Immunomodulatory factors involved in defense against plant diseases caused by Liberibacter species

Abstract

The present disclosure provides methods and compositions for increasing plant resistance to disease caused by infection with bacteria of the Liberibacter genus. TIFF2024506316000099.tif76128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 147,452, filed February 9, 2021, which is incorporated by reference for all purposes. [Background technology]

[0002] background Citrus greening disease (Huanglongbing (HLB)), associated with the bacterium Candidatus Liberibacter asiaticus (CLas) and transmitted by the Asian citrus psyllid (ACP), is the most destructive disease of citrus and causes significant reductions in citrus quality and yield. HLB causes billions of dollars in losses in citrus produce annually and severely impacts the vitality of the citrus industry. Control is achieved in part, primarily by cutting down infected trees and chemical treatments against the insect vector. No efficient and sustainable disease control method for HLB has been found. In Florida, more than 80% of citrus orchards have been infected with CLas since the first discovery of HLB-positive trees in 2005. Since then, HLB has spread to Texas and California. Felling all infected trees is no longer a viable strategy, and application of control chemicals only temporarily suppresses the disease and is not an environmentally friendly approach to long-term solutions.

[0003] Another important disease caused by Liberibacter species is potato zebra chip (ZC) disease (also called potato zebra complex disease). ZC disease is caused by Candidatus Liberibacter solanacearum ( C andidatusL Iberibacter so lanacearum (CLso), which is transmitted by potato psyllids (e.g., Bactericera cockerelli). ZC disease reached epidemic levels in northern Texas in 2006 and has spread to Arizona, California, Colorado, Idaho, Oregon, Kansas, Nebraska, and New Mexico. ZC disease has caused millions of dollars in losses to the potato industry in the southwestern United States, especially Texas. In addition to potatoes, other solanaceous crops, including tomatoes, eggplants, and peppers, can also be infected by it. Summary of the Invention

[0004] overview By comparative analysis of small RNA pools between the HLB-resistant / tolerant US942 and HLB-susceptible Cleopatra, we identified regulators that responded to HLB in US942 but not Cleopatra. We predicted and annotated potential immune negative and positive regulators and down-regulated or assessed expression levels in US942 compared to Cleopatra, and also in another closely related HLB-resistant citrus, Sydney hybrid (Microcitrus virgata), with a different genetic and geographical background. Since functional validation of potential regulators in tree crops is always challenging and time-consuming, the present inventors have developed a rapid functional screening method using a similar and comparable C. liberibacter solanacearum (CLso) / potato psyllid / Nicotiana benthamiana interaction system to mimic the natural transmission and infection circuitry of HLB, and this cost-effective screening method allows for the rapid identification and functional characterization of regulators involved in plant immune responses to HLB. To identify positive defense regulators or negative immune suppressors, the present inventors performed functional tests in this pathosystem. Thereby, methods and compositions are provided herein for increasing the expression of positive defense regulators and / or inhibiting the expression of negative immune regulators so as to enhance plant resistance to Liberibacter infection, such as resistance to HLB or potato zebra chip disease.

[0005] In one aspect, a method for enhancing resistance to HLB is provided herein, the method comprising genetically modifying a plant, for example a plant of the citrus family or a solanaceous crop, to reduce the expression of an endogenous gene encoding a negative regulator of immune response polypeptide, wherein the negative regulator of immune response polypeptide is a polypeptide listed in Table 1. In some embodiments, the negative regulator of immune response polypeptide is VAD1, PRT6, PUB26, PAO1, LIN2, CRWN, or GPX8. In some embodiments, reducing the expression of the negative regulator polypeptide comprises contacting the plant with an siRNA that targets the endogenous nucleic acid encoding the negative regulator polypeptide. In some embodiments, reducing the expression of the negative regulator polypeptide comprises viral vector-mediated gene silencing. In some embodiments, reducing the expression of the negative regulator polypeptide comprises knocking out the expression of the endogenous gene encoding the negative regulator. In some embodiments, the method comprises gene editing the endogenous gene to reduce or knock out expression, for example by gene editing with CRISPR / CAS. In some embodiments, the negative regulator of immune response polypeptide comprises an amino acid sequence identical to the polypeptide sequence listed in Table 3, or comprises an amino acid sequence that is at least 70%, 75%, 80%, or 85% identical, or at least 90% or at least 95% identical to the polypeptide sequence listed in Table 3. In some embodiments, the negative regulator of immune response polypeptide comprises an amino acid sequence identical to the polypeptide sequence of VAD1, PRT6, PUB26, PAO1, LIN2, CRWN, or GPX8 listed in Table 3, or comprises an amino acid sequence that is at least 70%, 75%, 80%, or 85% identical, or at least 90% or at least 95% identical to the polypeptide sequence.In some embodiments, the plant is a Citrus maxima plant, a Citrus medica plant, a Citrus micrantha plant, a Citrus reticulata plant, a Citrus aurantiifolia plant, a Citrus aurantium plant, a Citrus latifolia plant, a Citrus limon plant, a Citrus limonia plant, a Citrus paradisi plant, a Citrus clementina plant, a Citrus unshu plant, a Citrus serrata ... In some embodiments, the plant is a variety of potato or tomato. In some embodiments, the plant is a variety of pepper.

[0006] In a further aspect, a method for enhancing resistance to HLB is provided herein, comprising genetically modifying a plant, for example a citrus plant or a solanaceae crop, to highly express a gene encoding a positive defense regulator polypeptide as described in Table 2. In some embodiments, the positive defense regulator peptide is BRAP2, NDR1-like, or PSL4. In some embodiments, the method comprises genetically modifying a plant to highly express a polypeptide comprising an amino acid sequence identical to a polypeptide as described in Table 4, or an amino acid sequence having at least 70%, 75%, 80%, or 85% identity to a polypeptide as described in Table 4; or at least 90% or 95% identity. In some embodiments, the polypeptide is identical to the polypeptide sequence of BRAP2, NDR1-like, or PSL4 as described in Table 4, or has at least 70%, 75%, 80%, or 85% identity, or at least 90% identity, or at least 95% identity. In some embodiments, the polypeptide is endogenous to the plant. Alternatively, the polypeptide can be heterologous to the plant. In some embodiments, the plant is a Citrus maxima plant, a Citrus medica plant, a Citrus micrantha plant, a Citrus reticulata plant, a Citrus aurantiifolia plant, a Citrus aurantium plant, a Citrus latifolia plant, a Citrus limon plant, a Citrus limonia plant, a Citrus paradisi plant, a Citrus clementina plant, a Citrus unshu plant, a Citrus sinensis plant, a Citrus tangerina plant, a Citrus itangensis plant, an Atalantia baccifolia plant, or a Poncirus trifoliata plant. In some embodiments, the plant is a potato or tomato variety. In some embodiments, the plant is a Capsicum variety.

