Derivative of cinnamic acid inhibits T3SS of Xanthomonas oryzae pv. oryzae through the HrpG-HrpX regulatory cascade.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has a significant impact on rice yield and quality worldwide. Traditionally, bactericide application has been commonly used to control this devastating disease. However, the overuse of fungicides has led to a number of problems such as the development of resistance and environmental pollution. Therefore, the development of new methods and approaches for disease control are still urgent. In this paper, a series of cinnamic acid derivatives were designed and synthesized, and three novel T3SS inhibitors A10, A12 and A20 were discovered. Novel T3SS inhibitors A10, A12 and A20 significantly inhibited the hpa1 promoter activity without affecting Xoo growth. Further studies revealed that the title compounds A10, A12 and A20 significantly impaired hypersensitivity in non-host plant tobacco leaves, while applications on rice significantly reduced symptoms of bacterial leaf blight. RT-PCR showed that compound A20 inhibited the expression of T3SS-related genes. In summary, this work exemplifies the potential of the title compound as an inhibitor of T3SS and its efficacy in the control of bacterial leaf blight.

Discovery of Epipodophyllotoxin-Derived B2 as Promising XooFtsZ Inhibitor for Controlling Bacterial Cell Division: Structure-Based Virtual Screening, Synthesis, and SAR Study.

The emergence of phytopathogenic bacteria resistant to antibacterial agents has rendered previously manageable plant diseases intractable, highlighting the need for safe and environmentally responsible agrochemicals. Inhibition of bacterial cell division by targeting bacterial cell division protein FtsZ has been proposed as a promising strategy for developing novel antibacterial agents. We previously identified 4'-demethylepipodophyllotoxin (DMEP), a naturally occurring substance isolated from the barberry species Dysosma versipellis, as a novel chemical scaffold for the development of inhibitors of FtsZ from the rice blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Therefore, constructing structure-activity relationship (SAR) studies of DMEP is indispensable for new agrochemical discovery. In this study, we performed a structure-activity relationship (SAR) study of DMEP derivatives as potential XooFtsZ inhibitors through introducing the structure-based virtual screening (SBVS) approach and various biochemical methods. Notably, prepared compound B2, a 4'-acyloxy DMEP analog, had a 50% inhibitory concentration of 159.4 µM for inhibition of recombinant XooFtsZ GTPase, which was lower than that of the parent DMEP (278.0 µM). Compound B2 potently inhibited Xoo growth in vitro (minimum inhibitory concentration 153 mg L-1) and had 54.9% and 48.4% curative and protective control efficiencies against rice blight in vivo. Moreover, compound B2 also showed low toxicity for non-target organisms, including rice plant and mammalian cell. Given these interesting results, we provide a novel strategy to discover and optimize promising bactericidal compounds for the management of plant bacterial diseases.

The CC-NB-LRR OsRLR1 mediates rice disease resistance through interaction with OsWRKY19.

Nucleotide-binding site-leucine-rich repeat (NB-LRR) resistance proteins are critical for plant resistance to pathogens; however, their mechanism of activation and signal transduction is still not well understood. We identified a mutation in an as yet uncharacterized rice coiled-coil (CC)-NB-LRR, Oryza sativa RPM1-like resistance gene 1 (OsRLR1), which leads to hypersensitive response (HR)-like lesions on the leaf blade and broad-range resistance to the fungal pathogen Pyricularia oryzae (syn. Magnaporthe oryzae) and the bacterial pathogen Xanthomonas oryzae pv. oryzae, together with strong growth reduction. Consistently, OsRLR1-overexpression lines showed enhanced resistance to both pathogens. Moreover, we found that OsRLR1 mediates the defence response through direct interaction in the nucleus with the transcription factor OsWRKY19. Down-regulation of OsWRKY19 in the rlr1 mutant compromised the HR-like phenotype and resistance response, and largely restored plant growth. OsWRKY19 binds to the promoter of OsPR10 to activate the defence response. Taken together, our data highlight the role of a new residue involved in the NB-LRR activation mechanism, allowing identification of a new NB-LRR downstream signalling pathway.

