Nucleic acid constructs for delivery of beneficial agents to plant cells

Abstract

Circular nucleic acid constructs of less than 1000 nucleotides are disclosed for manipulating plant traits. At least a portion of the constructs are single stranded.

Description

[0001] NUCLEIC ACID CONSTRUCTS FOR DELIVERY OF BENEFICIAL AGENTS TO PLANT

[0002] CELLS

[0003] RELATED APPLICATION / S

[0004] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 667,175 filed on 3 July 2024, the contents of which are incorporated herein by reference in their entirety.

[0005] SEQUENCE LISTING STATEMENT

[0006] The XML file, entitled 104096. xml, created on 2 July 2025, comprising 21,905 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0007] FIELD AND BACKGROUND OF THE INVENTION

[0008] The present invention, in some embodiments thereof, relates to circularized nucleic acid constructs useful for delivery of beneficial agents to plant cells.

[0009] RNA interference refers to the process of sequence-specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA in animal). The corresponding process in plants is commonly referred to as post transcriptional gene silencing or RNA silencing. ’The process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla. Such protection from foreign gene expression may have evolved in response to the production of double stranded RNAs (dsRNA) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded RNA or viral genomic RNA.

[0010] The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA). Short interfering RN As derived from Dicer activity are typically about 21-23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex. Chemically synthesized double- stranded RNAs are unstable in vivo, namely, susceptible to degradation with enzymes (nucleases) in cells which limits their use in vivo. To solve this problem, RNA strands with non-natural nucleic acids have been developed in order to enhance stability of double- stranded RNAs in cells. However use of non-natural nucleic acids may reduce biological activity, there exists another problem that its biological activity reduces while the stability is improved.

[0011] Background art includes US Patent Application No. US2010 / 0137407, US Patent Application No. US2010 / 0305191 and WO2017165724; Abe N, Abe H, Nagai C, et al (2011) Bioconjugate Chem 22:2082-2092. https: / / doi(dot)org / 10.1021 / bc2003154; Xie J, Ye F, Deng X, et al (2023) Journal of Translational Internal Medicine 11:372-381. https: / / doi(dot)org / 10.2478 / jtim-2023- 0122; Zhang L, Liang D, Chen C, et al (2017) Mol Ther Nucleic Acids 10:237-244. https: / / doi(dot)org / 10.1016 / j.omtn.2017.12.007; Zhang P, Li S, Chen M (2020) Characterization and Function of Circular RNAs in Plants. Front Mol Biosci 7 : .https : / / doi(dot)org / 10.3389 / fmolb.2020.00091.

[0012] SUMMARY OF THE INVENTION

[0013] According to an aspect of some embodiments of the present invention there is provided a circular nucleic acid construct being no more than 1000 nucleotides in length comprising:

[0014] (i) at least one double-stranded sequence; and

[0015] (ii) at least two single- stranded loop sequences, wherein at least one of the at least two single-stranded loop sequences comprises a plant cell penetration sequence and / or a promoter sequence, wherein the at least one double- stranded sequence, or the at least one of the at least two single-stranded loop sequences comprises or encodes a plant beneficial product.

[0016] According to some embodiments of the invention, the circular nucleic acid construct is no more than 500 nucleotides in length.

[0017] According to some embodiments of the invention, the plant cell penetration sequence comprises an inverted repeat sequence.

[0018] According to some embodiments of the invention, the inverted repeat sequence is a viral inverted repeat sequence.

[0019] According to some embodiments of the invention, less than 50 % of the construct comprises viral sequences or bacterial sequences.

[0020] According to some embodiments of the invention, the circular nucleic acid construct is devoid of a viral protein encoding sequence. According to some embodiments of the invention, the circular nucleic acid construct is devoid of a capsid protein encoding sequence.

[0021] According to some embodiments of the invention, the circular nucleic acid construct is composed of ribonucleotides.

[0022] According to some embodiments of the invention, the circular nucleic acid construct is composed of deoxyribonucleotides.

[0023] According to some embodiments of the invention, a portion of the double stranded sequence encodes a gene silencing agent or a genome editing agent.

[0024] According to some embodiments of the invention, the portion of the double stranded sequence comprises a gene silencing agent.

[0025] According to some embodiments of the invention, the nucleic acid construct comprises two loop sequences, wherein a length of the first loop sequence is the same as a length of the second loop sequence.

[0026] According to some embodiments of the invention, the sequence of the first loop sequence at least 90 % identical to a sequence of the second loop sequence.

[0027] According to some embodiments of the invention, the portion of one of the at least one single- stranded loop sequence is complementary to a target sequence in a plant genome.

[0028] According to some embodiments of the invention, the portion of the at least one single- stranded loop sequence encodes a gene silencing agent.

[0029] According to some embodiments of the invention, the gene silencing agent is an antisense, an anti-miRNA molecule or an RNA interference-inducing molecule.

[0030] According to some embodiments of the invention, the portion of the at least one single- stranded loop sequence encodes a genome editing agent.

[0031] According to some embodiments of the invention, at least a portion of the at least one single-stranded loop sequence comprises a sequence of interest and at least one homology arm being at least 90 nucleotides in length.

[0032] According to some embodiments of the invention, the sequence of interest is flanked by two homology arms.

[0033] According to some embodiments of the invention, at least a portion of one of the loop sequences comprises an internal ribosome entry site (IRES) operatively linked to a sequence encoding a plant beneficial expression product.

[0034] According to some embodiments of the invention, the plant beneficial expression product is a protein or peptide product. According to some embodiments of the invention, the peptide product is an anti-microbial peptide.

[0035] According to some embodiments of the invention, the plant beneficial expression product is an RNA product.

[0036] According to some embodiments of the invention, the at least one single- stranded loop sequence comprises the promoter sequence, the promoter sequence being operatively linked to a plant beneficial expression product.

[0037] According to some embodiments of the invention, the promoter sequence is a polymerase II promoter or a polymerase III promoter.

[0038] According to some embodiments of the invention, the promoter sequence comprises a TATA box element.

[0039] According to an aspect of some embodiments of the present invention there is provided single- stranded circular (ssc) nucleic acid construct being no more than 1000 nucleotides in length comprising:

[0040] (i) a sequence which is, or encodes, at least one plant beneficial product; and

[0041] (ii) a plant cell penetration sequence and / or a promoter sequence.

[0042] According to some embodiments of the invention, the ssc nucleic acid construct is no more than 500 nucleotides in length.

[0043] According to some embodiments of the invention, the plant cell penetration sequence comprises an inverted repeat sequence.

[0044] According to some embodiments of the invention, the inverted repeat sequence is a viral inverted repeat sequence,

[0045] According to some embodiments of the invention, the less than 50 % of the construct comprises viral sequences.

[0046] According to some embodiments of the invention, the at least one plant beneficial product is selected from the group consisting of:

[0047] (i) a gene silencing agent which hybridizes to a plant RNA;

[0048] (ii) a genome editing agent which edits the genome of a plant; and

[0049] (iii) a protein or a peptide.

[0050] According to some embodiments of the invention, the sequence encodes the plant beneficial product.

[0051] According to some embodiments of the invention, the promoter sequence is operatively linked to the sequence which encodes the at least one plant beneficial product. According to some embodiments of the invention, the ssc nucleic acid construct comprises an inverted repeat of a virus.

[0052] According to some embodiments of the invention, the gene silencing agent is an antisense agent or an anti-miRNA agent.

[0053] According to some embodiments of the invention, the genome editing agent is an sgRNA or a sequence of interest comprising at least one homology arm.

[0054] According to some embodiments of the invention, the ssc nucleic acid construct is composed of ribonucleotides.

[0055] According to some embodiments of the invention, the ssc nucleic acid construct is composed of deoxyribonucleotides.

[0056] According to some embodiments of the invention, the plant beneficial product is an RNA.

[0057] According to some embodiments of the invention, the plant beneficial product is a protein or peptide.

[0058] According to some embodiments of the invention, the ssc nucleic acid construct further comprises an IRES sequence.

[0059] According to some embodiments of the invention, the peptide is an anti-microbial peptide.

[0060] According to another aspect, there is provided an agricultural composition comprising the nucleic acid construct described herein and an agriculturally acceptable carrier.

[0061] According to another aspect, there is provided a method of affecting a plant trait or performance comprising contacting the plant with at least one of the nucleic acid constructs described herein, thereby affecting the plant trait or performance.