[0007] In a further aspect, the present disclosure provides plants having enhanced resistance to HLB produced by the methods of targeting the genes described herein, such as the genes described in the immediately preceding two paragraphs. [Brief description of the drawings]

[0008] [Figure 1] Figure 1a-1c: Nb / psyllid / CLso pathosystem combined with viral-induced gene silencing (VIGS) showed that VAD is a negative regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and VAD expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) Detail of the leaf from panel a. c) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. Significant differences are analyzed by Student's t-test (*P < 0.01). [Diagram 2]Figure 2a-2d: Carrizo plants with VAD knockdown showed higher expression of defense marker genes, including pathogenesis-related PR-2 and chitinase (CHI). a. One of the cuttings from the Carrizo plants with VAD knockdown. VAD was knocked down by RNA silencing. Carrizo plants were transfected with pHellsgate8, a VAD hairpin RNA expression vector. b. The expression level of VAD in the VAD-silenced Carrizo plants was analyzed by qRT-PCR and normalized to the ubiquitin gene (CsUbi). Significant differences were analyzed by T-test (*P < 0.01). c and d. The expression levels of defense marker genes PR2 (c) and CHI (d) in the VAD-silenced Carrizo plants were analyzed by qRT-PCR and normalized to the ubiquitin gene (CsUbi). Significant differences were analyzed by T-test (*P < 0.01). [Diagram 3] Figure 3a-3c: The Nb / psyllid / CLso pathosystem combined with VIGS showed that PAO1 is a negative regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and PAO1 expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) Detail of the leaf from panel a. c) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. Significant differences are analyzed by Student's t-test (*P < 0.05). [Figure 4]Figure 4a-4c: Nb / psyllid / CLso pathosystem combined with VIGS showed that CRWN is a negative regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and expression of CRWN was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) Detail of the leaf from panel a. c) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. Significant differences are analyzed by Student's t-test (*P < 0.05). [Diagram 5] Figure 5a-5c: Nb / psyllid / CLso pathosystem combined with VIGS showed that GPX8 is a negative regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and GPX8 expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) Detail of the leaf from panel a. c) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. Significant differences are analyzed by Student's t-test (*P < 0.05). [Figure 6] Figure 6a-6b: Nb / psyllid / CLso pathosystem combined with VIGS showed that PRT6 is a negative regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and PRT6 expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) CLso bacterial titer measured by probe-based qPCR in 50 ng of host genomic DNA. [Figure 7]Figure 7a-7c: Nb / psyllid / CLso pathosystem combined with VIGS showed that PUB26 is a negative regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and PUB26 expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) Detail of the leaf from panel a. c) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. [Figure 8] Figure 8a-8c: Nb / psyllid / CLso pathosystem combined with VIGS showed that LIN2 is a negative regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and LIN2 expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) Detail of the leaf from panel a. c) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. Significant differences are analyzed by Student's t-test (*P < 0.05). [Figure 9] Figure 9a-9c: Nb / psyllid / CLso pathosystem combined with VIGS showed that BRAP is a positive regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and BRAP expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) Detail of the leaf from panel a. c) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. Significant differences are analyzed by Student's t-test (*P < 0.05). [Figure 10]Figure 10a-10b: Nb / psyllid / CLso pathosystem combined with VIGS showed that PSL4 is a positive regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and PSL4 expression was knocked down by VIGS. Silencing of the RB gene (iRB control) was used as a control in non-silenced plants. b) CLso bacterial titer measured by probe-based qPCR in 50 ng of host genomic DNA. [Figure 11] Figure 11a-11b: Nb / psyllid / CLso pathosystem combined with VIGS showed that NDR1-like is a positive regulator in the response to CLso infection. a) Two-week-old Nb plants were exposed to CLso-positive potato psyllids for 5 days and PSL4 expression was knocked down by VIGS. Silencing of RB gene (iRB control) was used as a control in non-silenced plants. b) Titer of CLso bacteria measured by probe-based qPCR in 50 ng of host genomic DNA. Significant differences are analyzed by Student's t-test (*P < 0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description The present disclosure provides targets for altering immune response pathways such that resistance to HLB is enhanced.

[0010] The present invention utilizes various recombinant nucleic acid techniques that are commonly used. In general, the terminology and experimental procedures in recombinant DNA technology described below are those that are commonly adopted in the art. Many manuals that provide instructions for carrying out recombinant DNA manipulation are available, such as Sambrook and Russell, "Molecular Cloning, A Laboratory Manual" (3rd Edition, 2001); and "Current Protocols in Molecular Biology" (Ausubel et al., John Wiley and Sons, New York, 2009-2014).

[0011] As used herein, the terms "citrus greening disease" and "yellow dragon disease (HLB)" refer to a bacterial infection of plants (e.g., citrus plants) caused by bacteria of the genus Candidatus Liberibacter (Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, and Candidatus Liberibacter americanus). The Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae both vector and transmit the infection. Currently, three different HLB types are known: Asian types, which are tolerant to high temperatures, and African and American types, which are sensitive to high temperatures.

[0012] The term "HLB resistant / tolerant" or "HLB resistance / resistance" refers to the increased ability of a citrus plant comprising one or more of the genetic modifications described herein to prevent or resist HLB infection or to prevent or resist HLB-induced infection symptoms, compared to a corresponding control citrus plant not comprising the genetic modification. Thus, an "HLB resistant" plant may have increased resistance to HLB compared to a control citrus plant. Thus, unless otherwise specified, the term "HLB resistant" includes plants that are resistant to HLB, such as citrus plants that are able to grow and produce fruit despite being infected with HLB. Thus, the terms "HLB resistant / tolerant" and "HLB resistant" are used interchangeably herein to refer to plants that have increased ability to prevent HLB infection or have reduced one or more HLB-induced infection symptoms.

[0013] The term "negative immune suppressor" or "negative immune response regulator" or "negative immune response regulator" refers to a gene or a polypeptide encoded by the gene that reduces the host defense response, i.e., reduces one or more aspects of the plant's immune response to CLas infection, such that the plant has increased susceptibility to HLB. A list of negative immune suppressors is provided in Table 1. Exemplary polypeptide sequences are provided in Table 3.

[0014] The term "positive defense regulator" refers to a gene or a polypeptide encoded by the gene that enhances the host defense response, i.e., enhances one or more aspects of the plant's immune response to CLas infection, such that the plant has increased resistance / tolerance to HLB. A list of positive defense regulators is provided in Table 2. Exemplary polypeptide sequences are provided in Table 4.

[0015] The term "nucleic acid" or "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. A nucleic acid may also contain modified nucleotides that allow for accurate read-through by a polymerase without significantly altering expression of a polypeptide encoded by the nucleic acid.

[0016] The phrases "nucleic acid encoding..." or "polynucleotide encoding..." refer to a nucleic acid that directs the expression of a particular protein or peptide. Nucleic acid sequences include both DNA strand sequences that are transcribed into RNA and RNA sequences that are translated into proteins. Nucleic acid sequences include both full length nucleic acid sequences and non-full length sequences derived from the full length nucleic acid sequences. It should further be understood that a sequence includes degenerate codons of the naturally occurring sequence or sequences that may be introduced to provide codon preferences in a particular host cell.

