Pesticidal proteins and metohds of use

Abstract

Provided herein are methods of reducing a psyllid pest population comprising contacting the psyllid with a bacterial pesticidal protein.

Description

PESTICIDAL PROTEINS AND METOHDS OF USEGOVERNMENT SUPPORT CLAUSE

[0001] This invention was made with government support under Grant No. 2017-70016- 26755 and 2020-70029-33177 awarded by the National Institute of Food and Agriculture. The Government has certain rights in the invention.BACKGROUND

[0002] The Asian citrus psyllid, Diaphorina citri is the most important citrus pest because it vectors the bacterium that causes citrus greening disease or huanglongbing. This disease is a major threat to the citrus industry. Management of D. citri with chemical pesticides has not effectively reduced the impact of citrus greening. The deployment of pesticidal proteins derived from bacteria, such as Bacillus thuringiensis (Bt), represents a sustainable alternative to the application of chemical pesticides and is a promising tool for use within an integrated management program for citrus greening. Although Bt-derived pesticidal proteins have been successfully employed for management of agricultural insect pests, relatively few proteins have demonstrated activity against D. citri.INCORPORATED BY REFERENCE OF INFORMATION SUBMITTED ELECTRONICALLY

[0003] This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 59651_SeQlisting.xml; Size: 25,245 Bytes; Created: January 8, 2025), which is incorporated by reference in its entirety.SUMMARY

[0004] In one aspect, described herein are methods of method for reducing a psyllid pest population comprising contacting the psyllid with a bacterial pesticidal protein. In some embodiments, the pesticidal protein is Tpp78Aa1 , Xpp37Aa1 , App6Aa2, Mpp23Aa1 or Tpp78Ba1.

[0005] In another aspect, described herein is a modified pesticidal protein comprising a bacterial pesticidal protein, wherein the pesticidal protein has been modified to include at least one psyllid gut binding peptide.BRIEF DESCRIPTION OF THE FIGURES

[0006] Figures 1A-C. Coomassie-stained SDS PAGE gels of Bt-derived pesticidal proteins expressed in E. coli and purified from inclusion bodies (Figure 1A), expressed in B. thuringiensis and purified from crystals (Figure 1 B), and expressed in E. coli and purifiedusing His-tag affinity chromatography (Figure 1 C). Proteins of the expected size are indicated by black arrow heads. Mr., molecular mass marker (kDa).

[0007] Figure 2. Mortality of D. citri adults fed on a single dose of Bt-derived pesticidal protein. Average psyllid mortality at day 7 and standard error are shown. Adult psyllids (4 days old) were fed with 500 pg / mL of Cry proteins and App proteins or 200 pg / mL of Tpp, Xpp and Mpp proteins. Asterisks above the error bars indicate statistical difference relative to the buffer control (P < 0.05; Student's t-test).

[0008] Figure 3. D. citri mortality on exposure to different concentrations of Tpp78Aa1 or Xpp37Aa1 . For each protein, psyllids were challenged with seven protein concentrations. A carbonate buffer only was used as the buffer control. Average mortality at seven days and the standard error are shown. Xpp37Aa1 at higher concentrations may inhibit D. citri feeding.

[0009] Figure 4. Determination of the LC50 for Tpp78Aa1 modified with gut binding peptide DcNy6. Modification of Tpp78Aa1 with the DcNy6 resulted in a four-fold increase in toxicity against first instar D. citri.DETAILED DESCRIPTION

[0010] The present disclosure is based on the discovery of several bacterial pesticidal proteins being toxic to Diaphorina citri (D. citri). The data provided herein show the results from the largest screen of bacterial pesticidal proteins against D. citri, representing the largest screen reported for any hemipteran pest. Tpp78Aa1 was identified as being toxic to D. citri. This protein along with a few others with identified activity against D. citri provide promising control agents for D. citri.

[0011] In one aspect, described herein are methods of reducing a psyllid pest population comprising contacting the psyllid with a bacterial pesticidal protein. Exemplary bacterial pesticidal proteins include, but are not limited to, Tpp78Aa1 , Xpp37Aa1 , Tpp78Ba1 , App6Aa2, Cry1 , such as members of the Cry1 A, Cry1 B, Cry1 C, Cry1 D, Cry1 E, and Cry1 F families; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc, Mpp23Aa1 , Mpp64Ba, Mpp64Ca, VpblAe, Vpb2Ae, Pra, and Prb. In some embodiments, the pesticidal protein is Tpp78Aa1 , Xpp37Aa1 , App6Aa2, Mpp23Aa1 orTpp78Ba1. In some embodiments, the psyllid is an Asian citrus psyllid (D. citri).

[0012] In some embodiments, the methods comprise contacting the psyllid with at least two bacterial pesticidal proteins. In some embodiments, the methods comprise contacting the psyllid with Mpp23Aa1 and Xpp37Aa1 .

[0013] In another aspect, described herein is a modified pesticidal protein comprising a bacterial pesticidal protein that has been modified by substitution or addition to include apsyllid gut binding peptide. In some embodiments, the psyllid gut binding peptide is an Asian citrus psyllid (D. citri) gut binding peptide. Exemplary D. c / tr / gut binding peptides include, but are not limited to, DcAd4 (nucleotide sequence set forth in SEQ ID NO: 1 , amino acid sequence set forth in SEQ ID NO: 2), DcNy6 (nucleotide sequence set forth in SEQ ID NO: 3, amino acid sequence set forth in SEQ ID NO: 4) or DcNy9 (nucleotide sequence set forth in SEQ ID NO: 5, amino acid sequence set forth in SEQ ID NO: 6). In some embodiments, the modified pesticidal protein comprises the gut binding peptide set forth in SEQ ID NO: 2. In some embodiments, the modified pesticidal protein comprises the gut binding peptide set forth in SEQ ID NO: 4. In some embodiments, the modified pesticidal protein comprises the gut binding peptide set forth in SEQ ID NO: 6.

[0014] Also contemplated are nucleotide sequences encoding the modified pesticidal protein. In some embodiments, the modified pesticidal protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 7 and a nucleotide sequence set forth in SEQ ID NO: 1 , 3, or 5.

[0015] The disclosure further provides a vector comprising one or more nucleotide sequences encoding the pesticide proteins, modified pesticide proteins, and / or gut binding peptides described herein.

[0016] The term “vector” as used herein encompasses (but is not limited to) a phage, plasmid, viral or retroviral vector, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. The vector comprising the polynucleotides as described herein may comprise selectable markers for propagation and / or selection in a host. Further, the vector may be prepared from native (endogenous) and / or foreign (exogenous, heterologous) sequences with respect to the host.

[0017] Preferably, the vector referred to herein is suitable as a cloning vector, i.e., replicable in microbial systems. Such vectors ensure efficient cloning in bacteria, yeasts or fungi and make possible the stable transformation of plants.

[0018] Suitable vector backbones are, in some embodiments, derived from vectors known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNAI , pcDNA3 (Invitrogene) or pSPORTI (GIBCO BRL). Further examples of typical fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith, D.B., and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), where glutathione S transferase (GST), maltose E-binding protein and protein A, respectively, are fused with the nucleic acid of interest encoding a protein to be expressed.

