RNA-BASED BIOCONTROL METHODS TO PROTECT PLANTS AGAINST PATHOGENIC BACTERIA AND / OR PROMOTE BENEFICIAL EFFECTS OF SYMBIOTIC AND COMMENSAL BACTERIA

Abstract

The present invention pertains to the field of agriculture. The invention relates to a method to inhibit gene expression in bacteria, which is referred to here as Antibacterial Gene Silencing (AGS). In particular embodiments, the method is used to protect plants against pathogenic bacteria by targeting pathogenicity factors and/or essential genes in a sequence-specific manner via small non-coding RNAs. The method can also be used to enhance beneficial effects and/or growth of plant-associated symbiotic or commensal bacteria. The invention involves either the generation of stable transgenic plants that constitutively express antibacterial small RNAs or, alternatively, the exogenous delivery of these small RNA entities onto plants, either in the form of RNA extracts or embedded into plant extracellular vesicles (EVs), which were found to be effective in reducing bacterial pathogenicity. The invention also describes a method to identify in a rapid, reliable and cost-effective manner, small RNAs that possess antibacterial activity and that can be further exploited for RNA-based biocontrol applications to confer plant protection against pathogenic bacteria. In addition, the latter approach is instrumental to rapidly characterize any genes from any bacterial species.

Claims

1. An in vitro method for inhibiting the expression of at least one gene in a target bacterial cell, said method comprising the step of contacting said target bacterial cell with small RNAs, or with compositions containing small RNAs.

2. The method of claim 1, wherein said small RNA is a siRNA or a miRNA inhibiting specifically the expression of a bacterial essential gene or a bacterial virulence gene or an antibacterial resistance gene of a phytopathogenic bacterium.

3. The method according to claim 1 or 2, wherein said target bacterial cell is a cell from a phytopathogenic bacteria which is for example chosen among: Ralstonia solanacearum, Xanthomonas oryzae pathovars, Xanthomonas campestris pathovars, Xanthomonas axonopodis pathovars, Xanthomonas euvesicatoria pathovars, Xanthomonas hostorum pathovars, Pseudomonas syringae pathovars, Pseudomonas viridiflava pathovars, Pseudomonas savastonoi pathovars, Candidatus liberibacter asiaticus, Candidatus liberibacter solanacearum, Acidovorax citrulli, Acidovorax avenae pathovars, Pectobacterium atrosepticum pathovars, Pectobacterium carotovorum pathovars, Pectobacterium sp., Agrobacterium tumefaciens, Dickeya (dadantii and solani), Erwinia amylovora, Clavibacter michiganensis (michiganensis and sepedonicus), Xylella fastidiosa, Pectobacterium (carotovorum and atrosepticum), Streptomyces scabies, Phytoplasma sp., and Spiroplasma sp.

4. The method according to claim 1 or 2, wherein said target bacterial cell is a cell from a plant beneficial bacteria, which is for example chosen among: Bacillus (e.g. Bacillus subtilis), Pseudomonas (e.g. Pseudomonas putida, Pseudomonas stuzeri, Pseudomonas fluorescens, Pseudomonas protegees, Pseudomonas brassicacearum), Rhizobia (e.g. Rhizobium meliloti), Burkholderia (e.g. Burkholderia phytofirmans), Azospirillum (e.g. Azospirillum lipoferum), Gluconacetobacter (e.g. Gluconacetobacter diazotrophicus), Serratia (e.g. Serratia proteamaculans), Stenotrophomonas (e.g. Stenotrophomonas maltophilia), Enterobacter (e.g. Enterobacter cloacae).

5. The method according to any of claims 1 to 4, wherein said small RNAs have a size comprised between 15 and 30 base pairs.

6. In vitro use of a small RNA or of a composition comprising small RNAs, for inhibiting the expression of at least one gene in a target bacterial cell, wherein said target bacterial cell is contacted directly with said small RNA or with said composition.

7. The in vitro use of claim 6, wherein said small RNA is single-stranded or double-stranded.

8. The in vitro use of claim 6, wherein said composition contains plant extracts obtained from producer plant cells that express at least one long dsRNA that exhibit sequence homologies with at least one gene of said bacterial cell.

9. The in vitro use of claim 8, wherein said composition contains total RNAs, or total small RNAs, or apoplastic fluids, or extracellular vesicles, or extracellular free small RNAs, from said plant cells.

10. The in vitro use of claim 8 or claim 9, wherein said producer plant cells are cells from plants chosen in the group consisting of: Tobacco (e.g. Nicotiana benthamiana, Nicotiana tobaccum); Taro (Colocasia esculenta); Giger (Zingiber officinale), Arabidopsis (e.g. Arabidopsis thaliana); Tomato (e.g. Lycopersicon esculentum or Solanum lycopersicum); Potato (Solanum tuberosum); Rice (Oryza sativa); Maize (Zea mays); Barley (Hordeum vulgare); Wheat (e.g. Triticum aestivum, Triticum durum), Cottonseed, Cotton, Bean, Banana/plantain, Sorghum, Pea, Sweet potatoes, Soybeans, Cabbage, Cassava, Onion, Melon, Oats, Peanut, Sunflower, Palm oil, Rye, Citrus, Wheat, Peppers, Yams, Olives, Grapes, Sesame, Sugarcane, Sugarbeet, Pea and Coffee, Orange trees, Apple trees, Citrus trees, Olive trees Chrysanthemum, Impatiens, Geranium, Pelargonium, Phlox, Rhododendron anthurium spp, Rose tree, Curcumas, Anthuriums, Begonia, Hibiscus rosa-sinensis, Amaryllis, Calla, Cyclamen, and Dracaena.

11. The in vitro use according to any one of claims 6-10, wherein said small or long RNA inhibits at least one gene encoding a virulence factor or an essential gene or an antibacterial resistance gene if said bacterial cell is pathogenic, or inhibits at least one gene encoding a repressor of growth or a negative regulator of a pathway that is useful for the host if said bacterial cell is beneficial for the host.

12. A method for treating target plants against bacterial infection, said method comprising the step of introducing into a cell of said target plant a long dsRNA molecule targeting specifically at least one virulence bacterial gene or at least one essential bacterial gene or at least one antibacterial resistance gene.

13. A method for treating target plants against bacterial infection, said method comprising the step of delivering small RNAs inhibiting at least one essential or virulence or antibacterial resistance bacterial gene, or a composition containing such small RNAs, on target plant tissues prior to and/or after bacterial infection.

14. The method of claim 13, wherein said composition contains plant extracts obtained from plant cells expressing at least one long dsRNA that is specific to at least one virulence or essential or antibacterial resistance bacterial gene.

15. The method of any one of claim 13-14, wherein said composition contains apoplastic fluids, or extracellular vesicles, or extracellular free small RNAs recovered from said plant extracts.

16. The method of any one of claim 13-15, wherein said composition is a liquid sprayable composition.

17. A recombinant plant RNA virus triggering the in planta production of small RNAs that can inhibit the expression of at least one bacterial gene target.

18. A DNA recombinant vector comprising a DNA polynucleotide sequence encoding long RNAs inhibiting the expression of at least one essential, virulence or antibacterial resistance bacterial gene, wherein said polynucleotide sequence is expressible in plant cells.

19. A transgenic plant comprising the recombinant plant RNA virus of claim 17, or the recombinant vector of claim 18.

20. The transgenic plant of claim 19, stably or transiently expressing a DNA polynucleotide sequence encoding long RNAs inhibiting the expression of at least one essential bacterial gene, virulence bacterial gene or antibacterial resistance gene.

21. The transgenic plant of claim 19, stably or transiently expressing functional small RNAs inhibiting the expression of at least one essential bacterial gene, virulence bacterial gene or antibacterial resistance gene.

22. A phytotherapeutic composition containing a significant amount of small RNAs inhibiting the expression of an essential bacterial gene, or of a virulence bacterial gene or of an antibacterial resistance bacterial gene.

23. The phytotherapeutic composition of claim 22, containing small RNAs that are contained within total RNA extracts, or extracellular vesicles, or apoplastic fluids or extracellular free small RNA extracts from the transgenic plant of claim 19.

24. The phytotherapeutic composition of claim 22 or claim 23, further containing a bactericidal compound.

25. A combination product comprising the phytotherapeutic composition as defined in claim 22 or claim 23, and a bactericidal compound.

26. The use of the phytotherapeutic composition of claim 22-24, or of the combination product of claim 25, for inhibiting or preventing the growth or pathogenicity of bacteria on target plants.

27. The use of claim 26, wherein said phytopathogenic bacteria are chosen among: Ralstonia solanacearum, Xanthomonas oryzae pathovars, Xanthomonas campestris pathovars, Xanthomonas axonopodis pathovars, Xanthomonas euvesicatoria pathovars, Xanthomonas hostorum pathovars, Pseudomonas syringae pathovars, Pseudomonas viridiflava pathovars, Pseudomonas savastonoi pathovars, Candidatus liberibacter asiaticus, Candidatus liberibacter solanacearum, Acidovorax citrulli, Acidovorax avenae pathovars, Pectobacterium atrosepticum pathovars, Pectobacterium carotovorum pathovars, Pectobacterium sp., Agrobacterium tumefaciens, Dickeya (dadantii and solani), Erwinia amylovora, Clavibacter michiganensis (michiganensis and sepedonicus), Xylella fastidiosa, Pectobacterium (carotovorum and atrosepticum), Streptomyces scabies, Phytoplasma sp., and Spiroplasma sp.

28. The use of claim 26 or 27, wherein said target plants are chosen among Rice, Maize, Barley, Cottonseed, Cotton, Bean, Banana/plantain, Sorghum, Pea, Sweet potatoes, Soybeans, Cabbage, Cassava, Potato, Tomato, Onion, Melon, Oats, Peanut, Sunflower, Palm oil, Rye, Citrus, Wheat, Peppers, Yams, Olives, Grapes, Taro, Tobacco, Sesame, Sugarcane, Sugarbeet, Pea and Coffee, Orange trees, Apple trees, Citrus trees, Olive trees, Chrysanthemum, Impatiens, Geranium, Pelargonium, Phlox, Rhododendron anthurium spp, Rose tree, Curcumas, Anthuriums, Begonia, Hibiscus rosa-sinensis, Amaryllis, Calla, Cyclamen, and Dracaena.