[0017] Two nucleic acid sequences or two polypeptides are said to be "identical" when the sequence of nucleotides or amino acid residues in the two sequences are the same when aligned to the maximum extent as described below. "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over the entire comparison window, where a portion of a polynucleotide sequence or a polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) in order to optimally align two sequences. The percentage is calculated by: determining the number of positions where the same nucleic acid base or the same amino acid residue is present in both sequences to derive the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to derive the percentage of sequence identity. When the percentage of sequence identity is used in relation to proteins or peptides, it is understood that non-identical residue positions often differ by conservative amino acid substitutions, in which amino acids are replaced with other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), so that the functional properties of the molecule are not changed. When sequences differ due to conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1.Scoring of conservative substitutions is calculated, for example, using the algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17 (1988), for example, as implemented in the PC / GENE program (Intelligenetics, Mountain View, California, USA).

[0018] When used in the context of a polynucleotide or polypeptide sequence, the term "substantial identity" or "substantially identical" refers to a sequence having at least 60% sequence identity to a reference sequence. Alternatively, the percent identity can be any integer between 60% and 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% when compared to a reference sequence using a program described herein; preferably using BLAST with standard parameters, as described below. Those skilled in the art will understand that these values ​​can be appropriately adjusted to account for codon degeneracy, amino acid similarity, reading frame alignment, and the like, to determine the corresponding identity of proteins encoded by two nucleotide sequences.

[0019] For sequence comparison, typically one sequence serves as a reference sequence, to which test sequence is compared.When sequence comparison algorithm is used, test sequence and reference sequence are input into computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.Sequence comparison algorithm then calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0020] As used herein, the term "comparison window" refers to a segment having a contiguous sequence of any one of the groups consisting of 20 to 600, generally any one of the groups consisting of about 50 to about 200, more generally any one of the groups consisting of about 100 to about 150, in which a sequence and a reference sequence having the same number of contiguous sequences can be optimally aligned and then compared. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman, Add. APL. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the method of searching similarity of Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package by the Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by manual alignment and visual inspection.

[0021] Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410, and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm first involves identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which, when aligned with words of the same length in database sequences, either match or fall within some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al. supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a matched residue pair; always > 0), and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score.Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by a value X from the maximum achieved; when the cumulative score falls below 0 due to the accumulation of one or more negatively scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M = 1, N = -2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). For purposes of this application, amino acid sequence identity is determined using BLASTP with default parameters.

[0022] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or two amino acid sequences will occur by chance. For example, in a comparison of a test nucleic acid with a reference nucleic acid, the smallest sum probability is less than about 0.01, more preferably about 10 -5 and most preferably less than about 10 -20 A nucleic acid is considered to be similar to a reference sequence if it is less than

[0023] The term "complementary" is used herein to mean that a polynucleotide sequence is complementary to all or a part of a reference polynucleotide sequence.In some embodiments, a polynucleotide sequence is complementary to at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200 or more consecutive nucleotides of a reference polynucleotide sequence.In some embodiments, a polynucleotide sequence is "substantially complementary" to a reference polynucleotide sequence when at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the polynucleotide sequence is complementary to the reference polynucleotide sequence.

[0024] A polynucleotide sequence is "heterologous" if it is of heterologous origin to an organism, or to a second polynucleotide sequence, or if it is from the same species and has been modified from its original form. For example, when a heterologous coding sequence is said to be operably linked to a promoter, it means that the coding sequence is from one species while the promoter sequence is from another, different species; or, if both are from the same species, it means that the coding sequence is not naturally associated with the promoter (e.g., the coding sequence is a genetically engineered coding sequence, e.g., from another gene in the same species or from an allele of a different ecotype or variant).

[0025] "Expression cassette" refers to a nucleic acid construct that, when introduced into a host cell, causes transcription and / or translation of an RNA or polypeptide, respectively. Non-translatable or non-translatable antisense or sense constructs are also expressly included in this definition. Those skilled in the art will understand that in both the case of transgene expression and the case of endogenous gene suppression (e.g., by antisense or by sense suppression), the inserted polynucleotide sequence does not need to be identical to the sequence of the gene from which it is derived, and may only be substantially identical to the sequence of the gene from which it is derived.

[0026] As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, the promoter used in the polynucleotide construct of the present invention includes cis-acting transcriptional control elements and regulatory sequences, which are involved in regulating or changing the timing and / or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, which includes the following involved in regulating transcription: enhancer, promoter, transcription terminator, origin of replication, chromosomal integration sequence, 5' and 3' untranslated regions, or intron sequences. Typically, these cis-acting sequences interact with proteins or other biomolecules to effect transcription of a gene (start / stop, regulate, change, etc., transcription of a gene). A "plant promoter" is a promoter capable of initiating transcription in a plant cell. A "constitutive promoter" is a promoter capable of initiating transcription in almost all tissue types, while a "tissue-specific promoter" initiates transcription only in one or a few specific tissue types. An "inducible promoter" is a promoter that initiates transcription only under certain environmental or developmental conditions.

[0027] The term "plant" includes the following: entire plants, vegetative organs and / or structures of shoots (e.g., leaves, stems, and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg cells and central cells), seeds (including zygotes, embryos, endosperms, and seed coats), fruits (e.g., mature ovaries), seedlings, plant tissues (e.g., vascular tissue, ground tissue, etc.), cells (e.g., guard cells, egg cells, trichomes, etc.), and their progeny.

[0028] Detailed Description of the Invention Introduction As described in the Examples section below, negative regulators of immune response against Liberibacter infection, such as HLB or potato zebra chip disease, and positive defense regulators of immune response against Liberibacter infection have been identified using screening techniques. Described herein are methods and compositions for enhancing the resistance / tolerance of citrus plants to HLB by genetically modifying the citrus plant to silence, inhibit, or reduce the expression or activity of a negative regulator of immune response; and / or by genetically modifying the citrus plant to increase the expression or activity of a positive defense regulator. Similarly, solanaceous crop plants, such as potato or tomato, can be modified to reduce and / or increase the expression of the immune modulator polypeptides described herein.

[0029] While any plant species can be used in any of the compositions or methods described herein, in preferred embodiments, the plant is a member of the Rutaceae family, such as a Citrus maxima plant, a Citrus medica plant, a Citrus micrantha plant, a Citrus reticulata plant, a Citrus aurantiifolia plant, a Citrus aurantium plant, a Citrus latifolia plant, a Citrus limon plant, a Citrus limonia plant, a Citrus paradisi plant, a Citrus clementina plant, a Citrus unshu plant, a Citrus sinensis plant, a Citrus tangerina plant, a Citrus itangensis plant, an Atalantia baccifolia plant, or a Poncirus trifoliata plant. In some embodiments, the plant is a potato or tomato variety. In some embodiments, the plant is a Capsicum variety.

[0030] Negative regulator of immune response In some embodiments, provided herein are methods and compositions for inhibiting expression of one or more of the negative regulators of plant immunity genes listed in Table 1. Exemplary polypeptide sequences for various species of citrus are provided in Table 3.

