RNA-BASED THERAPEUTIC METHODS TO PROTECT ANIMALS AGAINST PATHOGENIC BACTERIA AND / OR PROMOTE BENEFICIAL EFFECTS OF SYMBIOTIC AND COMMENSAL BACTERIA
20220288230 · 2022-09-15
Inventors
Cpc classification
A61K31/7088
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A01H3/00
HUMAN NECESSITIES
A61K31/713
HUMAN NECESSITIES
C12N15/8218
CHEMISTRY; METALLURGY
International classification
A61K48/00
HUMAN NECESSITIES
A61K31/7088
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
C12N15/82
CHEMISTRY; METALLURGY
Abstract
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 and animals 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 symbiotic or commensal bacteria. The invention involves the exogenous delivery of small RNA entities onto bacteria, either in the form of RNA extracts or embedded into plant extracellular vesicles (EVs), so as to reduce bacterial growth, survival and/or 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 have the potential to be further developed as anti-infective agents. In addition, the latter method is instrumental to rapidly characterize any gene from any bacterial species.
Claims
1-43. (canceled)
44. 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, said small RNAs having a length comprised between 15 and 30 base pairs.
45. The method of claim 1, wherein said bacteria are animal pathogenic bacteria.
46. The method of claim 1, wherein said bacteria are chosen from the group consisting of: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bordetella pertussis, Borrelia sp. (burgdorferi, garinii, afzelii, recurrentis, crocidurae, duttonii, hermsii etc.), Brucella sp. (abortus, canis, melitensis, suis), Campylobacter jejuni, Chlamydia sp. (pneumoniae, trachomatis), Chlamydophila psittaci, Clostridium sp. (botulinum, difficile, perfringens, tetani), Corynebacterium diphtheriae, Ehrlichia sp. (canis, chaffeensis), Enterococcus (faecalis, faecium), Escherichia coli O157:H7, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira sp., Listeria monocytogenes, Mycobacterium sp. (leprae, tuberculosis), Mycoplasma pneumoniae, Neisseria (gonorrhoeae, meningitidis), Pseudomonas aeruginosa, Porphyromonas gingivalis, Nocardia asteroides, Rickettsia rickettsii, Salmonella sp. (typhi, typhimurium), Shigella sp. (sonnei, dysenteriae), Staphylococcus (aureus, epidermidis, saprophyticus), Streptococcus sp. (agalactiae, mutans, pneumoniae, pyogenes, viridans), Tannerella forsythia, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.
47. The method of claim 1, wherein said composition contains extracellular free small RNAs, or extracellular vesicles containing said small RNAs or apoplastic fluid containing said small RNAs or nanoparticles coupled to said small RNAs.
48. A therapeutical composition containing, as active principle, the small RNA as defined in claim 1.
49. The therapeutic composition according to claim 48, containing extracellular free small RNAs, or extracellular vesicles containing said small RNAs or apoplastic fluid containing said small RNAs or nanoparticles coupled said small RNAs, and a pharmaceutically acceptable excipient.
50. The therapeutic composition according to claim 48, wherein it is formulated for an oral, topical or systemic administration, preferably as a pill, a cream, or an oral spray.
51. A method for treating and/or preventing a bacterial infection in a subject in need thereof, said method comprising administering to said subject the therapeutic composition of claim 5.
52. The method according to claim 51, wherein said composition is administered orally, topically or systemically to said subject.
53. The method according to claim 51, wherein said bacterial infection is due to human pathogenic bacteria chosen from: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bordetella pertussis, Borrelia sp. (burgdorferi, garinii, afzelii, recurrentis, crocidurae, duttonii, hermsii etc.), Brucella sp. (abortus, canis, melitensis, suis), Campylobacter jejuni, Chlamydia sp. (pneumoniae, trachomatis), Chlamydophila psittaci, Clostridium sp. (botulinum, difficile, perfringens, tetani), Corynebacterium diphtheriae, Ehrlichia sp. (canis, chaffeensis), Enterococcus (faecalis, faecium), Escherichia coli O157:H7, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira sp., Listeria monocytogenes, Mycobacterium sp. (leprae, tuberculosis), Mycoplasma pneumoniae, Neisseria (gonorrhoeae, meningitidis), Pseudomonas aeruginosa, Porphyromonas gingivalis, Nocardia asteroides, Rickettsia rickettsii, Salmonella sp. (typhi, typhimurium), Shigella sp. (sonnei, dysenteriae), Staphylococcus (aureus, epidermidis, saprophyticus), Streptococcus sp. (agalactiae, mutans, pneumoniae, pyogenes, viridans), Tannerella forsythia, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.