29. A method for manufacturing the phytotherapeutic composition of claim 23, comprising the steps of: a) generating a recombinant transgenic plant cell producing a siRNA or a miRNA inhibiting specifically a bacterial essential gene or a bacterial virulence gene or an antibacterial resistance gene of a phytopathogenic bacterium, b) recovering the cell plant extract, or the total RNAs, or apoplastic fluids, or extracellular vesicles, or extracellular free small RNAs from said recombinant plant cells, c) optionally, adding an excipient or another active principle in said phytotherapeutic composition.

30. The method of claim 29, wherein said recombinant transgenic plant cell is derived from Tobacco (e.g. Nicotiana benthamiana, Nicotiana tobaccum); Taro (Colocasia esculenta); Giger (Zingiber officinale), Arabidopsis (e.g. Arabidopsis thaliana); Tomato (e.g. Lycopersicon esculentum or Solanum lycopersicum); Potato (Solanum tuberosum); Rice (Oryza sativa); Maize (Zea mays); Barley (Hordeum vulgare); Wheat (e.g. Triticum aestivum, Triticum durum).

31. The method of claim 29 or 30, wherein step a) is performed by expressing plant cells with at least one long dsRNA that is specific to said at least one bacterial gene.

32. An in vitro method to identify candidate small RNAs with antibacterial activity, said method comprising the steps of: a) expressing in plant cells at least one long dsRNA inhibiting at least one bacterial gene, b) contacting said plant cells with a lysis buffer or with the apoplastic fluid of said plant cells, c) incubating said plant cell lysates or fluid with target bacterial cells, and d) assessing the viability, growth, metabolic activity, of said bacterial cells.

33. The method of claim 32, wherein said plant cells are issued from tobacco leaves.

34. An in vitro method to identify candidate genes that affect the proliferation of bacterial cells, said method comprising the steps of: a) generating small RNAs inhibiting at least one bacterial gene, b) incubating said small RNAs with bacterial cells, and c) assessing the viability, growth, metabolic activity, of said bacterial cells.

Description

FIGURE LEGENDS

[0228] FIG. 1. Phenotypical and molecular characterization of Arabidopsis transgenic plants expressing the inverted repeat IR-CFA6/HRPL in both untreated and bacterial challenged conditions [0229] A. Schematic representation of the Pto DC3000 genes Cfa6 and HrpL. The 250 bp regions of Cfa6 (1-250 nt) and HrpL (99-348 nt) genes were used to generate the chimeric hairpin construct under the control of the constitutive 35S promoter. [0230] B. Representative pictures of five-week old Col-0 plants and of independent homozygous transgenic plants expressing the 35 S.sub.pro:IR-CYP51 (Control vector: CV) or the 35S.sub.pro:IR-CFA6/HRPL construct. [0231] C. Accumulation level of anti-Cfa6 and anti-HrpL siRNAs detected by low molecular weight Northern blot analysis of the Arabidopsis plants depicted in B. U6 was used as a loading control. [0232] D. Pto DC3000 HrpL mRNA accumulation is significantly decreased on IR-CFA6/HRPL-infected plants compared to Col-0- and CV-infected plants. Arabidopsis plants depicted in B. were dip-inoculated with Pto DC3000 WT strain and at 3 days post-infection (dpi), bacterial transcript levels of ProC, Cfa6 and HrpL were monitored by quantitative RT-PCR analysis. These mRNA levels are quantified relative to the level of bacterial GyrA transcript. Error bars indicate the standard deviations of mRNA values obtained in three independent experiments. Statistically significant differences were assessed using ANOVA test (ns: p-value>0.05; *: p-value<0.05, **: p-value<0.01, ***: p-value<0.001).

[0233] FIG. 2. Phenotypical and molecular characterization of Arabidopsis transgenic plants expressing the inverted repeat IR-LuxA/LuxB in both untreated and bacterial challenged conditions [0234] A. Schematic representation of the luxCDABE operon inserted into Pto DC3000 WT genome. The 250 bp regions of LuxA (1-250 nt) and LuxB (1-250 nt) genes were used to generate the chimeric hairpin construct under the control of the constitutive 35S promoter. [0235] B. Accumulation level of anti-LuxA/LuxB detected by low molecular weight Northern blot analysis of the Arabidopsis transgenic plants. U6 was used as a loading control. [0236] C. A significant impact on the luminescence of Pto DC3000 luciferase (Pto Luc) was observed in the transgenic lines expressing the IR-LuxA/LuxB as compared to Col-0 upon infection. The two independent transgenic lines of IR-LuxA/LuxB #18 and #20, along with Col-0 were syringe-infiltrated with Pto Luc at a concentration of 10.sup.6 cfu/ml and the luminescence was measured at 24 hours-post infiltration. [0237] D. The in planta growth of Pto DC3000 is unaltered in IR-LuxA/LuxB transgenic plants compared to Col-0 plants. Leaf discs from the plants used in C. were grinded and plated in a serial dilution to count Pto Luc for each condition at 24 hours post-infection.

[0238] FIG. 3. Arabidopsis transgenic plants expressing the IR-CFA6/HRPL construct suppress Pto DC3000-induced stomatal reopening [0239] A. The Pto Δcfa6 and ΔhrpL strains, but not the ΔhrcC strain, were impaired in their ability to reopen stomata and these phenotypes were rescued upon addition of exogenous COR. Sections of unpeeled leaves of Col-0 plants were incubated with mock solution (water) or Pto DC3000 WT, Δcfa6, ΔhrpL or ΔhrcC strains for 3 hours. Stomata aperture was assessed by measuring the width and length using ImageJ software. [0240] B. Pto DC3000 WT no longer induced stomatal reopening in Arabidopsis transgenic lines overexpressing the IR-CFA6/HRPL hairpin. Stomatal aperture measurement was conducted in Col-0 and 35S.sub.pro:IR-CFA6/HRPL #4, #5, #10 transgenic lines infected with Pto WT strain as described in A. [0241] C. The Pto DC3000-induced stomatal reopening response was unaltered in CV compared to Col-0 plants. Stomatal aperture measurement was conducted in Col-0 and CV plants infected with Pto WT strain as described in A.
Note: For all these experiments, n=number of stomata analyzed per condition and statistical significance was assessed using the ANOVA test (ns: p-value >0.05; ****: p-value <0.0001).

[0242] FIG. 4. Arabidopsis transgenic plants expressing the IR-CFA6/HRPL construct exhibit a reduced vascular spreading and growth of Pto DC3000 in adult leaves [0243] A. IR-CFA6/HRPL #4, #5 and #10-infected plants exhibit reduced vascular spreading of Pto WT compared to Col-0- and CV-infected plants. Plants were wound-inoculated in midveins with Pto WT-GFP and Col-0 was wound-inoculated with PtoΔcfa6-GFP. GFP fluorescence signal was observed under UV light and pictures were taken at 3 days post-infection (dpi). To index the spreading of bacteria from the inoculation sites, GFP fluorescence was observed under UV light. When the bacteria propagated away from any of the three inoculation sites, it was indexed as propagation with 4 corresponding to the highest propagation index. Pictures from three biological replicates were taken into consideration. [0244] B. Representative picture of infected leaves of conditions used in A. are depicted. White circles indicate the site of wound-inoculation in the leaf midvein. [0245] C. IR-CFA6/HRPL #4, #5 and #10 transgenic lines exhibit a significantly reduced Pto WT titer when compared to Col-0 and CV-infected plants. Col-0, CV and IR-CFA6/HRPL #4, #5 and #10 plants were dip-inoculated with Pto WT and Col-0 plants were dip-inoculated with the PtoΔcfa6-GFP strain. Bacterial titers were monitored at 2 days post-infection (dpi). Four leaves from three plants per condition and from three independent experiments (n) were considered for the comparative analysis. [0246] D. IR-CFA6/HRPL #4, #5 and #10 transgenic plants exhibit reduced water-soaking symptoms in comparison to Col-0 and CV plants. Representative leaf pictures of water-soaking symptoms were taken 24 hours after dip-inoculation.
Note: For all the above experiments, statistical significance was assessed using the two-way ANOVA test (ns: p-value>0.05; *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001; ****: p-value<0.0001).

[0247] FIG. 5. Phenotypical characterization of Arabidopsis transgenic plants expressing the inverted repeat IR-HRPG/HRPX/RSMA in both untreated and Xanthomonas campestris pv. campestris challenged conditions [0248] A. IR-HRPG/HRPX/RSMA #1- and #6-infected plants exhibit reduced vascular spreading of the virulent XccΔXopAC (GUS/GFP) strain compared to Col-O-infected plants. Plants were wound-inoculated in midveins with XccΔXopAC (GUS/GFP) at OD=0.01. GFP fluorescence signal was observed under UV light and pictures were taken at 3 days post-infection (dpi). The indexing was done as described in 4A. [0249] B. Representative picture of infected leaves of conditions used in B. are depicted. White circles indicate the site of wound-inoculation in the leaf midvein.

