A LuxR homologue of Xanthomonas oryzae pv. oryzae is required for optimal rice virulence

In Gram-negative bacteria a typical quorum sensing (QS) system usually involves the production and response to acylated homoserine lactones (AHLs). An AHL QS system is most commonly mediated by a LuxI family AHL synthase and a LuxR family AHL response regulator. This study reports for the first time the presence of a LuxR family-type regulator in Xanthomonas oryzae pv. oryzae ( Xoo ), which has been designated as OryR. The primary structure of OryR contains the typical signature domains of AHL QS LuxR family response regulators: an AHL-binding and a HTH DNA binding motif. The oryR gene is conserved among 26 Xoo strains and is also present in the genomes of close relatives X. campestris pv. campestris and X. axonopodis pv. citri . Disrupting oryR in three Xoo strains resulted in a significant reduction of rice virulence. The wild-type Xoo strains do not seem to produce AHLs and analysis of the Xoo sequenced genomes did not reveal the presence of a LuxI-family AHL synthase. The OryR protein was shown to be induced by macerated rice and affected the production of two secreted proteins: a cell-wall-degrading cellobiosidase and a 20-kDa protein of unknown function. By expressing and purifying OryR it was then observed that it was solubilized when grown in the presence of rice extract indicating that there could be a molecule(s) in rice which binds OryR. The role of OryR as a possible in planta induced LuxR family regulator is discussed.     

Xanthomonas campestris pv. campestris possesses a single gluconeogenic pathway that is required for virulence

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.     

The Xanthomonas oryzae pv. oryzae PhoPQ two-component system is required for AvrXA21 activity, hrpG expression, and virulence

The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the type one system-secreted molecule, AvrXA21. X. oryzae pv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eight rax (required for AvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in a raxR gene knockout strain. This survey led to the identification of the phoP gene encoding a response regulator that is up-regulated in the raxR knockout strain. Next we generated a phoP knockout strain and found it to be impaired in X. oryzae pv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca²⁺. The impaired virulence of the phoP knockout strain can be partially complemented by constitutive expression of hrpG, indicating that PhoP controls a key aspect of X. oryzae pv. oryzae virulence through regulation of hrpG. A gene encoding the cognate putative histidine protein kinase, phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in a phoQ knockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that the X. oryzae pv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH.     

Characterization of Xanthomonas oryzae pv. oryzae recX, a gene that is required for high-level expression of recA

A universally primed (UP)-PCR cross hybridization assay was developed for rapid identification of isolates of Rhizoctonia solani into the correct anastomosis group (AG). Twenty-one AG tester isolates belonging to 11 AGs of R. solani were amplified with a single UP primer which generated multiple PCR fragments for each isolate. The amplified products were spotted onto a filter, immobilized and used for cross hybridization against amplification products from the different isolates. Isolates within AG subgroups cross hybridize strongly, whereas between different AGs little or no cross hybridization occurs. Sixteen Rhizoctonia isolates from diseased sugar beets and potatoes were identified using the assay. The results were supported by restriction fragment length polymorphism analysis of the ITS1-5.8S-ITS2 region of the nuclear encoded ribosomal DNA. Through standardization and use of quick non-radioactive labeling techniques, the UP-PCR cross hybridization assay has potential for routine use by modern DNA chip technology.     

The diffusible signal factor synthase,RpfF, in Xanthomonas oryzae pv. oryzae is required for the maintenance of membrane integrity and virulence

