METHODS AND COMPOSITIONS FOR PREVENTING OR REDUCING INFECTIONS OF CROP PLANTS BY BACTERIAL AND FUNGAL PATHOGENS

Abstract

The present invention teaches methods and compositions useful for treating, preventing, or curing pathogen infections of living plants. In particular, the present invention teaches methods of enhancing plant response to pathogen-associated molecular patterns. The methods and compositions described herein are effective at treating biotrophic pathogens, including Liberibacters.

Claims

1. A recombinant nucleic acid molecule comprising a nucleic acid sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 operably linked to a heterologous promoter, wherein transcription of the nucleic acid molecule reduces expression of a BAG6 gene in a plant cell.

2. The recombinant nucleic acid molecule of claim 1, operably linked to a movement leader sequence.

3. The recombinant nucleic acid molecule of claim 2, wherein the movement leader sequence comprises at least 18 contiguous nucleotides of SEQ ID NO: 7.

4. The recombinant nucleic acid molecule of claim 1, operably linked to a GUS loop sequence or an intron sequence.

5. A recombinant vector comprising the recombinant nucleic acid molecule of claim 1.

6. A transgenic plant cell comprising the recombinant nucleic acid molecule of claim 1.

7. A transgenic plant comprising the recombinant nucleic acid molecule of claim 1.

8. A transgenic seed comprising the recombinant nucleic acid molecule of claim 1.

9. A method of repressing, preventing or otherwise reducing a bacterial or fungal infection of a plant comprising expressing the nucleic acid construct of claim 1 in said plant.

10. The method of claim 9, wherein said plant is a citrus tree.

11. The method of claim 9, wherein said infection is caused by a biotrophic plant pathogen.

12. The method of claim 9, wherein said infection is caused by a Liberibacter.

13. The method of claim 12 wherein said infection is caused by a Liberibacter infecting citrus.

14. The method of claim 12, wherein said infection is caused by Ca. Liberibacter asiaticus (Las).

15. A recombinant or nonrecombinant nucleic acid molecule comprising a nucleic acid sequence comprising at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10

16. A recombinant or nonrecombinant nucleic acid molecule of claim 1 or 15, wherein said molecule is comprised of a dsRNA or siRNA.

17. A method of repressing, preventing or otherwise reducing a bacterial or fungal infection of a plant comprising topical application of the nucleic acid of claim 15 or 16 to said plant.

18. The method of claim 17, wherein said plant is a citrus tree.

19. The method of claim 17, wherein said infection is caused by a biotrophic plant pathogen.

20. The method of claim 17, wherein said infection is caused by a Liberibacter.

21. The method of claim 20 wherein said infection is caused by a Liberibacter infecting citrus.

22. The method of claim 20, wherein said infection is caused by Ca. Liberibacter asiaticus (Las).

23. A method of identifying a functional homolog equivalent to SEQ ID NO: 1 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 in other plant species.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1. Schematic diagrams of the constructs used to silence the CiBag6 gene. Carrizo Cibag6 cDNA is diagrammed above the constructs. Constructs pIPG1150 and pIPG1160 are antisense (aRNA) and constructs pIPG1157, pIPG1159 and pIPG1174 are inverted repeat constructs. The CaMV 35S promoter (35S) and Arabidopsis sucrose transporter AtSuc2 gene promoter (Suc2) were used as indicated. In addition, a 104 nucleotide movement leader (ML) was used to construct pIPG1160, and two uidA fragments (labeled “GUS frag. 1”, 516 bp and “GUS frag. 2”, 1848 bp) taken from the 5′ end of the uidA gene from pCAMBIA2301 were used as indicated. Finally, a 190 bp catalase intron (“intron”) from pCAMBIA was used to construct pIPG1174 as shown.

[0035] FIG. 2. CiBAG6 expression levels in different transgenic lines by qPCR. To determine if native CiBAG6 expression in the transgenic Carrizo lines was actually silenced by the various silencing constructs used, DNA primers were designed based on the CiBAG6 sequence, but outside of the regions cloned and used for silencing purposes. cDNAs made from RNA extracts that were treated with DNase taken from each transformed plant line were used. qRT-PCR levels were normalized against the citrus gene, Elongation Factor 1 alpha. NT CK is nontransgenic check (i.e., control).

[0036] FIG. 3. Plant basal defense responses elicited by Las flagellin-22. To determine if several genes consistently associated with basal plant defense responses were affected by silencing CiBAG6, PCR primers known to be useful in examining three genes known to respond to PAMP elicitors in citrus were used (Pathogenesis Related 1 or PR1; Enhanced Disease Susceptibility 1 or EDS1, and Non race-specific Disease Resistance 1 or NDR1). Levels of expression of each of the three defense response genes in citrus were examined by qPCR. To elicit citrus plant defense responses, Las flagellin 22 (Las flg22) was used as a proxy for inoculation with Las. Defense responses in nontransgenic (NT) Carrizo inoculated with water (NT CK) was compared with NT Carrizo and CiBAG6 silenced Carrizo lines, all inoculated with Las flg22; again, qRT-PCR levels were normalized against the citrus gene, Elongation Factor 1 alpha. Controls also included determination of the levels of PR1, EDS1 and NDR1 in both NT Carrizo and CiBAG6 silenced lines that were not inoculated with Las flg22 as controls. Samples were taken at 24 hours post inoculation (24 hpi) and 48 hours post inoculation (48 hpi).

[0037] FIG. 4. CiBAG6 expression is suppressed in citrus leaves after root treatment using either long dsRNA or siRNA. Citrus rootstock Carrizo seedlings treated with root immersion into a solution with either dsRNA (800 nucleotides in length) or siRNA (average of 23 nucleotides in length) exhibited long distance silencing of ciBAG6 (in untreated leaves) within 48 hours. PCR (qRT-PCR) was used to determine levels of expression of CiBAG6 in all leaf samples, using primers well outside of the region of CiBAG6 used for the RNA treatments. qRT-PCR levels were normalized against the control mRNA expression level.

[0038] FIG. 5. CiBAG6 expression is suppressed in citrus leaves after spray treatment using either long dsRNA or siRNA. Citrus rootstock Carrizo seedlings sprayed with either 16 μg of long dsRNA or 1.4 μg of siRNA RNA was extracted from the leaves of the treated plants 80 hrs after spraying the leaves, and qRT-PCR used to determine levels of expression of CiBAG6 in all leaf samples, using primers well outside of the region of CiBAG6 used for the RNA treatments. qRT-PCR levels were normalized against the control mRNA expression level.

[0039] FIG. 6. Ca. Liberibacter asiaticus (Las) infection is reduced in commercially grown citrus after injection treatment using either long dsRNA or siRNA. An RNA-based, live cell assay was performed in four year old, commercially grown grapefruit trees in a field trial that were 100% infected with Las. Selected trees were injected with an aqueous formulation, 224, containing either long dsRNA or siRNA. Control plants were mock-injected with the aqueous formulation 224 without dsRNA or siRNA. qRT-PCR was used as in FIG. 4 to determine levels of expression of Las SC2_gp095 peroxidase. Plant cox1 was used to normalize expression SC2 peroxidase gene expression is an indicator of live Las cells, and was clearly suppressed after a single treatment application of dsRNA or siRNA in formula 224, one-month post treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Apoptosis

[0041] Type I programmed cell death (PCD) or apoptosis, is a genetically programmed and highly regulated cell death mechanism found in plant and animal cells that allows damaged cells to commit suicide. Apoptosis is critically important for elimination of damaged or infected cells that could compromise the function of the whole organism. Typical triggers of apoptosis are environmental insults or stresses that can damage cells or their DNA content. Reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide, nitric oxide and free hydroxide radicals are produced in response to stress, and particularly stress causing mitochondrial damage (Portt, et al., 2011). ROS production is a first line of defense in animal and plant cells against biotic disease agents, such as bacteria and fungi. ROS is also one of the major signals that can trigger apoptosis. In addition to ROS production, stress also activates production of the protein “Bax”, and the sphingolipid “ceremide”, and all three are direct proapoptotic messengers. These three major proapoptic messengers can to act independently of one another, since increases in the levels of any one of them (Bax, ROS, or ceramide) is sufficient to trigger apoptosis, but most often, they appear to act in concert.