[0019] In some embodiments, the vector comprising one or more nucleotide sequences described herein is propagated and amplified in a plant cell. In some embodiments, one copy of the vector is propagated and amplified in a plant cell. In some embodiments, two or more (e.g., 3, 4, 5, 6 7, 8 or more) copies of the vector are propagated and amplified in a plant cell.

[0020] In some embodiments, the vector described herein comprises a promoter (e.g., a phloem-specific promoter, a leaf-specific promoter, a light activated promoter or a leafdamage activated promoter) operably linked to a nucleotide sequence described herein. In some embodiments, the nucleotide sequence is further operably linked to termination signals and / or other regulatory elements.

[0021] The term "promoter" as used herein refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition site for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprised, in some cases, of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for enhancement of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements and that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence, which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements, derived from different promoters found in nature, or even be comprised of synthetic DNA segments.

[0022] In some embodiments, the promoter is a phloem cell-specific. Phloem cell-specific promoters can be used to express a modified pesticidal protein described herein in the phloem of transgenic plants. Exemplary phloem specific promoters include, but are not limited to, the CmGASI promoter described in U.S. Pat. No. 6,613,960; the Agrobacteriumrhizogenes RolC promoter (Graham et al., 1993); and the pumpkin PP2 promoter (Dinant et al. 2004).

[0023] Examples of additional useful phloem specific promoters include, but are not limited to, PP2-type gene promoters (U.S. Pat. No. 5,495,007), sucrose synthase promoters (Yang and Russell, 1990. Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), glutamine synthetase promoters (Edwards et al. 1990. Proc. Natl. Acad. Sci. USA 87:3459-3463, 1990), and phloem-specific plasma membrane H-i-ATPase promoters (DeWitt et al. 1991. Plant J. 1 , 121-128, 1991 ), prunasin hydrolase promoters (U.S. Pat. No. 6,797,859), and a rice sucrose transporter (U.S. Pat. No. 7,186,821).

[0024] For control of hemipteran pests that feed on xylem tissue, a variety of promoters that are active in xylem tissue including, but not limited to, protoxylem or metaxylem can be used. Promoters active in xylem tissue include, but are not limited to, promoters associated with phenylpropanoid biosynthetic pathways, such as the phenylalanine ammonia-lyase (PAL) promoters, cinnamate 4-hydroxylase (C4H) promoters, coumarate 3-hydroxylase promoters, O-methyl transferase (OMT) promoters, 4-coumarate:CoA ligase (4CL) promoters (U.S. Pat. No. 6,831 ,208), cinnamoyl-CoA reductase (CCR) promoters and cinnamyl alcohol dehydrogenase (CAD) promoters.

[0025] The terms “operably linked” or “functionally linked” refer to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0026] The term "constitutive promoter" as used herein refers to a promoter that is able to express the open reading frame (ORF) in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant. Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant tissues at a level of at least 1% reached in the plant tissue in which transcription is most active. "Constitutive expression" refers to expression using a constitutive promoter.

[0027] In some embodiments, an operable linkage comprises a sequential arrangement of a nucleotide sequence encoding a promoter, with a nucleic acid sequence to be expressed, and optionally, additional regulatory elements such as, for example, polyadenylation or transcription termination elements, enhancers, introns, etc., such that the nucleotidesequence of interest is expressed under the appropriate conditions (i.e., in a plant cell). Suitable arrangements include, e.g., those in which the nucleic acid sequence to be expressed is placed downstream (i.e., in 3’-direction) of the transcription regulating nucleotide sequence such that both sequences are covalently linked. Optionally, additional sequences may be inserted in-between the two sequences. Such sequences may be, for example, linker or multiple cloning sites. Furthermore, sequences can be inserted which encode parts of a fusion protein, in the event that a fusion protein comprising the product of the nucleic acid disclosed herein is desired. Preferably, the distance between the polynucleotide to be expressed and the transcription regulating nucleotide sequence is not more than 200 base pairs, such as not more than 100 base pairs or not more than 50 base pairs.

[0028] In another aspect, described herein is a method for expressing a polynucleotide of interest in a host cell comprising introducing a vector described herein into the host cell and expressing the polynucleotide of interest in the host cell. In some embodiments, the host cell is a plant cell.

[0029] The term "expression" refers to the transcription and / or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants. The "expression pattern" of a promoter (with or without enhancer) is the pattern of expression levels, which shows where in the plant and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter. The level of expression of a promoter can be determined by measuring the steady state concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of synthesis rates and degradation rates. When promoters are compared in this way, techniques available to those skilled in the art are hybridization S1 -RNAse analysis, northern blots and competitive RT-PCR. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA. The analysis of transcription start points in practically all promoters has revealed that there is usually no single base at which transcription starts, but rather a more or less clustered set of initiation sites, each of which accounts for some start points of the mRNA. Since this distribution varies from promoter to promoter the sequences of the reporter mRNA in each of the populations would differ from each other. Since each mRNA species is more or less prone to degradation, no single degradation rate can be expected for different reporter mRNAs. It has been shown forvarious eukaryotic promoter sequences that the sequence surrounding the initiation site (“initiator") plays an important role in determining the level of RNA expression directed by that specific promoter. This includes also part of the transcribed sequences. The direct fusion of promoter to reporter sequences would therefore lead to suboptimal levels of transcription. A commonly used procedure to analyze expression patterns and levels is through determination of the 'steady state' level of protein accumulation in a cell. Commonly used candidates for the reporter gene, known to those skilled in the art are betaglucuronidase (GUS), chloramphenicol acetyl transferase (CAT) and proteins with fluorescent properties, such as green fluorescent protein (GFP) from Aequora victoria. In principle, however, many more proteins are suitable for this purpose, provided the protein does not interfere with essential plant functions. For quantification and determination of localization a number of tools are suited. Detection systems can readily be created or are available which are based on, e.g., immunochemical, enzymatic, fluorescent detection and quantification. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression. Generally, individual transformed lines with one chimeric promoter reporter construct may vary in their levels of expression of the reporter gene. Also frequently observed is the phenomenon that such transformants do not express any detectable product (RNA or protein). The variability in expression is commonly ascribed to position effects, although the molecular mechanisms underlying this inactivity are usually not clear.

[0030] The expression of a polynucleotide of interest in a host cell (e.g., a plant cell) can be determined by various well known techniques, e.g., by Northern Blot or in situ hybridization techniques as described in WO 02 / 102970, the disclosure of which is incorporated by reference in its entirety.

[0031] In some embodiments, the host cell is from a plant (e.g., a plant cell, a plant seed or other plant part). To confirm the presence of the transferred polynucleotide of interest in transgenic cells and plants, a variety of assays may be performed. The expression of a polynucleotide of interest in a host cell (e.g., a plant cell) can be determined by various well known techniques, e.g., by Northern Blot or in situ hybridization techniques as described in WO 02 / 102970, the disclosure of which is incorporated by reference in its entirety. Such assays include, Northern Blot or in situ hybridization techniques as described in WO 02 / 102970, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT-PCR or TaqMan; immunological assays such as ELISAs and Western blots, and also, by analyzing the phenotype of the whole regenerated plant.