[0250] FIG. 6. Exogenously delivered total RNAs from IR-CFA6/HRPL transgenic plants reduce Pto DC3000 pathogenicity when applied on the surface of wild type Arabidopsis and tomato leaves [0251] A. In vitro AGS assay showing that total RNA extract from CFA6/HRPL #4 plants triggers silencing of both Cfa6 and HrpL genes. Pto WT cells were incubated in vitro for 4 and 8 hours with 20 ng/μl of total RNAs from CV or IR-CFA6/HRPL #4 plants. Significant reduction of the bacterial transcripts Cfa6 and HrpL was observed by RT-qPCR at both the timepoints, while accumulation of ProC and RpoB transcripts remained unaffected. GyrA was used as an internal control to quantify the accumulation of bacterial transcripts. Error bars indicate the standard deviations of values from three independent experiments. [0252] B. The ability of Pto WT to reopen stomata was altered upon exogenous application of total RNAs extract from IR-CFA6/HRPL plants compared to CV plants. Col-0 leaves were treated for 1 hour with water or 20 ng/μl of total RNAs extracted from CV or IR-CFA6/HRPL #4 plants and were incubated with Pto WT for 3 hours. Stomatal aperture was measured and analyzed as described in FIG. 3A. [0253] C. Treatment with IR-CFA6/HRPL, but not with CV, total RNAs compromised the ability of Pto DC3000 to multiply in the apoplast of leaves when compared to pretreatment with CV total RNAs. Col-0 leaves were treated with 20 ng/μl of total RNAs from CV or IR-CFA6/HRPL #4 plants for 1 hour, followed by dip-inoculation with Pto WT. Bacterial titers were monitored at 2 dpi. The number of leaves (n) corresponds to collective values from three independent experiments. [0254] D. The leaves treated with CV total RNAs displayed more necrotic symptoms as compared to the leaves treated with IR-CFA6/HRPL #4 total RNAs. The experiment was conducted as in C. but using five-week-old tomato (Solanum lycopersicum ‘Moneymaker’) plants. Representative pictures of infected leaves in the two conditions are depicted. [0255] E. A reduced number of Pto DC3000-GFP foci was observed in tomato leaves treated with total RNA extracts from IR-CFA6/HRPL #4 versus CV plants. Infected-leaves were observed at 3 dpi under UV light to estimate the number of GFP loci. On the left: Dot plot representing the number of GFP loci analyzed using ImageJ software from 3-4 different leaves per condition with at least 4 pictures per leaf. The values used for the analysis are from two different independent experiments. Student's t-test was performed for the comparative analysis. On the right: Representative picture of the tomato leaves described in D. [0256] F. Pto WT-GFP DNA content is decreased in tomato leaves treated with total RNA extracts from IR-CFA6/HRPL #4 versus CV plants. The level of bacterial DNA content was analyzed by qPCR using tomato Ubiquitin as a control. Student's t-test was performed for the comparative analysis.
Note: For A, B and C, statistically significant differences were assessed using ANOVA test (ns: p-value>0.05; **: p-value<0.01, ***: p-value<0.001).

[0257] FIG. 7. DCL-dependent antibacterial siRNAs, but not corresponding unprocessed dsRNA precursors, are the RNA entities responsible for AGS and for the suppression of stomatal reopening [0258] A. Upper panel: Accumulation level of IR-CFA6/HRPL transcripts in Col-0, dcl2-1 dcl3-1 dcl4-2 (dcl234), IR-CFA6/HRPL #4 (#4) and IR-CFA6/HRPL #4 in dcl234 mutant background (#4×dcl234) was performed by RT-qPCR. Ubiquitin was used as a control. The graph represents the mean and standard deviation of three independent experiments. Lower panel: Accumulation level of anti-Cfa6 and anti-HrpL siRNAs was performed by low molecular weight Northern blot analyses in the same genotypes. U6 was used as a loading control. [0259] B. Total RNA extract from #4×dcl234 plants does not alter the transcript accumulation levels of Cfa6 and HrpL. Pto WT cells were incubated in vitro for 8 hours with 20 ng/μl of total RNAs extracted from the same genotypes described in A. Accumulation levels of Cfa6 and HrpL transcripts was assessed by RT-qPCR analysis using GyrA as a control. Error bars indicate the standard deviations of values from three independent experiments. Statistically significant differences were assessed using ANOVA test (ns: p-value>0.05; *: p-value<0.05, **: p-value<0.01). [0260] C. Total RNA extract from #4×dcl234 plants does not suppress Pto DC3000-induced stomatal reopening response. Col-0 leaves were treated with water or 20 ng/μl of total RNA extracts from the same genotypes than the ones used in A. for 1 hour and incubated with Pto WT for 3 hours. Stomatal aperture was measured and analyzed as described in FIG. 2A. Two other biological replicates are presented in Supplementary FIG. 4B. [0261] D. Upper panel: Electrogram profiles representing the RNA size distribution of total, long and small RNAs from IR-CFA6/HRPL #4 plants determined with an agilent Bioanalyzer 2100 equipped with an RNA Nano chip. Low molecular weight RNA fractions are encircled for each sample. 18S and 25S ribosomal peaks are highlighted. Lower panel: Agarose gel picture of ethidium bromide stained total, long and small RNAs used in A. [0262] E. Small RNA species, but not the corresponding long RNA species, from IR-CFA6/HRPL plants suppress stomatal reopening to the same extent as total RNA extracts. The experiment was conducted as in D. but with total, long (>200 nt) or small (<200 nt) RNA fractions, which were separated from total RNAs of IR-CFA6/HRPL #4 plants.
Note: For all the stomata experiments, statistical significance was assessed using the ANOVA test (ns: p-value>0.05; ****: p-value<0.0001).

[0263] FIG. 8. A bacterially expressed small RNA resilient version of HrpL is refractory to gene silencing directed by anti HrpL siRNAs and exhibits a normal stomatal reopening phenotype upon exogenous application of anti HrpL siRNAs [0264] A. Schematic representation of the PtaΔhrpL strain along with the complementation strains generated upon transformation with the plasmids encoding WT HrpL or mut HrpL, respectively under the control of the constitutive promoter NptII. [0265] B. In vitro AGS assay showing that the PtaΔhrpL WT HrpL strain is sensitive to antibacterial RNAs while the PtaΔhrpL mut HrpL is refractory to these RNA entities. Bacterial PtaΔhrpL WT HrpL and PtaΔhrpL mut HrpL strains were incubated with total RNAs extracted from CV or IR-CFA6/HRPL #4 plants for 8 hours. Accumulation level of WT HrpL and mut HrpL transcripts was analyzed by RT-qPCR (the mRNA levels were relative to the level of GyrA transcript). Error bars indicate the standard deviations of values from three independent experiments. Statistically significant differences were assessed using ANOVA test (ns: p-value>0.05; *: p-value<0.05, **: p-value<0.01). [0266] C. Accumulation of anti-Cfa6 and anti-HrpL siRNAs was assessed by low molecular weight northern analysis using total RNA extracts from N. benthamiana plants transiently expressing 35S.sub.pro:IR-HRPL, 35S.sub.pro:IR-CFA6/HRPL and from non-transformed N. benthamiana leaves (Nb). U6 was used as a loading control. [0267] D. The PtaΔhrpL mut HrpL strain is refractory to anti HrpL siRNA action. Col-0 leaves were treated with total RNAs extracted either from N. benthamiana alone or from N. benthamiana expressing the inverted repeat IR-HRPL. Stomatal reopening response was assessed as described previously.
Note: For all the stomata experiments, statistical significance was assessed using the ANOVA test (ns: p-value>0.05; ****: p-value<0.0001).

[0268] FIG. 9. The apoplastic fluid of IR-CFA6/HRPL plants is composed of functional antibacterial siRNAs that are either embedded into EVs, and protected from micrococcal nuclease action, or in a free form, and sensitive to micrococcal nuclease digestion [0269] A. The ability of Pto WT to reopen stomata was also altered to similar levels upon exogenous application of Apoplastic fluid (APF) extract as compared to total RNAs derived from IR-CFA6/HRPL plants. Total RNAs and APF extracted from CV plants was used as negative control. Col-0 leaves were treated for 1 hour with water (Mock) or 20 ng/μl of total RNAs or 500 μl of APF extracted from CV or IR-CFA6/HRPL #4 plants and were incubated with Pto WT for 3 hours. Stomatal aperture was measured and analyzed as described in previous experiments. [0270] B. The two different vesicular fractions, P40 and P100, as well as the free RNA population present in the supernatent (SN) carry the antibacterial siRNAs and thus are involved in AGS. Apoplastic fluid extracted from both CV and IR-CFA6/HRPL #4 plants was subjected to ultracentrifugation at 40,000 g to pellet the larger population of EVs (P40) and the remaining supernatent was further subjected to ultracentrifugation at 100,000 g to pellet the smaller EVs (P100). SN was also restored. Col-0 leaves were treated for 1 hour with water (Mock) or P40, P100 and SN extracted from CV or IR-CFA6/HRPL #4 plants and were incubated with Pto WT for 3 hours. The P40, P100 and SN of #4 were treated with 20 units of Mnase and the SN of #4 was also treated with 20 units of Proteinase K. Stomatal aperture was measured and analyzed as described in previous experiments.
Note: For all the stomata experiments, statistical significance was assessed using the ANOVA test (ns: p-value>0.05; ****: p-value<0.0001).

[0271] FIG. 10. Exogenous delivery of in vitro synthesized antibacterial siRNAs reduces the pathogenicity as well as the viability of Pto DC3000 [0272] A. 2% Agarose gel of ethidium bromide stained in vitro synthesized long dsRNAs and RNase III digested siRNAs corresponding to IR-CYP51 and IR-CFA6/HRPL are depicted. [0273] B. The ability of Pto WT to reopen stomata was altered upon exogenous application of in vitro synthesized siRNAs, but not the long dsRNAs, corresponding to IR-CFA6/HRPL. Long dsRNAs and siRNAs from IR-CYP51 was used as negative control. Col-0 leaves were treated for 1 hour with water (Mock) or RNA presented in A. and then incubated with Pto WT for 3 hours. Stomatal aperture was measured and analyzed as described in previous experiments. [0274] C. In vitro AGS assay using the in vitro synthesized siRNAs from IR-CFA6/HRPL triggers silencing of both Cfa6 and HrpL genes. Pto WT cells were incubated in vitro for 8 hours with 2 ng/μl of in vitro synthesized siRNAs from IR-CYP51 or IR-CFA6/HRPL #4 plants. Significant reduction of the bacterial transcripts Cfa6 and HrpL was observed by RT-qPCR, while accumulation of ProC and RpoB transcripts remained unaffected. GyrA was used as an internal control to quantify the accumulation of bacterial transcripts. Error bars indicate the standard deviations of values from three independent experiments. [0275] D. and E. In vitro synthesized siRNAs against fusA or gyrB of Pto DC3000 have a significant impact on the growth of the Pto DC3000-GFP strain. siRNAs directed against secE, gyrB and fusA genes of Pto DC3000 were synthesized using in vitro transcription followed by RNaseIII digestion. The Pto DC3000-GFP strain was incubated with the indicated concentration of in vitro synthesized siRNAs. 96-well plate was set on the machine for the samples to be fractioned in droplets by the droplet-based microfluidic system (Millidrop). For each well, 10 droplets of ˜500 nl each were formed and incubated inside the instrument. For each droplet, measurements of biomass and of GFP fluorescence were acquired every ˜30 minutes.