[0062] According to some embodiments of the invention, the plant trait is selected from the group consisting of plant fitness, plant resilience, plant yield, plant growth, fruit size, fruit weight, vegetable size, vegetable weight, crop protection, plant health and stress resistance.

[0063] According to some embodiments of the invention, the contacting is effected by at least one of the following methods: seed treatment; seed priming; plant tissue culture media; plant cell culture media; plant spray; immersion; plant dipping; soaking; injection; tissue bombardment; tissue spraying; chemigation; or mechanical introduction.

[0064] According to some embodiments of the invention, the nucleic acid construct is contacted with the plant in combination with a nucleic acid cell delivery agent.

[0065] According to some embodiments of the invention, the nucleic acid cell delivery agent is selected from the group consisting of a nanoparticle, a hydroxide particle, chitosan and a polysaccharide.

[0066] According to some embodiments of the invention, the method further comprises contacting the plant with a plant protection agent.

[0067] According to some embodiments of the invention, the plant protection agent comprises at least one of the following agents: a fertilizer, an insecticide, a fungicide, a herbicide, a nematicide, a virucide, a biostimulant, an elicitor, a biopesticide, an antibiotic and a bacteriophage.

[0068] According to some embodiments of the invention, the contacting is effected a single time.

[0069] According to some embodiments of the invention, the contacting is effected a plurality of times.

[0070] According to some embodiments of the invention, the part thereof is a seed, root or a leaf.

[0071] According to some embodiments of the invention, the plant is at a flowering stage.

[0072] According to some embodiments of the invention, the plant is a plant culture.

[0073] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0074] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0075] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0076] In the drawings:

[0077] FIG. 1A-C are representative constructs according to embodiments of the invention and exemplary uses thereof. Letters in the figure describe representative sequences.

[0078] FIGs. 2A-B depict the SHR-miRNA schematic RNA structure. Figure 2A is a schematic illustration of the SHR-RNAi structure showing the various motif positions. Figure 2B is a picture of the predicted structure as generated by the RNAfold web server. An exemplary sequence that conforms to this structure is set forth in SEQ ID NO: 21.

[0079] FIG. 3 is a photograph depicting early seedling developments of SHR-RNAi treated A. thaliana seeds 8 days post sowing. Seeds were treated with either (1) circular single- stranded RNAi, or (2) linear single-stranded RNAi. Primed seeds with no RNAi addition served as control. All seeds were primed in a solution containing 25% PEG and 0.25% ZnEDTA, incubated for 3 days under continuous mixing, washed, desiccated, and cold- stratified prior to sowing on 0.5xMS agar medium. Plates were incubated under controlled growth conditions (25 °C, 16 / 8 h light / dark, -300 pE m2s-1). Representative images were captured 8 days after sowing.

[0080] FIGs. 4A-B are graphs illustrating SHR-RNAi effect on root development in treated A. thaliana seed 8 days post germination.

[0081] Figure 4A: Primary root length (cm) measured in plants treated with SHR-cssRNA (cRNA), SHR-LssRNA (LinRNA), or control (Ctrl) with no RNAi adddition. Bars represent the mean root length ± standard deviation (SD).

[0082] Figure 4B: Lateral root number (branching) in the same treatment groups. Bars represent the mean number of lateral roots ± SD.

[0083] FIGs. 5A-C depict LDLl-RNAi schematic structure. Figure 5A is a schematic illustration of the LDLl-RNAi structure showing the various motif positions. Figure 5B is a picture of the predicted structure as generated by the RNAfold web server. An exemplary sequence that conforms to this structure is set forth in SEQ ID NO: 22. Figure 5C is a schematic illustration of the dsRNA molecule.

[0084] FIG. 6 depicts early seedling development of LDLl-RNAi treated Arabidopsis thaliana seeds 11 days post-sowing, following RNAi-based priming treatments. Seeds were treated with either (#R22) LDL1 targeted circular single-stranded RNAi, or (#M0C) non-sense circular single- stranded RNAi (as a control). All seeds were primed in a solution containing 25% PEG and 0.25% ZnEDTA, incubated for 24 hr under continuous stratified prior to sowing on 0.5x MS agar medium. Plates were incubated under controlled growth conditions (25°C, 16 / 8 h light / dark, -300 pE m2s-1). Representative images were captured 11 days after sowing.

[0085] FIGs. 7A-D are graphs comparing the effects of LDL1- cssRNA RNAi (R22) and LDL1- dsRNA RNAi (R24) constructs on root length and LR formation to MOC treatment.

[0086] (Figures 7A-B): Primary root length (cm) of plants expressing the R24 (7 A) or R22 (7B) constructs, compared with the MOC treatment.

[0087] (Figures 7C-D): Eateral root number (branching) of plants treated with LDL7-dsRNA RNAi (R24) (7C) or LDL1- cssRNA RNAi (R22) (7D), compared with the MOC treatment. Bars represent mean values ± standard deviation (SD).

[0088] FIG. 8 depicts the expected molecule folding of the PDS-cssDNA serving as RNAi construct, generated by the DNAfold web server application (SEQ ID NO: 19).

[0089] FIGs. 9A-B are photographs of intact mature tomato leaves which were treated with either (Figure 9B) circular single- stranded DNA as PDS RNAi construct, or with water solution, as control (Figure 9A).

[0090] FIG. 10 is a graph summarizing the inhibition of PDS as measured by analysis of bleaching of infected tomato leaves.

[0091] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0092] The present invention, in some embodiments thereof, relates to circularized nucleic acid constructs useful for delivery of beneficial agents to plant cells.

[0093] Constructs that are effective for gene silencing in plants are of significant utility, as they provide a targeted and reliable means for downregulating gene expression in a wide range of species. The present invention provides novel and inventive circularized constructs that achieve efficient and specific gene silencing, offering valuable tools for functional genomics, crop trait development, and improved plant performance. These constructs represent a technical advancement in the field by enabling enhanced control over gene expression through stable or transient expression systems.

[0094] Whilst reducing the present invention to practice, the present inventors synthesized exemplary short, circularized RNA constructs, each of which were shown to effectively down- regulate expression of its target gene. The first construct (depicted in Figures 2A-B) was shown to be capable of transiently modulating SHR activity in A. thaliana. The second construct (depicted in Figures 5A-B) was shown to be capable of transiently modulating LDL1 activity in A. thaliana, Still a third circularized RNA construct (depicted in Figure 8) was shown to downregulate Phytoene desaturase (PDS) in tomato plants. The present inventors propose that employing circularized small nucleic acids to modulate gene expression in plants offers broad potential for diverse applications in plant biotechnology and crop improvement.

[0095] The circularized constructs described herein, each being less than 1000 nucleotides in length are different to viral-based vectors or bacterial-based vectors in that less than 50 %, less than 40 %, less than 30 %, less than 20 % or even less than 10 % of the construct is made up of viral sequences or bacterial sequences. Furthermore, the constructs are typically devoid of viral protein encoding sequences (e.g. capsid protein encoding sequence).

[0096] The circularized nucleic acid constructs described herein may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides or may be composed of a combination of deoxyribonucleotides and ribonucleotides.

[0097] The constructs are typically no greater than 1000 nucleotides in length, no greater than 900 nucleotides in length, no greater than 800 nucleotides in length, no greater than 700 nucleotides in length, no greater than 600 nucleotides in length, no greater than 500 nucleotides in length, no greater than 400 nucleotides in length, no greater than 300 nucleotides in length, no greater than 200 nucleotides in length, or even no greater than 100 nucleotides in length.

[0098] According to a particular embodiment the constructs are between 30-500 nucleotides in length, 50-500 nucleotides in length or 100-500 nucleotides in length.

[0099] As used herein, the term "construct" as used herein, refers to an artificially assembled nucleic acid molecule which comprises (or encodes) a beneficial agent which is introduced into a cell.

[0100] The phrase “circular nucleic acid construct” refers to a sequence of nucleic acids which forms a circular structure (i.e. devoid of a free phosphate group on one end and / or devoid of a free OH group on the other end).

[0101] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0102] According to a first aspect of the present invention, there is provided a circular nucleic acid construct being no more than 1000 nucleotides in length comprising:

[0103] (i) at least one double -stranded sequence: and

[0104] (ii) at least two single-stranded loop sequences, wherein at least one of the at least two single-stranded loop sequences comprise a plant cell penetration sequence and / or a promoter sequence, wherein the at least one double- stranded sequence, or the at least one of the at least two single-stranded loop sequences comprises or encodes a plant beneficial product.