[0032] Methods of producing a transgenic plant

[0033] The disclosure also provides a method for producing a transgenic plant comprising introducing one or more nucleotide sequences described herein into the genome of the plant. In some embodiments, the nucleotide sequence is operably linked to a phloemspecific promoter, leaf-specific promoter, stem-specific promoter, a light activated promoter or a leaf-damage activated promoter. In some embodiments, the promoter is a constitutive promoter.

[0034] A variety of techniques are available and known to those skilled in the art for introduction of constructs (e.g., vectors) into a host cell (e.g., plant cell). Exemplary techniques include transformation with DNA employing A. tumefaciens or A. rhizogenes as the transforming agent, liposomes, PEG precipitation, electroporation, DNA injection, direct DNA uptake, microprojectile bombardment, particle acceleration, CRISPR and the like (see, for example, EP 295959 and EP 138341 ). However, cells other than plant cells may be transformed with the vector described herein. The general descriptions of plant expression vectors and reporter genes, and Agrobacterium and Agrobacterium-mediated gene transfer, can be found in Gruber et al. (1993).

[0035] In some embodiments, the pesticidal proteins described herein (including modified pesticidal proteins) are introduced into a plant cell via a phloem-limited viral vector. Phloemlimited viruses have been described, including but not limited to the rice tungro virus (Bhattacharyya-Pakrasi et al. 1993. Plant J. 4, 71-79) and the commelina yellow mottle virus (Medberry et al. 1992. Plant Cell 4:185-192) also contain useful promoters that are active in vascular tissues. Others are described in U.S. Pat. No. 5,494,007; U.S. Pat. Nos. 5,824,857; 5,789,656; 6,613,960; and US Patent Publication 20100064394, and Guo et al. 2004;Graham et al. 1997, the disclosures of which are incorporated herein by reference in their entireties.

[0036] In some embodiments, the pesticidal proteins described herein (including modified pesticidal proteins) are introduced into a plant cell via a citrus tristeza viral vector (CTV).

[0037] Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see, e.g., EP 295959), techniques of electroporation (Fromm 1986) or high velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (e.g., U.S. Patent No. 4,945,050). Once transformed, the cells can be regenerated by those skilled in the art. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e., monocotyledonous or dicotyledonous, targeted for transformation.

[0038] In some embodiments, the construct is introduced into a host cell by introducing a genome editing component comprising: a) an enzyme inducing a double-stranded break(DSB) or a nucleic acid encoding same, and optionally a repair nucleic acid molecule, wherein the DSB-inducing enzyme optionally recognizes a predetermined site in the genome of said cell; b) an enzyme inducing a single-stranded break (SSB) or a nucleic acid encoding same, and optionally a repair nucleic acid molecule, wherein the SSB-inducing enzyme optionally recognizes a predetermined site in the genome of said cell; c) a base editor enzyme, optionally fused to a disarmed DSB- or SSB-inducing enzyme, wherein the base editor enzyme preferably recognizes a predetermined site in the genome of said cell; or d) an enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone ribosylation or histone citrullination, optionally fused to a disarmed DSB- or SSB-inducing enzyme, wherein the enzyme preferably recognizes a predetermined site in the genome of said cell.

[0039] In order to enable a break at a predetermined target site, the enzymes preferably include a binding / recognition domain and a cleavage domain. Particular enzymes capable of inducing double or single-stranded breaks are nucleases or nickases as well as variants thereof, including such molecules no longer comprising a nuclease or nickase function but rather operating as recognition molecules in combination with another enzyme. In recent years, many suitable nucleases, especially tailored endonucleases have been developed comprising meganucleases, zinc finger nucleases, TALE nucleases, Argonaute nucleases, derived, for example, from Natronobacterium gregoryi, and CRISPR nucleases, comprising, for example, Cas9, Cpf 1 , Csm1 , CasX or CasY nucleases as part of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. Thus, in some embodiments, the genome engineering component comprises a DSB- or SSB-inducing enzyme or a variant thereof selected from a CRISPR / Cas endonuclease, preferably a CRISPR / Cas9 endonuclease a CRISPR / Cpf1 endonuclease, or a CRISPR / Csm1 endonuclease, a zinc finger nuclease (ZFN), a homing endonuclease, a meganuclease and a TAL effector nuclease.

[0040] In some embodiments, the methods described herein comprise introducing one or more vectors comprising the one or more nucleotide sequences into the plant by transformation. In some embodiments, the methods described herein comprise introducing a vector comprising the nucleotide sequence described herein into the plant by transformation.

[0041] If desired, the vector may comprise a selectable marker, which may provide resistance to an antibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide (e.g., phosphinothricin), or a separate vector encoding a selectable marker may be utilized in conjunction with the vector comprising the nucleotide of interest described above. For certain plant species, different antibiotic or herbicide selection markers may be preferred. Selectionmarkers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics, the bar gene which confers resistance to the herbicide phosphinothricin, the hph gene which confers resistance to the antibiotic hygromycin, and the dhfr gene which confers resistance to methotrexate.

[0042] Methods for the production and further characterization of stably transformed plants are well-known to the person skilled in the art. As an example, transgenic plant cells are placed in an appropriate selective medium for selection of transgenic cells, which are then grown to callus. Shoots are grown from callus. Plantlets are generated from the shoot by growing in rooting medium. When a selection marker is used, the marker allows for selection of transformed cells as compared to cells lacking the DNA.

[0043] In some embodiments, the methods described herein comprise sexually crossing a plant with the transgenic plant described herein.

[0044] Transgenic plants

[0045] A transgenic plant or plant part comprising a vector (or nucleotide sequence) described herein is specifically contemplated.

[0046] The term “plant” refers to a photosynthetic, eukaryotic multicellular organism. The term “plant” encompasses whole plants, ancestors and progeny of the plants, wherein each of the aforementioned comprise the gene / nucleic acid of interest. The term “plant parts” encompasses all components of a plant including seeds, shoots, stems, leaves, roots, flowers, plant tissues, plant organs, plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, microspores and propagules. A “propagule” is any kind of organ, tissue, or cell of a plant capable of developing into a complete plant. A propagule can be based on vegetative reproduction (also known as vegetative propagation, vegetative multiplication, or vegetative cloning) or sexual reproduction. A propagule can therefore be seeds or parts of the non-reproductive organs, like stem or leaf. In particular, with respect to Poaceae, suitable propagules can also be sections of the stem, i.e., stem cuttings.

[0047] In some embodiments, the plant is a monocotyledonous plant, or the plant part is derived from a monocotyledonous plant. In some embodiments, the plant is a dicotyledonous plant, or the plant part is derived from a dicotyledonous plant.

[0048] A transgenic plant or plant part comprising a vector described herein is specifically contemplated. The vector may be present in the cytoplasm of the plant or may be incorporated into the genome either heterologous or by homologous recombination.