54. A method for promoting beneficial effects of commensal or symbiotic beneficial bacteria in a subject in need thereof, said method comprising administering to a subject in need thereof the composition as defined in claim 48.
55. The method according to claim 54, wherein said commensal or symbiotic beneficial bacteria is chosen from: Actinomyces naeslundii, Veillonella dispar, Faecalibacterium prausnitzii, Enterobacteriaceae, Bacteroides thetaiotaomicron, Escherichia coli K2, Bifidobacterium sp. (longum, bifidum, adolescentis, dentium, breve, thermophilum), Eggerthella lenta, Bacteroides sp. (xylanisolvens, thetaiotaomicron, fragilis, vulgatus, salanitronis), Parabacteroides distasonis, Faecalibacterium prausnitzii, Ruminococcus sp. (bromii, champanellensis, SR1/5), Streptococcus (parasanguinis, salivarius, thermophilus, suis, pyogenes, anginosus), Lactococcus (lactis, garvieae), Enterococcus (faecium, faecalis, casselflavus, durans, hirae, Melissococcus plutonius, Tetragenococcus halophilus, Lactobacillus sp. (casei, ruminis, delbrueckii, buchneri, reuteri, fermentum, pentosus, amylovorus, salivarius), Pediococcus (pentosaceus, claussenii), Leuconostoc (mesenteroides, lactis, carnosum, gelidum, citreum), Weissella (thailandensis, koreensis), Oenococcus oeni, Paenibacillus sp. (terrae, polymyxa, mucilaginosus, Y412MCI0), Thermobacillus composti, Brevibacillus brevis, Bacillus (amyloliquefaciens, subtilis, lichenformis, atrophaeus, weihenstephanensis, cereus, thuringiensis, coagulans, megaterium, selenitireducens), Geobacillus thermodenitrificans, Lysinibacillus sphaericus, Halobacillus halophilus, Listeria sp., Streptomyces sp., Eubacterium (rectale, eligens, siraeum), Clostridium saccharolyticum, and butyrate-producing bacterium (SS3/4 and SSC/2).
56. A method for improving the efficiency of an antibiotic treatment in a subject in need thereof, said method comprising administering to said subject the therapeutic composition of claim 48, wherein said small RNA inhibits specifically the expression of at least one bacterial antibiotic resistance gene.
57. The method according to claim 56, wherein said antibiotic resistance gene is chosen from: VIM-1, VIM-2, VIM-3, VIM-5, Case, OXA-28, OXA-14, OXA-19, OXA-145, PER-4, TEM-116, and GES-9.