Ginger Essential Oils-Loaded Nanoemulsions: Potential Strategy to Manage Bacterial Leaf Blight Disease and Enhanced Rice Yield.

The bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious rice diseases, causing huge yield losses worldwide. Several technologies and approaches have been opted to reduce the damage; however, these have had limited success. Recently, scientists have been focusing their efforts on developing efficient and environmentally friendly nanobactericides for controlling bacterial diseases in rice fields. In the present study, a scanning electron microscope (SEM), transmission electron microscope (TEM), and a confocal laser scanning microscope (CLSM) were utilized to investigate the mode of actions of ginger EOs on the cell structure of Xoo. The ginger EOs caused the cells to grow abnormally, resulting in an irregular form with hollow layers, whereas the dimethylsulfoxide (DMSO) treatment showed a typical rod shape for the Xoo cell. Ginger EOs restricted the growth and production of biofilms by reducing the number of biofilms generated as indicated by CLSM. Due to the instability, poor solubility, and durability of ginger EOs, a nanoemulsions approach was used, and a glasshouse trial was performed to assess their efficacy on BLB disease control. The in vitro antibacterial activity of the developed nanobactericides was promising at different concentration (50-125 µL/mL) tested. The efficacy was concentration-dependent. There was significant antibacterial activity recorded at higher concentrations. A glasshouse trial revealed that developed nanobactericides managed to suppress BLB disease severity effectively. Treatment at a concentration of 125 µL/mL was the best based on the suppression of disease severity index, AUDPC value, disease reduction (DR), and protection index (PI). Furthermore, findings on plant growth, physiological features, and yield parameters were significantly enhanced compared to the positive control treatment. In conclusion, the results indicated that ginger essential oils loaded-nanoemulsions are a promising alternative to synthetic antibiotics in suppressing Xoo growth, regulating the BLB disease, and enhancing rice yield under a glasshouse trial.

Whole Genome Sequencing and Tn5-Insertion Mutagenesis of Pseudomonas taiwanensis CMS to Probe Its Antagonistic Activity Against Rice Bacterial Blight Disease.

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Synthesis and bioactivity of 1,3-thiazolidine-2-thione derivatives against type III secretion system of Xanthomonas oryzae.

Targeting virulence factors of bacterial without affecting their growth and survival, has been an initiative strategy for the development of novel anti-microbial agents. The type III secretion system (T3SS), one of essential and highly conserved virulence factors in most Gram-negative pathogenic bacteria, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens on rice, which causes leaf blight disease. To discover potential anti-virulence agents against the pathogens, a new series of 1,3-thiazolidine-2-thione derivatives containing 5-phenyl-2-furan were designed and synthesized. Their structures were characterized by 1H NMR, 13C NMR, MS, and elemental analysis. All the title compounds inhibited the promoter activity of a harpin gene hpa1, significantly, that were further checked for the impact on bacterial growth. The results indicated that treatment of Xoo with the title compound III-7 did not affect bacterial growth or survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the inhibitor. The mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.

Synthesis and biological evaluation of 1,3,4-thiadiazole derivatives as type III secretion system inhibitors against Xanthomonas oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) infection directly leads to a severe disease known as leaf blight, which is a major cause of yield loss of rice. Use of traditional bactericides has resulted in severe resistance in pathogenic bacteria. A new approach screening compounds that target the virulence factors rather than killing bacterial pathogens is imperative. In gram-negative bacteria, the type III secretion system (T3SS) is a conserved and significant virulence factor considered as a target for drug development. Therefore, we designed and synthesized a new series of 5-phenyl-2-furan carboxylic acid derivatives stitched with 2-mercapto-1,3,4-thiadiazole. Bioassays revealed that the title candidates attenuated the hypersensitive response through suppressing the promoter activity of a harpin gene hpa1 without affecting bacterial growth. Quantitative real time polymerase chain reaction (qRT-PCR) analysis demonstrated reduced the expression of several genes associated with T3SS, when title compounds were applied. Additionally, hrp gene cluster members, including hrpG and hrpX, had reduced mRNA levels. In vivo greenhouse tests showed that candidate compounds could alleviate the effects of Xoo infection in rice (Oryza sativa) and possess better protective activity against rice bacterial leaf blight than bismerthiazol and thiodiazole copper. All tested compounds were safe to rice. This work suggests there are new safe options for Xoo control in rice from these 1,3,4-thiadiazole derivatives.