[0049] In some embodiments, the plant (or plant part) is derived from the Citrus genera.

[0050] In some embodiments, the plant (or plant part) is from an orange plant, lemon plant, lime plant, grapefruit plant, tangerine plant, pomelo plant, citron plant, mandarin orange plant, papeda plant, kaffir lime plant, ichang papeda plant, and kumquat plant.

[0051] In some embodiments, the plant (or plant part) is from an Indian curry plant.

[0052] Compositions

[0053] A composition comprising a fusion protein described herein is also contemplated.

[0054] Methods of controlling insect pests comprising contacting an insect with a pesticidal protein (or modified pesticidal protein) described herein (or a composition described herein, or a transgenic plant described herein), wherein upon ingestion, the pesticidal protein (or modified pesticidal protein) binds the gut epithelium, creates a pore, thereby killing the insect pest (e.g., Asian citrus psyllid).

[0055] Methods of controlling insect pests.

[0056] The disclosure also provides a method of controlling psyllid pests, the method comprising feeding a psyllid with a food source comprising pesticidal protein (or modified pesticidal protein) described herein or a composition described herein, wherein upon ingestion, the fusion protein binds the gut epithelium, creates a pore and kills the insect.

[0057] It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, plant species or genera, constructs, and reagents described as such.EXAMPLES

[0058] Materials and Methods

[0059] Bacterial strains, pesticidal proteins, and plasmids. A total of 17 Bt-derived pesticidal proteins from different structural groups were expressed and tested against D. citri adults. These proteins were either synthesized in vitro, acquired from the Bacillus Genetic Stock Center (BGST), or provided by other laboratories (Table 1 ). Details of the plasmids in which genes were inserted, the bacterial expression system, bacterial strains, and protein purification methods used for each protein are shown in Table 1.

[0060] Expression and purification of Bt proteins from inclusion bodies. The Bt proteins Cry1 Aa, Cry1 Ac, CrylCa, Cry1 Ea, and Cry3Aa were expressed in E. coli and purified from inclusion bodies as described elsewhere with modifications (70). Briefly, E. coli cells transformed with recombinant plasmids harboring each Bt gene were grown in 5 mL Luria- Bertani broth medium (LB) at 37 °C and 220 rpm for 16 h. These overnight cultures were then used to inoculate 400 mL of LB supplemented with 100 pg / mL of ampicillin. Cells were grown until the OD600 reached 0.8. Gene expression was induced with 1 mM isopropyl -D-1 -thiogalactopyranoside (IPTG) for four hours at 37 °C and 220 rpm. Cells were then harvested, resuspended in lysis buffer, and sonicated on ice. Inclusion bodies containing Bt proteins were pelleted by centrifugation and purified after washing three times in Triton X- 100 + 0.5 M NaCI, five times in 0.5 M NaCI, and two times in distilled water. Proteins were solubilized from inclusion bodies overnight in 50 mM Na2CO3 buffer containing 0.01 M dithiothreitol (DTT) at 37 °C. Protein refolding was achieved by dialysis against 6 L of 20 mM sodium carbonate buffer in 3,500 MWCO Slide-A-Lyzer dialysis cassette G2 (PIERCE, Rockford, IL, USA) at 4 °C under constant agitation. Refolded proteins were activated in a trypsin / protoxin ratio of 1 :10 (wt / wt) at 37 °C for 4 h with 120 rpm shaking. Amicon Ultra-0.5 centrifugal filters 30 kDa MWCO (Millipore Sigma, Burlington, MA, USA) were used to remove trypsin and concentrate protein before testing against D. citri. Protein concentration was determined by Bradford assay (71 ), and protein integrity was assessed by electrophoresis in 4-12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) (Fisher Scientific, Carlsbad, CA, USA). Proteins were stained with Coomassie brilliant blue R-250 (Fisher Scientific, Bremen, Germany).

[0061] Expression and purification of Bt proteins from crystals. The protein App6Aa2 was expressed in Bt sporulation medium as previously described (14, 72) with modifications. Briefly, overnight 5 mL cultures were used to inoculate 200 mL of Bt sporulation medium, and sporulation was induced with sodium phosphate buffer after overnight growth. Spores and crystals were harvested, suspended in crystal washing buffer (0.1 M NaCI, 2% Triton-X 100, 2% sodium deoxycholate, 20mM Bis-tris pH 6.5), and sonicated on ice. The pellet was then washed three times in the same buffer without deoxycholate, two times in 1 M NaCI, and two times in distilled water. The protein was then solubilized with 50 mM sodium hydroxide (NaOH) for one hour at 37 °C and 120 rpm shaking. The samples were then dialyzed against 6 L of 20 mM carbonate buffer (2 L overnight and 2 L every 2 h) using 3,500 MWCO dialysis cassettes (PIERCE, Rockford, IL). Protein concentration was determined via Bradford assay, and protein was resolved in a 4-12% SDS PAGE gel following staining with Coomassie brilliant blue R-250.

[0062] Expression and His-tag affinity purification of Bt proteins. A total of eight Bt proteins were expressed in E. coli and purified using His-tag affinity chromatography. DNA sequences for six of these proteins were cloned into pET30 for optimized protein yield (Table 1 , Table 2).Table 1. Proteins tested against D. citri including methods used for expression and purification.Protein Origin Plasmid Strain PurificationExpressed in E. coliCryl Aa BGSC (ECE52) pKK223-3 JM103 LB.Cryl Ac BGSC (ECE53) pKK223-3 JM103Cryl Ca BGSC (ECE125) pTZ19R DH5aCryl Ea BGSC (ECE127) pTZ19R DH5aCry3Aa BGSC (ECE131 ) pKK223-3 JM103Tpp78Aa1 Synthesized pET20(b) pET30 BL21 (DE3) His-tagTpp78Ba1 Synthesized pET20(b) pET30 BL21 (DE3)Tpp80Aa1 pET20 BL21 (DE3)Xpp22Aa1 pSTAB pET30 BL21 (DE3)Xpp37Aa1 pSTAB pET30 BL21 (DE3)Xpp55Aa1 pGEM pET30 BL21 (DE3)Xpp77Aa1 pSTAB pET30 BL21 (DE3)Mpp23Aa1 Synthesized pET30 BL21 (DE3)Expressed in B. thuringiensisApp6Aa2 pHT304-derived BMB171 CrystalBGSC, Bacillus Genetics Stock Center; I.B., Inclusion BodiesTable 2. Primers and PCR conditions used for cloning bacterial pesticidal protein genes into pET30(a).Primer Gene DNA sequence (5' -> 3') NkJ. ( U) R E.F, forward; R, Reverse; A.t., annealing temperature; R.E., Restriction Enzyme