EXAMPLES

Example 1: Materials and Methods

Generation of Transgenic Lines Carrying Inverted Repeats Constructs

[0276] The IR-CFA6/HRPL chimeric hairpin was designed to produce artificial siRNAs targeting a 250 bp region of Cfa6 (from nucleotide 1 to 250) and a 250 bp region of HrpL from nucleotide 99 to 348 (SEQ ID NO: 1, 2 and 3). The IR-CFA6-A and IR-CFA6-B are two independent inverted repeats that specifically target the Cfa6 gene from nucleotide 1 to 250 (SEQ ID NO: 4, 2 and 5) and from nucleotide 1 to 472 (SEQ ID NO: 6, 2 and 7), respectively. The IR-HRPL-A and IR-HRPL-B are two independent inverted repeats that specifically target HrpL from nucleotide 99 to 348 (SEQ ID NO: 8, 2 and 9) and from nucleotide 1 to 348 (SEQ ID NO: 10, 2 and 11), respectively. The IR-HRCC hairpin was designed to specifically target the HrcC gene (SEQ ID NO: 12, 2 and 13) and the IR-AvrPto/AvrPtoB to concomitantly target the type III effector AvrPto and AvrPtoB genes (SEQ ID NO: 14, 2 and 15). The IR-CYP51 hairpin was designed to produce siRNAs against three cytochrome P450 lanosterol C-14α-demethylase genes of the fungus F. graminearum, namely FgCYP51A, FgCYP51B and FgCYP51C as previously performed (SEQ ID NO: 16, 2 and 17), (19). This hairpin was used as a negative control for all the in planta assays of the invention. Additional inverted repeats were designed and cloned as part of this study to target virulence factors or essential genes from different strains of Pseudomonas, Xanthomonas and Ralstonia. These hairpins are described as follows: the IR-HrpG/HrpB/HrcC hairpin designed to concomitantly target the HrpG, HrpB and HrcC genes from Ralstonia species (SEQ ID NO: 18, 2 and 19), the IR-HrpB/HrcC/TssB/XpsR hairpin designed to concomitantly target the HrpB, HrcC, TssB and XpsR genes from Ralstonia species (SEQ ID NO: 20, 2 and 21), the IR-HrpG/HrpX/RsmA hairpin designed to concomitantly target the HrpG, HrpX and Rsma genes from Xanthomonas campestris pv. campestris (SEQ ID NO: 22, 2 and 23), the IR-RpoB/RpoC/FusA hairpin designed to concomitantly target the essential genes RpoB, RpoC and FusA from Pto DC3000 and Pseudomonas syringae strain CC440 (SEQ ID NO: 24, 2 and 25), the IR-SecE-RpoA-RplQ hairpin designed to concomitantly target the essential genes SecE, RpoA and RplQ from Pto DC3000 and Pseudomonas syringae strain CC440 (SEQ ID NO: 26, 2 and 27), the IR-NadHb/NadHd/NadHe hairpin designed to concomitantly target the essential genes NadHb, NadHd and NadHe from different Xanthomonas species including Xanthomonas campestris pv. campestris (SEQ ID NO: 28, 2 and 29), the IR-DnaA/DnaE1/DnaE2 hairpin designed to concomitantly target the essential genes NadHb, NadHd and NadHe from different Xanthomonas species including Xanthomonas campestris pv. campestris (SEQ ID NO: 30, 2 and 31). Furthermore, a chimeric inverted repeat was designed and cloned as part of this study to target the Photorhabdus luminescens luxCDABE operon chromosomally expressed in Pto DC3000 under the constitutive kanamycin promoter: the IR-LuxA/LuxB hairpin, designed to concomitantly target the LuxA and LuxB genes from Pto DC3000 luciferase strain (SEQ ID NO: 108, 2 and 109). All the above-described hairpins contain a specific intron sequence from the Petunia Chalcone synthase gene CHSA (SEQ ID NO: 2) and were cloned into a vector carrying the Cauliflower Mosaic Virus (CaMV) 35S constitutive promoter. More specifically, the following hairpin sequences: IR-CFA6/HRPL, IR-CYP51, IR-CFA6-B, IR-HRPL-B, IR-HrpG/HrpB/HrcC, IR-HrpB/HrcC/TssB/XpsR, IR-AvrPto/AvrPtoB, IR-HRCC, IR-HrpG/HrpX/RsmA and IR-LuxA/LuxB were cloned into a modified pDON221-P5-P2 vector carrying additional EcoRI and SalI restriction sites to facilitate the insertion of these long inverted-repeats into this vector. A double recombination between pDON221-P5-P2 carrying the hairpin sequence and pDON221-P1-P5r (Life Technologies, 12537-32), carrying the constitutive 35S promoter sequence, was conducted in the pB7WG GATEWAY compatible destination vector (binary vector carrying a BAR selection marker and gateway recombination sites). The remaining hairpins, namely the IR-CFA6-A, IR-HRPL-A, IR-RpoB/RpoC/FusA, IR-SecE-RpoA-RplQ, IR-NadHb/NadHd/NadHe and IR-DnaA/DnaE1/DnaE2 sequences were generated by PCR amplifications of the sense and antisense regions of the target genes using the bacterial genomic DNA as template and followed by the generation of modules required for the cloning into a final GreenGate destination vector pGGZ003. All the plasmids were then introduced into the Agrobacterium tumefaciens strains GV3101 or C58C1 and further used for either transient expression in Nicotiana benthamiana or stable expression in the Arabidopsis thaliana Columbia-0 (Col-0) reference accession.

Plant Material and Growth Conditions

[0277] Stable transgenic lines of IR-CFA6/HRPL and CV were generated by transforming Arabidopsis WT (accession Col-0) plants using Agrobacterium mediated-floral dip method. Three independent transgenic lines, #4, #5 and #10 expressing equal amount of anti-Cfa6 and anti-HrpL siRNAs were selected and propagated until T4 generation. Similarly, selected homozygous line of CV expresses abundant level of siRNAs against F. graminearum CYP51A/B/C genes was propagated until T4 generation for experimentation. Similarly, transgenic lines expressing IR-LuxA/LuxB and IR-HrpG/HrpX/RsmA were selected on the basis of siRNA production and propagated further. For genetic analysis, dcl2 dcl3 dcl4 (dcl234) triple mutant plant was crossed with the reference IR-CFA6/HRPL #4 line and the F3 plants were genotyped to select homozygous dcl234 mutant containing homozygous IR-CFA6/HRPL transgene. Sterilized seeds of Arabidopsis Col-0 and the selected homozygous transgenic lines were first grown for 12-14 days at 22° C. on plates containing ½×MS medium (Duchefa), 1% sucrose and 0.8% agar (with or without antibiotic selection) in 8 h photoperiod. Seedlings were then pricked out to soil pots and grown in environmentally controlled conditions at 22° C./19° C. with an 8 h photoperiod under light intensity of 100 μE/m.sup.2/s. Four- to five-week-old plants were used for all the experiments. Seeds of tomato (Solanum lycopersicum ‘Moneymaker’) and N. benthamiana were directly sown on soil pots and grown in environmentally controlled conditions at 22° C./19° C. (day/night) with a 16 h photoperiod under light intensity of 100 μE/m.sup.2/s. Four- to five-week old plants were used for all the experiments.

Bacterial Strains

[0278] The GFP expressing Pto DC3000-GFP and the Pto DC3000Δcfa6-GFP (Pto DC3118) strains were a gift from Dr. S. Y. He, while the Pto DC3000ΔhrpL strain was a gift from Dr. Cayo Ramos. The Pto DC3000 luciferase strain was a gift from Dr. Chris Lamb. The Pto DC3000ΔhrpL and Pto DC3000ΔhrcC strains expressing the GFP reporter gene were generated by transforming them with the same plasmid as in Pto DC3000-GFP by electroporation and then plated at 28° C. on NYGB medium (5 g/l bactopeptone, 3 g/l yeast extract, 20 ml/l glycerol) containing gentamycin (1 μg/ml) for selection. To generate the Pto DC3000-WT-HrpL and -mut-HrpL strains, the Pto DC3000ΔhrpL strain was transformed with the plasmids NPTII.sub.pro:WT-HrpL and NPTII.sub.pro:mut-HrpL, respectively, by electroporation and then plated in NYGB medium with gentamycin.

RNA Gel Blot Analyses

[0279] To perform northern blot analyses of low molecular weight RNAs, total RNA was extracted using TriZOL reagent and stabilized in 50% formamide. Around 30 μg of total RNA from the specified conditions were used to perform Northern blot analyses as previously described (49). Regions of 150 bp to 300 bp were amplified from the plasmids using gene specific primers and the amplicons were further used to generate specific 32P-radiolabelled probes synthesized by random priming. U6 probe was used as a control for equal loading of small RNAs.

Separation of Long and Small RNA Fractions

[0280] Total RNAs were extracted from Arabidopsis leaves of IR-CFA6/HRPL #4 using Tri-Reagent (Sigma, St. Louis, Mo.) according to the manufacturer's instructions. Using 100 μg of total RNA, long and small RNA fractions were separated using the mirVana miRNA isolation kit (Ambion, Life technologies) according to the manufacturer's instructions. The separation of long and small RNAs from the total RNAs was visualized using agarose gel electrophoresis and further analyzed using microfluidic based approach (Bioanalyzer 2100; Agilent Technologies, http://www.agilent.com). The total, long and small RNAs were further used to perform the stomatal reopening assay.