Small Molecule Inhibitors Specifically Targeting the Type III Secretion System of Xanthomonas oryzae on Rice.

The initiative strategy for the development of novel anti-microbial agents usually uses the virulence factors of bacteria as a target, without affecting their growth and survival. The type III secretion system (T3SS), one of the essential virulence factors in most Gram-negative pathogenic bacteria because of its highly conserved construct, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) causes leaf blight diseases and is one of the most important pathogens on rice. To find potential anti-virulence agents against this pathogen, a number of natural compounds were screened for their effects on the T3SS of Xoo. Three of 34 compounds significantly inhibited the promoter activity of the harpin gene, hpa1, and were further checked for their impact on bacterial growth and on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results indicated that treatment of Xoo with CZ-1, CZ-4 and CZ-9 resulted in an obviously attenuated HR without affecting bacterial growth and survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the three inhibitors. The mRNA levels of representative genes in the hypersensitive response and pathogenicity (hrp) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.

Two virulent sRNAs identified by genomic sequencing target the type III secretion system in rice bacterial blight pathogen.

BACKGROUND: Small non-coding RNA (sRNA) short sequences regulate various biological processes in all organisms, including bacteria that are animal or plant pathogens. Virulent or pathogenicity-associated sRNAs have been increasingly elucidated in animal pathogens but little is known about similar category of sRNAs in plant-pathogenic bacteria. This is particularly true regarding rice bacterial blight pathogen Xanthomonas oryzae pathovar oryzae (Xoo) as studies on the virulent role of Xoo sRNAs is very limited at present. RESULTS: The number and genomic distribution of sRNAs in Xoo were determined by bioinformatics analysis based on high throughput sequencing (sRNA-Seq) of the bacterial cultures from virulence-inducing and standard growth media, respectively. A total of 601 sRNAs were identified in the Xoo genome and ten virulent sRNA candidates were screened out based on significant differences of their expression levels between the culture conditions. In addition, trans3287 and trans3288 were also selected as candidates due to high expression levels in both media. The differential expression of 12 sRNAs evidenced by the sRNA-Seq data was confirmed by a convincing quantitative method. Based on genetic analysis of Xoo ΔsRNA mutants generated by deletion of the 12 single sRNAs, trans217 and trans3287 were characterized as virulent sRNAs. They are essential not only for the formation of bacterial blight in a susceptible rice variety Nipponbare but also for the induction of hypersensitive response (HR) in nonhost plant tobacco. Xoo Δtrans217 and Δtrans3287 mutants fail to induce bacterial blight in Nipponbare and also fail to induce the HR in tobacco, whereas, genetic complementation restores both mutants to the wild type in the virulent performance and HR induction. Similar effects of gene knockout and complementation were found in the expression of hrpG and hrpX genes, which encode regulatory proteins of the type III secretion system. Consistently, secretion of a type III effector, PthXo1, is blocked in Δtrans217 or Δtrans3287 bacterial cultures but retrieved by genetic complementation to both mutants. CONCLUSIONS: The genetic analysis characterizes trans217 and trans3287 as pathogenicity-associated sRNAs essential for the bacterial virulence on the susceptible rice variety and for the HR elicitation in the nonhost plant. The molecular evidence suggests that both virulent sRNAs regulate the bacterial virulence by targeting the type III secretion system.

Synthesis and bioactivity of thiazolidin-2-cyanamide derivatives against type III secretion system of Xanthomonas oryzae on rice.

