The rsmA-like gene rsmA(Xoo) of Xanthomonas oryzae pv. oryzae regulates bacterial virulence and production of diffusible signal factor.
ABSTRACT: The plant-pathogenic prokaryote Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, one of the most destructive diseases of rice. A nonpolar mutant of the rsmA-like gene rsmA(Xoo) of the Xoo Chinese strain 13751 was constructed by homologous integration with a suicide plasmid. Virulence tests on a host plant, namely the hybrid rice cultivar Teyou 63, showed that the mutant had lost its virulence almost completely, whereas tests on a nonhost, namely castor-oil plant (Ricinus communis), showed that the mutant had also lost the ability to induce a hypersensitive response in the nonhost. In addition, the rsmA(Xoo) mutant produced significantly smaller amounts of the diffusible signal factor, extracellular endoglucanase, amylase and extracellular polysaccharide, but showed significantly higher glycogen accumulation, bacterial aggregation and cell adhesion. The expression of most hrp genes, genes encoding AvrBs3/PthA family members, rpfB, xrvA, glgA, eglXoB and XOO0175 (encoding an ?-amylase) was down-regulated in the rsmA(Xoo) mutant. All phenotypes and expression levels of the tested genes in the rsmA(Xoo) mutant were restored to their levels in the wild-type by the presence of rsmA(Xoo) in trans. These results indicate that rsmA(Xoo) is essential for the virulence of Xoo.
Project description:BACKGROUND: Nonhost resistance is a generalized, durable, broad-spectrum resistance exhibited by plant species to a wide variety of microbial pathogens. Although nonhost resistance is an attractive breeding strategy, the molecular basis of this form of resistance remains unclear for many plant-microbe pathosystems, including interactions with the bacterial pathogen of rice, Xanthomonas oryzae pv. oryzae (Xoo). METHODS AND FINDINGS: Virus-induced gene silencing (VIGS) and an assay to detect the hypersensitive response (HR) were used to screen for genes required for nonhost resistance to Xoo in N. benthamiana. When infiltrated with Xoo strain YN-1, N. benthamiana plants exhibited a strong necrosis within 24 h and produced a large amount of H(2)O(2) in the infiltrated area. Expression of HR- and defense-related genes was induced, whereas bacterial numbers dramatically decreased during necrosis. VIGS of 45 ACE (Avr/Cf-elicited) genes revealed identified seven genes required for nonhost resistance to Xoo in N. benthamiana. The seven genes encoded a calreticulin protein (ACE35), an ERF transcriptional factor (ACE43), a novel Solanaceous protein (ACE80), a hydrolase (ACE117), a peroxidase (ACE175) and two proteins with unknown function (ACE95 and ACE112). The results indicate that oxidative burst and calcium-dependent signaling pathways play an important role in nonhost resistance to Xoo. VIGS analysis further revealed that ACE35, ACE80, ACE95 and ACE175, but not the other three ACE genes, interfered with the Cf-4/Avr4-dependent HR. CONCLUSIONS/SIGNIFICANCE: N. benthamiana plants inoculated with Xoo respond by rapidly eliciting an HR and nonhost resistance. The oxidative burst and other signaling pathways are pivotal in Xoo-N. benthamiana nonhost resistance, and genes involved in this response partially overlap with those involved in Cf/Avr4-dependent HR. The seven genes required for N. benthamiana-mediated resistance to Xoo provide a basis for further dissecting the molecular mechanism of nonhost resistance.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ?dgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases.