Bacterial Infection Assays in Plants

[0281] (a) Bacterial growth assay: Plants for this experiment were specifically used after three hours of beginning of the night cycle in growth chamber. Three plants per condition were dip-inoculated using the bacterium at 5×10.sup.7 cfu/ml with 0.02% Silwet L-77 (Lehle seeds). Plants upon bacterial dipping were immediately placed in chambers with high humidity to facilitate proper infection. Water-soaking symptoms upon dip-inoculation were observed 24 hours post-infection and pictures of leaves from three plants per condition were taken. Two days post-inoculation, bacterial titer for each mentioned condition was measured for individual infected leaf as described in (49). To quantify bacterial transcripts in infected plants, pool of infected leaf samples was collected three days post-inoculation.

[0282] (b) Wound-inoculation assay: To monitor the propagation of bacteria in the midveins, around 15 leaves from three plants per condition were manually inoculated with a toothpick dipped in GFP-tagged bacteria at a concentration of 5×10.sup.6 cfu/ml and then the plants were placed in chambers with high humidity for 3 days. Bacterial propagation was then analyzed by monitoring GFP signal under a UV light using an Olympus MV 10× macrozoom and pictures were taken with a CCD camera AxioCam Mrc Zeiss with a GFP filter.

[0283] (c) Plant protection assay: Prior to bacterial infection, four rosette leaves of three Arabidopsis plants per condition were individually treated by repeatedly soaking with mock solution or RNA solutions at a concentration of 20 ng/μl of specific total RNAs, both supplemented with Silwett L-77 (0.02%). One hour after pretreatment, leaves were dip-inoculated with Pto DC3000 WT or Pto DC3000Δcfa6 at a concentration of 5×10.sup.7 cfu/ml in similar way as that of RNAs. Bacterial titers were monitored two days post-inoculation, as specified earlier. In tomato, two leaves of three plants per condition were pretreated with a suspension having 20 ng/μl of specific total RNA supplemented with Silwett L-77 (0.02%) and then were dipped one hour after with GFP-tagged Pto DC3000 at 5×10.sup.7 cfu/ml. The plants were then placed in controlled conditions at 24° C./19° C. (day/night) with a 16 h photoperiod without lid cover for 3 days. Bacterial infection was then analyzed by monitoring GFP signal under a UV light using an Olympus MV 10× macrozoom and pictures were taken. Individual leaf samples were collected to quantify the amount of bacteria in each condition using ImageJ software.

Bacterial Luminescence Quantification

[0284] Three plants per condition were syringe-infiltrated with Pto DC3000 Luciferase (Pto Luc) strain at 1×10.sup.6 cfu/ml. Plants were placed in chambers with high humidity to facilitate proper infection. Leaf discs were placed in individual wells of a 96 well plate to quantify the luminescence using Berthold Centro LB 960 Microplate Luminometer. Four leaves per plant were taken into consideration. Leaf discs from individual leaves were pooled after to perform bacterial titer quantification as mentioned above.

Tomato Infection Quantification

[0285] (a) GFP loci quantification: Tomato leaves infected with Pto DC3000-GFP strain were subjected to GFP quantification under a UV light using an Olympus MV 10× macrozoom and pictures were taken with a CCD camera AxioCam Mrc Zeiss with a GFP filter. Number of GFP loci was quantified with ImageJ software for at least 10 pictures per condition.

(b) Bacterial Genomic DNA Quantification

[0286] To quantify bacterial infection in the infected tomato plants (Ross et al., 2006), the amount of bacterial genomic DNA (gDNA) was measured relative to plant gDNA. Genomic DNA was isolated from tomato leaf samples infected with Pto DC3000-GFP using the DNeasy plant mini kit (QIAGEN, Germany) according to the manufacturer's instructions. Using 1 ng of gDNA, qPCR was performed using Takyon SYBR Green Supermix (Eurogentec®) and GFP gene-specific primers. Amount of bacterial gDNA was normalized to that of tomato using Ubiquitin-specific primers.

Agrobacterium-Mediated Transient Expression of Inverted Repeats in N. benthamiana

[0287] To produce single hairpins, IR-CFA6 and IR-HRPL, and the chimeric hairpin IR-CFA6/HRPL, the A. tumefaciens strain carrying the plasmids were grown overnight in LB medium at 28° C. Cells were harvested by centrifugation and resuspended in a solution containing 10 mM MES, pH 5.6, 10 mM MgCl.sub.2 and 200 μM acetosyringone at a final density of 0.5 OD.sub.600. Cultures were incubated in the dark at room temperature for 5-6 hours before Agrobacterium-mediated infiltration in four-week old N. benthamiana. After 3 days of infiltration, leaf tissue was harvested and Northern blot analysis was performed to confirm the production of anti-Cfa6 and anti-HrpL siRNAs. The leaf samples were then used for total RNA extraction.

In Vitro Antibacterial Gene Silencing Assay

[0288] To assess whether the bacterial transcripts Cfa6 and HrpL can be directly targeted by the dsRNA and/or the siRNAs generated by the hairpin IR-CFA6/HRPL, 2 ml culture of Pto DC3000 WT, Pto DC3000-WT-HrpL and Pto DC3000-mut-HrpL at 10.sup.7 cfu/ml was treated for 4 and/or 8 hours, with 20 ng/μl of specified total RNA extracted from CV or IR-CFA6/HRPL #4 transgenic plants in a six-well plate, respectively. Similarly, to quantify the silencing of bacterial genes upon treatments with in vitro synthesized siRNAs, 2 ml of Pto DC3000-GFP at 1×10.sup.7 cfu/ml was treated for 6 hours with 2 ng/μl of in vitro synthesized IR-CYP51 siRNAs or IR-CFA6/HRPL siRNAs in a six-well plate, respectively. Bacteria were collected for each condition and further processed for molecular analyses.

Apoplastic Fluid (AF) and Extracellular Vesicles (EVs) Extraction

[0289] Extraction was done as previously described (44). Sixty leaves of 5 week-old CV or IR-CFA6/HRPL plants were infiltrated with Vesicle Isolation Buffer (VIB; 20 mM MES, 2 mM 324 CaCl.sub.2, 0.01 M NaCl, pH 6.0) with a syringe without needle. Leaves were then placed inside a 20 ml needless syringe. Syringe was then placed in 50 ml Falcon and centrifuged at 900 g for 15 minutes. The apoplastic fluid (APF) was collected and centrifuged subsequently at 2,000 g and 10,000 g for 30 minutes to get rid of any cell debris and then passed through a 0.45 μm filter. The APF was further subjected to ultracentrifugation step at 40,000 g to pellet EV fraction (P40). The pellet was resuspended in 2 ml of 2004 Tris buffer pH=7.5. The supernatant was then subjected to ultracentrifugation step at 100,000 g to pellet EV fraction (P100). The supernatant from this step was restored (SN).

Stomatal Aperture Measurements

[0290] Plants were kept under light (100 μE/m.sup.2/s) for at least 3 hours before subjecting to any treatment to assure full expansion of stomata. Intact leaf sections from three four-week-old plants were dissected and immersed in water (Mock) or bacterial suspension at a concentration of 1×10.sup.8 cfu/ml. After 3 hours of treatment, unpeeled leaf abaxial surface was observed under SP5 laser scanning confocal microscope and the pictures were taken from different regions. The stomatal aperture (width/length) was measured using ImageJ software for 30-70 stomata per condition. In case of RNA pretreatments, the leaf sections were incubated with total RNAs extracted from specified genotypes for one hour before incubation with the bacteria. When required in specified experiments, 1 μM of exogenous Coronatine (COR) (Sigma) (50) was supplemented to the bacterial suspension.

Real-Time RT-PCR Analyses

[0291] To monitor plant-encoded transcripts, total RNA was extracted from plant samples using RNeasy Plant Mini kit (Qiagen). 0.5 μg of DNA-free RNA was reverse transcribed using qScript cDNA Supermix (Quanta Biosciences). cDNA was then amplified by real time PCR reactions using Takyon SYBR Green Supermix (Eurogentec®) and transcript-specific primers. Expression was normalized to that of Ubiquitin. To monitor bacterial transcripts, total RNA was extracted from bacteria-infected plant samples or from in vitro treated bacteria as described previously. After DNAse treatment, 250 ng of total RNA was reverse transcribed using random hexamer primers and qScript Flex cDNA kit (Quanta Biosciences). cDNA was then amplified by real time PCR reactions using Takyon SYBR Green Supermix (Eurogentec®) and transcript-specific primers. Expression was normalized to that of GyrA. PCR was performed in 384-well optical reaction plates heated at 95° C. for 10 min, followed by 45 cycles of denaturation at 95° C. for 15 s, annealing at 60° C. for 20 s, and elongation at 72° C. for 40 s. A melting curve was performed at the end of the amplification by steps of 1° C. (from 95° C. to 50° C.).

In Vitro Synthesis of Inverted Repeat (IR) RNAs

[0292] In vitro synthesis of RNAs was generated following the instruction of the MEGAscript® RNAi Kit (Life Technologies, Carlsbad, Calif.). Templates like were amplified by PCR introducing the T7 promotor at both 5′ and 3′ end of the sequence. PCR amplification was done in two steps with two different annealing temperature to rise the specificity of primers annealing. After the amplification step, PCR products were purified by gel extraction thanks to the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) to eliminate any parasite amplification. Those purified PCR products were then used as templates for in vitro transcription: 2 μg was incubated for five hours at 37° C. with 2 μL of T7 polymerase (T7 enzyme Mix), 2 μL of 10×T7 Reaction Buffer and 2 μL of each 75 mM ATP, CTP, GTP and UTP. The total volume is adjusted to 20 μL with Nuclease free water. After the transcription reaction, dsRNAs were treated with 2 μL of DNaseI, 2 μL of RNase, 5 μL, of 10× reaction buffer to eliminate DNA templates and single stranded RNAs. Then, dsRNAs are purified with the filter cartridges provided with the kit. Long dsRNA obtained at this step are used for the following experiments (FIG. 1). siRNAs were obtained thanks to ShortCut® RNase III (NEB, Ipswich, Mass.). DsRNAs were digested for 20 minutes with RNaseIII and then purified thanks to the mirVana™ miRNA Isolation Kit (Life Technologies, Carlsbad, Calif.). After purification, siRNAs are used for the following experiments (FIG. 1). Each steps of the process were followed by gel electrophoresis (TAE 1×, 1% agarose gel for DNA amplification and 2% agarose gel for RNAs) to check the quality of RNAs.