[0042] Pathogens that benefit from plant cell death, such as Phytopthora, Ralstonia, Pseudomonas and Xyella are necrotrophic in lifestyle; that is, they kill host cells in order to provide nutrients to sustain in planta population growth. Such pathogens may do little to suppress apoptosis (type I) or necrotic (type III) programmed cell death (Portt, et al., 2011). Other pathogens, such as the obligate fungal parasites (rusts and mildews) and some bacteria, such as Rhizobium, are biotrophic, and must establish intimate cell membrane to membrane contact using haustoria or infection threads.

[0043] Programmed Cell Death for Controlling Infections

[0044] Liberibacters are the ultimate form of biotroph, living entirely within the living host cell and surrounded by host cell cytoplasm. For biotrophs, host cell death would be expected to severely limit growth in planta. Liberibacters, which must live entirely within living phloem cells, would have no options if their phloem host cells simply died. Biotrophic pathogens typically have multiple mechanisms available to suppress apoptosis or necrotic programmed cell death. In the case of necrotrophic pathogens (those that rely on killing plant cells in order to feed on the contents), suppressing host cell death results in denial of nutrients, and resistance is the result. Necrotrophic pathogens naturally trigger PCD.

[0045] Candidatus Liberibacter is a genus of Gram-negative bacteria in the Rhizobiaceae family. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Members of the genus are plant pathogens mostly transmitted by psyllids. The genus was originally spelled Liberobacter.

[0046] Liberibacters and Other Biotrophic Bacteria

[0047] By contrast, prototrophic pathogens rely on fully functional living cells to survive. For example, all species and strains of the genus Liberibacter live in plants entirely within living plant phloem cells. The first species described was Candidatus Liberibacter asiaticus (Las), the causal agent of Huanglongbing (HLB), commonly known as citrus “greening” disease. HLB is lethal to citrus and one of the top three most damaging diseases of citrus. The second species described was found in Africa, Ca. L. africanus (Laf), and the third, Ca. Liberibacter americanus (Lam) was found in Brazil. All three cause HLB in citrus. Beside the three citrus Liberibacters associated with HLB, three non-citrus Liberibacter species have been described. Ca. L. solanacearum (Lso), has been identified as the causal agent of serious diseases of potato (“Zebra chip”), tomato (“psyllid yellows”) and other solanaceous crops in the USA, Mexico, Guatemala, Honduras, and New Zealand (Hansen, et al., 2008; Abad, et al., 2009; Liefting, et al., 2009; Secor, et al., 2009). More recently, a different haplotype of Lso was found infecting carrots in Sweden, Norway, Finland, Spain and the Canary Islands (Alfaro-Fernandez, et al., 2012a, 2012b Munyaneza, et al., 2012a, 2012b; Nelson, et al., 2011). A fifth species of Liberibacter, Ca. L. europaeus (Leu) was recently found in the psyllid Cacopsylla pyri, the vector of pear decline phytoplasma. Finally, a sixth species of Liberibacter, Liberibacter crescens (Lcr), was recently characterized after isolation from diseased mountain papaya (Babaco). Except for Lcr, which is not known to be pathogenic, all other described Liberibacters are pathogenic and must be injected into living plant cells by specific insects. Furthermore, the pathogenic Liberibacters can only live within specific insect and plant cells; as obligate parasites, they do not have a free-living state—they are extreme biotrophs.

[0048] As an example of an ordinary biotroph, Xanthomonas citri, which causes citrus canker disease, invades the air spaces within a leaf and relies on inducing cell divisions in living cells in order to rupture the leaf surface (Brunings and Gabriel, 2003). Obviously, for biotrophs, host cell death would be expected to severely limit growth in planta. Liberibacters, which must live entirely within living phloem cells, would have no options if their phloem host cells simply died.

[0049] Mechanisms of Avoiding Programmed Cell Death

[0050] The genomes of Las and Lam differ (among other things) in that Las has 4 copies of peroxidase (Zhang, et al., 2011), and Lam has 2 (Wulff, et al., 2014). These are critical lysogenic conversion genes (conferring ability to colonize a plant or insect). With both Las and Lam (and likely Lso), these genes are amplified in copy number on a plasmid prophage to increase transcript copy number, and therefore, protein levels (Zhang, et al., 2011). Peroxidases degrade reactive oxygen species (ROS), like hydrogen peroxide. ROS production is one of the primary insect and plant host defenses against microbes. Since Liberibacters colonize living phloem cells and multiply within the plant cell cytoplasm, the ability to degrade ROS is a critical matter of survival to Liberibacter.

[0051] Peroxidases also suppress PCD, and since ROSS are strong pro-apoptotic inducers of PCD, particularly under certain nutrient deficiencies, the ability to absorb and degrade ROS is a matter of survival for the simple reason of keeping its host cell alive. Since Liberibacters can occupy a significant volume of cell cytoplasm, the ability to absorb and degrade ROS may be critical to suppressing citrus cell apoptosis. Lam causes a lot more disease than Las, but is always present in significantly lower titer (Lopes, et al., 2009). It is possible that the higher titer of Las is allowed by the activity of four peroxidases, which in combination with the higher titer allow a greater total absorption and inactivation of ROS, reducing the ROS induced proapoptotic effect on the citrus cell.

[0052] Plant pathogens provide a series of molecular signals that are detected by the plant and can trigger PCD. There are also built in brakes on the plant PCD system, and some of these have been exploited in transgenic plants in recent successful attempts to artificially suppress the cell death program. Avoidance of triggering PCD by biotrophs involves eliminating by evolution over time, to the greatest possible extent, production of “pathogen associated molecular patterns” (PAMPs). PAMPs are detected by plants as alien molecules and trigger strong defense responses called “innate immunity” in plants.

[0053] Innate immunity involves production of ROS, including superoxide anions, hydrogen peroxide, nitric oxide and free hydroxide radicals. ROS are produced in response to stress, and particularly stress causing mitochondrial damage (Portt, et al., 2011). ROS production is a first line of defense in animal and plant cells against biotic disease agents, such as bacteria and fungi. ROS is also one of the major signals that can trigger apoptosis. The genome sequences of both Las and Lam are highly reduced in size (both are 1.26 Mb) as compared with their closest Rhizobium relatives (genome sizes >6.4 Mb); significantly, both Las and Lam appear to lack flagella, a known PAMP, although both encode structural genes for flagellin. In addition, Lam lacks most of the genes needed to make lipopolysaccharide (LPS), a particularly potent PAMP and an important defensive barrier molecule integral to the outer membrane of most Gram negative bacteria.