[0031] Table 1. Negative regulators of plant immune responses to HLB These genes were targets of small RNAs that were induced by CLas infection in US942 but not in Cleopatra. TIFF2024506316000002.tif122151

[0032] The expression or activity of the negative regulator of immune response protein described herein can be inhibited or knocked out using known methods.Thus, in order to enhance HLB resistance, one or more of the genes provided in Table 1 can be knocked out or mutated.For example, in some embodiments, the native gene encoding the polypeptide that is identical or substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90% identical, or at least 95% identical) to the polypeptide sequence of VAD1, PRT6, OPT1, YSL6, PUB26, DMR6, PAO1, TPS5, ACA11, MPK1, CRT1, LIN2, CRWN (LINC4), GPX8, LOX2, or PI4K listed in Table 3 is mutated or knocked out in a citrus plant. In some embodiments, a native gene is mutated or knocked out in a Rutaceae plant that encodes a polypeptide identical or substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90% identical, or at least 95% identical) to the VAD1, PRT6, PUB26, PAO1, LIN2, CRWN (LINC4), or GPX8 polypeptide sequence set forth in Table 3. Gene sequences can be readily identified in other species of Citrus given the known genomic sequences and the conserved nature of the proteins.

[0033] In some embodiments, gene sequences are knocked out in plants. In the context of this application, "knockout" means that the plant does not produce the specific protein encoded by the gene. "Knockdown" means that the expression level or protein level or protein activity is reduced in plants compared to the corresponding control wild-type plants. Knockout and knockdown can be caused in various ways. For example, knockout plants can be made by deleting all or a substantial part (e.g., most of) or coding sequence of a polypeptide that is identical or substantially identical to the protein encoded by the gene listed in Table 1, or a polypeptide that is identical or substantially identical to any one of the polypeptide sequences of VAD1, PRT6, OPT1, YSL6, PUB26, DMR6, PAO1, TPS5, ACA11, MPK1, CRT1, LIN2, CRWN (LINC4), GPX8, LOX2, or PI4K listed in Table 3. In some embodiments, promoter sequences may be modified or deleted so that expression is eliminated or reduced. In some embodiments, target knockout or knockdown is achieved by introducing a mutation that prevents translation or transcription (such as a mutation that introduces a stop codon early in the coding sequence or a mutation that disrupts transcription). Knockout or knockdown can also be achieved by silencing or other suppression methods, such as silencing or other suppression methods that cause the plant to express substantially less of the native protein (e.g., less than 50, 25, 10, 5, or 1% of the native expression). Knockout or knockdown can also be achieved by CRISPR-CAS mediated mutation and deletion, or by using another gene editing technology, which will be described further below.

[0034] In some embodiments, the mutation introduced into the protein is as little as a single amino acid change that reduces or eliminates the activity of the protein. Alternatively, the mutation can include any number of amino acid changes, deletions, or insertions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) that reduce or eliminate the activity of the protein.

[0035] Methods for introducing genetic mutations into plant genes and selecting plants with desired traits are well known, and can be used to introduce mutations or knock out or knock down protein expression or activity. For example, seeds or other plant materials can be treated with mutagenic insertion polynucleotides (e.g., transposons, T-DNA, etc.) or with chemicals using standard techniques. Such chemicals include, but are not limited to, diethyl sulfate, ethyleneimine, ethyl methanesulfonate, and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from a source, such as X-rays or gamma rays, can be used. Plants with mutant proteins can then be identified, for example, by phenotype or molecular techniques.

[0036] Modified protein chains can also be readily designed using a variety of recombinant DNA techniques well known to those skilled in the art and described, for example, in Sambrook et al., supra. Hydroxylamine can also be used to introduce single base mutations in the coding region of a gene (Sikorski et al., Meth. Enzymol., 194:302-318 (1991)). At the level of primary structure, the chains can vary from the native sequence, for example, by amino acid substitutions, additions, deletions, etc. These modifications can be used in any combination to create the final modified protein chain.

[0037] Alternatively, homologous recombination can be used to induce targeted gene modification or gene knockout by specifically targeting a gene in vivo (for review, see Grewal and Klar, Genetics, 146:1221-1238 (1997) and Xu et al., Genes Dev., 10:2411-2422 (1996)). Homologous recombination has been demonstrated in plants (Puchta et al., Experientia, 50:277-284 (1994); Swoboda et al., EMBO J., 13:484-489 (1994); Offringa et al., Proc. Natl. Acad. Sci. USA, 90:7346-7350 (1993); and Kempin et al., Nature, 389:802-803 (1997)).

[0038] When applying homologous recombination technology to gene, it is possible to use standard techniques to cause mutations in selected parts of gene sequence (including 5' upstream region, 3' upstream region and intragenic region) in vitro, and then introduce the mutations into desired plants.Since it is known that the efficiency of homologous recombination varies depending on the vector used, in order to increase the efficiency of selection for the gene expression of modified PP2A subunit A protein in transgenic plants, the dicistronic gene targeting vector described in Mountford et al., Proc. Natl. Acad. Sci. USA, 91:4303-4307 (1994); and Vaulont et al., Transgenic Res., 4:247-255 (1995) is advantageously used.Mutant gene interacts with target wild-type gene, and this interaction is in such a manner that homologous recombination and targeted replacement of wild-type gene occur in transgenic plant cells, resulting in suppression of the activity of target protein.

[0039] Any of a number of genome editing techniques known to those skilled in the art can be used to mutate or knock out target protein. The use of a specific genome editing technique is not essential, as long as it can provide site-specific mutation of the desired nucleic acid sequence. Exemplary genome editing proteins include targeted nucleases, such as engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and engineered meganucleases. In addition, a system can be used that utilizes engineered guide RNA (gRNA) to guide endonuclease to the target cleavage site. The most widely used of these systems is the CRISPR / Cas system, which has engineered guide RNA to guide Cas-9 or Cas12 endonuclease to the target cleavage site.

[0040] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) system is an adaptive defense system in prokaryotes that cleaves foreign DNA. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements that determine the specificity of CRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPR systems have been identified across a range of bacterial hosts. In a typical system, a Cas endonuclease (e.g., Cas9) is guided to a desired site in the genome using a small guide RNA that targets a sequence-specific single- or double-stranded DNA sequence. The CRISPR / Cas system has been used in plants to induce site-specific mutations, including deletions (see Miao et al., 2013 Cell Research 23:1233-1236).

[0041] The basic CRISPR system uses two non-coding guide RNAs (crRNA and tracrRNA), which form a crRNA:tracrRNA complex and direct the nuclease to the target DNA via Watson-Crick base pairing between the crRNA and the target DNA. Thus, it is possible to modify the guide RNA to recognize any desired target DNA sequence. Recently, it has been shown that chimeric single-stranded guide RNAs (sgRNAs) containing both crRNA and tracRNA elements can be used to direct Cas nucleases to the location of target genes. It has been shown that chimeric sgRNAs can be used to direct Cas9 or Cas12, etc., to the location of desired genes in various organisms (Cong et al., 2013 Science 339:819-23).

[0042] Zinc finger nuclease (ZFN) is a genetically engineered protein that contains a zinc finger DNA binding domain fused with a nucleic acid cleavage domain, such as a nuclease.The zinc finger binding domain provides specificity, and can be engineered to specifically recognize any desired target DNA sequence.For a review of the construction and use of ZFN in plants and other organisms, see Urnov et al., 2010 Nat Rev Genet. 11(9):636-46.