Droplet-Based Microfluidic Assay for the Monitoring of In Vitro Pto DC3000 Growth

[0293] Droplet-based microfluidic experiments were performed in NYGB medium at a temperature of 28° C. RNAi assays were prepared by pipetting directly in the 96 well plate the different solutions to obtain 200 μl final: 100 μl of medium, 20 μl of a GFP-tagged Pto DC3000 (Pto DC3000-GFP) at 10.sup.7 cfu/ml, 20 μl of in vitro synthesized candidate siRNAs to obtain a final concentration at 2 ng/μl or sterile water for the control sample followed by 60 μl of medium. In case of mix of different RNAs, a v/v ratio of the different RNAs was prepared and 20 μl of the mix was added to the corresponding wells. The 96-well plate was set on the machine for the samples to be fractioned in droplets by the Millidrop Analyzer (http://www.millidrop.com). For each well, 10 droplets of ˜500n1 each were formed and incubated inside the instrument for the 24 hours. For each droplet, measurements of biomass and GFP fluorescence were acquired every ˜30 minutes.

Example 2. Arabidopsis-Encoded siRNAs Directed Against Either Endogenous Virulence Factors or Artificial Reporter Genes from Pto DC3000 Trigger their Silencing in the Context of Bacterial Infection

[0294] To test whether host-encoded small RNAs could alter bacterial gene expression, we have generated Arabidopsis stable transgenic plants that constitutively express a chimeric inverted repeat bearing sequence homology to the ECF-family sigma factor HrpL gene and the coronatine (COR) biosynthesis, Cfa6 gene, both of which encode key virulent determinants of Pto DC3000 (FIG. 1A, (51, 52)). As negative controls, we have also generated transgenic lines overexpressing an inverted repeat bearing sequence homology to three cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes of the fungus F. graminearum, which was previously shown to confer full protection against this fungal phytopathogen in both Arabidopsis and barley (20,21). These stable transgenic lines are referred to as IR-CFA6/HRPL and IR-CYP51 (or CV, Control Vector plants), respectively; and do not exhibit any developmental defect (FIG. 1B), despite high accumulation of artificial siRNAs (FIG. 1C). To investigate whether artificial siRNAs directed against Cfa6 and HrpL could interfere with the expression of these virulence factors during bacterial infection, we dip-inoculated the above transgenic plants with Pto DC3000 and further monitored Cfa6 and HrpL mRNA levels by RT-qPCR analyses. While the Cfa6 mRNA levels were moderately altered in two out of three independent IR-CFA6/HRPL lines compared to Col-0 plants, the levels of HrpL transcripts were reproducibly reduced in all the three IR-CFA6/HRPL lines compared to Col-0 plants at this timepoint (FIG. 1D). By contrast, the down-regulation of Cfa6 or HrpL mRNAs was not observed in IR-CYP51-versus Col-0-infected plants (FIG. 1D), supporting a specific effect of these antibacterial RNAs in this regulatory process. Similarly, the mRNA level of the non-targeted ProC gene was unchanged in both IR-CFA6/HRPL- and IR-CYP51-infected lines compared to Col-0-infected plants (FIG. 1D). Collectively, these data indicate that the Arabidopsis-encoded IR-CFA6/HRPL inverted repeat can at least trigger sequence-specific silencing of the bacterial HrpL transcript in the context of infection.

[0295] Because the expression of HrpL and Cfa6 virulence factors is known to be regulated by various environmental cues (52, 53), we also tested whether AGS could be effective against the Photorhabdus luminescens luxCDABE operon chromosomally expressed in Pto DC3000 under the constitutive kanamycin promoter (54). This lux-tagged Pto DC3000 strain spontaneously emits luminescence because it co-expresses the luciferase catalytic components luxA and luxB genes along with the genes required for substrate production, namely luxC, luxD and luxE (55). Two independent Arabidopsis transgenic lines, IR-LuxA/LuxB lines, overexpressing anti-luxA and anti-luxB siRNAs were selected and syringe-infiltrated with the lux-tagged Pto DC3000 strain (FIG. 2A/B). The levels of luxA and luxB mRNAs as well as the luminescence activity were further monitored at 24 hours post-inoculation (hpi). By doing so, we found a significant reduction in both luxA and luxB mRNA abundance as well as in luminescence activity in IR-LuxA/LuxB-compared to Col-0-infected plants (FIG. 2C). By contrast, the growth of the bacterial reporter strain was unchanged in IR-LuxA/LuxB lines compared to Col-0 plants in those conditions (FIG. 2D), indicating that the above effects were not due to a decreased bacterial titer in these transgenic plants. Altogether, these data indicate that AGS is effective against both endogenous stress-responsive bacterial genes and exogenous constitutive bacterial reporter genes during Pto DC3000 infection.

Example 3. Host-Encoded siRNAs Directed Against Cfa6 and HrpL Prevent Pto DC3000-Induced Stomatal Reopening Presumably by Suppressing Coronatine Biosynthesis

[0296] Because Cfa6 and HrpL are known to regulate each other (53) and because HrpL and Cfa6 are both essential for coronatine (COR) biosynthesis (52, 53), we next investigated whether IR-CFA6/HRPL plants could be protected from COR-dependent virulence responses. For this purpose, we monitored Pto DC3000-triggered stomatal reopening at 3 hours post-inoculation (3 hpi), a phenotype that is fully dependent on COR biosynthesis and thus abolished upon inoculation with Pto DC3000 mutants that are either deleted in Cfa6 or HrpL genes (FIG. 3A, (50)). It is noteworthy that this phenotype is not dependent on type III effectors at this timepoint of infection because a normal stomatal reopening response was observed upon treatment with the Pto DC3000 hrcC mutant (FIG. 3A, (50)), which is impaired in the assembly of the type III secretion system. Significantly, we found that Pto DC3000-induced stomatal reopening was fully abolished in the three independent IR-CFA6/HRPL transgenic lines infected with the virulent Pto DC3000 strain as compared to Col-0-infected leaves (FIG. 3B), thereby mimicking the phenotype observed on Col-0 leaves inoculated with the Pto DC3000 cfa6- or hrpl-deleted strains (FIG. 3A). By contrast, a normal Pto DC3000-induced stomatal reopening was observed in IR-CYP51-infected plants (FIG. 3C), indicating that the observed effect is specific to siRNAs directed against Cfa6 and HrpL genes. Furthermore, the compromised stomatal reopening phenotype detected in IR-CFA6/HRPL-infected transgenic plants was fully rescued upon exogenous application of COR (FIG. 3B). These data provide thus pharmacological evidence that the reduced Pto DC3000 pathogenesis manifested at infected IR-CFA6/HRPL stomata is likely caused by an altered ability of the associated and/or surrounding bacterial cells to produce COR.

Example 4. Arabidopsis Stable Transgenic Plants Expressing Small RNAs Against Key Virulence Factors from Pto DC3000 or Xanthomonas campestris pv. Campestris are Protected from Bacterial Infections

[0297] To further monitor the possible effects that anti-Cfa6 and anti-HrpL siRNAs could have on Pto DC3000 pathogenicity, we next monitored the ability of this bacterium to spread in the leaf vasculature of Arabidopsis IR-CFA6/HRPL transgenic plants. For this purpose, we scored the number of bacterial spreads occurring at three sites from the midvein of individual leaves wound-inoculated with a virulent GFP-tagged Pto DC3000 (Pto DC3000-GFP) strain. Using this quantification method, we observed an index of bacterial propagation that was significantly decreased in the three independent IR-CFA6/HRPL transgenic lines as compared to Col-0 plants (FIG. 4A). This suggests that siRNAs directed against Cfa6 and HrpL can reach xylem vessels and further dampen the virulence activity of Pto DC3000 in Arabidopsis leaf vasculature. By contrast, a normal Pto DC3000 vascular spreading was observed in the IR-CYP51 transgenic line compared to Col-0-infected leaves (FIG. 4A), arguing for a specific effect of anti-Cfa6 and anti-HrpL siRNAs in this process. Collectively, these results indicate that siRNAs directed against the pathogenicity determinants Cfa6 and HrpL can specifically restrict the spreading of Pto DC3000 in Arabidopsis leaf vasculature. An enhanced vascular disease protection effect towards the Gram-negative bacterium Xanthomonas campestris pv. campestris (Xcc) was also found in Arabidopsis transgenic plants overexpressing siRNAs against the virulence factors HrpX, HrpG and RsmA (FIG. 5, data not shown, (56-60)). This demonstrates that AGS can additionally be used to protect plants against this well-characterized vascular bacterial pathogen of Arabidopsis, which is the causal agent of black rot, one of the most devastating diseases of crucifer crops worldwide (25, 61).