[0054] Plant Regulators of Programmed Cell Death

[0055] Anti-apoptosis proteins have distinct and often specific anti-apoptotic effects when expressed in transgenic plants. Overexpression of many of the genes encoding these anti-apoptotic factors, including genes encoding a variety of chaperones, heat shock proteins and ROS scavengers, such as superoxide dismutase and peroxidases, can prevent apoptosis and are therefore cytoprotective from the effects of biotic and abiotic stresses. The Bcl-2 family is a well-characterized group of “core” anti-apoptotic regulators with strong general effects; these proteins inhibit production of Bax, and without affecting production of ROS. The Bcl-2 proteins, if overexpressed, broadly inhibit apoptosis in plants caused by both biotic and abiotic stresses. For example, expression of antiapoptotic genes bcl-xL (derived from chickens) and ced-9 (derived from Caenorhabditis elegans) blocked apoptosis in tomato and enhanced tolerance to viral induced necrosis and abiotic stress (Xu, et al., 2004). Similarly, expression of antiapoptotic genes bcl-2 (derived from humans), and op-iap (derived from baculovirus) blocked apoptosis in tobacco and conferred resistance to several necrotrophic fungal pathogens (Dickman, et al., 2001). No plant homologs of Bcl-2 have been identified to date.

[0056] There are also reports of an anti-apoptotic role for basic, but not acidic, pathogenesis related (PR) proteins, and many of these affect both biotic and abiotic stress induced responses. For example, a basic PR-1 protein from pepper, when overexpressed in transgenic tobacco, was shown to enhance heavy metal tolerance and to provide significantly higher resistance against Phytopthora, Ralstonia solanacearum and Pseudomonas syringae (Sarowar, et al., 2005). In all cases, necrosis in the abiotically- or biotically-stressed transgenic lines was greatly reduced compared to control lines. Clearly, the necrosis elicited by these pathogens is not a direct effect of pathogenic enzymes, but an indirect effect that triggers PCD that results in necrosis. Similarly, a basic PR-1 protein from grape, when overexpressed in transgenic tobacco, was shown to provide significantly reduced disease lesions in challenge inoculations with P. syringae (Zhijian, et al., 2010). Gilchrist and Lincoln (2011) reported that a grape PR-1 protein, when expressed in several transgenic host species, would suppress apoptosis, and in so doing, provide greatly improved tolerance to Xylella fastidiosa by blocking symptom induction, which in turn leads to a major reduction in total bacterial growth in planta. It should be noted that only a few members from the plant PR-1 family showed inducible expression by pathogens and possessed inhibitory activity against pathogens (Li, et al., 2011). For example, Arabidopsis and rice contain 22 and 39 PR-1 type genes, but only 1 and 2 members, respectively, have been found to be inducible by pathogen or insect attacks (Van Loon, et al., 2006).

[0057] BAG6 as a Regulator of Programmed Cell Death

[0058] The present disclosure is based in part on the inventors' unexpected discovery that the down regulation of citrus BAG6 gene in plants leads to an increased programmed cell death response to pathogen incursions.

[0059] In some embodiments, the present invention teaches that the down-regulation of BAG6 in plants leads to increased production of defense response protein NDR-1. This increased NDR-1 production in BAG6 silenced citrus is particularly significant, since elevated levels of this gene are known to increase basal resistance to both bacteria and fungi in Arabidopsis, and is suggested to be likely to increase resistance to Las in citrus (Lu, et al., 2013).

[0060] Thus in some embodiments, the present disclosure teaches the down-regulation of BAG6 genes for increasing plant resistance to pathogens. In some embodiments, the present disclosure teaches the down-regulation of BAG6 genes to increase resistance of biotrophic pathogens. In other embodiments, the disclosure teaches down-regulation of Bag6 genes to increase resistance to citrus greening disease caused by Las, Lam, and Lso.

[0061] In some embodiments, the present disclosure teaches the down-regulation of Carizzo-CiBAG6 (SEQ ID NO: 1). In some embodiments, the present disclosure teaches the down-regulation of Clementine-CiBAG6 (SEQ ID NO: 8). In some embodiments, the present disclosure teaches the down-regulation of Hamlin-CiBAG6 (SEQ ID NO: 9). In some embodiments, the present disclosure teaches the down-regulation of Valencia-CiBAG6 (SEQ ID NO: 10).

[0062] It has not been anticipated, expected, nor suggested in any plant system that silencing of a BAG6 gene, which results in suppressing production of the corresponding BAG6 protein, could increase levels of expression of downstream defense response proteins and be protective against pathogen infection or the effects of pathogen infections.

[0063] The disclosure here that silencing of BAG6 in citrus (and not overexpression, like the other examples presented herein) enhances production of defense response proteins and is protective from the effects of pathogen infection is therefore a surprising discovery.

[0064] For gene silencing purposes, both antisense RNA or RNAi may be used instead activate the RNAi pathway, although the processes result in differing magnitudes of the same downstream effects.

[0065] Controlled Regulation of BAG6 by Selective Spatial Temporal Silencing

[0066] By artificially removing one of the natural plant inhibitors of programmed cell death (PCD), specifically the one engaged when plants detect the presence of a pathogen, the present disclosure teaches effective defenses in response to pathogens in an unusually rapid manner. If this brake removal occurred generally in all plant tissues, it could have the undesirable result of making the plant more susceptible to necrotrophic pathogens.

[0067] However, if the PCD brake removal can be limited to just the phloem and nearby cells, then there should be no necrotrophic pathogen advantage, since necrotrophs do not attack phloem as a first target. Since Liberibacter and Xanthomonas are two bacterial genera that cause major plant diseases and are prototrophic, selective removal of a PCD brake control can confer immunity to Liberibacter, Xanthomonas and other biotrophic pathogens, including fungi, without undesired effects of increasing susceptibility to necrotrophs.

[0068] In some embodiments, the present disclosure teaches two RNA-based methods for selectively silencing the Bag6 gene in such a way that: 1) only the rootstock was transgenic, leaving the scion grafted onto the rootstock nontransgenic; 2) the RNA used was limited to phloem cells only (where Las resides), and 3) the RNA used in another case allowed limited migration to cells outside the phloem. The present disclosure teaches that the selective silencing methods of the present disclosure are effective at increasing basal defense responses and disease resistance in both the transgenic rootstock and the nontransgenic scion. This disclosure is of significant benefit to U.S. fruit crop producers, since the resulting plants became resistant to severe disease, had nontransgenic scions, and importantly for marketing, nontransgenic plants. In some embodiments, the term recombinant and transgenic are used interchangeably in this application.

[0069] As used herein, the term “brake” for programmed cell death can be used interchangeably with the term inhibition of PCD, or inhibitor of PCD.

[0070] Sequence Identity

[0071] In some embodiments, the present invention teaches the down-regulation of a CiBAG6 homolog or ortholog, in which said homolog or ortholog shares at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98.9%, 98.8%, 98.7%, 98.6%, 98.5%, 98.4%, 98.3%, 98.2%, 98.1%, 98%, 97.9%, 97.8%, 97.7%, 97.6%, 97.5%, 97.4%, 97.3%, 97.2%, 97.1%, 97%, 96.9%, 96.8%, 96.7%, 96.6%, 96.5%, 96.4%, 96.3%, 96.2%, 96.1%, 96%, 95.9%, 95.8%, 95.7%, 95.6%, 95.5%, 95.4%, 95.3%, 95.2%, 95.1%, 95%, 94.9%, 94.8%, 94.7%, 94.6%, 94.5%, 94.4%, 94.3%, 94.2%, 94.1%, 94%, 93.9%, 93.8%, 93.7%, 93.6%, 93.5%, 93.4%, 93.3%, 93.2%, 93.1%, 93%, 92.9%, 92.8%, 92.7%, 92.6%, 92.5%, 92.4%, 92.3%, 92.2%, 92.1%, 92%, 91.9%, 91.8%, 91.7%, 91.6%, 91.5%, 91.4%, 91.3%, 91.2%, 91.1%, 91%, 90.9%, 90.8%, 90.7%, 90.6%, 90.5%, 90.4%, 90.3%, 90.2%, 90.1%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, or 40% sequence identity with SEQ ID NO:1 or SEQ ID NO: 8 or SEQ ID NO:9 or SEQ ID NO. 10.