[0043] Transcription activator-like effectors (TALEs) are proteins secreted by some species of the Xanthomonas genus to alter gene expression in host plants and to facilitate bacterial colonization and survival. In plants, TALEs act as transcription factors and alter the expression of resistance genes. Recent studies of TALEs have revealed a code that links the repeat regions of TALEs with their target DNA binding sites. TALEs contain highly conserved and repetitive regions, mostly consisting of tandem repeats of 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation has been found between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotides in the TALE binding sites. The simple relationship between amino acid sequence and DNA recognition by TALE binding domains allows the design of DNA binding domains with any desired specificity.

[0044] TALE can be linked with non-specific DNA cleavage domain to prepare genome editing protein called TALEN.Similar to ZFN, restriction endonuclease, such as FokI, can be used conveniently.For the description of the use of TALEN in plants, see Mahfouz et al., 2011 Proc Natl Acad Sci US A. 108:2623-8 and Mahfouz 2011 GM Crops. 2:99-103.

[0045] Meganucleases are endonucleases with recognition sites of 12-40 base pairs. Therefore, the recognition sites occur rarely in any genome. By modifying the recognition sequence using protein engineering, the target sequence can be changed, and the nuclease can be used to cleave the desired target sequence. (See Seligman et al. 2002 Nucleic Acids Research 30: 3870-9, WO06097853, WO06097784, WO04067736, or US20070117128).

[0046] In addition to the above methods, other methods are known for introducing genetic mutations into the genes of plants and selecting plants with desired traits. For example, seeds or other plant material can be treated with mutagenic chemicals using standard techniques. Such chemicals include diethyl sulfate, ethyleneimine, ethyl methanesulfonate (EMS), and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from a source, such as X-rays or gamma rays, can be used.

[0047] Also provided is a method of suppressing the expression or activity of a polypeptide in a citrus plant using an expression cassette that expresses an RNA molecule (or a fragment thereof) that inhibits the expression or activity of an endogenous target gene in a plant cell, wherein the polypeptide is identical or substantially identical, e.g., at least 70%, 75%, 80%, 85%, or 90% identical; or at least 95% identical, to the protein encoded by the gene listed in Table 1 or to the polypeptide sequence of VAD1, PRT6, OPT1, YSL6, PUB26, DMR6, PAO1, TPS5, ACA11, MPK1, CRT1, LIN2, CRWN (LINC4), GPX8, LOX2, or PI4K listed in Table 3. Suppression or silencing of gene function generally refers to suppressing the level of mRNA or protein expressed by an endogenous gene and / or suppressing the level of functionality of the protein in a cell. The term does not require a particular mechanism, and can include RNAi (e.g., short interfering RNA (siRNA) and microRNA (miRNA)), antisense, co-suppression, viral suppression, hairpin suppression, stem-loop suppression, and the like.

[0048] Numerous methods can be used to suppress or silence gene expression in plants.The method of suppressing gene function using double-stranded RNA in various organisms, including plants, is well known.The expression cassette that codes for RNAi typically comprises a polynucleotide sequence that is at least substantially identical to target gene, which is linked to a complementary polynucleotide sequence.The sequence and its complement are often linked by a linker sequence, which allows the transcribed RNA molecule to fold so that the two sequences hybridize with each other.

[0049] RNAi (e.g., siRNA, miRNA) appears to function by base pairing with complementary RNA or DNA target sequences. When bound to RNA, the inhibitory RNA molecule causes either RNA cleavage or translational inhibition of the target sequence. When bound to a DNA target sequence, it is believed that the inhibitory RNA can mediate DNA methylation of the target sequence. Regardless of the specific mechanism, the result of these events is inhibition of gene expression. RNA silencing can also be achieved by expressing the target gene, or a part of the target gene, in a viral vector, such as tobacco rattle virus (TRV), Potato virus X (PVX), or Citrus Tristeza Virus (CTV), which can cause virus-induced gene silencing (VIGS) of the target gene.

[0050] MicroRNAs (miRNAs) are non-coding RNAs, approximately 19 to 24 nucleotides in length, that are processed from longer precursor transcripts that form stable hairpin structures.

[0051] In addition, antisense technology is available. To achieve this, a nucleic acid segment that is at least substantially identical to a desired gene is cloned and operably linked to a promoter so that an RNA antisense strand is transcribed. Then, the expression cassette is transformed into a plant, and the RNA antisense strand is produced. It has been suggested that antisense RNA inhibits gene expression in plant cells by preventing the accumulation of mRNA that codes for the protein of interest.

[0052] Another method of suppression is sense suppression: introducing an expression cassette in which the nucleic acid is oriented in a sense orientation relative to the promoter has been shown to be an effective means of blocking transcription of a target gene.

[0053] For these techniques, the sequence introduced in the expression cassette does not need to have complete identity to the target gene. In addition, the sequence does not need to be full length compared to either the primary transcript or the fully processed mRNA. Those skilled in the art will also recognize that these techniques can be used to suppress a gene family using one transcript. For example, if the transcript is designed to have a sequence that is conserved in the gene family, multiple members of the gene family can be suppressed. Conversely, if the goal is to suppress only one member of a homologous gene family, the transcript should target the sequence that has the most diversity between the members of the family.

[0054] Expression of the gene can also be inactivated using recombinant DNA technology by transforming plant cells with constructs that contain transposons or T-DNA sequences. The mutants prepared by these methods are identified using standard techniques. For example, mutants can be detected by PCR, or mutants can be detected by detecting the presence or absence of PP2A subunit A mRNA, for example, by Northern blot or reverse transcription PCR (RT-PCR).

[0055] Catalytic RNA molecules or ribozymes can also be used to inhibit the expression of embryo-specific genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific position, thereby functionally inactivating the target RNA. When performing this cleavage, the ribozyme itself does not change, and therefore it can be reused and cleave other molecules, making it a true enzyme. Inclusion of a ribozyme sequence in antisense RNA confers RNA cleavage activity to the RNA, thereby increasing the activity of the construct. The design and use of ribozymes specific for target RNA is well known.

[0056] The recombinant construct that encodes genome editing protein or encodes the nucleic acid that suppresses expression can be introduced into plant cells using standard genetic engineering techniques well known to those skilled in the art.In a typical embodiment, recombinant expression cassette can be prepared using well known techniques.In the case of CRISPR / Cas nuclease, the expression cassette can also transcribe guide RNA.

[0057] In some embodiments, genome editing protein itself is introduced into plant cell.In these embodiments, the introduced genome editing protein is provided in sufficient amount to modify cell, but does not persist after intended period or after one or more cell divisions.In such embodiments, no additional steps are required to remove or separate genome editing protein and modified cell.

[0058] In these embodiments, the genome editing protein is prepared in vitro using a well-known recombinant expression system (bacterial expression, in vitro translation, yeast cells, insect cells, etc.) before being introduced into plant cells. After expression, the protein is isolated, refolded if necessary, purified, and optionally treated to remove any purification tag, such as His tag. Once the crude, partially purified, or more completely purified genome editing protein is obtained, the protein can be introduced into plant cells via electroporation, by bombardment of particles coated with the protein, by transfection with chemicals, or by any other means of transport through the cell membrane.

[0059] Positive regulator of immune response In some embodiments, provided herein are methods and compositions for enhancing expression of one or more of the plant positive defense genes listed in Table 2. Exemplary polypeptide sequences for various species of Citrus are provided in Table 4.