58. The method according to claim 56, wherein said antibiotic compound is chosen from: Aminoglycosides, Carbapenems, Ceftazidime (3rd generation), Cefepime (4th generation), Ceftobiprole (5th generation), Ceftolozane/tazobactam, Fluoroquinolones, Piperacillin/tazobactam, Ticarcillin/clavulanic acid, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Streptomycin, Spectinomycin, Geldenamycin, herbimycin, Rifaximin, Ertapenem, Doripenem, Imipenem, Meropenem, Cefadroxil, Cefazolin, Cephradine, Cephapirin, Cephalothin, Cefalexin, Cefaclor, Cefoxitin, Cefotetan, Cefamandole, Cefinetazole, Cefonicid, Loracarbef, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Moxalactam, Ceftriaxone, Cephalosporins, Cefepime, Cephalosporins, Ceftaroline fosamil, Ceftobiprole, Glycopeptides, Teicoplanin, Vancomycin, Telavancin, Dalbavancin, Oritavancin, Lincosamides(Bs), Clindamycin, Lincomycin, Lipopeptide, Daptomycin, Macrolides(Bs), Azithromycin, Clarithromycin, Erythromycin, Roxithromycin, Telithromycin, Spiramycin, Fidaxomicin, Monobactams, Aztreonam, Nitrofurans, Furazolidone, Nitrofurantoin(Bs), Oxazolidinones(Bs), Linezolid, Posizolid, Radezolid, Torezolid, Penicillins, Amoxicillin, Ampicillin, Azlocillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin, Piperacillin, Temocillin, Ticarcillin, Penicillin combinations, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, Ticarcillin/clavulanate, Polypeptides, Bacitracin, Colistin, Polymyxin B, Quinolones/Fluoroquinolones, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nadifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Sulfonamides(Bs), Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX), Sulfonamidochrysoidine (archaic), Tetracyclines(Bs), Demeclocycline, Doxycycline, Metacycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol(Bs), Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol(Bs), Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline(Bs), Tinidazole, and Trimethoprim(Bs).
59. The method of claim 56 comprising: a) administering to said subject the therapeutic composition of claim 5, and b) administering to said subject, simultaneously or separately or in a staggered manner, an antibiotic compound.
60. An in vitro method to identify candidate genes involved in bacterial antibiotic resistance, or that affect the proliferation of human pathogenic bacterial cells, said method comprising the steps of: a) generating small RNAs having a length comprised between 15 and 30 base pairs and inhibiting specifically the expression at least one bacterial gene, b) incubating bacterial cells with said small RNA, c) optionally, incubating said small RNA treated bacterial cells with an antibiotic compound, d) assessing the viability, growth, metabolic activity, of said small RNA treated bacterial cells in the presence or absence of said antibiotic compound, and optionally compare same with the viability, growth, metabolic activity, of said small RNA treated bacterial cells in the absence of said antibiotic compound.
61. The method of claim 60, wherein the candidate gene is involved in bacterial antibiotic resistance if the viability, growth, metabolic activity, of said small RNA treated bacterial cells in the presence of said antibiotic compound is lower than the viability, growth, metabolic activity, of said small RNA treated bacterial cells in the absence of said antibiotic compound.
62. The method of claim 60, wherein said bacterial cells are chosen from: Actinomyces israelii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bordetella pertussis, Borrelia sp. (burgdorferi, garinii, afzelii, recurrentis, crocidurae, duttonii, hermsii etc.), Brucella sp. (abortus, canis, melitensis, suis), Campylobacter jejuni, Chlamydia sp. (pneumoniae, trachomatis), Chlamydophila psittaci, Clostridium sp. (botulinum, difficile, perfringens, tetani), Corynebacterium diphtheriae, Ehrlichia sp. (canis, chaffeensis), Enterococcus (faecalis, faecium), Escherichia coli O157:H7, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira sp., Listeria monocytogenes, Mycobacterium sp. (leprae, tuberculosis), Mycoplasma pneumoniae, Neisseria (gonorrhoeae, meningitidis), Pseudomonas aeruginosa, Porphyromonas gingivalis, Nocardia asteroides, Rickettsia rickettsii, Salmonella sp. (typhi, typhimurium), Shigella sp. (sonnei, dysenteriae), Staphylococcus (aureus, epidermidis, saprophyticus), Streptococcus sp. (agalactiae, mutans, pneumoniae, pyogenes, viridans), Tannerella forsythia, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.
Description
FIGURE LEGENDS
[0279]
[0284]
[0289]
[0293] 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).
[0294]
[0299] 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).
[0300]
[0303]
[0310] 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).
[0311]
[0317]
[0319] Bacterial PtoΔhrpL WT HrpL and PtoΔhrpL 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). [0320] 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. [0321] D. The PtoΔhrpL 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.
[0322] Note: For all the stomata experiments, statistical significance was assessed using the ANOVA test (ns: p-value>0.05; ****: p-value<0.00001).