[0072] In some embodiments, the present invention the down-regulation of a CiBAG6 homolog or ortholog, in which said homolog or ortholog shares nucleotide sequence encoding an amino acid sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to SEQ ID NO:11 or SEQ ID No: 12 or SEQ ID NO. 13.

[0073] In some embodiments, the sequence identity of the CiBAG6 homolog or ortholog is calculated based on the alignment and comparison of each gene's nucleic acid sequence. In other embodiments, the sequence identity of the homolog or ortholog is calculated based on the alignment and comparison of the encoded protein.

[0074] In some embodiments, the alignments and sequence identity calculations of the present invention are calculated using ClustalOmega software with default settings.

[0075] In some embodiments, the CiBAG6 gene homologs and orthologs of the present disclosure will encode for conservative amino acid substitutions. Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Conservative substitutions generally maintain: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. Further information about conservative substitutions can be found, for instance, in Ben Bassat, et al. (J. Bacteriol., 169:751-757, 1987), O'Regan, et al. (Gene, 77:237-251, 1989), Sahin-Toth, et al. (Protein Sci., 3:240-247, 1994), Hochuli, et al. (Bio/Technology, 6:1321-1325, 1988) and in widely used textbooks of genetics and molecular biology. The Blosum matrices are commonly used for determining the relatedness of polypeptide sequences. The Blosum matrices were created using a large database of trusted alignments (the BLOCKS database), in which pairwise sequence alignments related by less than some threshold percentage identity were counted (Henikoff, et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992). A threshold of 90% identity was used for the highly conserved target frequencies of the BLOSUM90 matrix. A threshold of 65% identity was used for the BLOSUM65 matrix. Scores of zero and above in the Blosum matrices are considered “conservative substitutions” at the percentage identity selected. The following table shows exemplary conservative amino acid substitutions.

TABLE-US-00001 TABLE 1 BLOSUM substitution Matrix Highly Conserved Substitutions Very Highly- (from the Conserved Original Conserved Blosum90 Substitutions (from Residue Substitutions Matrix) the Blosum65 Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg Lys Gln, His, Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Arg, Asp, Gln, Glu, Lys, Ser, Thr His, Lys, Ser, Thr Asp Glu Asn, Glu Asn, Gln, Glu, Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, Arg, Asn, Asp, Glu, His, Lys, Met His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn, Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln, Arg, Asn, Gln, Glu, Tyr Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe, Val Leu Ile; Val Ile, Met, Phe, Ile, Met, Phe, Val Val Lys Arg; Gln; Glu Arg, Asn, Gln, Arg, Asn, Gln, Glu, Ser, Glu Met Leu; Ile Gln, Ile, Leu, Gln, Ile, Leu, Phe, Val Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu, Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys, Thr Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, Tyr Tyr Trp; Phe His, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala, Ile, Leu, Met, Thr

[0076] In some embodiments, orthologs and homologs of the present invention can have no more than 3, 5, 10, 15, 20, 25, 30, 40, 50, or 100 conservative amino acid changes (such as very highly conserved or highly conserved amino acid substitutions). In other examples, one or several hydrophobic residues (such as Leu, Ile, Val, Met, Phe, or Trp) in a variant sequence can are found to be replaced with a different hydrophobic residue (such as Leu, Ile, Val, Met, Phe, or Trp) to create a variant functionally similar to the disclosed an amino acid sequences encoded by the nucleic acid sequences of CiBAG6.

[0077] In some embodiments, variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced. In other embodiments, the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to the disclosed an amino acid sequences encoded by the nucleic acid sequences of CiBAG6.

[0078] Recombinant DNA Constructs

[0079] Another aspect of this invention provides a recombinant nucleic acid construct including a heterologous promoter operably linked to DNA including at least one segment of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 more contiguous nucleotides with a sequence of about 70% to about 100% identity with a segment of equivalent length of a DNA having a sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:8 and SEQ ID NO:9 and SEQ ID NO. 10 The recombinant nucleic acid constructs are useful in providing a plant having improved resistance to bacterial or fungal infections, e.g., by expressing in a plant a transcript of such a recombinant nucleic acid construct. The contiguous nucleotides can number more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater than 30, e.g., about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 760, about 770, about 780, about 790, about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, about 900, or greater than 900 contiguous nucleotides from SEQ ID NO:1 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10.

[0080] The contiguous nucleotides can number more than about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500 contiguous nucleotides from SEQ ID NO:1 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10.

[0081] In some embodiments, the recombinant nucleic acid construct of this invention is provided in a recombinant vector. By “recombinant vector” is meant a recombinant polynucleotide molecule that is used to transfer genetic information from one cell to another. Embodiments suitable to this invention include, but are not limited to, recombinant plasmids, recombinant cosmids, artificial chromosomes, and recombinant viral vectors such as recombinant plant virus vectors and recombinant baculovirus vectors.

[0082] RNA Interference

[0083] Sequence-selective, post-transcriptional inactivation of expression of a target gene can be achieved in a wide variety of eukaryotes by introducing double-stranded RNA (dsRNA) corresponding to the target gene, a phenomenon termed RNA interference (RNAi). RNAi occurs when an organism recognizes dsRNA molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments of 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length, called small interfering RNAs (siRNAs) or microRNAs (miRNAs). The siRNAs then diffuse or are carried throughout the organism, including across cellular membranes, where they hybridize to mRNAs (or other RNAs) and cause hydrolysis of the RNA. Most plant miRNAs show extensive base pairing to, and guide cleavage of their target mRNAs (Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol. 57, 19-53; Llave et al. (2002) Proc. Natl. Acad. Sci. USA 97, 13401-10406). In other instances, interfering RNAs may bind to target RNA molecules having imperfect complementarity, causing translational repression without mRNA degradation.

[0084] The term “RNAi” or “RNA interference” refers to the process of sequence-specific post-transcriptional gene silencing (e.g., in nematodes), mediated by double-stranded RNA (dsRNA). “DsRNA” refers to RNA that is partially or completely double stranded. Double stranded RNA is also referred to as small interfering RNA (siRNA), small interfering nucleic acid (siNA), microRNA (miRNA), and the like. In the RNAi process, dsRNA comprising a first (antisense) strand that is complementary to a portion of a target gene and a second (sense) strand that is fully or partially complementary to the first antisense strand is introduced into an organism (e.g., plants and/or crops), by, e.g., transformation, injection, spray, brush or immersion, etc. After introduction into the organism, the target gene-specific dsRNA is processed into relatively small fragments (siRNAs) and can subsequently become distributed throughout the organism, leading to a loss-of-function mutation having a phenotype that, over the period of a generation, may come to closely resemble the phenotype arising from a complete or partial deletion of the target gene.

[0085] This approach takes advantage of the discovery that siRNA can trigger the degradation of mRNA corresponding to the siRNA sequence. RNAi is a remarkably efficient process whereby dsRNA induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Hutvagner and Zamore (2002), Curr. Opin. Genet. Dev., 12, 225-232; Sharp (2001), Genes Dev., 15, 485-490).

[0086] The effects of RNAi can be both systemic and heritable in plants. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata. The heritability comes from methylation of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell. A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression. Detailed methods for RNAi in plants are described in David Allis et al (Epigenetics, CSHL Press, 2007, ISBN 0879697245, 9780879697242), Sohail et al (Gene silencing by RNA interference: technology and application, CRC Press, 2005, ISBN 0849321417, 9780849321412), Engelke et al. (RAN Interference, Academic Press, 2005, ISBN 0121827976, 9780121827977), and Doran et al. (RNA Interference: Methods for Plants and Animals, CABI, 2009, ISBN 1845934105, 9781845934101), which are all herein incorporated by reference in their entireties for all purposes.