[0060] Table 2 Positive regulators of plant immune responses to HLB These genes were targets of small RNAs that were downregulated by CLas infection in US942 but not in Cleopatra. TIFF2024506316000003.tif66148

[0061] The expression of the proteins described herein can be increased using known techniques. To enhance HLB resistance, any one or any multiple of the genes provided in Table 4 can be highly expressed in plants. Thus, in some embodiments, a plant can be genetically modified to highly express a gene native to itself, or to express a heterologous corresponding gene from another species. In some embodiments, a citrus plant is genetically engineered to highly express a polypeptide that is identical or substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90% identical, or at least 95% identical) to the BRAP2, CYP93, NDR1-like, PSL4, LYM2, SOT12, SCE1, GLY1, PAL1, WRKY70, or EFR-like polypeptide sequence described in Table 4. In some embodiments, citrus plants are genetically engineered to highly express a polypeptide that is identical or substantially identical (e.g., at least 70%, 75%, 80%, 85%, 90% identical, or at least 95% identical) to the BRAP2, NDR1-like, or PSL4 polypeptide sequences set forth in Table 4. Gene sequences can be readily identified in other citrus species given the known genomic sequences and the conserved nature of the proteins.

[0062] In some embodiments, citrus plants are genetically modified to introduce a recombinant expression cassette for expressing a native or heterologous BRAP2, CYP93, NDR1-like, PSL4, LYM2, SOT12, SCE1, GLY1, PAL1, WRKY70, or EFR-like polypeptide. It should be understood that a transgenic plant includes a plant or plant cell into which an expression cassette has been introduced, as well as the progeny of such a plant or plant cell that contains the expression cassette, including progeny that have the expression cassette stably integrated into a chromosome.

[0063] In some embodiments, the transgenic plant may have increased (e.g., at least 5%, 10%, 50%, or more) expression of a BRAP2, CYP93, NDR1-like, PSL4, LYM2, SOT12, SCE1, GLY1, PAL1, WRKY70, or EFR-like polypeptide compared to a corresponding control plant that has not been genetically modified to overexpress the protein.

[0064] In some embodiments, gene editing techniques such as CRISPR / Cas can be used to increase expression of a BRAP2, CYP93, NDR1-like, PSL4, LYM2, SOT12, SCE1, GLY1, PAL1, WRKY70, or EFR-like polypeptide, for example, by introducing additional copies of the sequence encoding the protein into the genome of the plant.

[0065] In some embodiments, a recombinant expression vector containing a promoter-driven protein coding sequence may be introduced into the genome of a desired plant; or it may be introduced by CRISPR-CAS knock-in as described above; or it may be expressed by a viral vector, such as a CTV viral vector. In some embodiments, a polynucleotide encoding a polypeptide may be introduced into a plant, e.g., recombinantly, such that expression is controlled by a promoter that is endogenous to the plant. Thus, for example, in some embodiments, a DNA construct may be directly introduced into the genomic DNA of a plant cell, e.g., using techniques such as electroporation and microinjection into protoplasts of the plant cell, or the DNA construct may be directly introduced into plant tissues using bombardment methods, e.g., DNA particle bombardment. Alternatively, the DNA construct may be combined with appropriate T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Although the invention encompasses transient expression of a polypeptide, generally, expression can result from insertion of the expression cassette into the plant genome, e.g., such that at least some progeny of the plant also contain the integrated expression cassette.

[0066] Expression cassette Plant expression cassettes (e.g., for expression of a positive defense protein described herein or for expression of an inhibitory nucleic acid or inhibitory gene editing protein that inhibits or eliminates expression of a negative immune response regulator described herein) can include a polynucleotide operably linked to: a promoter (e.g., that provides for inducible or constitutive expression, that provides for environmentally or developmentally regulated expression, or that provides for cell or tissue specific / selective expression), a transcription initiation site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

[0067] A large number of promoters are available. Plant promoter fragments are available that direct expression of a desired polynucleotide in all tissues of a plant. In some embodiments, the promoters described herein include 500 to 2 kb, or 500 to 1 kb, or 500 to 2.5 kb upstream (5') from the position where transcription of the gene begins. Such promoters are referred to herein as "constitutive" promoters, and are active under most environmental conditions and at most stages of development or cell differentiation. Examples of constitutive promoters include the 35S transcription initiation region of the cauliflower mosaic virus (CaMV).

[0068] Alternatively, the plant promoter can direct the expression of the polynucleotide under environmental control. Such promoters are referred to herein as "inducible" promoters. Environmental conditions that can affect transcription by inducible promoters include: biotic stress, abiotic stress, salt stress, drought stress, pathogen attack, anaerobic conditions, low temperature stress, high temperature stress, low oxygen stress, or the presence of light.

[0069] In addition, chemically inducible promoters can be used, examples include those induced by benzylsulfonamide, tetracycline, abscisic acid, dexamethasone, ethanol, or cyclohexenol.

[0070] Examples of promoters under developmental control include promoters that initiate transcription only in certain tissues or preferentially in certain tissues, such as leaves, roots, fruits, seeds, or flowers. These promoters are sometimes called tissue-preferred promoters. The action of a promoter can also vary depending on its location on the genome. Thus, developmentally regulated promoters can be fully constitutive or partially constitutive at a certain location. Developmentally regulated promoters can also be modified for weak expression, if necessary.

[0071] Selection of plants with enhanced HLB resistance / tolerance Plants with enhanced HLB resistance / tolerance can be selected in many ways. Those skilled in the art will understand that the following methods are only a few of the possible ones. Those skilled in the art will understand that plant resistance response varies depending on many factors, including the plant. In general, enhanced resistance is measured by the reduction or disappearance of disease symptoms (e.g., the reduction in the number or size of lesions, or the reduction in the amount of fungal biomass in the plant or in a part of the plant) in response to CLas infection when compared to control plants. However, in some cases, enhanced resistance can also be measured by the occurrence of plant hypersensitive response (HR) (see, for example, Staskawicz et al. (1995) Science 268(5211): 661-7). Plants with enhanced pathogen resistance can cause enhanced hypersensitive response compared to control plants.

[0072] Enhanced HLB resistance can also be determined by measuring the increased expression of genes functionally linked to positive defense regulators or by measuring the decreased expression or activity of negative immune regulator proteins. Such expression measurements can be measured by quantifying the accumulation of RNA or subsequent protein products (e.g., using Northern or Western blot techniques, respectively (see, e.g., Sambrook et al. and Ausubel et al.)). EXAMPLES

[0073] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0074] In this example, the identification of positive defense regulators and negative immune response regulators is described. The experimental methodology used to identify and test the function of the positive and negative regulators is described in Huang et al. (2020) Plant Biotechnol. J. doi.oprg / 10.1111 / pbi.13502, which is incorporated by reference. Huang et al. describe an effective host / vector / pathogen interaction system that uses C. liberibacter solanacearum (CLso), a close relative of CLas that infects solanaceous plants, the potato psyllid, a major pest of potato and tomato, and Nicotiana benthamiana, an ideal host for virus-induced gene silencing (VIGS) experiments. VIGS is an effective silencing method to knock down the expression of endogenous genes in plants using viral (TRV) vectors. This system closely resembles the natural citrus / psyllid / CLas interaction system, and it can be used to rapidly characterize the function of candidate regulators in plant defense responses against C. liberibacter species.

