FIG 2C-2F for reference). In addition to the phenotypic readout, the pool of siRNAs generated by these different genetic construct designs is assessed using small RNA sequencing (sRNA-seq).Example 19: MIGS Assay in Soy

[0163] Microbe-induced gene silencing provides a method to deliver relatively small amounts of microbes to the plant niche of interest and then deliver a sustained level of siRNA over an extended period of time.

[0164] Protection of soybean from Fusarium virguliforme challenge by treatment with microorganisms engineered as described herein was demonstrated.(1) Producing infested sorghum seed inoculum

[0165] Sorghum or “white millet seed” was soaked overnight in a plastic tub. Excess water was drained and seeds were washed up to 3 times in milliQ water. Soaked sorghum was autoclaved for 1 hour in a liquid cycle and cooled at room temperature for 24 hours. Approximately 300g of wet sorghum were placed into an autoclavable spawn incubation plastic bag with a 0.2 micron microporous filter patch. Bags were closed by folding the bag’s mouth two times and sealing with tape. Bags were autoclaved for 1 hour in a liquid cycle and cooled at room temperature for 24 hours.

[0166] Ten-day-old cultures of Fusarium virguliforme grown on PDA were used to inoculate the autoclaved seed. Under sterile conditions, 10 plugs were taken from the advancing edge of the fungal colony to maximize homogeneity and were used to inoculate the sorghum. Bags were sealed using an impulse sealer to create an airtight seal. Grain was mixed well and incubated at room temperature for 3 weeks. The bags were incubated for 3 weeks at 25 °C in the dark. After 3 weeks, the infected sorghum was used directly in the experiments.

[0167] Instead of using fresh inoculum, an alternative method exists to stabilize the infested sorghum for use in these experiments. Briefly, the 3 week old inoculated seeds were spread on paper towels inside a biosafety cabinet and dried for 4-5 days to reduce the relative humidity below 5% at 25 deg C. Dried seeds can be re-bagged and stored in a cold room for 3 months until use.(2) Inoculation with sorghum seeds

[0168] Sorghum inoculum was prepared as indicated above. The 3 week old infested bag was opened on the day of planting and introduced at a rate of 3-5g per pot. On the same day as planting, CFU assays were conducted. One gram of sorghum inoculum was soaked in a 15 ml screw cap tubecontaining 1 ml of sterile PBS. Three tubes (replications) were done for each bag of infested sorghum. The seed suspension was vortexed for 10 seconds. From each tube, 100 pl was taken and added to the first well of a column containing 900 pl of PBS to make the first 10-1 dilution. Subsequent serial dilutions were made until a final dilution of 10-4 was achieved. From the dilutions of 10-3 and 10-4, 100 pl were spread on a 9cm diameter PDA plate supplemented with 0.01% Triton X100. The plates were incubated for 2-3 days at room temperature in the dark until colonies can be distinguished and counted. Colonies of Fusarium virguliforme were identified and counted on each plate to determine the CFU values per gram of sorghum inoculum for each isolate.

[0169] Fafard Potting Mix (Sungro) was sterilized in an autoclave dry cycle for 30 minutes at 121°C, followed by 30 minutes at 150°C. 3-inch pots were filled two thirds full with sterile potting mixture. A layer of 3-5 grams of infested sorghum grain was added to each pot (to achieve IxlO7CFU total) and the remaining volume of the pot was filled with potting mix.

[0170] The 3-inch pots were placed in Phytatray Ill’s (Sigma) and 1g of Osmocote Smart-Release Plant Food Plus (Osmocote) was added between the bottom of the 3-inch pot and Phytatray. The potting mix was saturated with 100 ml of MilliQ Water by placing the water in the Phytatray and placing the 3 inch pot in the water.(3) Planting and plant maintenance

[0171] Two seeds of soybean (Williams 82 variety) were planted in ~2-3 cm-deep holes in the middle of the pot (~5 cm spacing between the holes). Immediately after covering the seeds with potting mix, 1 ml of RBX product was pipetted on top of the soil directly above the seed.

[0172] Plants were maintained in GEN1000 (Conviron) Growth Chambers set to the following conditions: 16hr light (25°C) / 8hr dark (20°C), 50% Relative Humidity, 100% Light Intensity. Pots were watered from the bottom and after 5 days of growth, the plants are thinned to one plant per pot.(4) Plant leaf area measurements as biomass proxy

[0173] After 10 days, excised leaves were individually photographed from below using a standardized stage and distance. The images were processed using ImageJ software to measure the pixels of green leaf area. Leaf area was plotted and used as a proxy for measuring biomass.

[0174] Each pot was imaged with a label and ruler for scale comparison. Images were acquired with a benchtop scanner (Epson). Images were transferred to ImageJ and analyzed using the “Color Thresholding” feature with the following settings: Hue: 46 - 255; Saturation: 46 - 255; Brightness: 32 - 255

[0175] The resulting pixels were selected using the “Select” button at the bottom of the Threshold Color Window. “Command + M” generated a measurement of the number of pixels which were selected. The resulting measurement of pixels were copied and pasted into Google Sheets. Graphing and Statistical Analysis was accomplished using JMP vl8 Software Suite.Example 20: Treatment of soybeans with engineered Bacillus subtilis

[0176] Williams 82 soybean seeds were planted into soil with a layer of 5g sorghum infested with Fusarium virguliforme (Fv) (~1E7 CFU per pot) and maintained in a growth chamber for 10 days with 12 hours photoperiod and 25 degrees Celsius light / 20 degrees Celsius dark temperature cycles and 60% air humidity. Control plants with no Fv and no Bacillus inoculum were used as a negative control (“Healthy plant”). Plants were allocated in to 10 plants / treatment group. Immediately after planting, the soil above the seed was inoculated with 1ml ~1 x 109CFU / ml wild type or engineered Bacillus spore suspension or 1ml IX phosphate buffered saline (“Pathogen only”). Treatment groups received engineered Bacillus subtilis expressing the following dsRNA targeting: non-targeting randomized control (SEQ ID NO: 58) (dsRNA41, “D41”), panspecific targets (SEQ ID NO: 24) (dsRNA4, “D4”), fksl (SEQ ID NO: 45) (dsRNA28, “D28”), rhol-rhoA-rho2-rho3 (SEQ ID NO: 46) (dsRNA29, “D29), ERG1 (SEQ ID NO: 47) (dsRNA30, “D30”), ERG3 / ERG5 (SEQ ID NO: 48) (dsRNA 31, “D31”), ERG11 / CYP51 (SEQ ID NO: 49) (dsRNA32, “D32”), ERG4 (SEQ ID NO: 50) (dsRNA33, “D33”), ERG24 (SEQ ID NO: 51) (dsRNA34, “D34”), SGE1 (SEQ ID NO: 52) (dsRNA 35, “D35”), FvToxl (SEQ ID NO: 53) (dsRNA36, “D36”), Myosin-5 (SEQ ID NO: 54) (dsRNA 37, “D37”), PMT1-PMT2-PMT4 (SEQ ID NO: 55) (dsRNA38, “D38”), Luc7 (SEQ ID NO: 56) (dsRNA39, “D39:), Tom40 (SEQ ID NO: 57) (dsRNA40, “D40”), and rhol-rhoA-rho2-rho3-fksl (SEQ ID NO: 60) (dsRNA43, “D43”). Leaf area was measured by pixel count as described in Example 10, measurements are shown in FIG. 15A and FIG. 15B. Genes from independent gene pathways are indicated on x-axis. Plants treated with Bacillus expressing dsRNA31, dsRNA32, dsRNA33, dsRNA34, dsRNA37, dsRNA38, dsRNA39, dsRNA40, and dsRNA43 showed increased leaf area compared to controls and are indicated in bold in figures.

[0177] Growth in plants treated with Bacillus subtilis modified to express dsRNA31, dsRNA32, dsRNA34, dsRNA40, and dsRNA43 compared to control groups and commercial chemical control Saltro® (pydiflumetofen) (Syngenta) applied as a root drench at commercial rates. Growth and treatment methods were the same as described above, with 22 plants / condition. Measured leaf sizes are shown in FIG. 15C. All modified bacterial strains showed statistically significant increase in leafarea compared to untreated (Fv-only), with p-values: < 0.001 ("**), < 0.005 (***), < 0.008, and <0.05 (*). Two strains showed a statistically significant increase in leaf area compared to the wild-type control (A, p < 0.05). All strains performed equal to or better than Saltro®.Example 21: Seed treatment of soybeans with engineered Bacillus subtilis

[0178] Bacillus subtilis engineered to express dsRNA targeting ERG3 / ERG5 (SEQ ID NO: 48) (dsRNA 31, “D31”) was applied to naked Williams 82 soybean seeds using the following seed treatment formulation: 10.5 mL of microbial formulation comprised of 5.25 ml of microbial suspension (1E10 CFU / mL B. subtilis), 5 ml sodium alginate (Sigma) , 0.25 ml Flo Rite Pro 2805 (BASF) per 1kg seed. Seeds were shaken with the formulated microbial suspension in a sealed container until the seeds appeared dry. Soybean seeds treated with engineered bacteria were planted into soil with a layer of 5g sorghum infested with Fusarium virguliforme (Fv) (~1E7 CFU per pot) and maintained in a growth chamber for 10 days with 12 hours photoperiod and 25 degrees Celsius light / 20 degrees Celsius dark temperature cycles and 60% air humidity. Control plants with no Fv and no Bacillus inoculum were used as a negative control (“Untreated”). Plants were allocated into 10 plants / treatment group.