[0105] According to this aspect of the invention, at least a portion of the construct is double stranded (e.g. forming a stem structure) and at least a portion of the construct is single-stranded (e.g. forming at least one loop structure). In one embodiment, the loop sequences do not form base pairing to itself.

[0106] The length of the double- stranded portion is typically at least 3 base pairs. In one embodiment, the length of the double-stranded portion is no longer than 400 base pairs, no longer than 300 base pairs, no longer than 200 base pairs, no longer than 100 base pairs or even no longer than 50 base pairs. According to a particular embodiment, the length of the double stranded portion is between 17 to 34 base pairs, e.g. 21 to 25 base pairs, e.g. 22 to 24 base pairs, e.g. 23 base pairs.

[0107] In another embodiment, no more than 90 % of the molecule is double-stranded, no more than 80 % of the molecule is double-stranded, no more than 70 % of the molecule is double- stranded, no more than 60 % of the molecule double-stranded, no more than 50 % of the molecule is double-stranded, no more than 40 % of the molecule is double -stranded, no more than 30 % of the molecule is double- stranded.

[0108] In one embodiment, the construct comprises at least two loop sequences, whereby the nucleotide sequences with unpaired nucleotides are present at the 5' end and 3' end of both the sense strand and the antisense strand of the double-stranded portion forming a dumbbell shaped nucleic acid construct, exemplified in Figures 1 A-C. The nucleotide at the 5' end of the nucleotide sequence with unpaired nucleotides in the sense strand and the nucleotide at the 3' end of the nucleotide sequence with unpaired nucleotides in the antisense strand are ligated together to form a loop. The nucleotide at the 3fend of the nucleotide sequence with unpaired nucleotides in the sense strand and the nucleotide at the 5' end of the nucleotide sequence with unpaired nucleotides in the antisense strand are ligated together to form a loop. The loop length is preferably between 2 to 950 bases, e.g. between 2-500 bases, e.g. between 2-400 bases, e.g. between 2-200 bases, e.g. between 2-100 bases, e.g. between 2-50 bases, e.g. between 2-20 bases.

[0109] In one embodiment, the first loop is of a similar length as the second loop - see for example Figure 1A and Figure IB. For example, the length of the two loops are no more than 20 % different, or no more than 10 % different. In one embodiment, the first loop is of the identical length as the second loop. In still another embodiment, the length of the first loop is at least 50 % longer than the length of the second loop (see for example Figure 1C.

[0110] ’The sequence of the first loop may be identical (see for example Figure 1 B) or non-identical (see for example Figure 1 A, Figure 1C). In a particular embodiment, the sequence of the first loop is at least 50 %, 60 %, 70 %, 80 %, 90 % identical to the sequence of the second loop.

[0111] An exemplary loop sequence may include UUCAAGAGA or UGUGCUGUC (M. Miyagishi el al., Oligonucleotides 2003, Vol. 13: pp. 1-7).

[0112] The loop sequence may be physically modified. For example, in vivo stability of the construct can be enhanced by modifying with polyethylene glycol whose molecular weight is approximately 2000 to 5000.

[0113] The loop sequence may be chemically modified. For example, using chemical methyl a tors (e.g. using DNA methyltransferases). At least one of the loop sequences of the construct comprises a plant cell penetration sequence and / or a promoter sequence and / or long-distance transport motifs such as polypyrimidine (poly-CU) sequence, transfer RNA (tRN A) -related sequence etc (Wang T, et al, 2021; Front Cell Dev Biol 9:doi(dot)org / 10dot3389 / fcelldot2021dot651278).

[0114] The phrase “plant cell penetration sequence” refers to a sequence of nucleic acids which enhance uptake of an associated nucleic acid molecule into plant cells.

[0115] Typically, the plant cell penetration sequence is at least 5 nucleic acids in length.

[0116] In one embodiment, the plant cell penetration sequence is an inverted repeat sequence (e.g. an inverted repeat sequence of a virus). Examples of such include the IR of Tomato Yellow Leaf Curled Virus (TYLCV) e.g. SEQ ID NO: 1 or SEQ ID NO: 14.

[0117] Other examples of plant cell penetration sequences include, RNA loop of Potato Spindle Tuber Viroid (PSTVd) (Owens, 2007, Mol. Plant Pathol. 8, 549-560. doi: lOdotl 11 l / jdotl364- 3703dot2007dot00418dotx; Ding, 2009, Annu. Rev. Phytopathol. 47, 105-131. doi: 10.1146 / annurev-phyto-080508-081927), tRNA or tRNA like hairpin motifs (Atabekova et al., 2017, Biochimie 132, 28-37. doi: 10dotl016 / jdotbiochidot2016dotl0dot009; Zhang et al. 2016, Plant Cell 28, 1237-1249. doi: 10dotl l05 / tpcdotl5dot01056), plant conserved sequence motifs such as UAGGUUA and ACUUCU (Rosin et al., 2003, Plant Physiol. 132, 106-117. doi: 10dotl l04 / ppdotl02dot015560), the contents of which being incorporated herein by reference.

[0118] Exemplary promoter sequences which can be used in the constructs described herein include polymerase type II promoter sequences and polymerase type III polymerase sequences. The promoter sequence may comprise a TATA box element. In one embodiment, the promoter sequence is less than 70 base pairs in length, less than 60 base pairs in length, less than 50 base pairs in length, less than 40 base pairs in length,

[0119] According to a particular embodiment, the promoter sequence is no longer than 600 base pairs in length, no longer than 500 base pairs in length, no longer than 400 base pairs in length.

[0120] Thus for example, the promoter sequence may be between 40-600 base pairs in length, 40- 500 base pairs in length, 40-200 base pairs in length or 100-300 base pairs in length. Examples of such promoter sequences are provided herein below:

[0121] U6-26 (from Arabidopsis; SEQ ID NO: 2)

[0122] Hl (SEQ ID NO: 3)

[0123] 35S: See for example, www(dot)researchgate(dot)net / figure / ector-construct-for- monitoring-stable-transgene-expression-A-Structure-of-the-binary_fig4_231741535 minimal 35S Promoter: (SEQ ID NO: 4)

[0124] Enhanced 35S Promoter:(SEQ ID NO: 5)

[0125] Nekrasove consensus polll sequence: (SEQ ID NO: 6)

[0126] Additional short synthetic promoters are listed in Cai Y-M, Kallam K, Tidd H, et al (2020) Nucleic Acids Research 48: 11845-11856. https: / / doi(dot)org / 10dotl093 / nar / gkaa682), the contents of which is incorporated herein by reference.

[0127] In another embodiment, the loop sequence of the construct comprises an enhancer sequence.

[0128] As used herein, the term "enhancer" refers to a nucleic acid sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance an activity (e.g. level or tissue-specificity) of a promoter.

[0129] In one embodiment, the double-stranded portion of the construct comprises the plant beneficial product (e.g. an inducer of RNA interference).

[0130] The double- stranded portion of the construct is typically composed of ribonucleotides, although deoxyribonucleotides are also contemplated.

[0131] The construct according to this aspect of the invention may be specifically recognized by in vivo enzymes such as Dicer in plant cells, and the loop regions at both sides may be cleaved to form a naturally occurring type double -stranded molecule (e.g. double-stranded RNA). As a result, it can have activity equivalent to that of a double- stranded RNA and exerts a more sustainable or slow-releasing effect than RNA interference effect by conventional double-stranded RNAs.

[0132] RNA interference is also known as RNAi and is a phenomenon in which a small RNA molecule having a sequence complementary to a target RNA binds to the target RNA, thereby degrading the target RNA or suppressing the translation of the target RNA.

[0133] As mentioned, the construct containing the double-stranded portion which is introduced into cells is cleaved by Dicer in the cells to generate a double- stranded RNA (siRNA) which has an RNA interference effect. The ends of the siRNA can be either blunt ends or protruding ends. The siRNA turns into a single chain to form an RNA-nuclease complex (RNA induced silencing complex (RISC)), which recognizes a target mRNA having a sequence complementary to the siRN A, and degrades the target mRNA, thereby suppressing the expression of the corresponding target gene. ’The sense strand and the antisense strand can be designed to suppress the function of a target gene, based on the nucleotide sequence of the target gene. The designs can be confirmed by producing multiple sense and antisense strands and testing for each suppression efficiency. For example, designing using an algorithm for siRNA design or the like can be applied (References: J. A. Jaeger et al., Methods in Enzymology (1989) 183: 281-306; D. H. Mathews et ah, J. Mol. Biol. (1999) 288: 911-940). When designing, it is preferable that the strands do not suppress the expression of genes other than a target gene, the genes having sequences similar to the target gene (which is known as the off target effect). The lengths of the sense and the antisense strands are preferably designed in the range of, for example, 19 to 31 bases, preferably 21 to 25 bases, more preferably 22 to 24 bases, and even more preferably 23 bases.