Project description:Rice-pathogenic Xanthomonas oryzae bacteria cause severe harvest loss and challenge a stable food supply. The pathogen virulence relies strongly on bacterial TALE (transcription activator-like effector) proteins that function as transcriptional activators inside the plant cell. To understand the plant targets of TALEs, we determined the genome sequences of the Indian X. oryzae pv. oryzae (Xoo) type strain ICMP 3125T and the strain PXO142 from the Philippines. Their complete TALE repertoire was analyzed and genome-wide TALE targets in rice were characterized. Integrating computational target predictions and rice transcriptomics data, we were able to verify 12 specifically induced target rice genes. The TALEs of the Xoo strains were reconstructed and expressed in a TALE-free Xoo strain to attribute specific induced genes to individual TALEs. Using reporter assays, we could show that individual TALEs act directly on their target promoters. In particular, we show that TALE classes assigned by AnnoTALE reflect common target genes, and that TALE classes of Xoo and the related pathogen X. oryzae pv. oryzicola share more common target genes than previously believed. Taken together, we establish a detailed picture of TALE-induced plant processes that significantly expands our understanding of X. oryzae virulence strategies and will facilitate the development of novel resistances to overcome this important rice disease.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) in rice (Oryza sativa L.). In this study, we investigated the effect of a mutation in opsX (XOO1056), which encodes a saccharide biosynthesis regulatory protein, on the virulence and bacterial chemotaxis of Xoo. We performed DNA microarray analysis, which showed that 63 of 2,678 genes, including genes related to bacterial motility (flagellar and chemotaxis proteins) were significantly downregulated (<-2 log2 fold changes) by the mutation in opsX. Indeed, motility assays showed that the mutant strain was nonmotile on semisolid agar swarm plates. In addition, a mutant strain (opsX::Tn5) showed decreased virulence against the susceptible rice cultivar, IR24. Quantitative real-time RT-PCR reaction was performed to confirm the expression levels of these genes, including those related to flagella and chemotaxis, in the opsX mutant. Our findings revealed that mutation of opsX affects both virulence and bacterial motility. These results will help to improve our understanding of Xoo and provide insight into Xoo-rice interactions.
Project description:ExpA (GacA) is a global response regulator that controls the expression of major virulence genes, such as those encoding plant cell wall-degrading enzymes (PCWDEs) in the model soft rot phytopathogen Pectobacterium wasabiae SCC3193. Several studies with pectobacteria as well as related phytopathogenic gammaproteobacteria, such as Dickeya and Pseudomonas, suggest that the control of virulence by ExpA and its homologues is executed partly by modulating the activity of RsmA, an RNA-binding posttranscriptional regulator. To elucidate the extent of the overlap between the ExpA and RsmA regulons in P. wasabiae, we characterized both regulons by microarray analysis. To do this, we compared the transcriptomes of the wild-type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray data for selected virulence-related genes were confirmed through quantitative reverse transcription (qRT-PCR). Subsequently, assays were performed to link the observed transcriptome differences to changes in bacterial phenotypes such as growth, motility, PCWDE production, and virulence in planta. An extensive overlap between the ExpA and RsmA regulons was observed, suggesting that a substantial portion of ExpA regulation appears to be mediated through RsmA. However, a number of genes involved in the electron transport chain and oligogalacturonide metabolism, among other processes, were identified as being regulated by ExpA independently of RsmA. These results suggest that ExpA may only partially impact fitness and virulence via RsmA.
Project description:It has been known that most regulation of pathogenicity factor (rpf) genes in xanthomonads regulates virulence in response to the diffusible signal factor, DSF. Although many rpf genes have been functionally characterized, the function of rpfE is still unknown. We cloned the rpfE gene from a Xanthomonas oryzae pv. oryzae (Xoo) Korean race KACC10859 and generated mutant strains to elucidate the role of RpfE with respect to the rpf system. Through experiments using the rpfE-deficient mutant strain, we found that mutation in rpfE gene in Xoo reduced virulence, swarm motility, and production of virulence factors such as cellulase and extracellular polysaccharide. Disease progress by the rpfE-deficient mutant strain was significantly slowed compared to disease progress by the wild type and the number of the rpfE-deficient mutant strain was lower than that of the wild type in the early phase of infection in the inoculated rice leaf. The rpfE mutant strain was unable to utilize sucrose or xylose as carbon sources efficiently in culture. The mutation in rpfE, however, did not affect DSF synthesis. Our results suggest that the rpfE gene regulates the virulence of Xoo under different nutrient conditions without change of DSF production.
Project description:BACKGROUND: Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc). RESULTS: Changes in gene expression of the African Xoo strain MAI1 in the susceptible rice cultivar Nipponbare were profiled, using an SSH Xoo DNA microarray. Microarray hybridization was performed comparing bacteria recovered from plant tissues at 1, 3, and 6 days after inoculation (dai) with bacteria grown in vitro. A total of 710 bacterial genes were found to be differentially expressed, with 407 up-regulated and 303 down-regulated. Expression profiling indicated that less than 20% of the 710 bacterial transcripts were induced in the first 24 h after inoculation, whereas 63% were differentially expressed at 6 dai. The 710 differentially expressed genes were one-end sequenced. 535 sequences were obtained from which 147 non-redundant sequences were identified. Differentially expressed genes were related to metabolism, secretion and transport, pathogen adherence to plant tissues, plant cell-wall degradation, IS elements, and virulence. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. The Xoo MAI1 non-redundant set of sequences was compared against several X. oryzae genomes, revealing a specific group of genes that was present only in MAI1. Numerous IS elements were also found to be differentially expressed. Quantitative real-time PCR confirmed 86% of the identified profile on a set of 14 genes selected according to the microarray analysis. CONCLUSIONS: This is the first report to compare the expression of Xoo genes in planta across different time points during infection. This work shows that as-yet-unidentified and potentially new virulence factors are appearing in an emerging African pathogen. It also confirms that African Xoo strains do differ from their Asian counterparts, even at the transcriptional level.