[0179] For comparison, untreated Williams 82 soybean seeds were planted into soil with a layer of 5g sorghum infested with Fv (~1E7 CFU per pot). Immediately after planting, the soil above the seed was inoculated with 1ml engineered Bacillus spore suspension at four different dosages (~1 x 106, ~1 x 107, ~1 x 108, or ~1 x 109CFU / ml) or 1ml IX phosphate buffered saline (“Pathogen only”). Treatment groups received engineered Bacillus subtilis expressing the following dsRNA targeting: non-targeting randomized control (SEQ ID NO: 58) (dsRNA41, “D41”) and ERG3 / ERG5 (SEQ ID NO: 48) (dsRNA 31, “D31”). Leaf area was measured by pixel count as described in Example 10, measurements are shown in FIG. 16. Genes from independent gene pathways are indicated on x-axis. Plants treated with Bacillus expressing dsRNA31 applied via the seed treatment at ~l x 107CFU / mL / seed and via the root drench at - l x 109CFU / mL / seed show the highest and equivalent leaf area of all treatments and are indicated in bold in figures.Example 22: Treatment of elite commercial soybean seed with engineered Bacillus subtilis

[0180] Four varieties of elite commercial soybean seeds were treated with different strains of engineered Bacillus subtilis as described in Example 18, with the addition of metalaxyl and imidacloprid incorporated into the formulation at recommended rates. Controls included a formulation control without Bacillus (“Formulation”), wildtype B. subtilis, a chemical control Saltro®(pydiflumetofen) (Syngenta), all applied as seed treatments, and a negative control with no Fv and no treatments (“Healthy plant”). Treated seeds were planted into soil with a layer of 5g sorghum infested with a 1 :1 mixture of two field isolates of Fusarium virguliforme (Fv) (each delivered at ~1E7 CFU per pot) and maintained in a growth chamber for 10 days with 12 hours photoperiod and 25 degrees Celsius light / 20 degrees Celsius dark temperature cycles and 60% air humidity. Plants were allocated into 10 plants / treatment group. Treatment groups received engineered Bacillus subtilis expressing the following dsRNA targeting: ERG3 / ERG5 (SEQ ID NO: 48) (dsRNA 31, “D31”), PMT1-PMT2- PMT4 (SEQ ID NO: 55) (dsRNA38, “D38”), Tom40 (SEQ ID NO: 57) (dsRNA40, “D40”), and rhol- rhoA-rho2-rho3-fksl (SEQ ID NO: 60) (dsRNA43, “D43”). Leaf area was measured by pixel count as described in Example 10, measurements are shown in FIG. 17A - 17D. Plants treated vn aBacillus expressing dsRNA31, dsRNA38, and dsRNA43 showed consistent increased leaf area compared to controls, including Saltro, across different cultivars and are indicated in bold in figures. Images of the first true leaves of soybean plants 10 days post planting are shown for the untreated (“Healthy”), inoculated with Fv (“Pathogen Only”), engineered Bacillus subtilis expressing dsRNA43 targeting rhol-rhoA-rho2-rho3-fksl (“Treated”), and Saltro® treated plants in FIG. 17E.Example 23: Optimization of dsRNA

[0181] Sequences were optimized to improve biocontrol efficacy while reducing off-target activity against non-target organisms. First generation dsRNA constructs, for example dsRNA4, were designed to maximize the number of on-target siRNA kmers targeting Fusarium spp. without consideration for off-target activity. SEQ ID NO: 27 (dsRNAlO, “D10) was optimized to reduce off- target activity while maintaining moderate on-target activity against Fusarium virguliforme (Fv). FIG. 18A is a graph representing the number of on-target hits (siRNA kmers with greater than 0.7 probability of activity knocking down the target gene) and the number of off-target hits (siRNA kmers with greater than 0.7 probability of activity against a database of 16 representative non-target organisms).

[0182] Williams 82 soybeans were grown as described in Example 12. Plants were allocated in to 15 plants / treatment. Control plants with no Fv and no Bacillus inoculum were used as a negative control (“Healthy plant”). Treatment groups were inoculated with Fv and received no treatment (“Pathogen only”) or engineered Bacillus subtilis expressing dsRNA targeting CYP51 designed to maximize on- target siRNA kmers without consideration for off-target hits (SEQ ID NO: 27, dsRNAlO, “D10”) and designed to maximize on-target siRNA kmers while minimizing off-target hits (SEQ ID NO: 49,dsRNA32, “D32”). FIG. 18B is a graph representing the leaf area of each treatment group, showing increased leaf size in both groups treated with engineered Bacillus subtilis expressing either dsRNAlO or dsRNA32.Example 24: Treatment with Bacillus subtilis expressing dsRNA targeting multiple genes

[0183] Williams 82 soybeans were planted into soil with a layer of 5g sorghum infested with F. virguliforme (Fv, ~1 E7 CFU per pot) and maintained in a growth chamber for 10 days with 12 hours photoperiod and 25 degrees Celsius light / 20 degrees Celsius dark temperature cycles and 60% air humidity. Plants were allocated in to 10 plants / treatment. Control plants with no Fv or Bacillus inoculum were used as a negative control (“Healthy plant”). Immediately after planting, the soil above the seed is inoculated with 1ml Bacillus spore suspension (~1 x 109CFU / ml) or 1ml 0.9% sodium chloride (“Pathogen only”). Treatment groups received engineered Bacillus subtilis expressing dsRNA targeting the following: rhol-rhoA-rho2-rho3 (SEQ ID NO: 46) (dsRNA 29, “D29”), fksl (SEQ ID NO: 45) (D28), and rhol-rhoA-rho2-rho3-fksl (SEQ ID NO: 60) (dsRNA43, “D43”). Leaf area in photographs of first true leaves were measured as described previously. Leaf area of controls and treatment groups is shown in FIG. 19A. All treatment groups showed increased leaf area compared to “Pathogen only.” Plants treated with Bacillus subtilis expressing dsRNA targeting rhol- rhoA-rho2-rho3-fksl showed the greatest gain in leaf size.

[0184] Salanova lettuce seeds were planted into soil with a layer of 3g sorghum infested with Fusarium oxysporum f. sp. lactucae (Fol) (~1E7 CFU per pot) and maintained in a growth chamber for 21 days with 12 hours photoperiod and 20 degrees Celsius light / 17 degrees Celsius dark temperature cycles and 60% air humidity. Plants were allocated in to 10 plants / treatment group. Control plants with Fol or Bacillus inoculum were used as a negative control (“Healthy plant”). Immediately after planting, the soil above the seed is inoculated with 1ml Bacillus spore suspension (~1 x 109CFU / ml) or 1ml 0.9% sodium chloride (“Pathogen only”). Treatment groups received engineered Bacillus subtilis expressing dsRNA targeting the following: rhol-rho2-rho3 (SEQ ID NO: 34) (dsRNA17, “D17”), fmkl (SEQ ID NO: 33) (dsRNA16, “D16”), and rhol-rho2-rho3-fksl (SEQ ID NO: 59) (dsRNA42, “D42”). Leaf area in photographs of first true leaves were measured as described previously. Leaf area of controls and treatment groups is shown in FIG. 19B. All treatment groups showed increased leaf area compared to “Pathogen only.” Plants treated with Bacillus subtilis expressing dsRNA targeting rhol-rhoA-rho2-rho3-fksl showed the greatest gain in leaf size.Example 25: MIGS Assay in Lettuce

[0185] Protection of lettuce from Fusarium oxysporum f. sp. lactucae challenge by treatment with microorganisms engineered as described herein was demonstrated.(1) Fusarium inoculation preparationSorghum or “white millet seed” was soaked overnight in a plastic tub with a drain plug at the bottom. Excess water was drained through the drain plug. Soaked sorghum was autoclaved for 1 hour in a liquid cycle and cooled at room temperature for 24 hours. Approximately 300g of wet sorghum was placed into an autoclavable spawn incubation plastic bag with a 0.2 micron microporous filter patch. Bags were closed by folding the bag’s mouth two times and sealing with tape. Bags were autoclaved for 1 hour in a liquid cycle and cooled at room temperature for 24 hours.