[0134] There are various methods for synthesizing nucleic acids such as in-vitro transcription synthesis method, methods using plasmids or viral vectors, and methods using PCR cassettes. Although a method of synthesizing nucleic acids is not specifically limited, a chemical synthesis method is preferred in terms of high purity, ability to produce in large quantities, safety for use in vivo, ability of chemical modification, and the like. Examples of chemical synthesis method include, but not limited to, H-phosphonate method and phosphoroamidite method. For this purpose, commercially available automatic nucleic acid synthesizers may be used.

[0135] The ends of the nucleotide sequences with unpaired nucleotides at both ends of the sense strand and the antisense strand are ligated with a ligase (for example, T4 RNA ligase or T4 DNA ligase) to form two loops simultaneously. The reaction conditions include, for example, incubating in a buffer containing polyethylene glycol (PEG), BSA and the like for 20 hours at a low temperature. The synthesized dumbbell-shaped single-chain circular RNA can be collected and purified by typical methods (for example, high-performance liquid chromatography and PAGE method).

[0136] Additionally, or alternatively, the loop portion of the construct comprises the plant beneficial product.

[0137] In one embodiment, the loop sequence is composed of ribonucleotides and / or deoxyribonucleotides and the plant beneficial product is a gene silencing agent (e.g. targeted antisense or sense gene sequence, anti-miRNA sequence).

[0138] In another embodiment, the loop sequence is composed of ribonucleotides and the plant beneficial product is a genome editing agent (e.g. at least one small guide sequence, sgRNA).

[0139] In still another embodiment, the loop sequence is composed of ribonucleotides and / or deoxyribonucleotides and the plant beneficial product is an expression product (e.g. beneficial peptide such as an anti-microbial peptide). When the loop sequence is composed of ribonucleotides, the sequence typically comprises an internal ribosome entry site (IRES) followed by the desired sequence to be expressed (either RNA or protein). When the loop sequence is composed of deoxyribonucleotides, the sequence typically comprises a promoter followed by the desired sequence.

[0140] In another embodiment, the loop sequence is composed of deoxyribonucleotides and the plant beneficial product is a sequence for homologous directed recombination. The sequence to be inserted is operatively linked to at least one homology arm (e.g. of a length of about 90 or more nucleic acids) such that it can be recombined into the plant genome. Optionally, the sequence to be inserted is flanked by homology arms.

[0141] According to another aspect of the invention there is provided a single-stranded circular (ssc ) nucleic acid construct being no more than 1000 nucleotides in length comprising:

[0142] (i) a sequence which is, or encodes, at least one plant beneficial product; and

[0143] (ii) a plant cell penetration sequence and / or a promoter sequence.

[0144] In contrast to the constructs described herein above, the constructs according to this aspect of the invention are substantially not intended to generate a stem shape or hairpin shape and can be considered as a fully single-stranded.

[0145] It will be appreciated that single-stranded nucleic acid sequences can fold in on itself by base pairing, Preferably, the sequence is selected so as to avoid this phenomenon as much as possible. Examples of RNA structure predicting programs which can be used to minimize internal base pairing include: RNA structure: https : / / rna.urmc.rochester(dot)edu / RNAstructure Web / ; Folding prediction: m fold web server: http: / / www(dot)unafold(dot)org / mfold / applications / rna-folding- form.php.

[0146] In one embodiment, at least 99 %, 98 %, 97 %, 96 %, 95 %, 94 %, 93 %, 92 %, 91 %, 90 % of the molecule of this aspect of the invention is fully single stranded.

[0147] It will be appreciated that the buffer in which the molecule is maintained, can be adapted to enhance the probability that the construct will remain single-stranded. For example, the buffer may include at least one of the following factors:

[0148] (i) comprise a denaturant;

[0149] (ii) pH may be above 8;

[0150] (iii) have a low ionic strength (e.g. tap water, solution with low salt content or low concentration of phosphate buffer; and / or

[0151] (i v) the constructs may be present in low concentration. Examples of plant beneficial products are descri bed herein above and include gene silencing agents, genome editing agents, expression products and homologous directed recombination agents.

[0152] Cell penetration sequences and promoter sequences are described herein above.

[0153] Depending on the particular plant beneficial products comprised or encoded in the constructs described herein, the constructs (once introduced into plants) are capable of affecting a plant trait or performance.

[0154] Thus, for example, the present inventors contemplate use of the constructs described herein to affect at least one, two, three or more plant trait such as plant fitness, plant resilience, plant yield, plant growth, fruit yield, fruit size, fruit weight, vegetable size, vegetable weight, crop protection, plant health and stress resistance.

[0155] In one embodiment, the “beneficial agents” of the constructs of the present enhance the growth of host plants.

[0156] The phrase “improving the growth” as used herein refers to enhancing the rate of growth and / or amount of the plant, or a component thereof (such as a seed, leaf, fruit, stem etc.) as compared to a plant grown under identical conditions, but in the absence of the construct.

[0157] Thus, for example, the present inventors contemplate that the “beneficial agents” of the constructs of the present invention may be used to enhance the germination of the seeds of the plant.

[0158] In another embodiment, the “beneficial agents” of the constructs of the present invention can bring about elongation of plant roots. An exemplary beneficial agent is one that suppresses Lateral Root Density (LRD) gene - see for example Placido DF, et al (2020) Plant Biotechnology Journal 18: 1955-1968. https: / / doi(dot)org / 10.1111 / pbi.l3355. The target sequence may be SEQ ID NO: 7 or OPRIII-oxophyodienoatereductase gene from subfamily III (OPRIII) (SEQ ID NO: 8) - see for example Gabay et al., 2023, Nat Commun 14:539.

[0159] In another embodiment, the “beneficial agents” of the constructs of the present invention increase the number and / or size of the seeds of the plant.

[0160] In another embodiment, the “beneficial agents” of the constructs of the present invention increase the number and / or size of the shoots of plant - (i.e. a tillering effect).

[0161] In another embodiment, the “beneficial agents” of the constructs of the present invention increase the amount of fruit and / or size and / or weight of the fruit produced by the plant.

[0162] In another embodiment, the “beneficial agents” of the constructs of the present invention increase the amount of foliage produced by the plant. In another embodiment, the “beneficial agents” of the constructs of the present invention increase the height of the plant.

[0163] In another embodiment, the “beneficial agents” of the constructs of the present invention increase the weight of the plant.

[0164] The growth enhancing effects of the constructs of the present invention may be apparent under stressful conditions or non-stressful conditions.

[0165] Thus, the constructs may enhance the growth of a plant under a stressful condition as compared to the growth of the plant under that identical condition but grown in the absence of the construct.

[0166] An example of a beneficial agent which enhances the growth of a plant is a gene silencing agent capable of down-regulating cyclin-dependent kinase inhibitor and / or a kip related protein (KRP) (see for example Ajadi AA, Tong X, Wang H, et al (2020) International Journal of Molecular Sciences 21:. https: / / doi(dot)org / 10.3390 / ijms21010245; Cameiro AK, Montessoro P da F, Fusaro AF, et al (2021) Plants 10: 1804. https: / / doi(dot)org / 10.3390 / plants 10091804; Veylder L, Beeckman T, Beemster G, et al (2001) The Plant Cell 13: 1653-1668. https: / / doi(dot)org / 10.1105 / TPC.010087).

[0167] Thus, the present invention contemplates agents that downregulate expression of KRP7 (e.g. SEQ ID NO: 9) or CDKC2 (SEQ ID NO: 10).

[0168] In one embodiment, growing the plant in the presence of the constructs provides tolerance to a stressful condition.

[0169] Exemplary stressful conditions under which the plant may be grown include but are not limited to abiotic stress conditions including drought conditions, heat, cold or salt stress, low nutrient stress and other stressful conditions such as stress induced by other plants (e.g. weeds, cultivated or native plants).