[0087] The term “dsRNA” or “dsRNA molecule” or “double-strand RNA effector molecule” refers to an at least partially double-strand ribonucleic acid molecule containing a region of at least about 19 or more nucleotides that are in a double-strand conformation. The double-stranded RNA effector molecule may be a duplex double-stranded RNA formed from two separate RNA strands or it may be a single RNA strand with regions of self-complementarity capable of assuming an at least partially double-stranded hairpin conformation (i.e., a hairpin dsRNA or stem-loop dsRNA). In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as RNA/DNA hybrids. The dsRNA may be a single molecule with regions of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule.

[0088] DsRNA-mediated regulation of gene expression in plants is well known to those skilled in the art. See, e.g., WIPO Patent Application Nos. WO1999/061631A and WO1999/053050A, each of which is incorporated by reference herein in its entirety.

[0089] In some embodiments, an RNAi agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0090] In some embodiments the present invention also teaches expression vectors capable of producing inhibitor nucleic acid molecules. In some embodiments, the present invention teaches the use of RNA interference (RNAi) for the down-regulation of CiBAG6 genes, or homologs or orthologs of CiBAG6 genes. Thus in some embodiments the present invention teaches the expression of antisense, inverted repeat, small RNAs, artificial miRNA, or other RNAi triggering sequences.

[0091] In some embodiments the RNAi constructs of the present invention comprise sequences capable of triggering RNAi suppression of BAG6 genes, including nucleic acid fragments comprising sequence identities higher than about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to a BAG6 gene target region, such as those disclosed in SEQ ID NO: 1 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO. 10.

[0092] In some embodiments, the antisense or small RNA molecules are targeted to a section of the coding portion of the target gene. In other embodiments, the RNAi sequences of the present invention are targeted to the 5′ or 3′ untranslated regions (UTRs) of the target gene. In yet other embodiments, the RNAi sequences of the present invention are targeted to the promoter of the target gene. Methods of selecting sequence target regions for RNAi molecule design are described in more detail in (Fougerolles, et al., 2007; U.S. Pat. No. 7,732,593).

[0093] In some embodiments, the RNAi molecules of the invention may be modified at various locations, including the sugar moiety, the phosphodiester linkage, and/or the base. For example, in order to further increase the stability of the molecules in vivo, the 3′-end of the hairpin structure may be blocked by protective group(s). For example, protective groups such as inverted nucleotides, inverted abasic moieties, or amino-end modified nucleotides may be used. Inverted nucleotides may comprise an inverted deoxynucleotide. Inverted abasic moieties may comprise an inverted deoxyabasic moiety, such as a 3′,3′-linked or 5′,5′-linked deoxyabasic moiety (U.S. Patent Publication 2011/251258).

[0094] In some embodiments, the present invention also teaches the down-regulation of genes via antisense technology. In some embodiments, the present invention can be practiced using other known methods for down-regulating gene expression including T-DNA knockout lines, tilling, TAL-mediated gene disruption, transcriptional gene silencing, and site-directed methylations.

[0095] Application of RNAi Formulation/Treatment

[0096] Plant recombinant technology is the vehicle for delivering gene silencing of target genes, either endogenous plant target genes or target genes of a plant pest organism. In general, a plant is transformed with DNA that is incorporated into the plant genome, and when expressed produces a dsRNA that is complementary to a gene of interest, which can be an endogenous plant gene or an essential gene of a plant pest. Plant recombination techniques to generate transgene and beneficial plant traits require significant investments in research and development, and pose significant regulatory hurdles. Methods and formulations for delivering dsRNA into plant cells by exogenous application to exterior portions of the plant, such as leaf, stem, and/or root surfaces for regulation of endogenous gene expression are known in the art. See, e.g., U.S. Pat. No. 9,433,217, U.S. Patent Publication 2013/0047298, Chinese Patent No. 103748230B and Chinese Patent Publication CN101914540A, each of which is incorporated by reference herein in its entirety. Such methods and formulations represent a significant development for gene silencing technology using RNAi.

[0097] In some embodiments, the present invention teaches methods and formulations to topically apply exogenous RNA molecules to external tissue surfaces of plants. In some embodiments, the application exogenous RNA molecules, including dsRNA, siRNA, miRNA and aRNA, causes silencing of plant endogenous target genes or of the target genes of plant pests in the plant cells nearby the external tissue surfaces. In some embodiments, the application exogenous RNA molecules, including dsRNA, siRNA, miRNA and aRNA, causes silencing of plant endogenous target genes or of the target genes of plant pests in the plant cells that is located in a long distance from the external tissue surfaces.

[0098] In some embodiments, the present invention provides that applying dsRNA formulations (and/or treatments) by spray, brush, immersion of the dsRNA molecules, or other non-tissue invasive techniques, leads to absorption and assimilation of the exogenous RNA molecules into nearby or distant plant cells, thus causing endogenous and/or pest gene silencing. In some embodiments, pest genes are introduced into host plants by bacterial, fungal, or viral infection.

[0099] In some embodiments, the present invention teaches methods of repressing, preventing, eliminating, reducing, or otherwise ameliorating a bacterial or fungal infection of a plant comprising topical application of nucleic acid including DNA molecules as well as RNA molecules including dsRNA, siRNA, miRNA and aRNA

Example 1: Identification of Citrus CiBAG6 Homolog and Demonstration of Single Identical Homologs in Carrizo, Hamlin and Valencia

[0100] BLAST-P searches were performed using the predicted AtBAG6 protein sequence to identify a single citrus locus within the Clementine genome available online (http://www.phytozome.net/), referred to herein as CiBag6 (clementine0.9_012925m, coding region included in SEQ ID NO: 8). The DNA sequence of this locus, which is an AtBag6 homolog, was then used to determine how closely related it might be to other members of the Bag6 gene family in Clementine or Sweet Orange. Neither Clementine nor Sweet Orange varieties Hamlin or Valencia appeared to have any other sequences with high sequence similarity (>50%) over a stretch of more than 18 base pairs to AtBag6, other than a single CiBag6. This demonstrated not only that citrus did not carry multiple Bag6 homologs, but also that any region on CiBAG6 chosen as a probe or for silencing purposes would likely be specific to the target CiBag6 gene. Citrus mRNA was isolated from all three citrus varieties, and reverse transcriptase PCR (rtPCR) was performed using standard methods (Jiang, et al., 2012) to amplify a 930 bp exon fragment appropriate PCR primers. These DNA fragments were cloned into pGEM-T and three clones from each of the three citrus varieties were sequenced. The DNA sequences were 98% identical over the entire 930 bp stretch of CiBAG6 from Carrizo (SEQ ID NO: 1), and nearly 100% identical (1 bp difference between Carrizo and Hamlin or Valencia) over the first 200 bp of SEQ ID NO: 1.

Example 2: Antisense RNA (aRNA) Constructs with CaMV Promoter

[0101] The 828 bp region of Carrizo CiBAG6 (SEQ ID NO: 1) from the ATG start codon to the coding end of the fragment (position 103-930 of SEQ ID NO: 1) was cloned in the antisense direction into pIPG973 (WO 2013/032985 A1), which carries a single CaMV promoter. The CiBAG6 antisense fragment was then transcriptionally fused with 516 bp (in the sense orientation) from the uidA gene from pCAMBIA2301, forming pIPG1150 (see Table 2 and FIG. 1; SEQ ID NO: 2). This clone was used to transform Carrizo seedlings, resulting in multiple transformation events confirmed by PCR. Each transgenic event was numbered and expression of the CiBAG6 antisense construct was confirmed by rtPCR, using RNA extracted from each confirmed transgenic Carrizo event. Each confirmed expressing transgenic event was then evaluated to determine if CiBAG6 in Carrizo was actually silenced or not. DNA primers were designed based on the CiBAG6 transcript (cDNA) sequence, but outside of the regions cloned and used for silencing purposes. From FIG. 2, it may be seen that the control Carrizo exhibited relative CiBAG6 expression levels that were much higher than any of the silenced lines (labeled 1150-12, -34, -39, -40, -44 and -45). These results clearly demonstrated that the aRNA construct in pIPG1150 was effective in silencing CiBAG6.