[0186] Ten-day-old cultures of Fusarium oxysporum f. sp. lactucae grown on PDA were used to inoculate the autoclaved seed. Under sterile conditions, 10 plugs were taken from the advancing edge of the fungal colony to maximize homogeneity and were used to inoculate the sorghum. Bags were sealed using an impulse sealer to create an airtight seal. Grain was mixed well and incubated at room temperature for 3 weeks. The bags were incubated for 2 weeks at 20°C in the dark. After 3 weeks, the infected sorghum was used directly in the experiments.

[0187] Instead of using fresh inoculum, an alternative method exists to stabilize the infested sorghum for use in these experiments. Briefly, the 3 week old inoculated seeds were spread on paper towels inside a biosafety cabinet and dried for 4-5 days. Dried seeds can be re-bagged and stored in a cold room for 3 months until use.(2) Inoculation with sorghum seeds

[0188] Sorghum inoculum was prepared as indicated above. The 3 week old infested bag was opened on the day of planting and introduced at a rate of 3-5g per pot. On the same day as planting, CFU assays were conducted. One gram of sorghum inoculum was soaked in a 15 ml screw cap tube containing 1 ml of sterile PBS. Three tubes (replications) were done for each bag of infested sorghum. The seed suspension was vortexed for 10 seconds. From each tube, 100 pl was taken and added to the first well of a column containing 900 pl of PBS to make the first 10-1 dilution. Subsequent serial dilutions were made until a final dilution of 10-4 was achieved. From the dilution 10-3 and 10-4, 100 pl were spread on a 9cm diameter PDA plate supplemented with 0.01% Triton X100. The plates were incubated for 2-3 days at room temperature in the dark until colonies can be distinguished and counted. Colonies of Fusarium oxysporum f. sp. lactucae WQXQ identified and counted on each plate to determine the CFU values per gram of sorghum inoculum for each isolate.

[0189] Premium Playground Sand (Home Depot) was sterilized in an autoclave dry cycle for 30 minutes at 121 °C, followed by 30 minutes at 150 °C.

[0190] Sterilized playground sand and Fafard Potting Mix (Sungro) were mixed at a 1 :1 ratio. 3-inch pots were filled two thirds full with sterile potting / sand mixture. A layer of 1-3 grams of infested sorghum grain was added to each pot (to achieve 1x107CFU total) and the remaining volume of the pot was filled with potting / sand mix.

[0191] The 3-inch pots were placed in Phytatray Ill’s (Sigma) and 1g of Osmocote Smart-Release Plant Food Plus (Osmocote) was added between the bottom of the 3-inch pot and Phytatray. The potting mix was saturated with 100 ml of MilliQ Water by placing the water in the Phytatray and placing the 3 inch pot in the water.(3) Planting and plant maintenance

[0192] Two seeds of lettuce (Salanova Green Oakleaf variety) were planted in ~1 cm-deep holes in the middle of the pot (~3 cm spacing between the holes). Immediately after covering the seeds with potting mix, 1 ml of RBX product was pippetted on top of the soil directly above the seed.

[0193] Plants were maintained in GEN1000 (Conviron) Growth Chambers set to the following conditions: 16hr light (20°C) / 8hr dark (16°C), 50% Relative Humidity, 100% Light Intensity. Pots were watered from the bottom and after 7 days of growth, the plants were thinned to one plant per pot.(3) Plant leaf area measurements as biomass proxy

[0194] After 21 days, plants were individually photographed from above using a standardized stage and distance. The images were processed using ImageJ software to measure the pixels of green leaf area, as shown in FIG. 20A. Leaf area was plotted and used as a proxy for measuring biomass.

[0195] Each pot was imaged with a label and ruler for scale comparison. Images were acquired with an iPhone 16 at a fixed height and camera settings. Images were transferred to ImageJ and analyzed using the “Color Thresholding” feature with the following settings: Hue: 46 - 255; Saturation: 46 - 255; Brightness: 32 - 255

[0196] The resulting pixels were selected using the “Select” button at the bottom of the Threshold Color Window. “Command + M” generated a measurement of the number of pixels which were selected. The resulting measurement of pixels was copied and pasted into Google Sheets. Graphing and Statistical Analysis was accomplished using JMP vl8 Software Suite.

[0197] To assess treatment using bacterial delivery of dsRNA, Salanova® lettuce was inoculated with Fol or untreated (PBS), then treated with wild-type (WT s) or engineered Bacillus subtilis expressing dsRNA targeting PMT2 (SEQ ID NO: 20, GEBs-dsRNA3, “3”), multiple genes (SEQ ID NO: 24,GEBs-dsRNA4, “4”), or CYP51 (SEQ ID NO: 22 GEBs-dsRNA7, “7” and SEQ ID NO: 26 GEBs- dsRNA8, “8”) (n = 11 plants I condition). The individual pixel counts for each plant and average with standard deviation for each treatment condition are shown in FIG. 20B. Photographs of plants treated with B. subtilis expressing pan specific dsRNA comprised significantly more pixels than photos of plants treated with either WTAs, having a P-value of 0.0017, or Fol alone, with a P-value <0.0001, indicating a recovery of viability after inoculation with Fusarium oxysporum f. sp. lactucae by treatment with B. subtilis expressing a pan specific dsRNA. Photos of plants treated with bacteria expressing dsRNA targeting PMT2 or CYP51 had similar measured leaf areas, slightly smaller than the pan specific dsRNA.

[0198] A subset of parameters using bacterial delivery of dsRNA was repeated and results are shown in a scatterplot in FIG. 20C. Plants treated with A subtilis expressing dsRNA targeting multiple genes showed a significant increase in pixel count, representative of biomass, compared to plants treated with Fol, with P-value equal to 0.0001. The same treatment group was also significantly different from plants treated with wild-type B. subtilis with a P-value of 0.002. Images of lettuce plants from each treatment are shown in FIG. 20D.

[0199] To assess treatment using fungal delivery of dsRNA, Salanova® lettuce was inoculated with Fol or untreated (NaCl), then treated with wild-type (WT) or engineered Trichoderma harzianum expressing dsRNA targeting 0-glucuronidase (SEQ ID NO: 25 GUS, GETh-dsRNA5, “5”), or PMT2 (SEQ ID NO: 28 GETh-dsRNAl 1 “11,” SEQ ID NO: 29 GETh-dsRNA12 “12,” and SEQ ID NO: 30 GETh-dsRNA13 “13”) (n = 12 plants I condition). The individual pixel counts for each plant and average with standard deviation for each treatment condition are shown in FIG. 20E. The size of plants treated with T. harzianum expressing dsRNA targeting PMT2 were not measured significantly different than plants expressing wild-type T. harzianum.Example 26: Recovery of biomass in lettuce

[0200] Salanova Green Oakleaf lettuce seeds were planted into soil with a layer of 3g sorghum infested with ~1E7 CFU per pot Fusarium oxysporum f. sp. lactucae (Fol) and maintained in a growth chamber for 21 days with 12 hours photoperiod and 20 degrees Celsius light / 16 degrees Celsius dark temperature cycles and 60% air humidity. Control plants with no Fol and no Bacillus inoculum were used as a negative control (“Healthy plant”). Plants were allocated into 10 plants / treatment group. Immediately after planting, the soil above the seed was inoculated with 1ml ~1 x 109CFU / ml wild type or engineered Bacillus spore suspension or 1ml 0.9% sodium chloride (“Pathogen only”).Treatment groups received engineered Bacillus subtilis expressing the following dsRNA targeting: non-targeting randomized control (SEQ ID NO: 58) (dsRNA41, “D41”), panspecific targets (SEQ ID NO: 24) (dsRNA4, “D4”), fksl (SEQ ID NO: 33) (dsRNA16, “D16”), rhol-rhoA-rho2-rho3 (SEQ ID NO: 34) (dsRNA17, “D17”), ERG1 (SEQ ID NO: 35) (dsRNA18 “D18”), ERG3 / ERG5 (SEQ ID NO: 36) (dsRNA19, “D19”), CYP51 (SEQ ID NO: 37) (dsRNA20, “D20”), ERG4 (SEQ ID NO: 38) (dsRNA21, “D21”), ERG24 (SEQ ID NO: 39) (dsRNA22, “D22”), SGE1 (SEQ ID NO: 40) (dsRNA23, “D23”), Myosin-1 (SEQ ID NO: 41) (dsRNA24, “D24”), PMT1-PMT2-PMT4 (SEQ ID NO: 42) (dsRNA25, “D25”), Luc7 (SEQ ID NO: 43) (dsRNA26, “D26”), Tom40 (SEQ ID NO: 44) (dsRNA27, “D27”), and rhol-rhoA-rho2-rho3 -fksl (SEQ ID NO: 59) (dsRNA42, “D42”). Engineered strains were compared to two commercial biological controls, Asparello® T34 (Biobest Group, Westerlo, Belgium) and Rootshield® (BioWorks, Victor, NY) applied as root drenches at commercial rates.