[0063] The conditions for expression varied depending on the protein. For Xpp37Aa1 , Xpp55Aa1 and Tpp80Aa1 , E. coli cells harboring the recombinant plasmid were grown in LB medium supplemented with 10 pg / mL of kanamycin at 37 °C and 220 rpm until GD600 reached 0.5. Gene transcription was induced with 1 mM of IPTG. The cultures were grown overnight at 20 °C with 150 rpm shaking. For Tpp78Aa1 , Tpp78Ba1 , Xpp22Aa1 , Xpp77Aa1 and Mpp23Aa1 , cells were grown for 4 h at 37 °C and 220 rpm after IPTG induction. Cells were then harvested by centrifugation (2,600 x g, 20 min, 4 °C) and lysed using xTractorTMbuffer (Takara, Kusatsu, Japan) supplemented with 1 mg / mL of lysozyme plus 1 mM phenylmethylsulphonyl fluoride (PMSF) and 1x protease inhibitor (PI) followed by sonication on ice. All proteins were purified using HisPurTM Ni-NTA agarose (Fisher Scientific, Rockford, IL, USA). Briefly, lysates were centrifuged [10,000 rpm (Beckman J2-21 centrifuge), 30 min, 4°C], equilibrated with an equal volume of equilibration buffer (10 mM imidazole, 300 mM NaCI, and 50 mM Tris pH 8), and passed through columns packed with Ni-NTA agarose beads. Beads were previously equilibrated with six-column volumes of equilibration buffer. Columns were then washed with six-column volumes of wash buffer (25 mM Imidazole, 300 mM NaCI, and 50 mM Tris pH 8), and protein was eluted with a gradient concentration of imidazole (50, 100, 250, and 500 mM) in 300 mM NaCI and 50 mM Tris pH 8. Protein concentration was determined (71 ), and protein integrity was assessed in 4-12% SDS PAGE gels following Coomassie brilliant blue staining and further corrected with bovine serum albumin (BSA) in SDS-PAGE gel (17). Before bioassays were performed, imidazole was removed by dialysis against 6 L of 20 mM carbonate buffer in 3,500 MWCO dialysis cassettes (PIERCE, Rockford, IL, USA).

[0064] D. citri adults used for bioassay. Bioassays were performed with CLas-free psyllids maintained on orange jasmine plants, Murraya paniculata. Four-day old psyllids were kindly provided by the Florida Division of Plant Industry (DPI). Insects were kept at 26 ± 2 °C, 50 ± 5% relative humidity, and 14 h:10 h light-dark cycle.

[0065] Single dose assays. To identify proteins with pesticidal activity, we first challenged D. citri adults with a single dose of Bt-purified proteins. A membrane feeding bioassay was performed as previously described (14) with modifications. Four-day old D. citri adults were fed on membrane sachets consisting of 30% sucrose solution in carbonate buffer containing 500 pg / mL of Cry1 Aa, Cry1 Ac, CrylCa, Cry1 Ea, Cry3Aa, or App6Aa2. A dose of 200 pg / mL or 400 pg / mL was used for testing Tpps, Xpps, and Mpp23Aa1 proteins, because of low protein yield. Carbonate buffer (20 mM, pH 10.5) with 30% sucrose was used as the negative control. The bioassays were performed with three biological replicates, with each replicate using approximately 25 psyllids per tube. Mortality was recorded daily, and data at seven days were used for statistical analysis.

[0066] Determination of LC50 values. Based on the single dose mortality results and reports of their toxicity to hemipteran insects, we assayed two proteins (Tpp78Aa1 , and Xpp37Aa1 ) to determine their LC50 (the concentration that is lethal to 50% of the tested individuals). The LC50 assays were performed as described for the single dose assay, except that seven concentrations of each protein were used (6.25, 12.5, 25, 50, 100, 200, and 400 pg / mL) plus the buffer control. Each biological replicate consisted of approximately 20 insects, and the experiment was repeated for a total of three biological replicates.

[0067] Data analysis. Statistical differences in psyllid mortality in single dose assays between each Bt protein and its respective buffer control were determined by Student’s t-test (P < 0.05). The assumptions of normality and homogeneity of variance were checked before the t-test was performed in SigmaPlot (v. 14.5). LC50 was calculated with corrected psyllid mortality (73) at seven days using the PRO PROBIT model in SAS (SAS PROC PROBIT) (74).Example 1 - Expression and purification of bacterial pesticidal proteins

[0068] A total of 17 Bt proteins from five structural groups were expressed, purified, and used for D. citri bioassays. The four Cry1 proteins, (Cry1 Aa, Cry 1 Ac, CrylCa and Cry1 Ea), and Cry3Aa, were expressed in E. coli and purified from inclusion bodies. Trypsin-activated Cry1 proteins and Cry3Aa attained the expected protease-resistant cores of approximately 65 and 55 kDa, respectively (Fig.1 A).

[0069] App6Aa2 was expressed in Bt strains and purified from spore / crystal mixtures at high pH. The non-activated molecular mass of the Cry2 proteins was -70 kDa, and App6Aa2 was -55 kDa (Fig. 1 B).

[0070] In addition to the Cry proteins and App6Aa2, proteins from the Tpp, and Mpp structural groups and the Xpp holding class for which structures have yet to be determined (2), were also expressed for testing against D. citri. The proteins Tpp78Aa1 and Tpp78Ba1 were initially synthesized in pET-20b(+), expressed in the E. coli BL21 (DE3) strain, and purified using His-tag affinity purification. To improve protein yield, both Tpp78Aa1 and Tpp78Ba1 genes were cloned into pET30(a) for the addition of two His-tags. With these two tags, the molecular mass of Tpp78Aa1 and Tpp78Ba1 was -48 and -52 kDa, respectively (Fig. 1C). Tpp80Aa1 was also expressed in E. coli. This protein has an expected molecular mass of 42 kDa, and -55 kDa with the two His-tags (Fig. 1 C). From the Xpp group, we expressed and then His-tag purified Xpp22Aa1 , Xpp37Aa1 , Xpp55Aa1 , and Xpp77Aa1 . The expected molecular sizes were 79, 14, 40, and 21 kDa, respectively. The observed sizes in the SDS PAGE gel were correspondingly larger due to the His tags (Fig. 1C). From the Mpp group, we expressed and His-tag purified Mpp23Aa1 with expected and observed sizes of 29 and 34 kDa, respectively (Fig. 1C).Example 2 - Five Bt pesticidal proteins were toxic to D. citri in single dose assays

[0071] Single-dose bioassays were performed with individual proteins to identify proteins with pesticidal activity against four-day old D. citri adults. The proteins Cry1 Aa, Cry1 Ac, Cryl Ca, Cry1 Ea, Cry3Aa, Cry2Aa, Cry2Ab, and Cry2Ac were fed to psyllids at 500 pg / mL. The Cry1 proteins and Cry3Aa were trypsin-activated. Toxicity was significantly higher for Cry1 Ca and Cry1 Ea relative to the buffer control (P<0.05, Student’s t-test, Fig. 2, Table 3).