Targeting virulence factors of bacterial without affecting their growth and survival, has been an initiative strategy for the development of novel anti-microbial agents. The type III secretion system (T3SS), one of essential and highly conserved virulence factors in most Gram-negative pathogenic bacteria, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most Important bacterial pathogens on rice, which causes leaf blight disease. To discover potential anti-virulence agents against the pathogens, a new series of thiazolidin-2-cyanamide derivatives containing 5-phenyl-2-furan were designed and synthesized. Their structures were characterized by 1H NMR, 13C NMR, MS, and elemental analysis. All the title compounds inhibited the promoter activity of a harpin gene hpa1, significantly, that were further checked for the impact on bacterial growth and on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results indicated that treatment of Xoo with the title compounds II-2, II-3 and II-4 resulted in significantly attenuated HR without affecting bacterial growth or survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the three inhibitors. The mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.

Rice BiP3 regulates immunity mediated by the PRRs XA3 and XA21 but not immunity mediated by the NB-LRR protein, Pi5.

Plant innate immunity is mediated by pattern recognition receptors (PRRs) and intracellular NB-LRR (nucleotide-binding domain and leucine-rich repeat) proteins. Overexpression of the endoplasmic reticulum (ER) chaperone, luminal-binding protein 3 (BiP3) compromises resistance to Xanthomonas oryzae pv. oryzae (Xoo) mediated by the rice PRR XA21 [12]. Here we show that BiP3 overexpression also compromises resistance mediated by rice XA3, a PRR that provides broad-spectrum resistance to Xoo. In contrast, BiP3 overexpression has no effect on resistance mediated by rice Pi5, an NB-LRR protein that confers resistance to the fungal pathogen Magnaporthe oryzae (M. oryzae). Our results suggest that rice BiP3 regulates membrane-resident PRR-mediated immunity.

Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.

D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d-alanyl-d-alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation.

Design and Enantioselective Synthesis of Chiral Pyranone Fused Indole Derivatives with Antibacterial Activities against Xanthomonas oryzae pv oryzae for Protection of Rice.

A new class of chiral pyranone fused indole derivatives were prepared by means of N-heterocyclic carbene (NHC) organocatalysis and demonstrated notable antibacterial activity against Xanthomonas oryzae pv oryzae (Xoo). Bioassays showed that compounds (3S,4R)-5b, (3S,4R)-5d, and (3S,4R)-5l exhibited promising in vitro efficacy against Xoo, with EC50 values of 9.05, 9.71, and 5.84 mg/L, respectively, which were superior to that of the positive controls with commercial antibacterial agents, bismerthiazol (BT, EC50 = 27.8 mg/L) and thiodiazole copper (TC, EC50 = 70.1 mg/L). Furthermore, single enantiomer (3S,4R)-5l was identified as an optimal structure displaying 55.3% and 52.0% curative and protective activities against Xoo in vivo tests at a concentration of 200 mg/L, which slightly surpassed the positive control with TC (curative and protective activities of 47.2% and 48.8%, respectively). Mechanistic studies through molecular docking analysis revealed preliminary insights into the distinct anti-Xoo activity of the two single enantiomers (3S,4R)-5l and (3R,4S)-5l, wherein the (3S,4R)-configured stereoisomer could form a more stable interaction with XooDHPS (dihydropteroate synthase). These findings underscore the significant anti-Xoo potential of these chiral pyranone fused indole derivatives, and shall inspire further exploration as promising lead structures for a novel class of bactericides to combat bacterial infections and other plant diseases.

Fine mapping and sequence analysis reveal a promising candidate gene encoding a novel NB-ARC domain derived from wild rice (Oryza officinalis) that confers bacterial blight resistance.