[0323]
[0326] Note: For all the stomata experiments, statistical significance was assessed using the ANOVA test (ns: p-value>0.05; ****: p-value<0.00001).
[0327]
[0332]
[0337] Note: For B and C statistical significance was assessed using the ANOVA test (ns: p-value>0.05; **: p-value<0.001).
[0338]
[0339] In vitro synthesized antibacterial siRNAs were tested against several essential genes of P. aeruginosa PAO1 strain and were screened for having a significant impact on the growth of the bacteria. siRNAs directed against SecE, GyrB, DnaN, DnaA, RpoB or SodB genes of P. aeruginosa were synthesized using in vitro transcription followed by RNaseIII digestion. PAO1 strain at 10.sup.8 cfu ml.sup.−1 was treated with 5 ng/μl concentration of individual gene targeting siRNAs. 96-well plate was set on the machine for the samples to be fractioned in droplets by the Millidrop Analyzer. For each well, 10 droplets of ˜500 nL each were formed and incubated inside the instrument. For each droplet, measurements of biomass were acquired every ˜30 minutes for 14 hours. Median of scattering signal acquired from 30 droplets/condition at each time point is plotted.
EXAMPLES
Example 1: Materials and Methods
[0340] Generation of Transgenic Lines Carrying Inverted Repeats Constructs
[0341] The IR-HRPL/CFA6 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). Inverted repeats were designed and cloned as part of this study to target virulence factors or essential genes from different strains of Pseudomonas aeruginosa and Shigella. These hairpins are described as follows: the IT13 hairpin targeting the DnaA, DnaN and GyrB genes (SEQ ID NO: 108-109), the IT14 hairpin targeting the RpoC, SecE and SodB genes (SEQ ID NO: 110-111), the IT16 hairpin targeting the XcpQ, PscF and PscC genes (SEQ ID NO: 112-113), the IT18 hairpin XcpQ, ExsA and HphA genes of P. aeruginosa (SEQ ID NO: 114-115), the IT21 hairpin targeting the FtsA, Can and Tsf genes (SEQ ID NO: 116-117), the IT26 hairpin of targeting the AccD, Der and Psd genes (SEQ ID NO: 118-119), and the IT27 hairpin targeting the VirF, VirB and IcsA genes of Shigella flexneri (SEQ ID NO: 120-121). 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 as well as P. aeruginosa luciferase strain (SEQ ID NO: 248, 2 and 249). 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-HRPL/CFA6, 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.
[0342] Plant Material and Growth Conditions
[0343] 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 12×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/m2/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/m2/s. Four- to five-week old plants were used for all the experiments.
[0344] Bacterial Strains
[0345] 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 DC3000 ΔhrpL 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. The PAK and PAO1 strains of P. aeruginosa were availed from other labs in collaboration.
[0346] RNA Gel Blot Analyses
[0347] 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 (51). 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.
[0348] Separation of Long and Small RNA Fractions
[0349] 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.
[0350] Bacterial Infection Assays in Plants
[0351] (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 (51). To quantify bacterial transcripts in infected plants, pool of infected leaf samples was collected three days post-inoculation.
[0352] (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 MVrc Zeiss with a GFP filter.
[0353] (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.
[0354] In Vitro Synthesis of dsRNAs and sRNAs
[0355] 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. 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. Each steps of the process were followed by gel electrophoresis (TAE 1X, 1% agarose gel for DNA amplification and 2% agarose gel for RNAs) to check the quality of RNAs.
[0356] Bacterial Luminescence Quantification
[0357] 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. Luminiscence quantification assay with lux-tagged PAK strain was performed in LB medium with an inoculum of 1×10.sup.7 cfu/ml incubated with specific RNA extracts to obtain a final concentration of 20 ng/μl in at four individual wells per condition. The 96-well plate was set on the Berthold Centro LB 960 Microplate Luminometer and the luminescence was recorded every 30 minutes for a period of 4 hours.
[0358] Tomato Infection Quantification
[0359] (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.
[0360] (b) Bacterial genomic DNA Quantification
[0361] 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.