[0072] Table 3. Bioassay summary with statistical significance values. r, . . . .. .. . .. No. No. Dlff. P-Protem Activation ConcentrationD. _. . . * p insects Ave.aa .BioRe valueb0CrylAa yes (trypsin) 500 3 75 20.04 0.090Cry 1 Ac yes (trypsin) 500 3 74 16.60 0.144CrylCa yes (trypsin) 500 3 75 26.71 0.007*Cryl Ea yes (trypsin) 500 3 72 17.16 0.002*Cry3Aa yes (trypsin) 500 3 76 21.22 0.246App6Aa2 no 500 3 75 34.67 0.013*Tpp78Aa1 no 200 3 73 27.50 0.026*Tpp78Ba1 no 200 3 64 4.59 0.572Tpp78Ba1 no 400 3 63 20.01 0.195Tpp80Aa1 no 200 3 59 1.03 0.922Tpp80Aa1 no 400 3 58 6.63 0.604Xpp22Aa1 no 200 3 69 4.30 0.732Xpp22Aa1 no 400 3 72 1.64 0.832Xpp37Aa1 no 200 3 73 41.10 0.003*Xpp55Aa1 no 200 2 48 -3.46 0.779Xpp55Aa1 no 400 3 76 11.57 0.314Xpp77Aa1 no 200 3 72 5.74 0.571Xpp77Aa1 no 400 3 74 -3.51 0.511Mpp23Aa1 no 200 3 75 -5.48 0.405Mpp23Aa1 no 400 2 50 -0.68 0.936 difference of the mean relative to the buffer control;bP-value of Student’s t-test between each protein and the respective buffer control. *Statistically significant

[0073] Of the Cry1 proteins, the highest mortality was 34% for CrylCa. Psyllid mortality in Cry3Aa was <30%, and not significantly different from the buffer control (P>0.05, Student’s t- test, Fig. 2, Table 3). None of the three Cry2 proteins tested resulted in significant mortality relative to the buffer control (P>0.05, Student’s t-test, Fig. 2, Table 3). The protein App6Aa1 , also tested at 500 pg / mL, resulted in approximately 45% mortality, significantly higher than the buffer control (P<0.05, Student’s t-test, Fig. 2, Table S3).

[0074] For the Tpp, Xpp, and Mpp proteins, psyllids were initially fed on 200 pg / mL. Among these, significant mortality relative to the control was only observed for Tpp78Aa1 and Xpp37Aa1 with 53 and 45% mortality, respectively (P<0.05, Student’s t-test, Fig. 2, Table 3). For the remaining Tpp, Xpp, and Mpp proteins, we performed an additional test at 400 pg / mL, without significant mortality observed (P>0.05, Student’s t-test, Fig. S1 , Table 3).

[0075] Proteins Tpp78Aa1 and Xpp37Aa1 were selected for LC50 determination based on the results from single dose bioassays at 200 pg / mL. Although App6Aa2 killed 45% of psyllids, it was not included in the LC50 assays as the 500 pg / mL concentration used in the single dose assay was high relative to that needed for practical applications. Four-day old D. citri adults were fed with seven concentrations of each protein or buffer control (20 mM carbonate buffer, pH 10.5). The LC50 was calculated using day 7 mortality. As psyllid mortality at the highest concentration tested (400 pg / mL) for Xpp37Aa1 was 19% (Fig. 3), the LC50 value could not be determined. The LC50 value for Tpp78Aa1 was 204.84 pg / mL with a 95% confidence limit (95% CL) of 112.31 - 414.41 (Fig. 3; Table 1).

[0076] Table 4. Toxicity of Tpp78Aa1 to D. citri adultsN, number of insects tested; SE, standard error; LC50, lethal concentration in pg / mL for 50% of psyllids; 95% CL, 95% confidence interval.

[0077] Discussion:

[0078] The goal of this study was to screen for additional bacterial-derived pesticidal proteins with potential application in D. citri and citrus greening disease management. Five out of the 17 Bt proteins screened showed pesticidal activity against D. citri m single dose assays. Based on LC50 determination, Tpp78Aa1 was toxic to D. citri, thereby representing a promising pesticidal protein for use in D. citri management.

[0079] Toxicity related to protein activation: The ultimate result for susceptible insects that feed on Bt proteins is death following pore formation in the gut epithelium. The mechanism by which Bt proteins kill target insects is poorly understood, but several steps are proposed (7, 39, 40). Cry proteins are ingested in their solubilized form or in crystalline inclusions that undergo solubilization, proteolytic activation, receptor binding and post-binding events (e.g., oligomerization) that result in pore formation. Though this multi-step mechanism of action confers high specificity of Bt proteins to their target insects, it can also limit toxicity.Proteolytic activation of Bt proteins by digestive enzymes in the gut lumen may determine or significantly increase specificity and toxicity of some Bt proteins toward target insects. This step has been best characterized in Cry proteins, which are digested by trypsin and chymotrypsin (37). These two enzymes are found in the gut lumen of coleopteran and lepidopteran species but not in the gut lumen of hemipteran insects, which instead rely on cysteine peptidases recruited from the lysosome (37, 41 ). Two cysteine peptidases, cathepsin B and L, have been identified and characterized in D. citri (42, 43). Whether cathepsin B and L relate to Cry protein activation and toxicity in D. citri has yet to be determined. Cry1 Ac processing by cysteine peptidases in pea aphids was shown to producea protein of around 60 kDa (44). However, others have shown that pea aphid gut contents failed to activate Cry4Aa, Cry3Aa1 , and Cry1 Ab (12, 44, 45). In this study, all Cry proteins tested against D. citri, except for the Cry2 proteins, were trypsin processed before feeding assays due to the lack of tryptic enzymes in the psyllid gut. Despite this activation, low toxicity was observed for the four tested Cry1 proteins and the Cry3Aa protein, indicating that other factors limit the activity of Cry proteins against D. citri. Indeed, the significant difference in mortality for Cry1 Ca and Cry1 Ea in single dose bioassays could result from variation within the assay as observed from mortality in the buffer control treatments.

[0080] Recently characterized Bt proteins from the Mpp and Tpp groups have shown toxicity against hemipteran insects without the need for activation before feeding, indicating that proteins in these groups either do not require proteolysis or are activated by cathepsins in the gut (9, 14-16, 18, 46, 47). It is unknown whether hemipterans can activate proteins in the Xpp and App groups or whether proteolytic activation is required. Trypsin processing of App6Aa results in the loss of internal sequences, but the impact of trypsin processing on toxicity has not been demonstrated (48). Protease digestion was not observed for Xpp37Aa1 after treatment with gut juice proteases of sweet potato weevil (Cylas puncticollis) or trypsin (49), so proteolytic processing might not be required for toxicity of this protein to C. puncticollis. It is unknown whether proteolytic activation is required for Xpp22Aa1 or Xpp77Aa1 .