[0170] An example of a beneficial agent that may increase drought resistance is a sequence that acts as a sponge and base pairs with Triticum aestivum tae-miRl 127b-3p mature miRNA (see for example Han R et al., BMC Genomics (2014) 15:289). The miR sequence is: SEQ ID NO: 11. Therefore, a complementary sequence that can serve as a sponge is set forth in SEQ ID NO: 12 (if supplied as RNA) or SEQ ID NO: 13 (if supplied as DNA).

[0171] The plants of this aspect of the invention may be grown in areas which are prone to stressful conditions. Alternatively, or additionally, the plants of this aspect of the invention may be grown at times of year which are stressful to the plants.

[0172] In one embodiment, growing the plant in the presence of the “beneficial agents” of the constructs enhances plant nutrient uptake. Methods for introducing the constructs according to the present invention into a plant cell are known in the art.

[0173] As used herein the term “introduction” or “introducing” describes a process by which a foreign DNA, such as the nucleic acid constructs described herein, enters into a recipient cell and expressed therein. The expression may be stable throughout the plants' life. According to another embodiment, the nucleic acid sequence of the present invention is transiently transformed into a plant cell, and thus the regenerated plant is not defined as genetically modified organism (non- GMO).

[0174] The present inventors contemplate introducing more than one of the constructs described herein into the same plant. In one embodiment, the plurality of constructs (e.g. two, three, four or more) are introduced into the plant substantially at the same time. In another embodiment, one construct is introduced into a plant at a first time and a second construct may be introduced into the same plant at a later time. The first and second construct may be identical or may be designed for introducing different beneficial agents.

[0175] Exemplary methods for introducing the nucleic acid constructs into plant cells include, but are not limited to seed treatment; seed priming; plant tissue culture media; plant cell culture media; plant spray; immersion; plant dipping; soaking; injection; tissue bombardment; tissue spraying; chemigation; and mechanical introduction.

[0176] Transient transformation of, for example, leaf cells, meristematic cells, or the whole plant is also envisaged by the present invention. Transient transformation can be affected by any of the direct DNA transfer methods described above or by mechanical or vector mediated viral infection using the plant viruses derived plasmid of the present invention.

[0177] According to certain typical embodiments, the expression constructs of the present invention are introduced into the cells of the plant slightly trimmed roots by soaking roots of seedlings or young plantlets in a solution comprising the desired construct or constructs as described in International Application Publication No. WO 2010 / 004561.

[0178] According to other typical embodiments, the expression constructs of the present invention are introduced into the cells of a plant embryo within a plant seed by soaking the seeds in a seed priming solution comprising the desired construct or constructs as described in International Application Publication No. WO 2011 / 001434. According to a particular embodiment, the seeds are intact (e.g. have not been pierced using an injection needle). Further, preferably the seeds which are primed have not undergone imbibition. Preferably the DNA is not taken up by the seeds using agrobacterium tumefaciens.

[0179] The advantage of using the above-described methods of introduction is in that the nucleic acid construct is introduced into an intact plant (when introduced via the roots) or to the embryo from which an intact plant will be developed.

[0180] Alternatively, the nucleic acid construct of the present invention can be introduced into isolated cells or tissues by direct nucleic acid (NA) transfer. There are various methods of direct DNA or RNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to pulses of strong electric field, opening up mini-pores to allow NA to enter. In microinjection, the NA is mechanically injected directly into the cells using micropipettes. In microparticle bombardment, the NA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues. Additional direct NA transfer techniques include glass or silicon carbide whiskers (carborundum) (see, for example, Dunwell, Methods Mol. Biol. 1999; 111:375-82). The cells or tissues into which the NA constructs are introduced are then regenerated into a plant by methods known to a person skilled in the art.

[0181] Plants may be contacted with the constructs of the invention at any stage of the plant life cycle e.g. a post-blossom stage, a blossom stage, a pre-blossom stage, or any combination thereof.

[0182] The contacting may be affected in the vicinity of or onto: a root, a stem, a trunk, a seed, a fruit, a flower, a leaf, or any combination thereof of the plant.

[0183] According to an embodiment, application is carried out in an open field. According to an embodiment, application is carried out in a greenhouse. According to an embodiment, the application is carried out as a single treatment. According to an embodiment, at least two applications are carried out under any regimen or for any duration.

[0184] According to an embodiment, the applying comprises repeated application (2 or more applications e.g., every week, seasonal, bi-weekly, bi-monthly etc.). Repeated applications are especially envisaged for field / greenhouse treatments.

[0185] According to an embodiment, repeated application comprises weekly, daily, monthly, or bi-monthly administration during blossom, post-blossom, pre-blossom, or any combination thereof. For example, suggested regimen may include but is not limited to, spraying plants in open fields and green house, adding to irrigation of plants grown in the open field, green house, nurseries, closed controlled system (vertical farming) and in pots.

[0186] As used herein the term "plant" refers to whole plants, a plant tissue, a plant organ, a fruit, a vegetable, an eatable portion of a plant, a grafted plant including seeds, shoots, stems, roots (including tubers), rootstock, scion, and plant cells, tissues, fruit, flower and organs. The plant may be in any form including cuttings and harvested material (e.g., fruit).

[0187] In one embodiment, the plant is an agricultural plant. The phrase “agricultural plants", or "plants of agronomic importance", refers to plants that are cultivated by humans for food, feed, fiber, and fuel purposes. In one embodiment, the plant is not a wild plant.

[0188] In one embodiment, the constructs are introduced into a monocotyledonous plant. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales. In a particular embodiment, the monocotyledonous plant can be selected from the group consisting of maize, rice, wheat, barley, sorghum and sugarcane.

[0189] In another embodiment, the constructs are introduced into a dicotyledonous plant, including those belonging to the orders of the Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales, and Violates. In a particular embodiment, the dicotyledonous plant can be selected from the group consisting of cotton, bean, pepper, and tomato.

[0190] Preferably, the plant is an agricultural plant. Agricultural plants include monocotyledonous species such as: maize (Zea mays), common wheat (Triticum aestivum), spelt (Triticum spelta), einkorn wheat (Triticum monococcum), emmer wheat (Triticum dicoccum), durum wheat (Triticum durum), Asian rice (Oryza sativa), African rice (Oryza glabaerreima), wild rice (Zizania aquatica, Zizania latifolia, Zizania palustris, Zizania texana), barley (Hordeum vulgare), Sorghum (Sorghum bicolor), Finger millet (Eleusine coracana), Proso millet (Panicum miliaceum), Pearl millet (Pennisetum glaucum), Foxtail millet (Setaria italica), Oat (Avena sativa), Triticale (Triticosecale), rye (Secale cereal), Russian wild rye (Psathyrostachys juncea), bamboo (Bambuseae), or sugarcane (e.g., Saccharum arundinaceum, Saccharum barberi, Saccharum bengalense, Saccharum edule, Saccharum munja, Saccharum officinarum, Saccharum procerum, Saccharum ravennae, Saccharum robustum, Saccharum sinense, or Saccharum spontaneum)-, as well as dicotyledonous species such as: soybean (Glycine max), canola and rapeseed cultivars (Brassica napus). cotton (genus Gossypium), alfalfa (Medicago sativa), cassava (genus Manihot), potato (Solanum tuberosum), tomato (Solanum lycopersicum), pea (Pisum sativum), chick pea (Cicer arietinum), lentil (Lens culinaris), flax (Linum usitatissimum) and many varieties of vegetables.

[0191] Following introduction of the construct into the plants, the plants are typically grown under conditions (e.g. light and water) and for a time to allow the plant to flourish (i.e. grow). The plant may be analyzed after 2 days of growth, 3 days of growth, 4 days of growth, 5 days of growth, 6 days of growth, 1 week of growth, 2 weeks of growth, 3 weeks of growth, 1 month of growth or more.

[0192] The constructs may be provided per se or may be formulated in agriculturally acceptable carriers.

[0193] As used herein the term "agriculturally acceptable carrier" refers to a material that facilitates application of the bacteria (or agent isolated therefrom) to the intended target, which may be for example a plant, a plant material, compost, earth, surroundings or equipment, or that facilitates storage, transport or handling. Carriers used in compositions for application to plants and plant material are preferably non-phytotoxic or only mildly phytotoxic. A suitable carrier may be a solid, liquid or gas depending on the desired formulation. In one embodiment the carriers include polar liquid carriers such as water, mineral oils and vegetable oils. In one embodiment the carrier enhances the stability of the active ingredient as described herein.