[0075] By comparing sRNA profiles between the uninfected HLB-resistant hybrid US-942 and the uninfected HLB-susceptible mandarin Cleopatra, conserved miRNAs were found that were constitutively more abundant in US-942 compared to the HLB-susceptible Cleopatra. Additional miRNAs were also found that were constitutively less abundant in US-942 compared to Cleopatra. We predicted and annotated potential immune negative and positive regulators and assessed their expression levels in US942 and Cleopatra, as well as in another closely related HLB-resistant citrus, the Sydney hybrid (Microcitrus virgata), which has a different genetic and geographical background. We also performed functional studies in the Nicotiana benthamiana (Nb) / Potato psyllid / Candidatus liberibacter solanacearum (CLso) pathosystem described in Huang et al., 2020. BRAP2, CYP93, NDR1-like, PSL4, LYM2, SOT12, SCE1, GLY1, PAL1, WRKY70, and EFR-like were identified as positive immune response regulators; and VAD1, PRT6, OPT1, YSL6, PUB26, DMR6, PAO1, TPS5, ACA11, MPK1CRT1, LIN2, CRWN (LINC4), GPX8, LOX2, and PI4K were identified as negative immune response regulators.

[0076] Functional validation of the candidate regulators in defense responses against CLso was performed by TRV-based VIGS, which knocked down Nb orthologous / homologous genes of those listed in Tables 1 and 2 in CLso-infected Nb plants. Two-week-old Nb plants were exposed to CLso-positive potato psyllid larvae for 5 days. Three to four days after removing the psyllid larvae, Agrobacterium tumefaciens carrying a TRV vector containing a 200-300 bp gene fragment silencing the target gene was used to inoculate Nb leaves by infiltration. Seventeen days after infiltration, the yellowing symptoms of plants and greening of vascular tissues were observed and compared with siRB control. Plant leaf tissues were harvested to detect CLso DNA, and the expression of the target gene was analyzed by quantitative real-time polymerase chain reaction. A TRV construct containing a fragment of RB, a late-blight resistance gene specific to Solanum bulbocastanum, was used as a negative control (siRB). Nb does not have an orthologous gene and therefore does not contain the target RB gene.

[0077] Figures 1a-1c provide data showing that mutant plants in which VAD expression had been knocked down by VIGS showed reduced CLso bacterial titers compared to control plants in which the RB gene had been silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0078] Figures 2a-2d provide data showing that carizo plants in which VAD was knocked down (knocked down by RNA silencing) showed higher expression of defense marker genes including infection-specific (PR-2) and chitinase (CHI).

[0079] Figures 3a-c provide data showing that PAO1 is a negative regulator in the response to CLso infection. PAO1 is a polyamine oxidase that regulates reactive oxygen species homeostasis. Mutant plants in which PAO1 expression was knocked down by VIGS showed reduced CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0080] Figures 4a-4c provide data showing that CRWN is a negative regulator in the response to CLso infection. CRWN is a nuclear lamina protein. Loss of CRWN protein induces expression of salicylic acid biosynthetic genes. Mutant plants in which expression of CRWN was knocked down by VIGS showed reduced CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0081] Figures 5a-5c provide data showing that GPX8 is a negative regulator in the response to CLso infection. GPX8 is a glutathione peroxidase. Reduced expression of GPX impairs resistance to photooxidative stress but increases resistance to pathogenic bacteria (see, e.g., Chang et al., Plant Physiol. 150: 670-683, 2009). Mutant plants in which GPX8 expression has been knocked down by VIGS showed reduced CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0082] Figures 6a-6b provide data showing that PRT6 is a negative regulator in the response to CLso infection. PRT6 is an E3 ubiquitin protein ligase. Arabidopsis and barley prt6 mutant plants are resistant to Pst and Ps. japnoica, as well as Blumeria graminis f. sp. hordei (see, for example, Christopher et al., Plant Direct 3:12 e00194, 2019). Mutant plants in which expression of PRT6 has been knocked down by VIGS showed reduced CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0083] Figures 7a-7b provide data showing that PUB25 / 26 is a negative regulator in the response to CLso infection. PUB25 / 26 is an E3 ligase that targets the inactivated immune kinase BIK1 for degradation. Mutant plants in which expression of PUB25 / 26 was knocked down by VIGS showed reduced CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0084] Figures 8a-8c provide data showing that LIN2 is a negative regulator in the response to CLso infection. LIN2 encodes coproporphyrinogen III oxidase, a key enzyme in the chlorophyll and heme biosynthetic pathways of the tetrapyrrole pathway. Molecular markers associated with defense responses are more highly expressed in LIN2 mutants (see, e.g., Guo et al., Plant Cell Rep 32:687-702, 2013). Mutant plants in which LIN2 expression has been knocked down by VIGS showed reduced CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0085] The positive regulators identified in the above screens were also analyzed as regulators of immune response.

[0086] Figures 9a-9c provide data showing that BRAP is a positive regulator in the response to CLso infection. BRAP is an E3 ligase that positively regulates pathogen-associated molecular patterns triggered in plant defense responses (see, e.g., Xie et al., PLoS Pathog 12: 1005529, 2016). Mutant plants in which BRAP expression was knocked down by VIGS showed increased CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0087] Figures 10a-10b provide data showing that PSL4 is a positive regulator in the response to CLso infection. PSL4 is essential for controlling the stable accumulation and disposition of EFR, the receptor for elf18. Mutant plants in which PSL4 expression was knocked down by VIGS showed increased CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0088] Figures 11a-11b provide data showing that NDR1-like is a positive regulator in the response to CLso infection. NDR1-like (NON RACE-SPECIFIC DISEASE RESISTANCE 1) is required for cultivar non-specific resistance to bacterial and fungal pathogens. It mediates the systemic acquired resistance response (see, e.g., Day et al., Plant Cell. 18:2782-91, 2006). Mutant plants in which NDR1-like expression has been knocked down by VIGS showed increased CLso bacterial titers compared to control plants in which the RB gene was silenced, as measured by probe-based qPCR in 50 ng of host genomic DNA.

[0089] All references, publications, and accession numbers are incorporated by reference to the same extent as if each individual accession number was specifically and individually indicated to be incorporated by reference. Although the foregoing disclosure has been described in some detail for purposes of illustration and example for purposes of clear understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present disclosure that certain changes and modifications can be made to the present invention without departing from the spirit or scope of the invention.