[0102] To determine if the CiBAG6 silenced lines were affected in their response to a known PAMP elicitor (Shi, et al., 2013, 2014), levels of expression of each of the three defense response genes were examined by qPCR. PCR primers known to be useful in examining these responses in citrus were used (Pathogenesis Related 1 or PR1; Enhanced Disease Susceptibility 1 or EDS1, and Non race-specific Disease Resistance 1 or NDR1) (Shi, et al., 2014).

[0103] To elicit citrus plant defense responses, Las flagellin 22 (Las flg22) was used as an elicitor and proxy for inoculation with Las, using 10 μM of the Las flg22 elicitor dissolved in water (Shi, et al., 2013). Defense responses in nontransgenic (NT) Carrizo inoculated with water (NT CK) was compared with NT Carrizo and CiBAG6 silenced Carrizo lines, all inoculated with Las flg22. Controls also included determination of the levels of PR1, EDS1 and NDR1 in both NT Carrizo and CiBAG6 silenced lines that were not inoculated with Las flg22 as controls. From FIG. 3, the expression levels of NDR1 in line 1150-12 (silenced with pIPG1150) was particularly striking, since it was significantly higher both 24 and 48 hours after elicitation (>10× higher) than in NT controls. The increased expression of NDR-1 is significant since elevated levels of this gene are known to increase basal resistance to both bacteria and fungi in Arabidopsis, and is suggested to be likely to increase resistance to Las in citrus (Lu, et al., 2013). This result clearly demonstrated that silencing of CiBAG6 in Carrizo occurred, accompanied by increased expression of defense response genes, and would likely increase resistance in Carrizo to Las (source organism for flg22), and likely to additional biotrophic bacteria and fungi.

TABLE-US-00002 TABLE 2 Summary of CiBAG6 silencing constructs used. pIPG1150 CaMV promoter; aRNA (no movement Example 2 leader) pIPG1157 CaMV promoter; RNAi construct with Example 4 1848 bp GUS loop pIPG1159 AtSuc2 promoter; RNAi construct with Example 5 1848 bp GUS loop pIPG1160 AtSuc2 promoter with movement leader; Example 3 aRNA pIPG1174 CaMV promoter; RNAi construct with Example 6 190 bp intron

Example 3: Antisense RNA (aRNA) Constructs with AtSuc2 Promoter

[0104] As in Example 2, the 828 bp region of Carrizo CiBAG6 (SEQ ID NO: 1) from the ATG start codon to the coding end of the fragment (position 103-930 of SEQ ID NO: 1) was cloned in the antisense direction into pIPG980 (WO 2013/032985 A1), which carries a single CaMV promoter, forming pIPG1160 (see Table 2 and FIG. 1; SEQ ID NO: 3). A potential movement leader sequence from Arabidopsis thaliana, flowering locus T (FT) from nucleotide position 326 to nucleotide 429 in GenBank: GQ395494.1 (SEQ ID NO: 7) was transcriptionally fused in the sense orientation at the 5′ end of the aRNA construct utilized in construction of pIPG1160 (FIG. 1).

[0105] As provided in Example 2, each transgenic event was confirmed by PCR and expression of the CiBAG6 antisense construct was confirmed by rtPCR, using RNA extracted from each confirmed transgenic Carrizo event. Each confirmed expressing transgenic event was then evaluated to determine if CiBAG6 in Carrizo was actually silenced or not. From FIG. 2, it may be seen that the control Carrizo exhibited relative CiBAG6 expression levels that were much higher than 1160-13, again clearly demonstrated that the pIPG1160 construct, this time using the AtSuc2 promoter and providing a potential long distance transport leader, was effective in silencing CiBAG6.

[0106] Again to determine if the CiBAG6 silenced lines were affected in their response to the known PAMP elicitor Las flg22, levels of expression of each of the three defense response genes PR1, EDS1 and NDR1 were examined by qPCR as in Example 2. From FIG. 3, the expression levels of NDR1 in line 1160-13 (silenced with pIPG1160) was significantly higher (5× higher) 24 hours after elicitation than in NT controls. Expression levels of EDS1 were 1.7× higher 24 hours after elicitation than in NT controls. By 48 hours, the effects from silencing due to this construct were not significantly different from the controls. Overall, the effects on silencing of CiBAG6 by this construct were less than the effects observed using pIPG1150, and likely was due to the expected reduced promoter strength and tissue specificity of the AtSuc2 promoter used to construct pIPG1160, compared to the CaMV promoter used to construct pIPG1150 (Table 2). This result clearly demonstrated that silencing of CiBAG6 in Carrizo occurred, accompanied by increased expression of defense response genes, and would likely increase resistance in Carrizo at least to Las (source organism for flg22), and likely to additional biotrophic bacteria and fungi. This result also indicated that the AtSuc2 promoter might be useful in preventing unintended consequences of too much silencing of CiBAG6 or overly generalized silencing of CiBAG6.

Example 4: RNAi Constructs with CaMV Promoter and GUS Loop

[0107] For silencing purposes, a region of 200 bp in length was selected from the first exon, based on a high level of sequence identity (nearly 100% as explained in Example 1) among the three CiBAG6 cDNA clones obtained from citrus rootstock cultivar Carrizo citrange (Citrus sinensis L. Osbeck x Poncirus trifoliata L. Raf.), and from sweet orange cultivars (Citrus sinensis) Hamlin and Valencia. Homology with Hamlin and Valencia was important in order to be able to graft transmit the silencing RNA used to nontransgenic Hamlin and Valencia scions (to be grafted later after regeneration of transgenic Carrizo rootstock), with the expectation that silencing would occur in the grafted scion.

[0108] The first 200 bp of Carrizo CiBAG6 (SEQ ID NO: 1) was cloned in sense orientation into pIPG980 (WO 2013/032985 A1), which carries a single CaMV promoter, followed by a 1,848 bp fragment from the uidA gene that forms a GUS loop, followed by the same 200 bp CiBAG6 sequence, but in an antisense orientation, forming pIPG1157 (see Table 2 and FIG. 1; SEQ ID NO: 4). Upon expression, each mRNA formed from pIPG1157 will anneal to form a stem-loop structure. This clone was used to transform Carrizo seedlings, resulting in multiple transformation events confirmed by PCR.

[0109] As provided in Example 2, expression of each transgenic event was confirmed by rtPCR, using RNA extracted from each confirmed transgenic Carrizo event. Each confirmed expressing transgenic event was then evaluated to determine if CiBAG6 in Carrizo was actually silenced or not. From FIG. 2, it may be seen that the control Carrizo exhibited relative CiBAG6 expression levels that were much higher than the three 1157 transgenic events examined (1157-6, 1157-9 and 1157-16), again clearly demonstrated that the pIPG1157 construct, this time using RNAi rather than aRNA for silencing, was effective in silencing CiBAG6.

