Biosynthesis of Amino Acids in Xanthomonas oryzae pv. oryzae Is Essential to Its Pathogenicity.
ABSTRACT: Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease, which causes a large reduction in rice production. The successful interaction of pathogens and plants requires a particular nutrient environment that allows pathogen growth and the initiation of both pathogen and host responses. Amino acid synthesis is essential for bacterial growth when bacteria encounter amino acid-deficient environments, but the effects of amino acid synthesis on Xoo pathogenicity are unclear. Here, we systemically deleted the essential genes (leuB, leuC, leuD, ilvC, thrC, hisD, trpC, argH, metB, and aspC) involved in the synthesis of different amino acids and analyzed the effects of these mutations on Xoo virulence. Our results showed that leucine, isoleucine, valine, histidine, threonine, arginine, tryptophan, and cysteine syntheses are essential to Xoo infection. We further studied the role of leucine in the interaction between pathogens and hosts and found that leucine could stimulate some virulence-related responses and regulate Xoo pathogenicity. Our findings highlight that amino acids not only act as nutrients for bacterial growth but also play essential roles in the Xoo and rice interaction.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) causes the serious disease bacterial blight in rice. The pepA (Xoo0834) gene from Xoo is one of around 100 genes that have been selected for the design of antibacterial drugs. The pepA gene encodes leucine aminopeptidase (LAP), an exopeptidase that catalyzes the hydrolysis of leucine residues from the N-terminus of a protein or peptide. This enzyme was expressed in Escherichia coli, purified and crystallized, and preliminary X-ray structural studies have been carried out. The LAP crystal diffracted to 2.6 A resolution and belonged to the cubic space group P2(1)3. The unit-cell volume of the crystal was compatible with the presence of two monomers in the asymmetric unit.
Project description:Rice (Oryza sativa) plants expressing the XA21 cell-surface receptor kinase are resistant to Xanthomonas oryzae pv. oryzae (Xoo) infection. We previously demonstrated that expressing a chimeric protein containing the ELONGATION FACTOR Tu RECEPTOR (EFR) ectodomain and the XA21 endodomain (EFR:XA21) in rice does not confer robust resistance to Xoo. To test if the XA21 ectodomain is required for Xoo resistance, we produced transgenic rice lines expressing a chimeric protein consisting of the XA21 ectodomain and EFR endodomain (XA21:EFR) and inoculated these lines with Xoo. We also tested if the XA21:EFR rice plants respond to a synthetic sulfated 21 amino acid derivative (RaxX21-sY) of the activator of XA21-mediated immunity, RaxX. We found that five independently transformed XA21:EFR rice lines displayed resistance to Xoo as measured by lesion length analysis, and showed that five lines share characteristic markers of the XA21 defense response (generation of reactive oxygen species and defense response gene expression) after treatment with RaxX21-sY. Our results indicate that expression of the XA21:EFR chimeric receptor in rice confers resistance to Xoo. These results suggest that the endodomain of the EFR and XA21 immune receptors are interchangeable and the XA21 ectodomain is the key determinant conferring robust resistance to Xoo.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) causes rice bacterial blight (BB), one of the most widespread and destructive diseases in rice-growing regions worldwide. Melatonin enhances pathogen resistance by inducing plant innate immunity, but the direct effect of melatonin on plant pathogenic bacteria is poorly understood. In this study, we investigated the direct effects of melatonin on Xoo. Exogenous melatonin at 200 ?g/mL significantly inhibited the proliferation of Xoo and reduced the mRNA expression of five genes involved in cell division. This concentration of melatonin also inhibited the motility and biofilm formation of Xoo. Notably, melatonin was observed to alter the length of Xoo cells. To provide deeper insights into the mechanisms underlying this antibacterial activity, we examined global gene expression changes in Xoo strain PXO99 in response to the application of 200 ?g/mL melatonin using RNA sequencing (RNA-Seq). A wide range of differentially expressed genes (DEGs) related to catalytic activity and metal-binding activity were downregulated in Xoo cells in response to the melatonin treatment. In addition, DEGs responsible for carbohydrate and amino acid metabolism were also downregulated. These results suggest that the inhibitory mechanism of melatonin on Xoo proliferation may involve the regulation of cell division in combination with a reduction in the concentration or activity of enzymes involved in metabolism.
Project description:BACKGROUND:Disease resistance is an important factor that impacts rice production. However, the mechanisms underlying rice disease resistance remain to be elucidated. RESULTS:Here, we show that a robust set of genes has been defined in rice response to the infections of Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae (Mor). We conducted a comprehensive analysis of the available microarray data from a variety of rice samples with inoculation of Xoo and Mor. A set of 12,932 genes was identified to be regulated by Xoo and another set of 2709 Mor-regulated genes was determined. GO enrichment analysis of the regulated genes by Xoo or Mor suggested mitochondrion may be an arena for the up-regulated genes and chloroplast be another for the down-regulated genes by Xoo or Mor. Cytokinin-related processes were most frequently repressed by Xoo, while processes relevant to jasmonic acid and abscisic acid were most frequently activated by Xoo and Mor. Among genes responsive to Xoo and Mor, defense responses and diverse signaling pathways were the most frequently enriched resistance mechanisms. InterPro annotation showed the zinc finger domain family, WRKY proteins, and Myb domain proteins were the most significant transcription factors regulated by Xoo and Mor. KEGG analysis demonstrated pathways including 'phenylpropanoid biosynthesis', 'biosynthesis of antibiotics', 'phenylalanine metabolism', and 'biosynthesis of secondary metabolites' were most frequently triggered by Xoo and Mor, whereas 'circadian rhythm-plant' was the most frequent pathway repressed by Xoo and Mor. CONCLUSIONS:The genes identified here represent a robust set of genes responsive to the infections of Xoo and Mor, which provides an overview of transcriptional reprogramming during rice defense against Xoo and Mor infections. Our study would be helpful in understanding the mechanisms of rice disease resistance.