[0194] The carrier can include a dispersant, a surfactant, an additive, water, a thickener, an anti- caking agent, residue breakdown, a composting formulation, a granular application, diatomaceous earth, an oil, a coloring agent, a stabilizer, a preservative, a polymer, a coating, or a combination thereof. One of ordinary skill in the art can readily determine the appropriate carrier to be used taking into consideration factors such as a type of soil, climate conditions, and the like.

[0195] The additive can comprise an oil, a gum, a resin, a clay, a polyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester, a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, an alcohol sulfate, a sodium alkyl butane diamate, a polyester of sodium thiobutant dioate, a benzene acetonitrile derivative, a proteinaceous material, or a combination thereof.

[0196] The surfactant can contain a heavy petroleum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an aryl alkyl polyoxyethylene glycol, an alkyl amine acetate, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or a combination thereof.

[0197] The anti-caking agent can include a sodium salt such as a sodium sulfite, a sodium sulfate, a sodium salt of monomethyl naphthalene sulfonate, a sodium salt of dimethyl naphthalene sulfonate, or a combination thereof; or a calcium salt such as calcium carbonate, diatomaceous earth, or a combination thereof.

[0198] Exemplary agriculturally acceptable carriers include, but are not limited to, vermiculite, charcoal, sugar factory carbonation press mud, rice husk, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, a starch, talc, polyvinyl pyrrolidone, or a combination thereof.

[0199] The constructs may be provided to the plants in combination with other nucleic acid delivery agents (including for example nanoparticles, hydroxide particles, chitosan and polysaccharide).

[0200] Additionally, or alternatively, the constructs may be provided to the plants in combination with at least one plant protection agent or agrochemical, examples of which include but are not limited to a fertilizer, an insecticide, a fungicide, a herbicide, a nematicide, a plant growth regulator, a virucide, a biostimulant, an elicitor, a biopesticide, an antibiotic and a bacteriophage.

[0201] In some instances, the fertilizer is a liquid fertilizer. The agrochemical can either be applied to a plant growth medium or to plants and / or seeds. Liquid fertilizer can include, without limitation, ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates, magnesian limestone, magnesia, urea, urea- formaldehydes, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, KiSCL-SMgSCh, kainite, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, or a combination thereof.

[0202] The micronutrient fertilizer material can comprise boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron ammonium sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese chelate, a manganese chloride, a manganese frit, a sodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination thereof. The insecticide can include an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination thereof.

[0203] The herbicide can comprise a chlorophenoxy compound, a nitrophenolic compound, a nitrocresolic compound, a dipyridyl compound, an acetamide, an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoic acid derivative, anisic acid, an anisic acid derivative, a benzonitrile, benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate, chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzene derivative, a fluorodinitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, an isopropylamine derivative, oxadiazolinone, a phosphate, a phthalate, a picolinic acid compound, a triazine, a triazole, a uracil, a urea derivative, endothall, sodium chlorate, or a combination thereof.

[0204] The fungicide can comprise a substituted benzene, a thiocarbamate, an ethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, an organomercury compound, an organotin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione, metlaxyl, thiamimefon, triforine, or a combination thereof.

[0205] In one embodiment, the plant growth regulator is selected from the group consisting of: Abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikegulac, dimethipin, 2,6- dimethylpuridine, ethephon, flumetralin, flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione (prohexadione-calcium), prohydrojasmon, thidiazuron, triapenthenol, tributyl phosphorotrithioate, 2,3,5-tri-iodobenzoic acid, trinexapac-ethyl and uniconazole. Other examples of plant growth regulators which can be comprised in the article of manufacture include those based on dichlorophene and benzylalcohol hemi formal (Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK from Rohm & Haas) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (Acticide® MBS from Thor Chemie). Other plant growth regulators that can be incorporated the agricultural compositions are described in US 2012 / 0108431, which is incorporated by reference in its entirety.

[0206] Preferred nematode-antagonistic biocontrol agents include ARF18; Arthrobotrys spp.; Chaetomium spp.; Cylindrocarpon spp.; Exophilia spp.; Fusarium spp.; Gliocladium spp.; Hirsutella spp.; Lecanicillium spp.; Monacrosporium spp.; Myrothecium spp.; Neocosmospora spp.; Paecilomyces spp.; Pochonia spp.; Stagonospora spp.; vesicular-arbuscular mycorrhizal fungi, Burkholderia spp.; Pasteuria spp., Brevibacillus spp.; Pseudomonas spp.; and Rhizobacteria. Particularly preferred nematode-antagonistic biocontrol agents include ARF18, Arthrobotrys oligospora, Arthrobotrys dactyloides, Chaetomium globosum, Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia pisciphila, Fusarium aspergilus, Fusarium solani, Gliocladium catenulatum, Gliocladium roseum, Gliocladium vixens, Hirsutella rhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii, Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora phaseoli, vesicular- arbuscular mycorrhizal fungi, Burkholderia cepacia, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae, Pasteuria ramosa, Pastrueia usage, Brevibacillus laterosporus strain G4, Pseudomonas fluorescens and Rhizobacteria.

[0207] In another embodiment, the nucleic acid constructs are contacted with the plants in combination with a nutrient. The nutrient can be selected from the group consisting of a nitrogen fertilizer including, but not limited to Urea, Ammonium nitrate, Ammonium sulfate, Non-pressure nitrogen solutions, Aqua ammonia, Anhydrous ammonia, Ammonium thiosulfate, Sulfur-coated urea, Urea-formaldehydes, IBDU, Polymer-coated urea, Calcium nitrate, Ureaform, and Methylene urea, phosphorous fertilizers such as Diammonium phosphate, Monoammonium phosphate, Ammonium polyphosphate, Concentrated superphosphate and Triple superphosphate, and potassium fertilizers such as Potassium chloride, Potassium sulfate, Potassium- magnesium sulfate, Potassium nitrate. Such compositions can exist as free salts or ions within the seed coat composition. Alternatively, nutrients / fertilizers can be complexed or chelated to provide sustained release over time.

[0208] In one embodiment, the nucleic acid constructs are contacted with the plants in combination with a rodenticide selected from the group of substances consisting of 2- isovalerylindan- 1,3 -dione, 4-(quinoxalin-2-ylamino) benzenesulfonamide, alpha-chlorohydrin, aluminum phosphide, antu, arsenous oxide, barium carbonate, bisthiosemi, brodifacoum, bromadiolone, bromethalin, calcium cyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor, coumafuryl, coumatetralyl, crimidine, difenacoum, difethialone, diphacinone, ergocalciferol, flocoumafen, fluoroacetamide, flupropadine, flupropadine hydrochloride, hydrogen cyanide, iodomethane, lindane, magnesium phosphide, methyl bromide, norbormide, phosacetim, phosphine, phosphorus, pindone, potassium arsenite, pyrinuron, scilliroside, sodium arsenite, sodium cyanide, sodium fluoroacetate, strychnine, thallium sulfate, warfarin and zinc phosphide. In another embodiment, the nucleic acid constructs are contacted with the plants in combination with a bicyclic corboxamide pesticide or fungicide such as those disclosed in WO2024126650, the contents of which are incorporated herein by reference.

[0209] As used herein the term “about” refers to ± 10 %

[0210] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0211] The term “consisting of means “including and limited to”.

[0212] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0213] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0214] Throughout this application, various embodiments of this invention may 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 the invention. 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. 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 numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0215] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0216] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0217] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0218] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0219] EXAMPLES

[0220] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0221] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

[0222] Example 1: Transient Silencing of the SHORT-ROOT Gene in Arabidopsis thaliana

[0223] Background

[0224] The SHORT-ROOT (SHR) gene in Arabidopsis thaliana (AF233752) is known to be a key regulator of root and shoot development. The SHR gene encodes a mobile transcription factor that orchestrate developmental processes, tissue patterning, and stem cell maintenance within the plant. Due to its essential function, complete silencing of SHR has been previously associated with severe developmental abnormalities, underscoring its essential role in plant viability.

[0225] SHR-RNAi constructs, designed for transient activity, were introduced into A. thaliana seeds via an osmo-priming technique. Upon germination, the constructs lead to a distinct root phenotype characterized by increased lateral root (LR) formation, as observed 8 days post- germination.

[0226] Construct design:! wo RNAi constructs were designed, one as a linear version of single- stranded RNA and one as a circular single stranded RNA. Both constructs contain the following sequence design, as illustrated in Figure 2A:

[0227] A. An IR motif (e.g. SEQ ID NO: 14) generating a hairpin structure on one edge, that is expected to assist with the penetration of the RNAi molecule to the plant tissue.