[0110] Again, to determine if the CiBAG6 silenced lines were affected in their response to the known PAMP elicitor Las flg22, levels of expression of each of the three defense response genes PR1, EDS1 and NDR1 were examined by qPCR as in Examples 2 and 3. From FIG. 3, the expression levels of NDR1 in line 1157-9 (silenced with pIPG1157) was particularly striking, since it was significantly higher at 24 hours after elicitation (9.6× higher) and by 48 hours had climbed to 30× higher after elicitation than in NT controls. Both EDS1 and PR1 had climbed to 4.8× and 25× higher, respectively, than NT controls elicited with the same amount of Las flg22. Again, the increased expression of NDR-1 is significant since elevated levels of this gene are known to increase basal resistance to both bacteria and fungi in Arabidopsis, and is suggested to be likely to increase resistance to Las in citrus (Lu, et al., 2013). This result clearly demonstrated that silencing of CiBAG6 in Carrizo occurred using an RNAi construct driven by the same promoter as used the aRNA construct in Example 2 (pIPG1150), but accompanied by even greater increased expression of defense response genes than seen using aRNA, and would likely strongly increase resistance in Carrizo to Las (source organism for flg22), and likely to additional biotrophic bacteria and fungi.

Example 5: RNAi Constructs with AtSuc2 Promoter and GUS Loop

[0111] A construct identical to that used in Example 4 was created, except that a single AtSuc2 promoter was substituted for the single CaMV promoter of pIPG1157, to form pIPG1159 (see Table 2 and FIG. 1; SEQ ID NO: 5). This clone was used to transform Carrizo seedlings, resulting in multiple transformation events confirmed by PCR.

[0112] As provided in Example 2, expression of each transgenic event was confirmed by rtPCR, using RNA extracted from each confirmed transgenic Carrizo event. Each confirmed expressing transgenic event was then evaluated to determine if CiBAG6 in Carrizo was actually silenced or not. From FIG. 2, it may be seen that the control Carrizo exhibited relative CiBAG6 expression levels that were much higher than the seven 1159 transgenic events examined (1159-1, 1159-2, 1159-5. 1159-6, 1159-9, 1159-16 and 1159-21), again clearly demonstrated that the pIPG1159 construct, again using RNAi rather than aRNA for silencing, but RNAi gene expression driven by the AtSuc2 promoter, was effective in silencing CiBAG6.

[0113] Again, to determine if the CiBAG6 silenced lines were affected in their response to the known PAMP elicitor Las flg22, levels of expression of each of the three defense response genes PR1, EDS1 and NDR1 were examined by qPCR as in Examples 2, 3 and 4. From FIG. 3, the expression levels of NDR1 in line 1159-6 (silenced with pIPG1159) was particularly striking, since it was significantly higher than controls at 24 hours after elicitation (19× higher), but as with the aRNA construct operationally driven by the AtSuc2 promoter in Example 3 (pIPG 1160-13), by 48 hours the effect was greatly reduced (to 7× higher expression of NDR-1), but still significantly different from all controls. Unlike the antisense construct, however, expression levels of both EDS1 and PR1 of the 1159-6 line was 7.6× and 6.6× higher, respectively, than NT controls at 24 hours after elicitation by Las flg22. This result clearly demonstrated that silencing of CiBAG6 in Carrizo occurred using an RNAi construct driven by the same promoter as used the aRNA construct in Example 3 (pIPG1160), but accompanied by even greater increased expression of defense response genes than seen using aRNA, and would likely strongly increase resistance in Carrizo to Las (source organism for flg22), and likely to additional biotrophic bacteria and fungi. As with Example 3 (pIPG1160) the effect on expression of elicited defense response genes was stronger at 24 hours than 48 hours after elicitation, indicating that the AtSuc2 promoter may provide a better regulated response.

Example 6: RNAi Constructs with 35S Promoter and Intron Loop

[0114] The use of introns has shown to be more effective than random stretches of DNA loops in the creation of RNAi constructs (Stoutjesdijk, et al., 2002). Therefore the 190 bp catalase intron was PCR amplified from pIPG973 and used to replace the GUS fragment 2 loop used in pIPG1157 to form pIPG1174 (see Table 2 and FIG. 1; SEQ ID NO: 6). This clone was used to transform Carrizo seedlings, resulting in multiple transformation events confirmed by PCR.

[0115] As provided in Example 2, expression of each transgenic event was confirmed by rtPCR, using RNA extracted from each confirmed transgenic Carrizo event. Each confirmed expressing transgenic event was then evaluated to determine if CiBAG6 in Carrizo was actually silenced or not. From FIG. 2, it may be seen that the control Carrizo exhibited relative CiBAG6 expression levels that were much higher than the six 1174 transgenic events examined (1174-1, 1174-11, 1174-12, 1174-29, 1174-30 and 1174-35), again clearly demonstrating that the pIPG1174 construct, again using RNAi rather than aRNA for silencing, but the catalase intron as a loop, was effective in silencing CiBAG6.

[0116] Again, to determine if the CiBAG6 silenced lines were affected in their response to the known PAMP elicitor Las flg22, levels of expression of each of the three defense response genes PR1, EDS1 and NDR1 were examined by qPCR as in Examples 2, 3, 4 and 5. From FIG. 3, the expression levels of NDR1 in line 1174-29 (silenced with pIPG1174) was significantly higher than controls at both 24 and 48 hours after elicitation (6-7.7× higher). Consistent with the other silencing lines driven by the CaMV promoter, the effect is almost as pronounced at 48 hours as at 24 hours after elicitation by Las flg22. This result clearly demonstrated that silencing of CiBAG6 in Carrizo occurred using an RNAi construct driven by the same promoter as used in the RNAi construct in Example 4 (pIPG1157).

Example 7: Movement of Both RNAi and aRNA from Transgenic Rootstock to Nontransgenic Citrus Scion

[0117] Thirty-two nontransgenic mature Hamlin (sweet orange) citrus scions were grafted onto silenced, transformed Carrizo rootstock lines expressing pIPG1150, pIPG1159, pIPG1160 or pIPG1174. Four grafted plants representing each of the four different silencing constructs were tested in these assays. The quality of the RNA extracted from each line was verified by PCR to ensure uniformity. Using two of the constructs (pIPG1150, an aRNA construct, and pIPG1159 an RNAi construct), the silencing signal failed to move. One aRNA construct that failed to move, pIPG1150, was engineered to move moderately well in construct pIPG1160 with addition of the movement leader (SEQ ID NO. 7). One RNAi construct (expressed from IPG1174) moved very efficiently into nontransgenic Hamlin sweet orange scions.

Example 8: CiBAG6-Silenced Lines were Highly Resistant to Las and Conferred this Resistance Long Distance to Nontransgenic Scions Heavily Infected with Las

[0118] Las infected citrus (both C. sinensis sweet orange and C. paradisi grapefruit) exhibiting symptoms of HLB and also tested by real time PCR and showing strong positive PCR signals for Las bacterial infection were approach grafted to non-infected transgenic CiBAG6-silenced lines. After several months, many of the transgenic CiBAG6 silenced lines became PCR positive, indicating Las bacterial infection through the graft union. After exhibiting at least two positive PCR tests over a period of two months, such CiBAG6-silenced lines were considered to be infected by Las and the graft union from the infected source tree was severed. In most cases where the transgenic Carrizo became infected, the Las-infected scion remained intact from the approach graft union, leaving the infected scion attached to an infected but CiBAG6 silenced Carrizo rootstock.

[0119] In many cases, the CiBAG6 silenced lines never became infected, while the nontransgenic, infected scion remained infected and still attached to its infected rootstock through the approach graft union. After six months, the infected nontransgenic tree was severed from the transgenic Carrizo rootstock, again leaving the infected nontransgenic scion still attached to a noninfected but CiBAG6 silenced Carrizo rootstock.