[0090] Table 3. Polypeptide sequences of citrus plant negative immune response regulators TIFF2024506316000004.tif20897TIFF2024506316000005.tif222155TIFF2024506316000006.tif138155TIFF2024506316000007.tif223156TIFF2024506316000008.tif222156TIFF2024506316000009.tif218156TIFF2024506316000010.tif222156TIFF2024506316000011.tif222156TIFF2024506316000012.tif222156TIFF2024506316000013.tif222156TIFF2024506316000014.tif76136TIFF2024506316000015.tif222155TIFF2024506316000016.tif222156TIFF2024506316000017.tif222156TIFF2024506316000018.tif222155TIFF2024506316000019.tif54155TIFF2024506316000020.tif222156TIFF2024506316000021.tif222156TIFF2024506316000022.tif39128TIFF2024506316000023.tif223155TIFF2024506316000024.tif101155TIFF2024506316000025.tif223155TIFF2024506316000026.tif160156TIFF2024506316000027.tif222156TIFF2024506316000028.tif222109TIFF2024506316000029.tif215156TIFF2024506316000030.tif171155TIFF2024506316000031.tif222109TIFF2024506316000032.tif222156TIFF2024506316000033.tif222156TIFF2024506316000034.tif222156TIFF2024506316000035.tif222156TIFF2024506316000036.tif218156TIFF2024506316000037.tif131155TIFF2024506316000038.tif215156TIFF2024506316000039.tif9 5156TIFF2024506316000040.tif222156TIFF2024506316000041.tif10155TIFF2024506316000042.tif186155TI FF2024506316000043.tif222156TIFF2024506316000044.tif222156TIFF2024506316000045.tif219155TIFF20 24506316000046.tif222156TIFF2024506316000047.tif222156TIFF2024506316000048.tif215156TIFF2024506 316000049.tif84155TIFF2024506316000050.tif219155TIFF2024506316000051.tif186155TIFF202450631600 0052.tif222107TIFF2024506316000053.tif215156TIFF2024506316000054.tif222156TIFF2024506316000055. tif222156TIFF2024506316000056.tif222156TIFF2024506316000057.tif222156TIFF2024506316000058.tif2 4156TIFF2024506316000059.tif222156TIFF2024506316000060.tif222156TIFF2024506316000061.tif116156.

[0091] Table 4. Polypeptide sequences of positive defense regulators in citrus plants TIFF2024506316000062.tif212155TIFF2024506316000063.tif108156TIFF2024506316000064.tif223155TIFF2024506316000065.tif47155 TIFF2024506316000066.tif175155TIFF2024506316000067.tif222156TIFF2024506316000068.tif218156TIFF2024506316000069.tif6155TIFF2024506316000070.tif223156TIFF2024506316000071.tif135155TIFF2024506316000072.tif223155TIFF2024506316000073.tif39156TIFF2024506316000074.tif223156TIFF2024506316000075.tif47155TIFF2024506316000076.tif222105TIFF2024506316000077.tif222156TIFF2024506316000078.tif222156TIFF2024506316000079.tif222156TIFF2024506316000080.tif28155TIFF2024506316000081.tif189155TIFF2024506316000082.tif222155TIFF2024506316000083.tif222156TIFF2024506316000084.tif222156TIFF2024506316000085.tif222156TIFF2024506316000086.tif215156TIFF2024506316000087.tif223156TIFF2024506316000088.tif222156TIFF2024506316000089.tif218156TIFF2024506316000090.tif219156TIFF2024506316000091.tif222156TIFF2024506316000092.tif222156TIFF2024506316000093.tif222156TIFF2024506316000094.tif222155TIFF2024506316000095.tif222156TIFF2024506316000096.tif222156TIFF2024506316000097.tif222156TIFF2024506316000098.tif10155

Claims

1. 1. A method for enhancing resistance of a plant to a disease caused by bacteria of the genus Liberibacter, the method comprising the steps of: genetically modifying the plant to reduce expression of an endogenous gene encoding a negative regulator of an immune response polypeptide, wherein the negative regulator of the immune response polypeptide is PAO1, VAD1, PRT6, PUB26, LIN2, CRWN, or GPX8.

2. 2. The method of claim 1, wherein the disease is HLB and the plant is a citrus plant.

3. The plant may be Citrus maxima plant, Citrus medica plant, Citrus micrantha plant, Citrus reticulata plant, Citrus aurantiifolia plant, Citrus aurantium plant, Citrus latifolia plant, Citrus limon plant, Citrus limonia plant, Citrus paradisi plant, Citrus clementina plant, Citrus unshiu plant, Citrus sinensis plant, Citrus 3. The method of claim 2, wherein the plant is a Citrus sinensis plant, a Citrus tangerina plant, a Citrus ichangensis plant, an Atalantia buxifolia plant, or a Poncirus trifoliata plant.

4. 2. The method of claim 1, wherein the disease is potato zebra chip disease and the plant is a potato plant.

5. 5. The method of any one of claims 1 to 4, wherein reducing the expression of the negative regulator comprises contacting the plant with an siRNA that targets an endogenous nucleic acid encoding the negative regulator.

6. The method of any one of claims 1 to 4, wherein reducing the expression of the negative regulatory factor comprises viral vector-mediated gene silencing.

7. 5. The method of any one of claims 1 to 4, wherein reducing expression of the negative regulator comprises knocking out expression of an endogenous gene encoding the negative regulator.

8. 5. The method of any one of claims 1 to 4, comprising genetic editing of the endogenous gene to reduce or knock out expression.

9. The method of claim 8, wherein the gene editing technology is CRISPR / CAS gene editing.

10. The method of any one of claims 1 to 9, wherein the negative regulator of an immune response polypeptide comprises an amino acid sequence having at least 70% identity to a polypeptide sequence of PAO1, VAD1, PRT6, PUB26, LIN2, CRWN, or GPX8 as set forth in Table 3.

11. The method of claim 10, wherein the negative regulator of an immune response polypeptide comprises an amino acid sequence having at least 90% or at least 95% identity to a polypeptide sequence of PAO1, VAD1, PRT6, PUB26, LIN2, CRWN, or GPX8 listed in Table 3.

12. 1. A method for enhancing plant resistance to a disease caused by bacteria of the Liberibacter genus, comprising the steps of: genetically modifying a plant to enhance expression of a gene encoding a positive defense regulator polypeptide as set forth in Table 2, wherein the positive defense regulator peptide is BRAP2, NDR1-like, or PSL4.

13. 13. The method of claim 12, wherein the disease is HLB and the plant is a member of the Citrus family.

14. 14. The method of claim 13, wherein the plant is a Citrus maxima plant, a Citrus medica plant, a Citrus micrantha plant, a Citrus reticulata plant, a Citrus aurantiifolia plant, a Citrus aurantium plant, a Citrus latifolia plant, a Citrus limon plant, a Citrus limonia plant, a Citrus paradisi plant, a Citrus clementina plant, a Citrus unshu plant, a Citrus sinensis plant, a Citrus tangerina plant, a Citrus itangensis plant, an Atalantia baccifolia plant, or a Poncyrus trifoliata plant.

15. 13. The method of claim 12, wherein the disease is potato zebra chip disease and the plant is a potato plant.

16. 16. The method of any one of claims 12 to 15, comprising genetically modifying a plant to highly express a polypeptide comprising an amino acid sequence having at least 70% identity to a BRAP2, NDR1-like, or PSL4 polypeptide sequence set forth in Table 4.

17. 17. The method of claim 16, comprising genetically modifying a plant to highly express a polypeptide comprising an amino acid sequence having at least 95% identity to a BRAP2, NDR1-like, or PSL4 polypeptide sequence set forth in Table 4.

18. The method according to any one of claims 12 to 17, wherein the polypeptide is endogenous to the plant.

19. The method according to any one of claims 12 to 17, wherein the polypeptide is heterologous to the plant.

20. A plant having enhanced resistance to HLB produced by the method according to any one of claims 1 to 19.

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