[0201] 11 engineered strains of Bacillus, shown in bold in FIGs 21A and 21B, show a statistically significant increase in leaf area compared to untreated (Pathogen only), with p-values: < 0.0002 (***), < 0.005 (**), and < 0.05 (*). Two strains show a statistically significant increase in leaf area compared to the wild-type control (A, p < 0,05). Bacillus expressing dsRNA42 (“D42”) outperforms Asperello® and Rootshield® (p < 0.05).

[0202] Growth in Fol-infected lettuce treated with Bacillus subtilis engineered to express dsRNA4, having pan-specific targeting, was repeated under similar conditions, and compared to Healthy plant, Pathogen only and wild-type Bacillus, with 16 plants in each group. Growth results are shown in FIG. 21C, demonstrating a significant increase in growth compared to lettuce treated with pathogen only (p<0.0001) and wild-type Bacillus (p=0.002).

[0203] Salanova Green Oakleaf lettuce was inoculated with one of two field isolates of Fol, Fol-01 and Fol-02. Groups of 10 infected plants each were treated with wild-type or engineered Bacillus subtilis expressing dsRNA targeting panspecific targets (SEQ ID NO: 24) (dsRNA4, “D4”) and compared to growth of infected plants treated with commercial biological control Rootshield® (BioWorks). Measured leaf area in pixels is shown in FIG. 21D. Data indicates growth in infected plants treated with engineered B. subtilis expressing panspecific dsRNA equal to or greater than growth in infected plants treated with Rootshield.Example 27: Additional MIGS screening methods

[0204] Inoculation with the pathogen Fol can be achieved by preparing a sorghum infested seed inoculum in the same way that is indicated for F. virgulifarme in Example 10 and applying it as a layer in the middle of the soil. Treatment with an engineered microbe uses a seed or seedling drench with a cell or spore suspension as indicated in Example 10.

[0205] Planting is done by directly sowing seeds in water saturated Fafard Professional Potting Mix (Sungro). The plants are maintained in a growth chamber or greenhouse for 3-4 weeks with 12 hours photoperiod and 20 degrees Celsius light / 16 degrees Celsius dark temperature cycles and 60% air humidity. Alternatively, plants can be grown in a greenhouse to yield with similar environmental settings.

[0206] Depending on their developmental stage, plants receive 50 to 250 ml water with one Tablespoon per gallon of Jack’s solution (20:20:20 - sulfurphosphorus :potassium) per 4 inch square pot. Alternatively, plants are fertilized by applying a teaspoon of Osmocote Professional nutrient pellets in between each pot and Phytatray and watered with plain reverse osmosis water.

[0207] The plants are organized inside the growth chamber or greenhouse in a completely randomized spatial arrangement. The number of plant replicates used per treatment is determined using a power analysis that uses data generated through the assay development, aiming at detecting differences in disease index scoring and biomass (see below) between treatments of at least 10% in magnitude with an 80% power and a 95% confidence. Based on previous experience, the number of replicates can vary between 5 and up to 30 plants per treatment. Treatments consist of plants treated with the engineered microbes described herein with an active payload against the pathogen (e.g. dsRNA hairpin to generate silencing RNAs against PMT2 gene in Fol) and several controls that can include nontreated plants, plants treated with a formulation and versions of the engineered microbes that lack their active payloads.(1) Disease index scoring

[0208] A 1-4 scale wilt score is used: 1) No symptom s / heal thy plant, 2) mild stunting, 3) severe stunted growth and chlorosis / necrosis, and 4) dead plant. The ratings are analyzed using a Kruska- Wallis test, followed by pairwise comparisons using the Wilcoxon rank-sum test. MIGS is indicated by a lower disease scoring in plants treated with the engineered microorganism.(2) Plant biomass measurements and root disease severity

[0209] The above ground parts of the plants are excised at the crown with ethanol sterilized scissors (make sure to clean in between treatments) and weighed. Roots are pulled gently by hand out of the soil mix in the pots and the soil attached is removed by shaking. Further cleaning is accomplished byrinsing under tap water. Excess moisture is removed by patting with paper towels. Roots are visually inspected for disease incidence and severity is recorded using a 1 to 5 scale. The percentage of rotted roots are estimated and assigned a score of: 1=1-20%, 2=21-40%, 3=41-60%, 4=61-80%, or 5 = 81- 100%. The entire root system from each plant is placed in a pre-labeled waxed paper bag and dried in the oven in the same way as the above ground parts. Above ground parts and roots are weighed using a laboratory scale with decigram level precision. The ratings are analyzed using a Kruska-Wallis test, followed by pairwise comparisons using the Wilcoxon rank-sum test. MIGs are indicated by a lower root disease scoring in plants treated with the engineered microorganism.(3) Fol biomass estimation

[0210] Total DNA is extracted from the soil mix using the Zymo quick DNA fecal / soil microbiome kit. The quality and yield of DNA is quantified using a Nanodrop. Quantitative PCR is performed using primers Fol la-F 5’-TGTACCCTGATAATCCTGGTAC-3’ (SEQ ID NO: 163) and Folla-R 5’- AGCTTGACTCTATCGTTGTCGA-3’ (SEQ ID NO: 164) and Folla-P 5’-6FAM- CCAGCAGGCTGAAGGATGCTTTGTA-QSY7-3’ (SEQ ID NO: 165). 20 microliter reactions are prepared, consisting of IX PerfeCTa Multiplex qPCR ToughMix (Quantabio, Beverly, MA), 400nM of each of Folla-F and Folla-R primers, 50nM of the probe and 2 pl of DNA template (10-20 ng). The qPCR conditions are initialized with 1 cycle of incubation at 95 degrees Celsius for 10 min and 40 cycles at 95 degrees Celsius for 15 s and 60 degrees Celsius for 1 min, with fluorescent data collection in the annealing and extension step. Extrapolation of CT values to CFU per gram of soil is achieved by developing a standard curve. Estimates of biomass are compared among treatments using ANOVA and a post hoc pairwise comparison, such as Tuckey’s honest test. MIGS is indicated by reductions in biomass of Fol in the rhizosphere of plants treated with the engineered microorganism.Example 28: MIGS Assay in Strawberry

[0211] Protection of strawberries from Fusarium oxysporum f. ^.ftagariae challenge by treatment with microorganisms engineered as described herein is demonstrated.(1) Spore suspension, Seedling inoculation and planting

[0212] The method is similar to Example 11 but for the following:

[0213] a cultivar susceptible to Fusarium oxysporum f. sp. fragariae (Fof) infection, such as Monterey, which is a susceptible and marketable cultivar, is chosen for the assay. Seedlings are planted in a water saturated 1: 1 mix of autoclaved playground sand and “Fafard Professional Potting Mix”. The recommended environmental conditions for plant growth either in a growth chamber orgreenhouse are 25 degrees Celsius light / 18 degrees Celsius dark temperature cycles and 50% air humidity.(2) Disease index scoring

[0214] A 0-3 scale is used: 0) No symptom s / healthy plant, 1) a leaf or two displaying wilting and / or moderate stunting; 2) many leaves wilted, severe stunted growth, and 3) dead plant.(3) Plant biomass measurements and root disease severity

[0215] The above ground parts of the plants are excised at the crown with ethanol sterilized scissors (make sure to clean in between treatments) and placed in pre-labeled waxed paper bags for drying in an oven at 50 degrees Celsius, until weights are unchanged after 24 h by further drying. Roots are pulled gently by hand out of the soil mix in the pots and the soil attached is removed by shaking. Further cleaning is accomplished by rinsing under tap water. Excess moisture is removed by patting with paper towels. Roots are visually inspected for disease incidence and severity is recorded using a 1 to 5 scale. The percentage of rotted roots are estimated and assigned a score of: 1=1-20%, 2=21- 40%, 3=41-60%, 4=61-80%, or 5 = 81-100%. The entire root system from each plant is placed in a pre-labeled waxed paper bag and dried in the oven in the same way as the above ground parts. Above ground parts and roots are weighed using a laboratory scale with decigram level precision. The ratings for both disease index and root rotting are analyzed using a Kruska-Wallis test, followed by pairwise comparisons using the Wilcoxon rank-sum test. MIGS is indicated by a lower disease scoring and / or higher plant or root biomass in plants treated with the engineered microorganism.(4) F of biomass estimation