[0081] Toxicity of Bt proteins against D. citri and other hemipterans. Although several Bt strains produce a wide array of pesticidal proteins with potential applications against hemipterans, the number of studies reporting toxicity against hemipteran insects is low relative to those against lepidopterans and coleopterans. The recent identification of highly active Bt proteins against planthoppers (15, 17, 47), for instance, suggests that bacterial pesticidal proteins are promising tools for the management of hemipterans and that more effort is required for testing and screening proteins across structural groups (2). Only four individual proteins had been tested in vitro or expressed in transgenic plants against D. citri prior to the current study (13, 14, 21 -24). Transgenic citrus plants or curry leaf plants expressing Cryl Ba resulted in significant reduction in the survival of psyllids (23) or severe damage to the gut epithelium (24). Despite the high identity, especially between Cry1 Ab and the Cry1 proteins (Cry1 Aa and Cry1 Ac) tested in this study, low toxicity was observed at single-dose assays for all Cry1 proteins. For instance, Cry1 Aa (92.6% of similarity with Cry1 Ab) (50) killed only 28% of psyllids fed with 500 pg / mL of protein. Although unlikely to cause such a difference in toxicity, it is important to note that Cry1 Ab was expressed in Bt strains and purified from crystals, whereas the Cry1 proteins used in this study were expressed in E. coli and purified from inclusion bodies. A critical step for the latter type ofpurification is protein refolding after solubilization, which was accomplished in this study by dialysis. Though an impairment in toxicity would be expected if the protein does not refold properly (51 ), we do not see that as the case here. We expect that trypsin-digestion of unfolded proteins would result in a degraded product, as internal cleavage sites would be exposed if the protein was improperly folded. However, a trypsin-resistant core around 60-65 kDa was observed for all Cry1 proteins tested in this study.

[0082] Bt proteins included in the Tpp group are toxic to planthoppers (15, 17, 47). Similarly, this study showed pesticidal activity of Tpp78Aa1 against D. citri. Although Tpp78Ba1 shares 68.6% similarity to Tpp78Aa1 , psyllid mortality at 400 pg / mL was only 32% and insufficient for LC50 determination. Interestingly, both proteins were highly toxic to L. striatellus (15, 17). We also tested Tpp80Aa1 , which is toxic to 3rd instar mosquito larvae (i.e., Culex pipiens), against D. citri. As with Tpp78Ba1 , psyllid mortality at 400 pg / mL was less than 50%. Differences in toxicity among proteins with high similarity are expected if changes in amino acids occur in regions that are critical for toxicity, such as regions involved in binding to the gut receptor(s). Amino acids critical to gut binding have been well characterized using a site- directed mutagenesis approach in which replacing one or a few amino acids can decrease or even abolish toxicity (9, 52). Interestingly, the effects of substitution of critical amino acids can differ in insect species from the same genus. For instance, substitution of Mpp51 Aa2 native amino acids at positions 217 and 219 produced modified variants that were 29-fold more toxic to Lygus lineolaris, but 12-fold less toxic towards L. hesperus (9). These results highlight how small changes in critical sites can significantly affect toxicity. This observation can explain the differences in toxicity levels observed here in proteins from similar groups, including Cry1 and Tpp proteins.

[0083] Along these same lines, a pesticidal protein active against one sap sucking insect, will not necessarily be toxic to other sap-sucking insects. For example, Cry1 Ac is toxic to Acyrthosiphon pisum, but not to D. citri. Cry3Aa is toxic to other sap-sucking insects, but not to D. citri (Table 5 below).

[0084] Table 5. Known efficacy of tested pesticidal proteins against other hemipteran pests.. Tested against Active against Protein . . ® ® >TInsect Toxicity hemipterans? hemipterans?CrylAa noSignificant at 500CrylAc yes yes Acyrthisiphon pisum pg / mLCrylCa noCrylEa noCry3Aa yes yes M. euphorbiae 60% at 500 pg / mLApp6Aa2 noNilaparvata lugens LC50 = 15.78 pg / mLTpp78Aal yes eSLaodelphax striaellus LC50 = 6.89 pg / mLTpp78Bal yes yes Laodelphax striaellus LC50 = 9.72 pg / mLTpp80Aal noXpp22Aal noXpp55Aal noXpp77Aal noMpp23Aal no

[0085] These examples illustrate that empirical testing is required with each species to identify pesticidal proteins with toxicity. Another key aspect is that pesticidal protein screens using adult insects may overlook toxicity of proteins to nymphs. As an example, Tpp78Ba1 was not toxic in single dose adult D. citri bioassays at 7 days (Figure 2), but was toxic to D. citri nymphs at 9 days (Table 7 below). Similar toxicity to nymphs is possible for additional Bt proteins.

[0086] Pesticidal proteins from the App and Xpp groups have not previously been tested against hemipterans. The protein App6Aa (formerly Cry6Aa) is toxic to nematodes (53). As demonstrated herein, App6Aa2 is toxic to D. citrial a single dose. Xpp37Aa1 forms a binary toxin with Mpp23Aa with both genes located in the same operon. Mpp23Aa / Xpp37Aa proteins are highly toxic to Anthonomus grandis and C. puncticollis (49, 54). However, the presence of both proteins is not required for pesticidal activity. This was demonstrated in C. puncticollis, where individual proteins exerted toxicity toward C. puncticollis larvae (49). Although other proteins in the Mpp group have demonstrated toxicity against hemipterans (14, 46), no toxic activity was observed for Mpp23Aa1 toward D. citrim this study.

[0087] Toxicity of Tpp78Aa1 relative to others D. citri- and hemipteran-active proteins The 50% lethal concentration (LC50) of Tpp78Aa1 for psyllids is high relative to that of other D. c / t / 7-active proteins including Mpp51Aa1 (110 pg / mL), CrylAb and Cryl Ba (-120 pg / mL). Tpp78Aa1 has a remarkable toxicity against planthoppers with an LC50 as low as 6.89 pg / mL (17, 20). As observed with the protein Mpp51 Aa1 , we expect greater toxicity of Tpp78Aa1 and the other D. c / f / 7-active proteins against nymphs, particularly first instar nymphs. In wipe feeding assays, Mpp51 Aa1 is ~2-fold more toxic to 4th instar nymphs relative to D. citri adults (14), which correlates with the severe impact of Mpp51 Aa1 expressed by transgenic plants on D. citri nymphs with >90% reduction in emergence to adult (22). We are unable to determine the toxicity of pesticidal proteins against D. citri neonates, the most susceptible stage, with the current feeding strategies. In contrast to adult psyllids, first instar nymphs move short distances, require constant feeding for hydration and nutrient intake, and survive for a much shorter period without food (~24h). Bioassays toassess efficacy of lepidopteran and coleopteran target pests of Bt proteins are typically performed with neonates (<24h) (55-62). Indeed, the most active pesticidal proteins against hemipterans have also been tested with nymphs (20). These include Mpp64Ba / Ca (LC50 as low as 2.14 pg / mL against Sogatella furcifera) and others from the Vpb1 and Vpb2 structural classes (LC50 of 0.0875 against Aphis gossypii) (16, 20, 63, 64), which also represent good candidates for screening against D. citri.