Project description:Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage materials produced in various microorganisms under nutrient-limited conditions. PhaR is a regulatory protein involved in PHA synthesis. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens in rice and has PHA biosynthesis genes in its genome, but the biological function of phaR in Xoo is unknown. In this study, we investigated the effects of the mutagenesis of phaR gene in Xoo strain PXO99A. Compared to the wildtype, the PhaR gene knock-out mutant PXO99?phaR was hypermotile and showed decreased growth rates in both rich and limited nutrient media. PXO99?phaR also showed almost 75% decrease in extracellular polysaccharide (EPS) production. When inoculated in rice leaves by leaf-clipping method, PXO99?phaR displayed reduced virulence in terms of lesion length and bacterial multiplication compared with the wildtype strain. PXO99?phaR also showed enhanced hypersensitive response (HR) induction in the leaves of non-host Nicotiana benthamiana with elevated hpa1 gene expression. Introduction of a cosmid containing the phaR coding sequence restored the phenotypes of the mutant to those of the wildtype strain. These results suggest that PhaR gene is an important gene that affects multiple bacterial characteristics, including EPS production, growth rate, defense response induced harpin production and motility, related to its virulence in plant.
Project description:The rpf genes and colS XOO1207/colR XOO1208 were known to require for virulence of Xanthomonas oryzae pv. oryzae (Xoo). In Xoo KACC10331 genome, two more colS/colR genes, colS XOO3534 (raxH)/colR XOO3535 (raxR) and colS XOO3762/colR XOO3763 were annotated. The colS XOO3534/colR XOO3535 were known to control AvrXa21 activity and functions of colS XOO3762/colR XOO3763 were unknown in Xoo. To characterize the relationship between rpf and colS/colR genes, expression of colS/colR genes in Rpf mutants of Xoo were analyzed with quantitative reverse transcription PCR (qRT-PCR). Expressions of all three colS/colR genes increased in the rpfF mutant in which DSF synthesis is defective. Expression of colS XOO1207/colR XOO1208, colS XOO3534/colR XOO3535 and colS XOO3762/colR XOO3763 increased 2, 2-7, 3-13 folds respectively. Expression of colS XOO3534 and colS XOO3762 also increased 2-4 folds in the rpfG mutant in which the signal from DSF is no longer transferred to down-stream. Expression of the other colS/colR genes was not significantly changed in the rpfG mutant compared to the wild type. Since RpfF and RpfG are responsible for DSF synthesis and signal transfer from DSF to down-stream to regulate virulence gene expression, these results suggest that the DSF and DSF-mediated signal regulate negatively three colS/colR genes in Xoo.
Project description:Thiazole, isothiazole, thiadiazole and their derivatives are widely thought to induce host defences against plant pathogens. In this article, we report that bismerthiazol, a thiadiazole molecule, reduces disease by inhibiting the histidine utilization (Hut) pathway and quorum sensing (QS). Bismerthiazol provides excellent control of bacterial rice leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo), but does not greatly inhibit Xoo growth in vitro. According to RNA-sequencing analysis, the transcription of the Hut pathway genes of Xoo ZJ173 was inhibited after 4.5 and 9.0 h of bismerthiazol treatment. Functional studies of hutG and hutU indicated that the Hut pathway had little effect on the growth and bismerthiazol sensitivity of Xoo in vitro, but significantly reduced the aggregation of Xoo cells. Deletion mutants of hutG or hutU were more motile, produced less biofilm and were less virulent than the wild-type, indicating that the Hut pathway is involved in QS and contributes to virulence. The overexpression of the hutG-U operons in ZJ173 reduced Xoo control by bismerthiazol. Bismerthiazol did not inhibit the transcription of Hut pathway genes, QS or virulence of the bismerthiazol-resistant strain 2-1-1. The results indicate that bismerthiazol reduces Xoo virulence by inhibiting the Hut pathway and QS.