[0298] We next investigated whether stable expression of siRNAs against Cfa6 and HrpL could also impact growth of Pto DC3000 in planta, a phenotype known to be dependent on both COR and on a functional type III secretion system (52). To this end, we dip-inoculated IR-CFA6/HRPL, IR-CYP51 and WT plants with Pto DC3000 and further monitored bacterial titer at 48 hpi. Using this assay, we found a significant reduction in Pto DC3000 titer in the three independent IR-CFA6/HRPL transgenic lines compared to Col-0-infected plants, and this phenotype was reminiscent to the one observed in WT plants infected with a Cfa6-deleted strain (FIG. 4C). Interestingly, we additionally observed a reduced Pto DC3000-induced water soaking disease symptoms in the three independent IR-CFA6/HRPL plants compared to WT-infected plants at 24 hpi, which resemble the phenotype observed in WT leaves dip-inoculated with the Cfa6 mutant strain (FIG. 4D). By contrast, the bacterial growth and water soaking disease symptoms were unaltered in IR-CYP51 transgenic plants dip-inoculated with Pto DC3000 (FIG. 4C/D), indicating that the above effects are specific to siRNAs directed against Cfa6 and HrpL genes. Altogether, these data further support a major role for anti-Cfa6 and anti-HrpL siRNAs in dampening the virulence activity of Pto DC3000 in the context of infection. They also provide compelling evidence that AGS is an effective strategy that can be used to control bacterial pathogenicity in stable transgenic plants.

Example 5. Exogenous Delivery of Total RNAs Derived from IR-CFA6/HRPL Plants Protect WT Arabidopsis and Tomato Plants Against Pto DC3000

[0299] Environmental RNAi is a phenomenon by which (micro)organisms can uptake external RNAs from the environment, resulting in the silencing of genes containing sequence homologies to the RNA triggers (24). This RNA-based process has been initially characterized in C. elegans (26-29), and was further found to operate in other nematodes but also in insects, plants and fungi (26, 30). However, this approach has never been used against a bacterial phytopathogen that lacks a canonical eukaryotic-like RNAi machinery such as Pto DC3000. To test this possibility, we first assessed whether RNAs expressed from IR-CFA6/HRPL plants could trigger silencing of Cfa6 and HrpL genes in in vitro conditions. For this purpose, we extracted total RNAs from CV and IR-CFA6/HRPL plants, incubated them with Pto DC3000 cells, and further analyzed by RT-qPCR the levels of Cfa6 and HrpL mRNAs at 4 and 8 hours after RNA treatments. Results from these analyses revealed a reduced accumulation of both virulence factor mRNAs upon treatment with RNA extracts from IR-CFA6/HRPL plants, a molecular effect that was not observed with RNA extracts derived from CV plants (FIG. 6A). By contrast, the level of the non-targeted ProC and RpoB mRNAs remained unaltered in the same conditions (FIG. 6A). These data therefore imply that plant antibacterial RNAs are likely taken-up by Pto DC3000 cells and subsequently trigger sequence-specific silencing of Cfa6 and HrpL genes. It also suggests that exogenous application of these antibacterial RNAs could be used as a strategy to dampen Pto DC3000 pathogenesis in Col-0 plants. To test this intriguing hypothesis, we pre-treated Arabidopsis Col-0 leaf tissues with total RNA extracts from IR-CFA6/HRPL plants for one hour, subsequently challenged them with Pto DC3000 for 3 hours, and further monitored bacterial-induced stomatal reopening events. Strikingly, we found that RNA extracts from IR-CFA6/HRPL plants fully suppressed the ability of Pto DC3000 to reopen stomata (FIG. 6B), thereby mimicking the phenotype observed in infected IR-CFA6/HRPL transgenic plants (FIG. 3). We additionally investigated whether this approach could be used to control the growth of Pto DC3000 in planta. For this purpose, we first pre-treated for one hour Col-0 Arabidopsis plants with total RNA extracts from IR-CFA6/HRPL plants and further dip-inoculated them with Pto DC3000. We found that these RNA extracts triggered a decreased Pto DC3000 titer at 2 dpi (FIG. 6C), a phenotype that was comparable to the ones observed in infected IR-CFA6/HRPL transgenic plants (FIG. 4C), as well as in Col-0 plants inoculated with the PtoΔcfa6 strain (FIG. 6C). By contrast, application of total RNA extracts from CV plants did not alter growth of Pto DC3000 in the same conditions (FIG. 6C), supporting a specific effect of antibacterial RNAs in this process. To assess whether such RNA-based biocontrol approach could also be effective in cultivated plants, we repeated the same assay on tomato (Solanum lycopersicum, cultivar Moneymaker), which is the natural host of Pto DC3000. Pre-treatment of WT tomato leaves for one hour with RNA extracts from IR-CFA6/HRPL plants led to compromised Pto DC3000-induced necrotic disease symptoms and also to a reduction in bacterial content compared to leaves pre-treated with RNA extracts derived from CV plants (FIG. 6D-F). Collectively, these data provide evidence that external application of plant-derived antibacterial RNAs can trigger AGS and disease protection against Pto DC3000 in both Arabidopsis and tomato plants.

Example 6. Small RNA Species, but not their dsRNA Precursors, are Causal for the Compromised Stomatal Reopening Phenotype Observed Upon Exogenous Application of Total RNAs Derived from the IR-CFA6/HRPL Hairpin

[0300] Next, we interrogated which RNA entities are responsible for AGS and pathogenesis reduction upon external application of antibacterial RNAs. To address this question, we first crossed the IR-CFA6/HRPL #4 reference line with the dcl2-1 dcl3-1 dcl4-2 (dcl234) triple mutant and subsequently selected F3 plants that were homozygous for the three dcl mutations and for the IR-CFA6/HRPL transgene. Molecular characterization of these IR-CFA6/HRPL #4×dcl234 plants revealed an enhanced accumulation of IR-CFA6/HRPL inverted repeat transcripts (i.e. unprocessed dsRNAs) compared to the level detected in IR-CFA6/HRPL #4 parental line (FIG. 7A). Furthermore, this effect was associated with undetectable levels of anti-Cfa6 and anti-HrpL siRNAs (FIG. 7A). These data are thus consistent with a role of DCL2, DCL3 and DCL4 in the biogenesis of these siRNAs through the processing of the IR-CFA6/HRPL inverted repeat. We subsequently extracted total RNAs from these plants, incubated them with Pto DC3000 cells for 8 hours, and further monitored Cfa6 and HrpL mRNA levels by RT-qPCR analysis. Using this in vitro assay, we found that RNA extracts from IR-CFA6/HRPL #4×dcl234 plants were no longer able to trigger down-regulation of Cfa6 and HrpL mRNAs (FIG. 7B), despite high accumulation of artificial dsRNA precursors (FIG. 7A). By contrast, RNA extracts from the IR-CFA6/HRPL #4 parental line, which contain high levels of anti-Cfa6 and anti-HrpL siRNAs (FIG. 7A), triggered reduced accumulation of both targeted virulence factors (FIG. 7B). Moreover, while RNA extracts from IR-CFA6/HRPL #4 plants suppressed Pto DC3000-induced stomatal reopening events, we found that RNA extracts from IR-CFA6/HRPL #4×dcl234 plants were inactive in this process, such as control RNA extracts derived from Col-0 or dcl234 plants (FIG. 7C, data not shown). Collectively, these data provide compelling evidence that dsRNAs produced from the IR-CFA6/HRPL inverted repeat are neither involved in AGS nor in pathogenesis reduction. They rather suggested that small RNAs are likely the antibacterial RNA entities responsible for these molecular and physiological phenotypes. To verify this assumption, we further purified small RNA species from IR-CFA6/HRPL plant total RNAs using a glass fiber filter-based method (FIG. 7D), and subjected them to stomatal reopening assay. By doing so, we found that these small RNA species suppressed Pto DC3000-triggered stomatal reopening, to the same extent as IR-CFA6/HRPL plant total RNA extracts (FIG. 7E). By contrast, long RNA species (above 200 bp), which were not filtered through the above columns, were inactive (FIG. 7E), further supporting that antibacterial plant dsRNAs are not involved in this response. Altogether, these data provide solid evidence that DCL-dependent siRNAs produced from the inverted repeat IR-CFA6/HRPL are critical for AGS and pathogenesis reduction, while cognate dsRNA precursors are ineffective for both processes.

Example 7. A Bacterially Expressed Small RNA Resilient Version of HrpL is Insensitive to siRNA-Directed Silencing and Exhibits a Normal Stomatal Reopening Phenotype Indicating that Anti-HrpL siRNAs are Causal for AGS and Pathogenesis Reduction

[0301] Although the above findings indicate that external application of antibacterial siRNAs can trigger AGS and antibacterial activity, they do not firmly demonstrate that these RNA entities are causal for these phenomena. To address this issue, we decided to generate and characterize recombinant bacteria expressing a siRNA-resilient version of the HrpL gene, which was found to be subjected to AGS regulation in both in vitro and in planta conditions (FIGS. 1 and 6). To this end, we complemented the PtoΔhrpL mutant with either a WT HrpL transgene or a mutated version, mut HrpL that contains as many silent mutations as possible in the siRNA targeted region, which are predicted to alter the binding of siRNAs with the HrpL mRNA but to produce the same protein sequence. Furthermore, to assess the post-transcriptional regulatory control that anti-HrpL siRNAs might exert over these bacterial transgenes, we expressed them under the constitutive neomycin phosphotransferase II (NPTII) promoter. The two resulting recombinant bacteria are referred to as PtoΔhrpL WT HrpL and PtoΔhrpL mut HrpL, respectively, and were found to restored ability to reopen stomata when inoculated on Col-0 plants (FIG. 8A, data not shown), indicating that both transgenes are functional. We further assessed the sensitivity of each recombinant bacterium to AGS. For this purpose, we incubated PtoΔhrpL WT HrpL and PtoΔhrpL mut HrpL strains with total RNA extracts from CV and IR-CFA6/HRPL #4 plants for 8 hours and further monitored HrpL transgene mRNA levels by RT-qPCR analysis. We found a significant decrease in the accumulation of HrpL mRNAs expressed from the PtoΔhrpL WT HrpL strain, which was not detected upon treatment with control RNA extracts from CV plants (FIG. 8B). These data indicate that the WT HrpL transgene expressed from the PtoΔhrpL WT HrpL strain is fully sensitive to AGS despite its constitutive expression driven by the NPTII promoter. By contrast, the accumulation of HrpL mRNAs expressed from the PtoΔhrpL mut HrpL strain was unaltered in response to RNA extracts from IR-CFA6/HRPL #4 plants (FIG. 8B), indicating that siRNAs no longer exert their AGS effect towards this recombinant bacterium. Collectively, these findings demonstrate that anti-HrpL siRNAs are causal for the post-transcriptional silencing of the HrpL virulence factor gene within Pto DC3000 cells. Next, we investigated the responsiveness of each recombinant bacterial strain to siRNA-directed pathogenesis reduction by exploiting the Pto DC3000-induced stomatal reopening assay, which is highly sensitive to small RNA action. To assess the specific effect of siRNAs towards suppression of HrpL-mediated stomatal reopening function, we first cloned an IR-HRPL inverted repeat targeting the same HrpL sequence region than the one targeted by the IR-CFA6/HRPL hairpin, and further validated its capacity to produce HrpL siRNAs upon Agrobacterium-mediated transient transformation in Nicotiana benthamiana leaves (FIG. 8C). N. benthamiana total RNA extracts containing anti-HrpL siRNAs were found to fully suppress the ability of Pto DC3000 to reopen stomata (FIG. 8D). Importantly, similar results were obtained when N. benthamiana RNA extracts containing anti-HrpL siRNAs were incubated with the PtoΔhrpL WT HrpL strain (FIG. 8D), supporting a sensitivity of this bacterial strain to siRNA action. By contrast, the PtoΔhrpL mut HrpL strain was fully competent in reopening stomata in the same conditions (FIG. 8D), indicating that anti-HrpL siRNAs no longer exert their antibacterial effects towards this recombinant bacterial strain. These data provide thus evidence that anti-HrpL siRNAs are causal for the suppression of HrpL-mediated stomatal reopening function. They also further validate a novel role of HrpL in bacterial-induced stomatal reopening, indicating that AGS can be employed as a tool to characterize bacterial gene function.