[0362] Agrobacterium-Mediated Transient Expression of Inverted Repeats in N. benthamiana
[0363] 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 MgCl2 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 HrpL siRNAs. The leaf samples were then used for total RNA extraction.
[0364] In Vitro Antibacterial Gene Silencing Assay
[0365] 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 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.
[0366] Apoplastic Fluid (AF) and Extracellular Vesicles (EVs) Extraction
[0367] Extraction were done as previously described (46). 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 CaCl2), 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 20 μM 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).
[0368] Stomatal Aperture Measurements
[0369] 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 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) (52) was supplemented to the bacterial suspension.
[0370] Real-Time RT-PCR Analyses
[0371] 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.).
[0372] Droplet-Based Microfluidic Assay for the Monitoring of In Vitro Pto DC3000-GFP or P. aeruginosa PAO Growth
[0373] Droplet-based microfluidic experiments with Pto DC3000 were performed in NYGB medium at a temperature of 28° C., while the same experiments with P. aeruginosa PAO were performed in LB medium at a temperature of 37° 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 bacteria at 10.sup.7 cfu/ml, 20 μl of in vitro synthesized candidate siRNAs to obtain the final concentration wanted or sterile water for the control sample followed by 60 μl of medium. 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 ˜500 nl each were formed and incubated inside the instrument for the 24 hours. For each droplet, measurements of biomass (and GFP fluorescence for Pto DC3000-GFP) 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
[0374] 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 (
[0375] Because the expression of HrpL and Cfa6 virulence factors is known to be regulated by various environmental cues (54, 55), we also tested whether AGS could be effective against the Photorhabdus luminescens luxCDABE operon chromosomally expressed in Pto DC3000 under the constitutive kanamycin promoter (56). 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 (57). 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 (
Example 3. Host-Encoded siRNAs Directed Against Cfa6 and HrpL Prevent Pto DC3000-Induced Stomatal Reopening Presumably by Suppressing Coronatine Biosynthesis
[0376] Because Cfa6 and HrpL are known to regulate each other (55) and because HrpL and Cfa6 are both essential for coronatine (COR) biosynthesis (54, 55), 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 (
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
[0377] 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 (
[0378] 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 (54). 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 (
Example 5. Exogenous Delivery of Total RNAs Derived from IR-CFA6/HRPL Plants Protect WT Arabidopsis and Tomato Plants Against Pto DC3000
[0379] 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 (30-34), and was further found to operate in other nematodes but also in insects, plants and fungi (30, 35). 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 (
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
[0380] 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 F.sub.3 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 (
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
[0381] 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 (
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
[0382] 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 (
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
[0383] 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 CFA6HRPL dsRNAs of expected size (
[0384] 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 (
Example 10. Plant Small RNAs and In Vitro Synthesized Small RNAs can Trigger AGS in Pseudomonas aeruginosa, and this Regulatory Process can be Exploited to Reduce the Growth of this Bacterium by Targeting Some of its Essential Genes
[0385] The above findings, along with the fact that long dsRNAs expressed from mammalian cells are known to trigger potent antiviral interferon response (37), which is not the case in plant cells, prompted us to further assess whether plants could be employed to produce small RNAs against animal pathogenic bacteria. For this end, we have first transiently expressed the inverted repeat IR-LuxA LuxB construct described in the EXAMPLE 2 in N. benthamiana leaves using Agrobacterium-mediated transformation. As a negative control, we have also transiently expressed in N. benthamiana leaves an inverted repeat carrying sequence homologies with the GFP reporter gene. Total RNAs, containing either anti-LuxA B siRNAs or anti-GFP siRNAs, were incubated with a previously described Pseudomonas aeruginosa (PAK) strain expressing a lux reporter system (64), and the bioluminescence activity was further monitored in in vitro conditions on a microplate reader. Using this approach, we detected a specific decrease in bioluminescence activity in the presence of anti-LuxA and anti-LuxB plant siRNAs, which was not observed with anti-GFP siRNAs (
[0386] To further determine whether AGS could additionally be detected against PAK endogenous genes, we have further generated chimeric inverted repeats designed to concomitantly target DnaA, DnaN and GyrB genes, or RpoC, SecE and SodB genes. It is noteworthy that these P. aeruginosa targets were chosen because their individual deletion was known to alter the survival of this bacterium (38-40). Both inverted repeat constructs were found to overexpress small RNAs against these bacterial genes upon Agrobacterium-mediated transformation in N. benthamiana leaves (data not shown). Interestingly, when 20 ng/ul of each total RNA extracts were incubated with the lux-tagged PAK strain, we found a decrease in bioluminescence activity compared to total RNAs extracts derived from non-transformed N. benthamiana leaves (
[0387] Finally, we investigated whether in vitro synthesized siRNAs could also be active in these prokaryotic cells, as observed in the phytopathogenic bacterium Pto DC3000 (EXAMPLE 10,
Example 11. In Planta Production of EV-Embedded siRNAs Directed Against Essential or Virulence Genes from Pseudomonas aeruginosa, Shigella Flexneri and Staphylococcus aureus
[0388] To produce plant EV-embedded small RNAs that might be ultimately used as RNAi-based prophylactic or therapeutic agents, we generate inverted repeat constructs and express them in planta (preferentially in tobacco by using transient and/or stable Agrobacterium-mediated transformation methods).