[0120] After at least one full year of challenge inoculation, 4 out of 4 transgenic Carrizo rootstocks transformed with pIPG1150, 2 out of 3 transgenic Carrizo rootstocks transformed with pIPG1157, 2 out of 4 transgenic Carrizo rootstocks transformed with pIPG1159, and 5 out of 5 transgenic Carrizo rootstocks transformed with pIPG1160 were either cured of Las or had failed to become infected by Las. In the cases of pIPG1150 and pIPG1160, the attached, previously infected, nontransgenic scions had become cured of Las. These results clearly demonstrated that silencing of CiBAG6 in a rootstock conferred immunity from, or strong resistance to, infection by Las, depending upon the level of efficiency of the silencing. Furthermore, these results demonstrated long distance movement of the silencing signal from the transgenic rootstock, across a graft union, to provide immunity or strong resistance to nontransgenic citrus, to the level of curing of Las. Again, this curing appeared to depend upon the efficiency of the asRNA or siRNA construct in suppressing CiBAG6. Finally, these results are consistent with reports that the efficiency of RNAi increases with the length of the clones (asRNA or siRNA) used to create the dsRNA for RNAi purposes.

Example 9: Challenge Inoculations with Las of CiBAG6-Silenced Rootstock with Grafted Non-Transgenic Hamlin or Valencia Mature Scions

[0121] Grafted mature, nontransgenic Hamlin shoots from Example 7 that were micrografted onto transgenic Carrizo lines and demonstrated to exhibit mobile silencing will be challenge inoculated with Las as in Example 8.

Example 10. Silenced CiBAG6 Lines Exhibit Both Heat and Cold Tolerance

[0122] Two different transgenic lines (1150-53 and 1174-57) and two nontransgenic lines (NT) were put under high constant temperature stress (42° C. for 6 days); these levels of heat treatment are effective in curing citrus of HLB. Although all plants survived these treatments, the control plants were clearly damaged, partially defoliated and recovered slowly, while the two silenced Carrizo lines exhibited almost no damage or defoliation and needed no recovery period. Cold tolerance was also tested with two lines (−1° C.) for 5 days. Both the control and silenced plants exhibited cold damage (wilted leaves) from the treatment. After 7 days of recovery, the silenced line clearly had recovered fully with little leaf loss, while the control plant was nearly completely defoliated. Suppression of the target apoptotic regulatory gene CiBAG6 appears to strongly enhance the citrus plant's resistance to abiotic stress, and may provide citrus growers with a means to protect newly planted trees from frost damage.

Example 11. CiBAG6 Expression was Suppressed in Nontransgenic Citrus Leaves after Treating the Roots with an Aqueous Solution of CiBAG6 dsRNA or siRNA within 48 Hours of Such Treatment

[0123] In order to determine if exogenously supplied dsRNA or siRNA could be applied directly to citrus trees in order to silence CiBAG6 and avoid the use of transgenic rootstocks to achieve the desired effects, citrus rootstock Carrizo seedlings were germinated and grown hydroponically, the roots rinsed and immersed in a water solution containing either 224 μg of long dsRNA (800 nt in length) or 19.6 μg of siRNA (ca. 23 nt in length). The dsRNA used was PCR amplified from Carrizo citrange using primers designed from positions 637-1434 of SEQ ID NO. 10) and transcribed in vitro. The siRNA used was formed after digestion of the 800 nt Carrizo dsRNA product with RNAseIII from New England Biolabs.

[0124] Care was taken to protect the leaves from exposure to the dsRNA and siRNA root treatments. RNA was extracted from the leaves of the treated plants 48 hrs after treatment of the roots. Real time quantitative reverse transcription PCR (qRT-PCR) was used to determine levels of expression of CiBAG6 in all samples, using primers well outside of the region of CiBAG6 used for the RNA treatments. qRT-PCR levels were normalized against the control reaction (set at 100%), and relative expression levels of CiBAG6 in both treatments determined. As documented in FIG. 4, both dsRNA and RNAi moved long distances from the roots to the shoots and into the leaves within 48 hrs, as evidenced by the suppression of expression of CiBAG6 in the leaves.

Example 12. CiBAG6 Expression was Suppressed in Nontransgenic Citrus Leaves after Spraying the Leaves with an Aqueous Solution of CiBAG6 dsRNA or siRNA

[0125] In order to determine if exogenously supplied dsRNA or siRNA could be applied directly to citrus trees by spray treatment in order to silence CiBAG6 and avoid the use of root drench or transgenic rootstocks to achieve the desired effects, citrus rootstock Carrizo seedlings were germinated and grown hydroponically. Citrus leaves sprayed with an aqueous solution containing either 16 μg of long dsRNA (800 nt in length) or 1.4 μg of siRNA (ca. 23 nt in length), made as described in Example 11.

[0126] The spray concentration used was 2.8 μg/ml siRNA and 32 μg/ml dsRNA; plant weight range was 0.5 to 0.9 grams, so the amount applied was in the range of 2-20 μg/g plant tissue. RNA was extracted from the leaves of the treated plants 80 hrs after treatment of the leaves, and qRT-PCR used to determine levels of expression of CiBAG6 in all samples as described in Example 11. Clearly, from FIG. 5 it may be seen that both dsRNA and RNAi sequences from effectively suppressed expression of CiBAG6, and using levels of dsRNA and siRNA 20× less than those used for the root treatments.

Example 13. Treatment Using Either Long dsRNA or siRNA of CiBAG6 Results in Killing of Ca. Liberibacter asiaticus (Las) Cells

[0127] An RNA-based, live cell assay was performed in a field trial using commercially grown citrus. Four year old grapefruit trees planted in a commercial grove that was 100% infected (all trees in the grapefruit block were infected) with Las were used for the field trial. Selected trees were injected with an aqueous formulation containing either 720 μg of long dsRNA (800 nt in length) or 112.5 μg of siRNA (ca. 23 nt in length) made as described in Example 11.

[0128] Plant weight of these field grown trees was estimated to average 26 kg, and therefore the amount injected was in the range of 4.3 to 28 mg/kg plant tissue. Control plants were mock-injected with the aqueous carrier solution. Both DNA and RNA were extracted from leaf samples taken from the field, one month after treatments were applied. Not surprisingly, negative but small effect was noted on the apparent titer of Las as determined by DNA based qRT-PCR reactions one month after treatment. It is well understood that bacterial DNA, including DNA from dead bacteria killed by a treatment, is highly persistent in plants, and that since RNA degrades more rapidly after cell death, is a much more sensitive real time indicator of anti-bacterial therapies (Fittipaldi et al, 2012). Therefore, RNA based assays have been recently adapted for evaluation of treatments designed to cure Las infections of citrus (Gardner et al. 2016). To this end, an RNA based live cell assay for Las was utilized for initial evaluation of these field trials, using methodology similar to that described in Example 11, except that expression of the Las bacterial gene, SC2_gp095 peroxidase was compared to the standard plant housekeeping gene cox1.

[0129] The effects of CiBAG6 siRNA and dsRNA treatments of grapefruit trees on the transcriptional activity of Las prophage encoded locus SC2_gp095 peroxidase was determined. Plant cox1 was used to normalize expression As clearly observed in FIG. 6, SC2 peroxidase gene expression, an indicator of living Las cells, was suppressed after single trunk injection treatment of commercially grown, Las-infected grapefruit trees (4 years old) in a field trial using dsRNA or siRNA in formula 224, within one month post treatment. This result demonstrated that such treatments killed Las cells in commercially grown citrus trees in the field and confirmed the data provided in Example 8.

[0130] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0131] All publications, patents, patent publications, and nucleic acid and amino acid sequences cited are incorporated by reference herein in their entirety for all purposes.

[0132] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0133] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

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