[0216] Total DNA is extracted from the soil mix using the Zymo quick DNA fecal / soil microbiome kit. The quality and yield of DNA is quantified using a Nanodrop. Quantitative PCR is performed using primers Frag F 5’-GTGAGACGGACATCTTGAAG-3’ (SEQ ID NO: 166) and Frag-R 5’- AGGAACATTTCCAGCACCGA-3’ (SEQ ID NO: 167) and Frag_TaqMan_probe 5’-6FAM- CACTTGCCTTTGGGCTCTGGATACTAAC-BHQ1-3’ (SEQ ID NO: 168). 25 microliter reactions are prepared, consisting of IX PerfeCTa Multiplex qPCR ToughMix (Quantabio, Beverly, MA), 400nM of each ofFrag_F and Frag_R primers, 2 pl of DNA template (10-20 ng). The qPCR conditions are initialized with 1 cycle of incubation at 95 degrees Celsius for 3 min and 40 cycles at 95 degrees Celsius for 15 s and 62 degrees Celsius for 30 seconds, with fluorescent data collection in the annealing and extension step. Extrapolation of CT values to CFU per gram of soil is achieved by developing a standard curve. Estimates of biomass are compared among treatments using ANOVA and a post hocpairwise comparison, such as Tuckey’ s honest test. MIGS is indicated by reductions in biomass of Fof in the rhizosphere of plants treated with the engineered microorganism.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered microorganism, wherein the engineered microorganism comprises: at least one dsRNA expression cassette, wherein the at least one dsRNA expression cassette is integrated in a genome of the engineered microorganism, and wherein the at least one dsRNA expression cassette comprises: an expression region comprising a complementary pair of sequences encoding for a dsRNA about 20-5000 base pairs in length and comprising a plurality of small interfering RNAs (siRNAs), each 18-31 nucleotides in length, wherein one of the sequences of the complementary pair of sequences comprises a sequence of at least one region of at least one target gene in at least one target organism, and wherein the plurality of siRNAs suppresses expression of the at least one target gene; and two antisense promoters flanking the sequences encoding the dsRNA, wherein the promoters are in opposite orientation to each other.

2. The engineered microorganism of claim 1, wherein the genome of the engineered microorganism comprises from 1 to 40 dsRNA expression cassettes.

3. The engineered microorganism of claim 1, wherein the region of the at least one target gene is an exon region.

4. The engineered microorganism of claim 1, wherein the plurality of siRNAs suppresses expression of 2 to 40 target genes.

5. The engineered microorganism of claim 1, wherein the target genes are in more than one target organism.

6. The engineered microorganism of claim 5, wherein the target genes are in 2 to 40 target organisms.

7. The engineered microorganism of claim 1, wherein the plurality of siRNAs comprises a synthetic sequence.

8. The engineered microorganism of claim 7, wherein the synthetic sequence is generated based on factors comprising consensus splice variant target sequence, DICER activity sites, thermodynamics of siRNA binding to mRNA, reduction of off-target binding, and any combination thereof.

9. The engineered microorganism of claim 1, wherein the dsRNA comprises a plurality of DICER cleavage sites.

10. The engineered microorganism of claim 9, wherein the dsRNA comprises from 1 to 5000 DICER cleavage sites.

11. The engineered microorganism of claim 10, wherein a distribution of DICER cleavage sites provides for more than one cleavage motif to generate the plurality of siRNAs 18-31 nucleotides in length.

12. The engineered microorganism of claim 11, wherein the more than one cleavage motif comprises from 1 to 5000 cleavage motifs.

13. The engineered microorganism of claim 12, wherein the from 1 to 40 cleavage motifs provide for from 1 to about 70,000 distinct RNA sequences 18-31 nucleotides in length.

14. The engineered microorganism of claim 13, wherein the plurality of distinct RNA sequences are siRNAs.

15. The engineered microorganism of claim 14, wherein from about 1% to about 100% of the distinct RNA sequences comprise the plurality of siRNAs.

16. The engineered microorganism of 15, wherein the plurality of siRNAs suppress from about 1% to about 100% expression of a target gene.

17. The engineered microorganism of claim 1, wherein the dsRNA comprises a plurality of DICER modulating spacer regions (DMSR).

18. The engineered microorganism of claim 17, wherein the sequences encoding the dsRNA further comprise high-affinity DICER cut sites distinct from the DMSR.

19. The engineered microorganism of claim 1, wherein the engineered microorganism exhibits a characteristic comprising my coparasitism, biofilm formation, swarming, secretion, production of extracellular vesicles, or expression of natural antifungal or antibacterial defense toxic compounds or peptides.

20. The engineered microorganism of claim 19, wherein the engineered microorganism is further modified to exhibit the characteristic.

21. The engineered microorganism of claim 1, wherein the engineered microorganism further expresses Vesicle Nucleating peptide (NVp).

22. The engineered microorganism of claim 1, wherein the target organism is a fungus, an oomycete, a nematode, a plant, an insect, or a virus.

23. The engineered microorganism of claim 22, wherein the target organism is the fungus or the oomycete.

24. The engineered microorganism of claim 23, wherein the fungus or the oomycete is a Blumeria spp., Botrytis spp., Cadophora spp., Colletotrichum spp., Fusarium spp., Macrophomina spp., Mycosphaerella spp., Phakopsora spp., Phomposis spp., Phyllachora spp., Phytophthora spp., Puccinia spp., Pyricularia spp., Pythium spp., Rhizoctonia spp., Rhizomonas spp., Sclerotinia spp., Sclerotium spp., or Verticillium spp.

25. The engineered microorganism of claim 24, wherein the at least one target gene comprises CDC1, CDC2, CDC28, SIR2, RAD52, POL3, SEC14, HOG1, GAPDH, RPL, FMK1, PMT1, PMT2, PMT4, CYP51, HSP90, SOD1, FKS1, RHO1, ERG1, ERG3, ERG4, ERG5, ERG11, ERG24, SGE1, MYOSIN 5, LUC7, TOM40, FV-TOX1, GBP1, CUT3, HMP1,CESA, ENDO3, ENDOP, RMPK1, CRZ1, CAT, PG1, CHS1, CHS6, OBSP, GPA1, GPB1, GBS, GPR11, GPCR, GLS2, STP, LLS, TUB, THIT, NLP1, hydrophobinl, AAC, CD A, or any combination thereof.

26. The engineered microorganism of claim 24, wherein the dsRNA is complementary to a region of any one of SEQ ID NOs: 19-114.

27. The engineered microorganism of claim 22, wherein the target organism is the nematode.

28. The engineered microorganism of claim 27, wherein the nematode is a Heterodera spp. or Meloidogyne spp.

29. The engineered microorganism of claim 27, wherein the at least one target gene comprises Gr-EXPB2, Mi-SXPl, Mi-CRT, Hg30C02, Hsl0A06, Mj-col-45, Mj-col-13, Hg-cpa-6, CHS 1 & 2, Mj-ndk-1 (nucleoside diphosphate kinase), Hg-acs-1 (acyl-CoA synthetase), Mj-egl- 30 (Ras-like GTPase), Hg-kin-1 (serine / threonine-protein kinase), Mj-crt, Mj-ral-2, Mi-col- 1, Mi-drsh-1, Mincl6803, Mi-duoxl, Lemmi-5, Hg-snb-1 (synaptobrevin), Hg-cpn-1, Hg-rps- 23, Hg-Prp-17, Hg-cobp-1, Hg-rsr-2, or any combination thereof.

30. The engineered microorganism of claim 27, wherein the dsRNA is complementary to a region of any one of SEQ ID NOs: 115-146.

31. The engineered microorganism of claim 22, wherein the target organism is the plant.

32. The engineered microorganism of claim 31, wherein the at least one target gene comprises a gene encoding 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS), Acetolactate synthase (ALS), PPO (Protoporphyrinogen oxidase), ACCase (Acetyl-CoA carboxylase), Photosystem II (PSII) reaction center proteins (DI, D2), ALS (Alpha- and beta-tubulin), HPPD (4-hydroxyphenylpyruvate dioxygenase), HPPD (4-hydroxyphenylpyruvate dioxygenase), GS (Glutamine synthetase), ALS (ALS1 and ALS2, Arabidopsis thaliana homologs), AHAS (Acetohydroxy acid synthase), GAT (Glutamate- 1 -semialdehyde aminotransferase), RUBISCO (Ribulose- 1,5-bisphosphate carboxylase / oxygenase), PPGPP (Guanosine pentaphosphate synthetase), ATPase (Adenosine triphosphatase), MVA pathway genes (Mevalonate pathway), ADC (Arginine decarboxylase), DHPS (Dihydropteroate synthase), GTPCH (GTP cyclohydrolase I), GST (Glutathione S-transferase), ABC transporters (ATP -binding cassette transporters), Cytochrome P450 monooxygenases, CYP (Cytochrome P450 family), qSORl, or any combination thereof.

33. The engineered microorganism of claim 22, wherein the target organism is the insect.

34. The engineered microorganism of claim 33, wherein the at least one target gene comprises a gene encoding Chitin Synthase (CHS), Vitellogenin (Vg), Hexamerin, Juvenile Hormone Esterase (JHE), Acetylcholinesterase (AChE), Arginine Kinase, Cathepsin L, Trehalose-6- phosphate synthase (TPS), Snf7, Cullin-1, or Rabi 1, or any combination thereof.

35. The engineered microorganism of claim 1, wherein the target organism is found in a phyllosphere, a rhizosphere, soil, a root system, a shoot system, a flower, a leaf, a fruit, a stem, a root, or any combination thereof, of a plant.