[0389] More specifically, we have targeted the essential genes from P. aeruginosa, including LptH, LolA, TolB, LpxA, LpxD, dnaA, dnaN, gyrB, rpoC, secE and sodB, using the following constructs (all of them containing the intron of SEQ ID NO:2, apart from the target sequences): [0390] IR-LptH/LolA/TolB, SEQ ID NO: 250-251; [0391] IR-LpxA/LpxD/TolB, SEQ ID NO: 252-253; [0392] IR-dnaA/dnaB/gyrB, SEQ ID NO:108-109; [0393] IR-rpoC/secE/SodB, SEQ ID NO:110-111; and [0394] IR-secE/dnaN/gyrB, SEQ ID NO: 254-255.
[0395] We have also targeted the essential genes of Shigella flexneri, including FtsA, Can, Tsf AccD, Der, Psd using the constructs: [0396] IR-FtsA/Can/Tsf SEQ ID NO: 116-117; and [0397] IR-AccD/Der/Psd, SEQ ID NO: 118-119.
[0398] The same approach has been also used for the production of plant EV-embedded small RNAs directed against key virulence genes from P. aeruginosa, including genes involved in the regulation and/or assembly of type II or type III secretion systems, XcpQ, PscC, PcrV, PcrR, ExoS, ExoU, ExsA, Vrf the quorum sensing signaling factors LasR, RhlR, MvfR, VqsM, the GAC signaling components GacA, RsmA, by using the following constructs: [0399] IR-XcpQ ExsA/PcrV/LasR/RhlR/VqsM/RmsA, SEQ ID NO: 256-257; [0400] IR-XcpQ/PscF/PscC, SEQ ID NO: 258-259; [0401] IR-ExoS/ExsA/Vrf, SEQ ID NO: 260-261; [0402] IR-ExoU/ExsA/Vrf, SEQ ID NO: 262-263; [0403] IR-LasR/RhlR/VqsM, SEQ ID NO: 264-265; and [0404] IR-GacA/RmsA/MvfR, SEQ ID NO: 266-267.
[0405] We have also targeted the virulence genes of Shigella flexneri, including VirF, VirB, IcsA using the constructs IR-VirF/VirB IcsA, SEQ ID NO: 268-269, and the virulence genes of Staphylococcus aureus, including the genes encoding surface bound proteins fnbA, clfA, clfB, spa, atl, the leukotoxins lukF-PV, lukS-PV, lukE, lukD, HlgB, the alpha hemolysin hla, and the toxic shock syndrome toxin-1 tsst-1, by using the constructs [0406] IR-fnbA/clfA/clfB/spa, SEQ ID NO: 270-271; [0407] IR-lukF-PV/lukS-PV/lukE/lukD, SEQ ID NO: 272-273; and [0408] IR-HlgB/hla tsst-1/atl, SEQ ID NO: 274-275.
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