[0088] Practical use and field implementation There are several important points for the practical use of Tpp78Aa1 along with other D. c / tr / '-active proteins for sustainable field implementation for D. citri management. These include: (i) the toxicity level of the pesticidal protein, (ii) the delivery strategy, and (iii) the longevity of the technology. When compared to Bt proteins used in transgenic crops for management of insect target pests with toxicity at a few ppm or ppb (9, 56-59, 61), the D. c / 't / 7-active proteins appear to lack sufficient toxicity for practical application. However, as mentioned previously the D. c / 't / 7-active pesticidal proteins have been tested against adults and 4th instar nymphs rather than neonates (i.e., Mpp51Aa1 ). Additionally, the identification of active proteins is the initial step toward the use of molecular methods for modification of pesticidal proteins for enhanced activity (9, 18, 20, 65-67). Indeed, remarkable toxicity of a Mpp51 Aa2 variant protein (formerly Cry51Aa2) was achieved against Lygus spp. and thrips by modification using site-directed mutagenesis and resulted in the most successful and only example of a field-deployed transgenic cotton targeting a hemipteran pest (9). In terms of protein delivery to psyllids, several studies have demonstrated the successful delivery via transgenic plants including fruit-bearing citrus (21 - 23) and a non-crop plant that is highly attractive to psyllids, Indian curry leaf (24). However, despite the demonstrated safety of Bt proteins expressed in several crops over the last decades, public acceptance of transgenic citrus poses a challenge to the adoption of bioengineered citrus plants. Finally, resistance to the pesticidal protein affecting the longevity and usefulness of the transgenic plants producing pesticidal proteins is a major concern (68, 69). The primary considerations related to the development of insect resistance to transgenic plants have been well summarized by Gassmann (2023) (68) and discussed by Mishra et al. (2023) in the context of D. citri and citrus greening management (22).

[0089] In summary, the toxicity of 14 Bt-derived pesticidal proteins were tested against D. citri and identified Tpp78Aa as toxic to D. citri adults. Considering the suboptimal toxicity level of Tpp78Aa1 to psyllids for practical applications, further work should address (i) the level of susceptibility of first instar nymphs to Tpp78Aa1 , (ii) molecular modification of Tpp78Aa1 for enhanced toxicity, (iii) potential synergist impacts of Tpp78Aa1 when combined with other D. c / 't / 7-active pesticidal proteins with different modes of action. Finally, the proteins with demonstrated toxicity against D. citri represent a small subset of thebacterial pesticidal proteins characterized to date (50) and further screening is likely to identify additional proteins with greater toxicity against D. citri for use in the integrated management of citrus greening disease.Example 2 - Modification of Tpp78Aa1

[0090] Constructs of Tpp78Aa1 modified at different sites (Table 6) with gut binding peptide DcNy6 were expressed in E. coli strain BL21 and purified. Membrane feeding assays were conducted with first instar D. citri using the sandwich feeding assay (Tavares et al., Journal of Invertebrate Pathology:"! 08208, 2024) with 10 pg / ml of protein. Tpp78Aa1 was modified by replacement of amino acids with DcNy6 at 5 different sites in the protein (Table 6).

[0091] Table 6. Modification of Tpp78Aa1 by sequence replacement with DcNy6.

[0092] A significant increase in psyllid mortality was noted for Tpp78Aa1 modified at position 13-19 in these single dose bioassays. The LC50 value was then determined to be approximately 2.6 pg / ml (95% confidence limits; 0.73-4.94; Figure 3), indicating four-fold greater susceptibility of D. citri neonates when compared to wild type Tpp78Aa1 (LC50 of 10.3 pg / ml; Table 7).

[0093] Table 7. LC50 values for bacterial pesticidal proteins tested against D. citri.

[0094] We have demonstrated toxicity for Tpp78Aa1 and Tpp78Ba1 against D. citri (Table 6). Of these two proteins, Tpp78Aa1 is the more toxic. It is notable that first instar nymphs are significantly more susceptible to these proteins than adults. For example, the LC50 for Tpp78Aa1 against adults was >400 pg / ml, but only 20.5 pg / ml against first instars (Table 8).

[0095] Table 8. Summary of LC50 data for bacterial pesticidal proteins against D. citri.

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[0097] Informal Sequence Listing:

Claims

CLAIMSWhat is claimed is:1 . A method of reducing a psyllid pest population comprising contacting the psyllid with a bacterial pesticidal protein.

2. The method of claim 1 , wherein the bacterial pesticide protein is Tpp78Aa1 , Xpp37Aa1 , App6Aa1 or Tpp78Ba1 .

3. The method of claim 1 or claim 2, wherein the psyllid is an Asian citrus psyllid (D. citri).

4. A modified pesticidal protein comprising a bacterial pesticidal protein, wherein the pesticidal protein has been modified to include at least one psyllid gut binding peptide.

5. The modified pesticidal protein of claim 3, wherein the bacterial pesticidal protein is Tpp78Aa1 , Xpp37Aa1 , App6Aa1 or Tpp78Ba1 .

6. The modified pesticidal protein of claim 4 or claim 5, wherein the psyllid gut binding peptide is an Asian citrus psyllid gut binding peptide.

7. The modified pesticidal protein of any one of claims 4-6, wherein the gut binding peptide is DcAd4 (SEQ ID NO: 2), DcNy6 (SEQ ID NO: 4 or SEQ ID NO: 19) or DcNy9 (SEQ ID NO: 6).

8. The modified pesticidal protein of any one of claims 4-7, wherein the bacterial pesticidal protein is modified by addition to include the gut binding peptide.

9. The modified pesticidal protein of any one of claims 4-7, wherein the bacterial pesticidal protein is modified by substitution to include the gut binding peptide.

10. The modified pesticidal protein of any one of claims 4-9, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 20-24.

11. A nucleic acid encoding the modified pesticidal protein of any one of claims 4- 10.

12. A host cell comprising the nucleic acid of claim 11 .

13. The host cell of claim 12, wherein the host cell is a plant cell.

14. A vector comprising the nucleic acid of claim 11 operably liked to a promoter.

15. The vector of claim 14, that is a citrus tristeza viral vector.

16. A transformed plant comprising the vector of claim 14 or claim 15.

17. A method of providing pesticidal activity in a plant comprising introducing the vector of claim 14 or claim 15 into a host cell of the plant and expressing a polynucleotide of claim 11 in said host cell, thereby providing pesticidal activity in the plant.

18. A method of providing pesticidal activity in a plant comprising introducing a vector comprising a nucleic acid encoding a bacterial pesticidal protein into a host cell of the plant, and expressing the nucleic acid in said host cell, wherein the vector is a citrus tristeza viral vector and the bacterial pesticide protein is Tpp78Aa1 , Xpp37Aa1 , App6Aa1 or Tpp78Ba1.

19. A method of reducing a D. citri psyllid pest population, the method comprising providing a plant expressing the modified protein of any one of claims 4-10, and allowing the psyllid to feed on the plant.

20. The method of any one of claims 17-19, wherein the plant is a citrus plant.21 . The method of claim 20, wherein the citrus plant is an orange plant, lemon plant, lime plant, grapefruit plant, tangerine plant, pomelo plant, citron plant, mandarin orange plant, papeda plant, kaffir lime plant, ichang papeda plant, or kumquat plant.

22. The method of any one of claims 18-21 , wherein the plant is an Indian curry plant.

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