36. The engineered microorganism of claim 35, wherein the engineered microorganism is natively co-located with the target organism in the phyllosphere, the rhizosphere, the root system, soil, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant.

37. The engineered microorganism of claim 35, wherein the engineered microorganism is further modified to grow in the phyllosphere, the rhizosphere, the root system, soil, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant, and wherein the engineered microorganism is co-located with the target organism.

38. The engineered microorganism of claim 1, wherein the anti-sense promoters are head to head promoters.

39. The engineered microorganism of claim 1, wherein the anti-sense promoters are one or more bidirectional promoters.

40. The engineered microorganism of claim 1, wherein the two antisense promoters each independently comprise the sequence of any one of SEQ ID Nos: 1-11.

41. The engineered microorganism of claim 1, wherein the engineered microorganism is a sporeforming microorganism.

42. The engineered microorganism of claim 1, wherein the engineered microorganism is a fungus.

43. The engineered microorganism of claim 42, wherein the fungus is a Trichoderma spp.

44. The engineered microorganism of claim 42, wherein the antisense promoters each comprise a strong constitutive or inducible promoter.

45. The engineered microorganism of claim 44, wherein the antisense promoters each comprise independently Pural, Pcbhl, PceI7, Ppgll, Pamyl, Ptefl, PcDNAl, PgpdA, Prp2, U6, Peel, Pxyn, Ptcul, Pyatl, Pactin, PoliC, PtetR, Pxyl, or any combination thereof.

46. The engineered microorganism of claim 1, wherein the engineered microorganism is a bacteria.

47. The engineered microorganism of claim 46, wherein the bacteria is a Bacillus spp.

48. The engineered microorganism of claim 46, wherein the antisense promoters each comprise a strong constitutive or inducible promoter.

49. The engineered microorganism of claim 48, wherein the antisense promoters each comprise independently PphoA, P43, P45, PT7, P34, PxylA, Physpank, Pspac, Pveg, PamyE, PgrD, PbacA, PcotC, Pspank, PnrpE, PamyP, PespsA, PSP6, PrrnBl+2 WT, PrrnBl+2 A starts or any combination thereof.

50. The engineered microorganism of claim 1, wherein the at least one target gene comprises a gene that affects a plant trait.

51. The engineered microorganism of claim 50, wherein the at least one target gene comprises GmFAD3, El, ZmMADSl, GmMIPSl, ZLKR, SDH, ZmBCH2, ghSAD-1, ghFAD2-l, ANR, FaMYBl, TFL1, plC, FaPGl, FaWRKY29, FaWRKY64, AGPase, LsNCED4, qSORl, or any combination thereof.

52. An engineered microorganism comprising at least one dsRNA expression cassette encoding for a hairpin dsRNA comprising a plurality of small interfering RNAs (siRNAs) each 18-31 nucleotides in length, wherein the dsRNA expression cassette is integrated in a genome of the engineered microorganism and wherein the at least one dsRNA expression cassette comprises a sequence comprising: a promoter; a first coding sequence about 20-5000 nucleotides in length comprising a sequence of at least one region of at least one target gene in at least one target organism; a second coding sequence, wherein the second coding sequence is the reverse complement of the first sequence; andan intron region separating the first coding region and the second coding region about 50-400 nucleotides in length; wherein the plurality of siRNAs suppresses expression of the at least one target gene.

53. The engineered microorganism of claim 52, wherein the genome of the engineered microorganism comprises from 1 to 40 dsRNA expression cassettes.

54. The engineered microorganism of claim 52, wherein the region of the at least one target gene is an exon region.

55. The engineered microorganism of claim 52, wherein the intron region is a hairpin region.

56. The engineered microorganism of claim 52, wherein the plurality of siRNAs suppresses expression of 2 to 40 target genes.

57. The engineered microorganism of claim 52, wherein the target genes are in more than one target organism.

58. The engineered microorganism of claim 57, wherein the target genes are in 2 to 40 target organisms.

59. The engineered microorganism of claim 52, wherein the plurality of siRNAs comprises a synthetic sequence.

60. The engineered microorganism of claim 59, wherein the synthetic sequence is generated based on factors comprising consensus splice variant target sequence, DICER activity sites, thermodynamics of siRNA binding to mRNA, reduction of off-target binding, and any combination thereof.

61. The engineered microorganism of claim 52, wherein the dsRNA comprises a plurality of DICER cleavage sites.

62. The engineered microorganism of claim 61, wherein the dsRNA comprises from 1 to 500 DICER cleavage sites.

63. The engineered microorganism of claim 62, wherein a distribution of DICER cleavage sites provides more than one cleavage motif to generate the plurality of siRNAs 18-31 nucleotides in length.

64. The engineered microorganism of claim 63, wherein the more than one cleavage motif comprises from 1 to 5000 cleavage motifs.

65. The engineered microorganism of claim 64, wherein the from 1 to 5000 cleavage motifs provide for from 1 to about 70,000 distinct RNA sequences 18-31 nucleotides in length.

66. The engineered microorganism of claim 65, wherein the plurality of distinct RNA sequences are siRNAs.

67. The engineered microorganism of claim 66, wherein from about 1% to about 100% of the distinct RNA sequences comprise the plurality of siRNAs.

68. The engineered microorganism of 67, wherein the plurality of siRNAs suppress from about 1% to about 100% expression of a target gene.

69. The engineered microorganism of claim 52, wherein the dsRNA comprises a plurality of DICER modulating spacer regions (DMSR).

70. The engineered microorganism of claim 69, wherein the sequence encoding the dsRNA further comprises high-affinity DICER cut sites distinct from the DMSR.

71. The engineered microorganism of claim 52, wherein the engineered microorganism exhibits a characteristic comprising my coparasitism, biofilm formation, swarming, secretion, production of extracellular vesicles, or expression of natural antifungal or antibacterial defense toxic compounds or peptides.

72. The engineered microorganism of claim 71, wherein the engineered microorganism is further modified to exhibit the characteristic.

73. The engineered microorganism of claim 52, wherein the engineered microorganism further expresses Vesicle Nucleating peptide (NVp).

74. The engineered microorganism of claim 52, wherein the target organism is a fungus, an oomycete, a nematode, a plant, an insect, or a virus.

75. The engineered microorganism of claim 74, wherein the target organism is the fungus or the oomycete.

76. The engineered microorganism of claim 75, wherein the fungus or the oomycete is a Blumeria spp., Botrytis spp., Cadophora spp., Collet otrichum spp., Fusarium spp., Macrophomina spp., Mycosphaerella spp., Phakopsora spp., Phomposis spp., Phyllachora spp., Phytophthora spp., Puccinia spp., Pyricularia spp., Pythium spp., Rhizoctonia spp., Rhizomonas spp., Sclerotinia spp., Sclerotium spp., or Verticillium spp.

77. The engineered microorganism of claim 75, wherein the at least one target gene comprises CDC1, CDC2, CDC28, SIR2, RAD52, POL3, SEC14, H0G1, GAPDH, RPL, FMK1, PMT1, PMT2, PMT4, CYP51, HSP90, SOD1, FKS1, RH01, ERG1, ERG3, ERG4, ERG5, ERG11, ERG24, SGE1, MYOSIN 5, LUC7, TOM40, FV-TOX1, GBP1, CUT3, HMP1,CESA, END03, ENDOP, RMPK1, CRZ1, CAT, PG1, CHS1, CHS6, OBSP, GPA1, GPB1, GBS, GPR11, GPCR, GLS2, STP, LLS, TUB, THIT, NLP1, hydrophobinl, AAC, CD A, or any combination thereof.

78. The engineered microorganism of claim 75, wherein the dsRNA is complementary to a region of any one of SEQ ID NOs: 19-114.

79. The engineered microorganism of claim 74, wherein the target organism is the nematode.

80. The engineered microorganism of claim 79, wherein the nematode is a Heterodera spp. or Meloidogyne spp.

81. The engineered microorganism of claim 79, wherein the at least one target gene comprises Gr-EXPB2, Mi-SXPl, Mi-CRT, Hg30C02, Hsl0A06, Mj-col-45, Mj-col-13, Hg-cpa-6, CHS 1 & 2, Mj-ndk-1 (nucleoside diphosphate kinase), Hg-acs-1 (acyl-CoA synthetase), Mj-egl- 30 (Ras-like GTPase), Hg-kin-1 (serine / threonine-protein kinase), Mj-crt, Mj-ral-2, Mi-col- 1, Mi-drsh-1, Mincl6803, Mi-duoxl, Lemmi-5, Hg-snb-1 (synaptobrevin), Hg-cpn-1, Hg-rps- 23, Hg-Prp-17, Hg-cobp-1, Hg-rsr-2, or any combination thereof.

82. The engineered microorganism of claim 79, wherein the dsRNA is complementary to a region of any one of SEQ ID NOs: 115-146.