[0228] B. A dsRNA stem structure of miRNA (miRNA+miRNA* sequences) against the SHR gene (e.g. SEQ ID NO: 15). C. A Basal Stem (BS) sequence (e.g. SEQ ID NO: 16) at one side of the miRNA structure.

[0229] D. A Distal Stem loop (DSL) sequence (e.g. SEQ ID NO: 17) on the other side of the miRNA dsRNA stem, generating a stem loop at the other edge of the molecule. Both motifs (C+D) are used as natural plant motifs flanking the miRNAs, generating a pre-miRNA structure, and expected to assist in generating accurate miRNA dsRNA structure, once introduced into the plant cell.

[0230] Both RNAi structures, the linear and the circular molecules, are expected to fold into a dumbbell shape structure, one with open edges and one as a closed circle - see Figure 2B.

[0231] Methods:

[0232] Application via seed priming method:

[0233] Approximately 50 Arabidopsis thaliana seeds were placed into a sterile 2 mL tube. To each tube, 200 pL of a priming solution was added. This solution comprised 25% polyethylene glycol (PEG) and 0.25% zinc ethylenediaminetetraacetic acid (ZnEDTA).

[0234] Following the addition of the priming solution, the various RNAi molecules were introduced into the tubes according to the following treatment groups:

[0235] 1. Circular RNAi Treatment: 300 ng of circular single-stranded RNAi molecules.

[0236] 2. Linear RNAi Treatment: 300 ng of single- stranded RNAi molecules.

[0237] 3. Control Group: No RNAi molecules were added.

[0238] Each tube was sealed, wrapped in aluminum foil to prevent light exposure, and incubated at room temperature on a roller mixer to ensure continuous agitation for a period of three days.

[0239] Upon completion of the incubation period, the seeds were washed three times with 1 mL of sterile deionized water. Each wash involved gentle pipetting followed by removal of the wash solution. After the final wash, excess liquid was removed, and the seeds were dried under desiccation conditions for a minimum of 24 hours.

[0240] Subsequently, the seeds underwent a cold stratification step by incubation at 4°C for 48 hours in sterile conditions. Post-stratification, the seeds were sown on sterile agar plates containing 0.8% agar and 0.5x Murashige and Skoog (MS) growth medium.

[0241] The plates were maintained under controlled growth conditions: 25°C with a 16-hour light / 8- hour dark photoperiod and a light intensity of approximately 300 pE m2s '. Germination and seedling development were monitored and photographic documentation was performed eight days post-germination.

[0242] RESULTS

[0243] As illustrated in Figure 3, and summarized in Figures 4A-B, lateral root (LR) number was significantly higher in the circular SHR-cssRNA (cRNA) treatment as compared to the linear SHR-LssRNA (LinRNA) treatment. No significant effect was observed in root lengths across all three conditions.

[0244] Example 2: Transient Silencing of the LDL1 Gene in Arabidopsis thaliana

[0245] Background

[0246] LDL1 / SWP1 (Lysine-specific Demethylase 1) is known to play a crucial role in the epigenetic regulation of gene expression by demethylating histone proteins. This process is essential for various developmental and physiological processes in plants. Studies in Arabidopsis using knockdown mutation demonstrated that the LDL1 / SWP1 (AT1G62830.1), which is regularly expressed in both main root and LR meristem, regulates lateral root initiation and elongation.

[0247] A circular LDL1 -RNAi construct, designed for transient effect only, was introduced into A. thaliana seeds via a an osmo-priming technique. Upon germination, the construct lead to a distinct root phenotype characterized by increased primary root elongation and lateral root number formation, as observed 11 days post-germination.

[0248] Construct designPTwo RNAi constructs were designed as follows:

[0249] 1. A dsRNA at a size of about 200bp (#R24) containing a sequence identical to the 5’ region of the LDL1 mRNA.

[0250] 2. A circular single stranded RNA (#R22), having the following motifs:

[0251] A. An IR motif (e.g. SEQ ID NO: 14) generating a hairpin structure on one edge of the molecule, that is expected to assist with the penetration of the RNAi molecule to the plant tissue.

[0252] B. A dsRNA stem structure of miRNA (miRNA / miRNA* sequence) against the LDL1 gene (e.g. SEQ ID NO: 18).

[0253] C. A Basal Stem (BS) sequence (e.g. SEQ ID NO: 16) at one side of the miRNA structure.

[0254] D. A Distal Stem loop (DSL) sequence (e.g. SEQ ID NO: 17) at the other side of the miRNA dsRNA stem, generating a stem loop at the other edge of the molecule. Both motifs (C+D) are used as natural plant motifs flanking the miRNAs, generating a pre-miRNA structure, which is expected to assist in generating accurate miRNA dsRNA structure, once introduced into the plant cell.

[0255] E. Spacer sequence (~38 nt long - SEQ ID NO: 20) generating a space between the pre- miRNA structure and the IR loop.

[0256] A schematic illustration of the LDZJ-RNAi structure is provided in Figures 5A-C.

[0257] METHODS

[0258] Application via seed priming method:

[0259] Approximately 50 Arabidopsis thaliana Columbia seeds were placed into a sterile 2 mL tube. Each tube received 200 pL of a priming solution composed of 25% polyethylene glycol (PEG) and 0.25% zinc ethylenediaminetetraacetic acid (ZnEDTA).

[0260] RNA interference (RNAi) molecules targeting the LDL1 gene were added to the tubes according to the following treatment groups:

[0261] 1. Circular RNAi (#R22): 300 ng of circular ssRNA molecule. 2.dsRNA (#R24): 300 ng of long (~200bp) dsRNA molecule, with no motifs

[0262] 3.MOC: 300 ng of Nonsense linear ssRNA molecules (~200nt).

[0263] Tubes were sealed, wrapped in aluminium foil to prevent light exposure, and incubated at room temperature on a roller mixer for 24 hours to ensure continuous agitation. Following incubation, seeds were washed three times with 1 mL of sterile deionized water. After the final wash, excess liquid was removed, and seeds were dried under desiccation conditions for at least 24 hours.

[0264] The seeds were then cold-stratified at 4°C for 72 hours and subsequently sown on sterile agar plates containing 0.8% agar and 0.5x Murashige and Skoog (MS) medium. Plates were incubated under controlled growth conditions (25°C, 16 / 8 h light / dark cycle, -300 pE m2s ' light intensity). Seedling development was monitored during 11 days post germination (Figures 7A-D) and images were captured 11 days post-sowing (Figure 6).

[0265] RESULTS

[0266] As illustrated in Figure 6 and 7A-D, treatment with LDL1- cssRNA RNAi (R22) construct resulted in increased root length and significantly enhanced LR formation compared to the MOC as control, suggesting a strong positive effect on root development. The LDL1 -dsRNA RNAi (R24) construct showed a milder and not significant increase in root length and branching.

[0267] Example 3: Transient Silencing of the PDS Gene in tomato (Lycopersicon esculentum, M-82) plants

[0268] Background

[0269] Phytoene desaturase (PDS) plays a key role in the carotenoid biosynthesis pathway in plants. Its silencing disrupts carotenoid production, causing chlorophyll breakdown and a distinct bleached or albino phenotype in mature leaves. Due to this clear visual effect, PDS is commonly used as a marker to assess the efficiency of molecular genetic tools.

[0270] Construct design

[0271] A circular single- stranded DNA constructs harbouring an Inverted repeat (IR) motif and a miRNA like sequence was designed as illustrated in Figure 8. The IR motif (SEQ ID NO: 14) which generates a hairpin structure on one side, is expected to assist with the penetration of the RNAi molecule to the plant tissue. The construct full sequence is set forth in SEQ ID NO: 19.

[0272] METHODS

[0273] Application via infiltration into tomato leaves:

[0274] Approximately 100 pL of aqueous solution, containing either 50 pmoles of PDS-cssDNA (treated) or no DNA (control), was injected into the petiole of an intact mature leaf. Treated plants were incubated under controlled growth conditions: 25°C with a 16-hour light / 8-hour dark photoperiod and a light intensity of approximately 300 pE m2s '. Plants were monitored, and photographic documentation was performed seven days post-treatment.

[0275] Intact mature tomato leaves were infilter treated with either (1) circular single- stranded DNA as PDS RNAi construct, or (2) with water solution, as control.