Project description:Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae. Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae.
Project description:BACKGROUND:Xanthomonas oryzae pv. oryzae (Xoo) can cause destructive bacterial blight in rice. As an antibacterial, resveratrol may inhibit Xoo growth. This study focused on the potential structural-activity relationship of resveratrol and its derivatives against Xoo growth, and 1H-NMR-based metabolomic analysis was applied to investigate the global metabolite changes in Xoo after resveratrol treatment. RESULTS:Resveratrol showed the strongest inhibitory effects on Xoo growth compared with its derivatives, which lacked double bonds (compounds 4-6) or hydroxyls were substituted with methoxyls (compounds 7-9). The IC50 of resveratrol against Xoo growth was 11.67?±?0.58??g/mL. Results indicated that the double bond of resveratrol contributed to its inhibitory effects on Xoo growth, and hydroxyls were vital for this inhibition. Interestingly, resveratrol also significantly inhibited Xoo flagellum growth. Based on 1H-NMR global metabolic analysis, a total of 30 Xoo metabolites were identified, the changes in the metabolic profile indicated that resveratrol could cause oxidative stress as well as disturb energy, purine, amino acid, and NAD+ metabolism in Xoo, resulting in the observed inhibitory effects on growth. CONCLUSIONS:This study showed that the double bond of resveratrol contributed to its inhibitory effects on Xoo growth, and hydroxyls were also the important active groups. Resveratrol could cause oxidative stress of Xoo cells, and disturb the metabolism of energy, purine, amino acid and NAD +, thus inhibit Xoo growth.
Project description:The xa5 gene encodes a basal transcription factor (TFIIA?) protein with wide spectrum resistance to bacterial blight caused by Xanthomonas oryzae pv. Oryzae (Xoo) in rice. It was only found in a few rice ecotypes, and the recessive characteristics limited its application in breeding. Here, we employed a TALEN-based technique to edit its dominant allelic TFIIA?5 and obtained many mutant TFIIA?5 genes. Most of them reduced rice susceptibility to varying degrees when the plants were challenged with the Xoo. In particular, the knocked-out TFIIA?5 can reduce the rice susceptibility significantly, although it cannot reach the xa5-mediated resistance level, indicating TFIIA?5 is a major component involved in disease susceptibility. In addition, the mutant encoding the protein with deletion of the 32nd amino acid or amino acid insertion between 32nd and 33rd site confers rice with the similar resistance to that of the knocked-out TFIIA?5. Thus, the amino acids around 32nd site are also the important action sites of TFIIA?5 besides the 39th amino acid previously reported. Moreover, the integration of xa5 into TFIIA?5-knockout plants conferred them with a similar resistance as IRBB5, the rice variety containing the homozygous xa5 gene. Thus, TFIIA?5 was not simply regarded as a resistant or a susceptible locus, as the substitution of amino acids might shift its functions.
Project description: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:MicroRNAs (miRNAs) are crucial player in plant-pathogen interaction. While the evidence has demonstrated that rice miRNAs mediate immune response to pathogens invasion, the roles of miRNAs on Xanthomonas oryzae pv. oryzae (Xoo) attack remain be in place. Herein, we monitored the responsive changes of rice miRNAs at 0, 8, 24 h across Xoo strain PXO86 infection in its compatible rice variety IR24 and incompatible variety IRBB5 by small RNA sequencing, and the genes targeted by miRNAs were also detected via degradome technology. The faithfulness of sequencing data was validated through quantitative real-time stem-loop reverse transcription-polymerase chain reaction assay. Bioinformatic analysis showed that the differentially expressed miRNAs could be divided into three immunity-related clusters, and 80 regulatory units were emerged in infection process, which comprises 29 differentially expressed known miRNAs and 38 cleaved targets. Furthermore, the miRNA presumptive function of separate immunity cluster in rice-Xoo interplay was confirmed through overexpressing osa-miR164a, osa-miR167d and osa-miR159b, and the disruption of regulatory units, osa-miR164a/OsNAC60, osa-miR167d-5p/OsWD40-174 and osa-miR159b/OsMYBGA, OsLRR-RLK2, OsMPK20-4, may reset rice defense response to Xoo infestation in a controllable manner. These findings provide new insights into the complex roles of characteristic miRNAs and their targets in rice-Xoo interactions.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) lead to the devastating rice bacterial diseases and have a very close genetic relationship. There are tissue-specificity differences between Xoo and Xoc, i.e., Xoo only proliferating in xylem vessels and Xoc spreading in intercellular space of mesophyll cell. But there is little known about the determinants of tissue-specificity between Xoo and Xoc. Here we show that Xoc can spread in the intercellular spaces of mesophyll cells to form streak lesions. But Xoo is restricted to growth in the intercellular spaces of mesophyll cells on the inoculation sites. In vivo, Xoc largely breaks the surface and inner structures of cell wall in mesophyll cells in comparison with Xoo. In vitro, Xoc strongly damages the cellulose filter paper in comparison with Xoo. These results suggest that the stronger cell wall-degradation ability of Xoc than that of Xoo may be directly determining the tissue-specificity.