Bacterial blight disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious constraints in rice production. The most sustainable strategy to combat the disease is the deployment of host plant resistance. Earlier, we identified an introgression line, IR 75084-15-3-B-B, derived from Oryza officinalis possessing broad-spectrum resistance against Xoo. In order to understand the inheritance of resistance in the O. officinalis accession and identify genomic region(s) associated with resistance, a recombinant inbred line (RIL) mapping population was developed from the cross Samba Mahsuri (susceptible to bacterial blight) × IR 75084-15-3-B-B (resistant to bacterial blight). The F2 population derived from the cross segregated in a phenotypic ratio of 3: 1 (resistant susceptible) implying that resistance in IR 75084-15-3-B-B is controlled by a single dominant gene/quantitative trait locus (QTL). In the F7 generation, a set of 47 homozygous resistant lines and 47 homozygous susceptible lines was used to study the association between phenotypic data obtained through screening with Xoo and genotypic data obtained through analysis of 7K rice single-nucleotide polymorphism (SNP) chip. Through composite interval mapping, a major locus was detected in the midst of two flanking SNP markers, viz., Chr11.27817978 and Chr11.27994133, on chromosome 11L with a logarithm of the odds (LOD) score of 10.21 and 35.93% of phenotypic variation, and the locus has been named Xa48t. In silico search in the genomic region between the two markers flanking Xa48t identified 10 putatively expressed genes located in the region of interest. The quantitative expression and DNA sequence analysis of these genes from contrasting parents identified the Os11g0687900 encoding an NB-ARC domain-containing protein as the most promising gene associated with resistance. Interestingly, a 16-bp insertion was noticed in the untranslated region (UTR) of the gene in the resistant parent, IR 75084-15-3-B-B, which was absent in Samba Mahsuri. The association of Os11g0687900 with resistance phenotype was further established by sequence-based DNA marker analysis in the RIL population. A co-segregating PCR-based INDEL marker, Marker_Xa48, has been developed for use in the marker-assisted breeding of Xa48t.

Antibacterial, anti-biofilm, and anti-virulence potential of tea tree oil against leaf blight pathogen Xanthomonas oryzae pv. oryzae instigates disease suppression.

Bacterial leaf blight (BLB) disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), causes major annual economic losses around the world. Inorganic copper compounds and antibiotics are conventionally used to control BLB disease. They often cause environmental pollution, contributing to adverse effects on human health. Therefore, research is now leading to the search for alternative control methods. Tea tree oil (TTO) is obtained from a traditional medicinal plant, Melaleuca alternifolia, with antibacterial properties. In this study, we found that TTO showed antibacterial activity against Xoo with a minimum inhibitory concentration (MIC) of 18 mg/ml. These antagonistic activities were not limited only to planktonic cells, as further studies have shown that TTO effectively eradicated sessile cells of Xoo in both initial and mature biofilms. Furthermore, it was also observed that TTO reduced various key virulence properties of Xoo, such as swimming, swarming motility, and the production of extracellular polymeric substances, xanthomonadin, and exoenzymes. TTO triggered ROS generation with cell membrane damage as an antibacterial mode of action against Xoo. The in silico study revealed that 1,8-cineole of TTO was effectively bound to two essential proteins, phosphoglucomutase and peptide deformylase, responsible for the synthesis of EPS and bacterial survival, respectively. These antibacterial and anti-virulence activities of TTO against Xoo were further confirmed by an ex vivo virulence assay where TTO significantly reduced the lesion length caused by Xoo on rice leaves. All these data concluded that TTO could be a safe, environment-friendly alternative approach for the comprehensive management of BLB disease.

Application of Bacterial Endophytes to Control Bacterial Leaf Blight Disease and Promote Rice Growth.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) disease in rice (Oryza sativa L.) and it is among the most destructive pathogen responsible for severe yield losses. Potential bacterial biocontrol agents (BCAs) with plant growth promotion (PGP) abilities can be applied to better manage the BLB disease and increase crop yield, compared to current conventional practices. Thus, this study aimed to isolate, screen, and identify potential BCAs with PGP abilities. Isolation of the BCAs was performed from internal plant tissues and rhizosphere soil of healthy and Xoo-infected rice. A total of 18 bacterial strains were successfully screened for in vitro antagonistic ability against Xoo, siderophore production and PGP potentials. Among the bacterial strains, 3 endophytes, Bacillus sp. strain USML8, Bacillus sp. strain USML9, and Bacillus sp. strain USMR1 which were isolated from diseased plants harbored the BCA traits and significantly reduced leaf blight severity of rice. Simultaneously, the endophytic BCAs also possessed plant growth promoting traits and were able to enhance rice growth. Application of the selected endophytes (BCAs-PGP) at the early growth stage of rice exhibited potential in suppressing BLB disease and promoting rice growth.

Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application.

The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5'-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99A strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.

Characterization of Type VI Secretion System in Xanthomonas oryzae pv. oryzae and Its Role in Virulence to Rice.

Type VI secretion system (T6SS) is a contact-dependent secretion system, employed by most gram-negative bacteria for translocating effector proteins to target cells. The present study was conducted to investigate T6SS in Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight in rice, and to unveil its functions. Two T6SS clusters were found in the genome of Xoo PXO99A. The deletion mutants, Δhcp1, Δhcp2, and Δhcp12, targeting the hcp gene in each cluster, and a double-deletion mutant targeting both genes were constructed and tested for growth rate, pathogenicity to rice, and inter-bacterial competition ability. The results indicated that hcp in T6SS-2, but not T6SS-1, was involved in bacterial virulence to rice plants. However, neither T6SS-1 nor T6SS-2 had any effect on the ability to compete with Escherichia coli or other bacterial cells. In conclusion, T6SS gene clusters in Xoo have been characterized, and its role in virulence to rice was confirmed.

PXO_RS20535, Encoding a Novel Response Regulator, Is Required for Chemotactic Motility, Biofilm Formation, and Tolerance to Oxidative Stress in Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight of rice, possesses two-component regulatory systems (TCSs) as an intracellular signaling pathway. In this study, we observed changes in virulence, biofilm formation, motility, chemotaxis, and tolerance against oxidative stress of a knockout mutant strain for the PXO_RS20535 gene, encoding an orphan response regulator (RR). The mutant strain lost virulence, produced significantly less biofilm, and showed remarkably reduced motility in swimming, swarming, and twitching. Furthermore, the mutant strain lost glucose-guided movement and showed clear diminution of growth and survival in the presence of H2O2. These results indicate that the RR protein encoded in the PXO_RS20535 gene (or a TCS mediated by the protein) is closely involved in regulation of biofilm formation, all types of motility, chemotaxis, and tolerance against reactive oxygen species (ROS) in Xoo. Moreover we found that the expression of most genes required for a type six secretion system (T6SS) was decreased in the mutant, suggesting that lack of the RR gene most likely leads to defect of T6SS in Xoo.

Discovery of Ethyl 2-Nitro-3-Arylacrylates Molecules as T3SS Inhibitor Reducing the Virulence of Plant Pathogenic Bacteria Xanthomonas.

Xanthomonas oryzae pv. oryzae (Xoo) is a gram-negative pathogen which causes leaf blight disease. Known traditional bactericides are not much more effective in inhibiting this bacteria than before. Selecting the virulence factor of the bacteria as the target without affecting their growth has been considered as a novel method for developing new anti-microbial drugs. Type III secretion systems (T3SS) are one of the important and highly conserved virulence factors in most gram-negative pathogens, which has been considered as an effective target to develop new anti-microbial drugs. In order to discover potential anti-microbial drugs against Xoo pathogens, a series of ethyl 2-nitro-3-arylacrylates compounds were screened. Among them, the compounds I-9, I-12, and I-13 could highly inhibit the promoter activity of a harpin gene hpa1, which were used to further check for the influence on bacterial growth and on the hypersensitive response (HR) caused by Xoo bacteria on non-host plants. The results showed that above compounds could reduce HR without affecting bacterial growth and survival. Moreover, qRT-PCR analysis indicated that treatment with the three inhibitors (I-9, I-12, and I-13) could suppress the expression of the Xoo T3SS in different extent. The mRNA levels of representative genes in the hrp cluster, including the key regulatory genes hrpG and hrpX, were decreased. Last but not least, in vivo test ensured that the above compounds reduced the disease symptoms of Xoo on the rice and Xcc on the Chinese radish.