[0276] RESULTS

[0277] Seven days post-infiltration, distinct bleaching was observed along the veins and surrounding tissue, exclusively in leaves treated with PDS-cssDNA, illustrated in Figure 9A-B.

[0278] To determine phytoene desaturase gene silencing in plants, ImageJ software was used to analyze leaf images for pigment loss (Figure 10). The images were converted to grayscale. Using ImageJ, the mean pixel intensity was measured within defined Regions Of Interest (ROIs) on silenced and control areas of the leaf. An increase in pixel intensity representing chlorosis or bleaching due to reduced carotenoid synthesis was interpreted as an indicator of successful PDS silencing. The difference in grayscale values between silenced and non-silenced tissue was quantified as the Pigment Depletion Score (PDS), providing a semi-quantitative measure of gene silencing efficiency. This method enables reliable, image-based assessment of gene function in plant tissues

[0279] It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A circular nucleic acid construct being no more than 1000 nucleotides in length comprising:(i) at least one double- stranded sequence; and(ii) at least two single- stranded loop sequences, wherein at least one of said at least two single-stranded loop sequences comprises a plant cell penetration sequence and / or a promoter sequence, wherein said at least one double-stranded sequence, or said at least one of said at least two single-stranded loop sequences comprises or encodes a plant beneficial product.

2. The circular nucleic acid construct of claim 1, being no more than 500 nucleotides in length.

3. The circular nucleic acid construct of claim 1, wherein said plant cell penetration sequence comprises an inverted repeat sequence.

4. The circular nucleic acid construct of claim 3, wherein said inverted repeat sequence is a viral inverted repeat sequence.

5. The circular nucleic acid construct of any one of claims 1-4, wherein less than 50 % of the construct comprises viral sequences or bacterial sequences.

6. The circular nucleic acid construct of any one of claims 1-5, being devoid of a viral protein encoding sequence.

7. The circular nucleic acid construct of any one of claims 1-6, being devoid of a capsid protein encoding sequence.

8. The circular nucleic acid construct of any one of claims 1-7, being composed of ribonucleotides.

9. The circular nucleic acid construct of any one of claims 1-8, being composed of deoxyribonucleotides.

10. The circular nucleic acid construct of any one of claims 1-9, wherein a portion of said double stranded sequence encodes a gene silencing agent or a genome editing agent.

11. The circular nucleic acid construct of any one of claims 1-9, wherein a portion of said double stranded sequence comprises a gene silencing agent.

12. The circular nucleic acid construct of any one of claims 1-11, comprising two loop sequences, wherein a length of said first loop sequence is the same as a length of said second loop sequence.

13. The circular nucleic acid construct of claim 12, wherein a sequence of said first loop sequence at least 90 % identical to a sequence of said second loop sequence.

14. The circular nucleic acid construct of any one of claims 1-13, wherein a portion of one of said at least one single-stranded loop sequence is complementary to a target sequence in a plant genome.

15. The circular nucleic acid construct of claim 14, wherein said portion of said at least one single- stranded loop sequence encodes a gene silencing agent.

16. The circular nucleic acid construct of claim 15, wherein said gene silencing agent is an antisense, an anti-miRNA molecule or an RNA interference-inducing molecule.

17. The circular nucleic acid construct of claim 14, wherein said portion of said at least one single- stranded loop sequence encodes a genome editing agent.

18. The circular nucleic acid construct of any one of claims 1-13, wherein at least a portion of said at least one single- stranded loop sequence comprises a sequence of interest and at least one homology arm being at least 90 nucleotides in length.

19. The circular nucleic acid construct of claim 18, wherein said sequence of interest is flanked by two homology arms.

20. The circular nucleic acid construct of any one of claims 1-19, wherein at least a portion of one of said loop sequences comprises an internal ribosome entry site (IRES) operatively linked to a sequence encoding a plant beneficial expression product.

21. The circular nucleic acid construct of any one of claims 1-20, wherein said plant beneficial expression product is a protein or peptide product.

22. The circular nucleic acid construct of claim 21, wherein said peptide product is an anti-microbial peptide.

23. The circular nucleic acid construct of any one of claims 1-20, wherein said plant beneficial expression product is an RNA product.

24. The circular nucleic acid construct of claim 9, wherein said at least one single- stranded loop sequence comprises said promoter sequence, said promoter sequence being operatively linked to a plant beneficial expression product.

25. The circular nucleic acid construct of claim 24, wherein said promoter sequence is a polymerase II promoter or a polymerase III promoter.

26. The circular nucleic acid construct of claim 24, wherein said promoter sequence comprises a TATA box element.

27. A single-stranded, circular (ssc) nucleic acid construct being no more than 1000 nucleotides in length comprising:(i) a sequence which is, or encodes, at least one plant beneficial product; and(ii) a plant cell penetration sequence and / or a promoter sequence.

28. The ssc nucleic acid construct of claim 27, being no more than 500 nucleotides in length.

29. The ssc nucleic acid construct of claims 27 or 28, wherein said plant cell penetration sequence comprises an inverted repeat sequence.

30. The ssc nucleic acid construct of claim 29, wherein said inverted repeat sequence is a viral inverted repeat sequence.

31. The ssc nucleic acid construct of any one of claims 27-30, wherein less than 50 % of the construct comprises viral sequences.

32. The ssc nucleic acid construct of claim 27, wherein said at least one plant beneficial product is selected from the group consisting of:(i) a gene silencing agent which hybridizes to a plant RNA;(ii) a genome editing agent which edits the genome of a plant; and(iii) a protein or a peptide.

33. The ssc nucleic acid construct of any one of claims 27-32, wherein said sequence encodes said plant beneficial product.

34. The ssc nucleic acid construct of claim 33, wherein said promoter sequence is operatively linked to said sequence which encodes said at least one plant beneficial product.

35. The ssc nucleic acid construct of any one of claims 27-34, comprising an inverted repeat of a virus.

36. The ssc nucleic acid construct of claim 32, wherein said gene silencing agent is an antisense agent or an anti-miRNA agent.

37. The ssc nucleic acid construct of claim 32, wherein said genome editing agent is an sgRNA or a sequence of interest comprising at least one homology arm.

38. The ssc nucleic acid construct of any one of claims Tl-31, being composed of ribonucleotides.

39. The ssc nucleic acid construct of any one of claims Tl-31, being composed of deoxyribonucleotides.

40. The ssc nucleic acid construct of claim 27, wherein said plant beneficial product is an RNA.

41. The ssc nucleic acid construct of claim 27, wherein said plant beneficial product is a protein or peptide.

42. The ssc nucleic acid construct of claim 41, further comprising an IRES sequence.

43. The ssc nucleic acid construct of claims 41 or 42, wherein said peptide is an anti- microbial peptide.

44. An agricultural composition comprising the nucleic acid construct of any one of claims 1-43 and an agriculturally acceptable carrier.

45. A method of affecting a plant trait or performance comprising contacting the plant with at least one of the nucleic acid constructs of any one of claim 1-42, thereby affecting the plant trait or performance.

46. The method of claim 45, wherein said plant trait is selected from the group consisting of plant fitness, plant resilience, plant yield, plant growth, fruit size, fruit weight, vegetable size, vegetable weight, crop protection, plant health and stress resistance.

47. The method of claim 45 or 46, wherein said contacting is effected by at least one of the following methods: seed treatment; seed priming; plant tissue culture media; plant cell culture media; plant spray; immersion; plant dipping; soaking; injection; tissue bombardment;tissue spraying; chemigation; or mechanical introduction.

48. The method of any one of claims 45-47, wherein said nucleic acid construct is contacted with the plant in combination with a nucleic acid cell delivery agent.

49. The method of claim 48, wherein said nucleic acid cell delivery agent is selected from the group consisting of a nanoparticle, a hydroxide particle, chitosan and a polysaccharide.

50. The method of any one of claims 45-49, further comprising contacting the plant with a plant protection agent.

51. The method of claim 50, wherein said plant protection agent comprises at least one of the following agents: an insecticide, a fungicide, a herbicide, a nematicide, a virucide, a biostimulant, an elicitor, a biopesticide, an antibiotic and a bacteriophage.

52. The method of any one of claims 45-50, wherein said contacting is effected a single time.

53. The method of any one of claims 45-50, wherein said contacting is effected a plurality of times.

54. The method of any of claims 45-53, wherein said part thereof is a seed, root or a leaf.

55. The method of any one of claims 45-53, wherein said plant is at a flowering stage.

56. The method of any one of claims 45-53, wherein said plant is a plant culture.

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