83. The engineered microorganism of claim 74, wherein the target organism is the plant.

84. The engineered microorganism of claim 83, wherein the at least one target gene comprises a gene encoding 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS), Acetolactate synthase (ALS), PPO (Protoporphyrinogen oxidase), ACCase (Acetyl-CoA carboxylase), Photosystem II (PSII) reaction center proteins (DI, D2), ALS (Alpha- and beta-tubulin), HPPD (4-hydroxyphenylpyruvate dioxygenase), HPPD (4-hydroxyphenylpyruvate dioxygenase), GS (Glutamine synthetase), ALS (ALS1 and ALS2, Arabidopsis thaliana homologs), AHAS (Acetohydroxy acid synthase), GAT (Glutamate- 1 -semialdehyde aminotransferase), RUBISCO (Ribulose- 1,5-bisphosphate carboxylase / oxygenase), PPGPP (Guanosine pentaphosphate synthetase), ATPase (Adenosine triphosphatase), MVA pathway genes (Mevalonate pathway), ADC (Arginine decarboxylase), DHPS (Dihydropteroate synthase), GTPCH (GTP cyclohydrolase I), GST (Glutathione S-transferase), ABC transporters (ATP -binding cassette transporters), Cytochrome P450 monooxygenases, CYP (Cytochrome P450 family), qSORl, or any combination thereof.

85. The engineered microorganism of claim 74, wherein the target organism is the insect.

86. The engineered microorganism of claim 85, wherein the at least one target gene comprises a gene encoding Chitin Synthase (CHS), Vitellogenin (Vg), Hexamerin, Juvenile Hormone Esterase (JHE), Acetylcholinesterase (AChE), Arginine Kinase, Cathepsin L, Trehalose-6- phosphate synthase (TPS), Snf7, Cullin-1, or Rabi 1, or any combination thereof.

87. The engineered microorganism of claim 52, wherein the target organism is found in a phyllosphere, a rhizosphere, a root system, a shoot system, a flower, a leaf, a fruit, a stem, a root, or any combination thereof, of a plant.

88. The engineered microorganism of claim 87, wherein the engineered microorganism is natively co-located with the target organism in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant.

89. The engineered microorganism of claim 87, wherein the engineered microorganism is further modified to grow in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant, wherein the engineered microorganism is co-located with the target organism.

90. The engineered microorganism of claim 52, wherein the engineered microorganism is a spore-forming microorganism.

91. The engineered microorganism of claim 52, wherein the engineered microorganism is a fungus.

92. The engineered microorganism of claim 91, wherein the fungus is a Trichoderma spp.

93. The engineered microorganism of claim 91, wherein the promoter comprises a strong constitutive or inducible promoter.

94. The engineered microorganism of claim 93, wherein the promoter comprises Pural, Pcbhl, PceI7, Ppgll, Pamyl, Ptefl, PcDNAl, PgpdA, Prp2, U6, Peel, Pxyn, Ptcul, Pyatl, Pactin, PoliC, PtetR, Pxyl, or any combination thereof.

95. The engineered microorganism of claim 52, wherein the engineered microorganism is a bacteria.

96. The engineered microorganism of claim 95, wherein the bacteria is a Bacillus spp.

97. The engineered microorganism of claim 95, wherein the promoter comprises a strong constitutive or inducible promoter.

98. The engineered microorganism of claim 97, wherein the promoter comprises independently PphoA, P43, P45, PT7, P34, PxylA, Physpank, Pspac, Pveg, PamyE, PgrD, PbacA, PcotC, Pspank, PnrpE, PamyP, PespsA, PSP6, PrrnBl+2 WT, PrrnBl+2 A starts or any combination thereof.

99. The engineered microorganism of claim 52, wherein the promoter comprises the sequence of any one of SEQ ID Nos: 1-11.

100. The engineered microorganism of claim 52, wherein the at least one target gene is a gene that affects a plant trait.

101. The engineered microorganism of claim 100, wherein the at least one target gene comprises GmFAD3, El, ZmMADSl, GmMIPSl, ZLKR, SDH, ZmBCH2, ghSAD-1, ghFAD2-l, ANR, FaMYBl, TFL1, plC, FaPGl, FaWRKY29, FaWRKY64, AGPase, LsNCED4, qSORl, or any combination thereof.

102. An engineered microorganism, wherein the engineered microorganism comprises a plurality of dsRNA expression cassettes integrated in a genome of the engineered microorganism, wherein each dsRNA expression cassette comprises: a complementary pair of sequences encoding a small interfering RNA (siRNA) 18-31 nucleotides in length, wherein the siRNA comprises a sequence complementary to a region of a target gene in a target organism, and wherein the siRNA suppresses expression of the target gene; and two antisense promoters flanking the sequences encoding the siRNA, wherein the promoters are in opposite orientation to each other.

103. The engineered microorganism of claim 102, wherein the engineered microorganism comprises 2 to 40 genomic insertions.

104. The engineered microorganism of claim 102, wherein the region of the target gene is an exon region.

105. The engineered microorganism of claim 102, wherein the siRNA suppresses expression of 2 to 40 target genes.

106. The engineered microorganism of claim 102, wherein the siRNA targets genes in more than one target organism.

107. The engineered microorganism of claim 106, wherein the target genes are in 2 to 40 target organisms.

108. The engineered microorganism of claim 102, wherein the siRNA comprises a synthetic sequence.

109. The engineered microorganism of claim 108, wherein the synthetic sequence is generated based on factors comprising consensus splice variant target sequence, thermodynamics of siRNA binding to mRNA, reduction of off-target binding, and any combination thereof.

110. The engineered microorganism of claim 102, wherein the engineered microorganism exhibits a characteristic comprising my coparasitism, biofdm formation, swarming, secretion, production of extracellular vesicles, or expression of natural antifungal or antibacterial defense toxic compounds or peptides.

111. The engineered microorganism of claim 110, wherein the engineered microorganism is further modified to exhibit the characteristic.

112. The engineered microorganism of claim 102, wherein the engineered microorganism further expresses Vesicle Nucleating peptide (NVp).

113. The engineered microorganism of claim 102, wherein the target organism is a fungus, an oomycete, a nematode, a plant, an insect, or a virus.

114. The engineered microorganism of claim 113, wherein the target organism is the fungus or the oomycete.

115. The engineered microorganism of claim 114, wherein the fungus or the oomycete is a Blumeria spp., Botrytis spp., Cadophora spp., Collet otrichum spp., Fusarium spp., Macrophomina spp., Mycosphaerella spp., Phakopsora spp., Phomposis spp., Phyllachora spp., Phytophthora spp., Puccinia spp., Pyricularia spp., Pythium spp., Rhizoctonia spp., Rhizomonas spp., Sclerotinia spp., Sclerotium spp., or Verticillium spp.

116. The engineered microorganism of claim 114, wherein the at least one target gene comprises CDC1, CDC2, CDC28, SIR2, RAD52, POL3, SEC14, HOG1, GAPDH, RPL, FMK1, PMT1, PMT2, PMT4, CYP51, HSP90, SOD1, FKS1, RHO1, ERG1, ERG3, ERG4, ERG5, ERG11, ERG24, SGE1, MYOSIN 5, LUC7, TOM40, FV-TOX1, GBP1, CUT3, HMP1,CESA, ENDO3, ENDOP, RMPK1, CRZ1, CAT, PG1, CHS1, CHS6, OBSP, GPA1, GPB1, GBS, GPR11, GPCR, GLS2, STP, LLS, TUB, THIT, NLP1, hydrophobinl, AAC, CD A, or any combination thereof.

117. The engineered microorganism of claim 114, wherein the dsRNA is complementary to a region of any one of SEQ ID NOs: 19-114.

118. The engineered microorganism of claim 113, wherein the target organism is the nematode.

119. The engineered microorganism of claim 118, wherein the nematode is a Heterodera spp. or Meloidogyne spp.

120. The engineered microorganism of claim 118, wherein the at least one target gene comprises Gr-EXPB2, Mi-SXPl, Mi-CRT, Hg30C02, Hsl0A06, Mj-col-45, Mj-col-13, Hg-cpa-6, CHS 1 & 2, Mj-ndk-1 (nucleoside diphosphate kinase), Hg-acs-1 (acyl-CoA synthetase), Mj-egl- 30 (Ras-like GTPase), Hg-kin-1 (serine / threonine-protein kinase), Mj-crt, Mj-ral-2, Mi-col- 1, Mi-drsh-1, Mincl6803, Mi-duoxl, Lemmi-5, Hg-snb-1 (synaptobrevin), Hg-cpn-1, Hg-rps- 23, Hg-Prp-17, Hg-cobp-1, Hg-rsr-2, or any combination thereof.

121. The engineered microorganism of claim 118, wherein the dsRNA is complementary to a region of any one of SEQ ID NOs: 115-146.