Example 8. The Apoplastic Fluid of IR-CFA6/HRPL Plants is Composed of Functional Antibacterial siRNAs that are Either Embedded into EVs, and Protected from Micrococcal Nuclease Action, or in a Free Form, and Sensitive to Micrococcal Nuclease Digestion

[0302] The results from the phenotypical analyses described in EXAMPLES 3 and 4 imply that small RNA species that are constitutively expressed in IR-CFA6/HRPL transgenic lines, must be externalized from plant cells towards the leaf surface, the apoplastic environment and xylem vessels in order to reach epiphytic and endophytic bacterial populations. To get some insights into the small RNA trafficking mechanisms that could be implicated in this phenomenon, we have first extracted the apoplastic fluid (APF) from IR-CFA6/HRPL plants and tested its ability to dampen bacterial pathogenesis by monitoring its impact on Pto DC3000-induced stomatal reopening. We found that this extracellular fluid triggered a full suppression of stomatal reopening during infection, thereby mimicking the effect triggered by IR-CFA6/HRPL-derived total RNAs (FIG. 9A). By contrast, the APF from IR-CYP51 plants was inactive, supporting a specific effect of anti-Cfa6 and anti-HrpL siRNAs from the AFP of IR-CFA6/HRPL plants in this process (FIG. 9A). We further tested whether EVs from IR-CFA6/HRPL plants could contribute to AGS. For this end, we recovered APF from IR-CFA6/HRPL plants and further performed differential ultracentrifugation at 40,000 g or 40,000 g followed by 100,000 g, which allowed us to collect two fractions, named P40 and P100, respectively. Interestingly, we found that both fractions were capable of suppressing stomatal reopening, although P100 was moderately less effective in this process (FIG. 9B). Importantly, both fractions remained active in the presence of micrococcal nuclease (Mnase), indicating that small RNAs are protected from external degradation when embedded into EVs. Intriguingly, we also noticed that the supernatant fraction (SN), recovered after the sequential centrifugation at 40,000 g and 100,000 g, exhibited strong antibacterial activity, despite a lack of canonical EVs detected in this fraction (FIG. 9B, data not shown). This suggests that EV-free small RNAs that are either associated with proteins and/or in a free-form could additionally be competent for AGS. To determine which of the two small RNA entities could possess such antibacterial activity, we treated SN fractions from IR-CFA6/HRPL plants with Mnase or proteinase K and further subjected them to stomatal reopening assay. Interestingly, we found that the Mnase treatment abrogated the antibacterial effect triggered by the IR-CFA6/HRPL-derived SN fraction, while an unaltered antibacterial activity was detected in the presence of proteinase K, which globally degraded proteins (FIG. 9B, data not shown). Collectively, these data indicate that functional EV-free antibacterial small RNAs are unlikely associated with proteins and are thus referred to here as Extracellular Free Small RNAs or “efsRNAs”. Our results also indicate that efsRNAs are sensitive to Mnase action because they lost their antibacterial effect upon treatment with this nuclease (FIG. 9B). Based on these findings, we propose that the APF from IR-CFA6/HRPL plants is composed of at least three populations of functional antibacterial small RNAs, which are 1) embedded into large EVs (P40 fraction), 2) embedded into EVs of smaller size (P100 fraction), or 3) in a free form.

Example 9. The In Vitro Synthesis of Small RNAs is an Easy, Rapid and Reliable Approach to Screen for Candidate Small RNAs Possessing Antibacterial Activities

[0303] In order to develop a screening platform for the identification of candidate small RNAs with antibacterial activities, we aimed to produce in vitro synthesized siRNAs against specific bacterial gene transcripts and further test their activities on bacterial pathogenicity or survival. For this end, we first decided to generate in vitro synthesized anti-Cfa6 and anti-HrpL siRNAs targeting the same sequences than the plant siRNAs produced from the DCL-dependent processing of IR-CFA6/HRPL. To do so, we used primers carrying T7 promoter sequences to amplify either CYP51 or CFA6/HRPL DNA from plasmids containing the IR-CYP51 or IR-CFA6/HRPL sequences. The resulting PCR products were gel-purified and subsequently used as templates for in vitro RNA transcription using a T7 RNA polymerase, which led to the production of CYP51 or CFA6/HRPL dsRNAs of expected size (FIG. 10A). Small RNAs were further obtained by digesting these dsRNAs into 18-25 bp siRNAs using the ShortCut® RNase III, although other non-commercial RNase III can also be used for this process (data not shown). As revealed by agarose gel electrophoresis, these siRNAs were deprived of dsRNA (FIG. 10A), indicating that the RNase III used in these experiments fully processed the initial pool of dsRNA molecules. We next analyzed the ability of synthetic dsRNA and siRNAs to suppress stomatal reopening. Consistent with our previous data showing that plant dsRNAs are inactive in triggering AGS (FIG. 7), we found that in vitro synthesized CFA6/HRPL dsRNAs did not interfere with Pto DC3000-induced stomatal reopening, nor did in vitro synthesized CYP51 dsRNAs, which were used as negative controls (FIG. 10B). By contrast, in vitro synthesized siRNAs directed against Cfa6 and HrpL fully prevented Pto DC3000-induced stomatal reopening, while in vitro synthesized anti-CYP51 siRNAs were inactive in this process (FIG. 10B). The latter result suggested that in vitro synthesized anti-Cfa6 and anti-HrpL siRNAs were likely capable of triggering silencing of Cfa6 and HrpL genes. To test this hypothesis, we further incubated the in vitro synthesized CYP51 and CFA6/HRPL siRNAs at a concentration of 2 ng/ul with 1×10.sup.7 cfu/ml of Pto DC3000 for 6 hours and further monitored Cfa6 and HrpL mRNAs by RT-qPCR analyses. By doing so, we found that anti-Cfa6/HrpL siRNAs triggered a significant reduced accumulation of Cfa6 and HrpL mRNAs compared to anti-CYP51 siRNAs (FIG. 10B), a molecular effect which was comparable to the one observed in response to plant-derived total RNAs containing anti-Cfa6 and anti-HrpL siRNAs (FIG. 6A, 7B). By contrast, the levels of the non-targeted ProC and RpoB mRNAs remained unchanged in response to anti-Cfa6 and anti-HrpL siRNAs compared to anti-CYP51 siRNAs (FIG. 10B). Collectively, these data indicate that in vitro synthesized siRNAs can trigger AGS and antibacterial activity to the same extent as plant-derived anti-Cfa6 and anti-HrpL siRNAs.

[0304] We next decided to determine whether this approach could be instrumental for the identification of candidate siRNAs with bactericidal activities. To test this idea, we performed in vitro synthesis of siRNAs directed against three conserved and housekeeping genes from Pto DC3000, namely SecE (PSPTO_0613, preprotein translocase SecE subunit), FusA (PSPTO_0623, translation elongation factor G) and GyrB (PSPTO_0004, DNA gyrase subunit B) and further monitor their impact on the in vitro growth of this bacterium. To do so, we took advantage of an established droplet-based microfluidic system, which is suitable for the accurate measurements of bacterial biomass and bacterially-expressed fluorescence reporter activity. By using this approach, we found that 0.33 ng/μl of in vitro synthesized siRNAs directed against FusA was capable of reducing both the biomass and the GFP signal from a GFP-tagged Pto DC3000 (Pto DC3000-GFP), compared to the conditions in the absence of siRNAs or in the presence of anti-SecE siRNAs (FIG. 10E, F). Strikingly, we did not detect any GFP signal nor bacterial biomass when the Pto DC3000-GFP strain was incubated with 1 ng/ul of in vitro synthesized anti-FusA siRNAs, nor when in vitro synthesized siRNAs directed against GyrB were applied at concentrations of either 0.33 ng/μ1 or 1 ng/μ1 (FIG. 10E, F). These data indicate that siRNAs directed against FusA and GyrB possess a potent bactericidal activity that mimics the effect that would be detected in the presence of an antibiotic. Based on these proof-of-concept experiments, we conclude that the in vitro synthesis of siRNAs is an easy, rapid and reliable approach to screen for novel candidate small RNAs with antibacterial activities. They also unveil a role for FusA and GyrB in the survival of Pto DC3000, which has not previously been reported for this bacterium. These results therefore further support the fact that AGS can be employed as a tool to characterize bacterial gene function.

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