122. The engineered microorganism of claim 113, wherein the target organism is the plant.

123. The engineered microorganism of claim 122, wherein the at least one target gene comprises a gene encoding 5 -enolpyruvylshikimate-3 -phosphate synthase (EPSPS), Acetolactate synthase (ALS), PPO (Protoporphyrinogen oxidase), ACCase (Acetyl-CoA carboxylase), Photosystem II (PSII) reaction center proteins (DI, D2), ALS (Alpha- and beta-tubulin), HPPD (4-hydroxyphenylpyruvate dioxygenase), HPPD (4-hydroxyphenylpyruvate dioxygenase), GS (Glutamine synthetase), ALS (ALS1 and ALS2, Arabidopsis thaliana homologs), AHAS (Acetohydroxy acid synthase), GAT (Glutamate- 1 -semialdehyde aminotransferase), RUBISCO (Ribulose- 1,5-bisphosphate carboxylase / oxygenase), PPGPP (Guanosine pentaphosphate synthetase), ATPase (Adenosine triphosphatase), MVA pathway genes (Mevalonate pathway), ADC (Arginine decarboxylase), DHPS (Dihydropteroate synthase), GTPCH (GTP cyclohydrolase I), GST (Glutathione S-transferase), ABC transporters (ATP -binding cassette transporters), Cytochrome P450 monooxygenases, CYP (Cytochrome P450 family), qSORl, or any combination thereof.

124. The engineered microorganism of claim 113, wherein the target organism is the insect.

125. The engineered microorganism of claim 124, wherein the at least one target gene comprises a gene encoding Chitin Synthase (CHS), Vitellogenin (Vg), Hexamerin, Juvenile Hormone Esterase (JHE), Acetylcholinesterase (AChE), Arginine Kinase, Cathepsin L, Trehalose-6- phosphate synthase (TPS), Snf7, Cullin-1, or Rabi 1, or any combination thereof.

126. The engineered microorganism of claim 102, wherein the target organism is found in a phyllosphere, a rhizosphere, a root system, a shoot system, a flower, a leaf, a fruit, a stem, a root, or any combination thereof, of a plant.

127. The engineered microorganism of claim 126, wherein the engineered microorganism is natively co-located with the target organism in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant.

128. The engineered microorganism of claim 126, wherein the engineered microorganism is further modified to grow in the phyllosphere, the rhizosphere, the root system, the shoot system, the flowers, the leaf, the fruit, the stem, the root, or any combination thereof, of the plant, wherein the engineered microorganism is co-located with the target organism.

129. The engineered microorganism of claim 102, wherein the antisense promoters comprise head to head promoters.

130. The engineered microorganism of claim 102, wherein the antisense promoters comprise one or more bidirectional promoters.

131. The engineered microorganism of claim 102, wherein the two antisense promoters each independently comprise the sequence of any one of SEQ ID Nos: 1-11.

132. The engineered microorganism of claim 102, wherein the engineered microorganism is a spore-forming microorganism.

133. The engineered microorganism of claim 102, wherein the engineered microorganism is a fungus.

134. The engineered microorganism of claim 132, wherein the fungus is a Trichoderma spp.

135. The engineered microorganism of claim 102, wherein the antisense promoters each comprise a strong constitutive or inducible promoter.

136. The engineered microorganism of claim 135, wherein the antisense promoters each comprise independently Pural, Pcbhl, PceI7, Ppgll, Pamyl, Ptefl, PcDNAl, PgpdA, Prp2, U6, Peel, Pxyn, Ptcul, Pyatl, Pactin, PoliC, PtetR, Pxyl, or any combination thereof.

137. The engineered microorganism of claim 102, wherein the engineered microorganism is a bacteria.

138. The engineered microorganism of claim 137, wherein the bacteria is a Bacillus spp.

139. The engineered microorganism of claim 102, wherein the antisense promoters each comprise a strong constitutive or inducible promoter.

140. The engineered microorganism of claim 139, in the antisense promoters each comprise independently PphoA, P43, P45, PT7, P34, PxylA, Physpank, Pspac, Pveg, PamyE, PgrD, PbacA, PcotC, Pspank, PnrpE, PamyP, PespsA, PSP6, PrrnBl+2 WT, PrrnBl+2 A starts or any combination thereof.

141. The engineered microorganism of claim 102, wherein the target gene is a gene that affects a plant trait.

142. The engineered microorganism of claim 141, wherein the target gene comprises GmFAD3, El, ZmMADSl, GmMIPSl, ZLKR, SDH, ZmBCH2, ghSAD-1, ghFAD2-l, ANR, FaMYBl, TFL1, plC, FaPGl, FaWRKY29, FaWRKY64, AGPase, LsNCED4, qSORl, or any combination thereof.

143. A composition comprising the engineered microorganism of any one of claims 1 to 142 or a spore, mycelium, or vegetative cell thereof, and a plant.

144. The composition of claim 143, wherein the plant is of the genus Brassica, Solanum, Malus, Citrus, Vitis, Saccharum, Zea, Oryza, Triticum, Glycine, Gossypium or any combination, strain, or variant thereof.

145. A method of treating a plant, the method comprising introducing the engineered microorganism of any one of claims 1 to 101 or a spore, mycelium, or vegetative cell thereof, to an ecosystem of a plant.

146. The method of claim 145, wherein the engineered microorganism generates one or more copies of the dsRNA comprising the plurality of siRNAs.

147. The method of claim 146, wherein the engineered microorganism further comprises a DICER enzyme, and wherein the DICER enzyme cleaves the dsRNA to generate the plurality of siRNAs.

148. The method of claim 147, further comprising transferring the plurality of siRNAs to a cell of the target organism by passive transfer.

149. The method of claim 148, wherein the passive transfer is effected by diffusion, extracellular vesicles, and / or cell lysis.

150. The method of claim 146, further comprising transferring the dsRNA to a cell of the target organism, wherein the transfer is a passive transfer.

151. The method of claim 150, wherein the transfer is effected by diffusion, extracellular vesicles, and / or cell lysis.

152. The method of claim 151, wherein the cell of the target organism comprises a DICER enzyme, and wherein the DICER enzyme cleaves the dsRNA to generate the plurality of siRNAs.

153. A method of treating a plant, the method comprising introducing the engineered microorganism of any one of claims 102 to 142 or a spore, mycelium, or vegetative cell thereof, to an ecosystem of a plant.

154. The method of claim 153, wherein the engineered microorganism generates a plurality of siRNAs from the plurality of dsRNA expression cassettes.

155. The method of claim 154, further comprising transferring the plurality of siRNAs to a target organism by passive transfer.

156. The method of claim 155, wherein the transfer is effected by diffusion, extracellular vesicles, and / or cell lysis.

157. A formulation, wherein the formulation comprises: the engineered microorganism of any one of claims 1 to 142, spore, mycelium, or vegetative cell thereof, and an adjuvant.

158. The formulation of claim 157, wherein the adjuvant comprises, alone or in combination, a surfactant, an oil, a compatibility agent, a stability agent, a buffering agent, a conditioning agent, a defoaming agent, a deposition agent, a flowability agent, a granulation agent, a drift control agent, a binding agent, a UV protectant, an osmotic protectant, nutrients, or a thickener.

159. The formulation of claim 158, wherein the adjuvant comprises Tween 20, Triton X-100, Silwet L77, sucrose, dextrose, lactose, mannitol, maltodextrin, magnesium chloride, seed coating polymer, sodium alginate, cellulose, microcrystalline cellulose, starch, titanium dioxide, talcum powder, clay, soybean oil, xanthan gum, molasses, or peptone.

160. The formulation of any one of claims 157-159, wherein the formulation is an emulsion, a colloid, a dust, a granule, a pellet, a powder, a liquid, a spray, a mist, a gel, a paste, a fog or a solution.

161. A method of manufacture, wherein the method comprises: generating the engineered microorganism of any one of claims 1 to 142; growing the engineered microorganism; and formulating a composition comprising the engineered microorganism or a spore, mycelium, or vegetative cell thereof, and an adjuvant.

162. The method of claim 161, wherein the adjuvant comprises, alone or in combination, a surfactant, an oil, a compatibility agent, a stability agent, a buffering agent, a conditioning agent, a defoaming agent, a deposition agent, a drift control agent, binding agent, a flowability agent, a granulation agent, a UV protectant, an osmotic protectant, nutrients, or a thickener.

163. The method of claim 162, wherein the adjuvant comprises phosphate buffered saline.

164. The method of claim 162, wherein the adjuvant comprises Tween 20, Triton X-100, Silwet L77, sucrose, dextrose, lactose, mannitol, maltodextrin, magnesium chloride, a seed coating polymer, sodium alginate, cellulose, microcrystalline cellulose, starch, titanium dioxide, talcum powder, clay, soybean oil, xanthan gum, molasses, or peptone.

165. The method of any one of claims 161 to 164, wherein the composition is an emulsion, a colloid, a dust, a granule, a pellet, a powder, a liquid, a spray, a mist, a gel, a paste, a fog, or a solution.

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