The Xanthomonas group of phytopathogens communicate with a fatty acid‐like cell–cell signalling molecule, cis‐11‐2‐methyl‐dodecenoic acid, also known as diffusible signal factor (DSF). In the pathogen of rice, Xanthomonas oryzae pv. oryzae, DSF is involved in the regulation of several virulence‐associated functions, including production and secretion of several cell wall hydrolysing type II secretion effectors. To understand the role of DSF in the secretion of type II effectors, we characterized DSF synthase‐deficient (rpfF) and DSF‐deficient, type II secretion (xpsE) double mutants. Mutant analysis by expression analysis, secretion assay, fatty acid analysis, and physiological studies indicated that rpfF mutants exhibit hypersecretion of several type II effectors due to a perturbed membrane and DSF is required for maintaining membrane integrity. The rpfF mutants exhibited significantly higher uptake of 1‐N‐phenylnapthylamine and ethidium bromide, and up‐regulation of r poE (σ^E). Increasing the osmolarity of the medium could rescue the hypersecretion phenotype of the rpfF mutant. The rpfF mutant exhibited highly reduced virulence. We report for the first time that in X. oryzae pv. oryzae RpfF is involved in the maintenance of membrane integrity by playing a regulatory role in the fatty acid synthesis pathway.     

Cloning of a locus involved in pathogenicity and production of extracellular polysaccharide and protease in Xanthomonas campestris pv. campestris

Abstract A Tn5-induced mutant of Xanthomonas campestris pv campestris deficient in protease and extracellular polysaccharide production, and non-pathogenic to turnip seedlings, was isolated. Recombinant clones which restored all three characteristics concomitantly were obtained from a wild-type genomic library. One of the plasmid clones, pIJ3090, showed no hybridization to previously isolated clones involved In protease production. The mutant was not complemented by other known clones involved in pathogenicity, in xanthan gum production or in protease production.     

水稻条斑病菌胞外多糖相关基因的鉴定

摘要:【目的】前期研究中从Tn5 转座子插入的水稻条斑病菌突变体库中获得了17 个胞外多糖改变的突变体。【方法】本文对这些突变体的Tn5 插入位点和基因类型进行了鉴定。【结果】结果显示,胞外多糖减少的11 个突变体中多数为Tn5 插入在已知的gum、xan 和wxoc 基因簇上,Xoryp_4217、Xoryp_2488 和Xoryp_0918为未知的与胞外多糖产生有关的基因,属首次报道;6 个胞外多糖增多的突变体中,fimO、pilY 和xopQ 与胞外多糖产生有关,但在水稻条斑病菌中未见报道;Xoryp2392、Xoryp_4221 和Xoryp_3511 为首次鉴定,其中Xoryp_3511 仅在水稻黄单胞菌中存在。毒性测定结果显示,胞外多糖减少的突变体在水稻上的毒性变弱,而胞外多糖增加的突变体在水稻上的毒性没有显著变化。【结论】这些结果为进一步分析水稻条斑病菌胞外多糖代谢途径以及与水稻的互作关系奠定了基础。     

Pigment and virulence deficiencies associated with mutations in the aroE gene of Xanthomonas oryzae pv. oryzae

Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus Xanthomonas. We identified an ethyl methanesulfonate-induced Xanthomonas oryzae pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig(-) Vir(-) Aro(-)). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the aroE gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an Escherichia coli aroE mutant for prototrophy, indicating that aroE is functionally conserved in X. oryzae pv. oryzae and E. coli. The nucleotide sequence of the 2.9-kb region containing aroE revealed that a putative DNA helicase gene is located adjacent to aroE. Our results indicate that aroE is required for normal levels of virulence and xanthomonadin production in X. oryzae pv. oryzae.     

A high-molecular-weight outer membrane protein of Xanthomonas oryzae pv. oryzae exhibits similarity to non-fimbrial adhesins of animal pathogenic bacteria and is required for optimum virulence

Transposon insertions in a novel 3.798 kb open reading frame (ORF) of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo) cause virulence deficiency and altered colony/lawn morphology. This ORF encodes a protein, XadA, of 1,265 amino acids that exhibits significant similarity to non-fimbrial adhesins of animal pathogenic bacteria such as Yersinia YadA and Moraxella UspA1. An interesting feature is that the YadA similarity region is repeated six times within the XadA sequence and encompasses almost the entire length of the protein. Anti-XadA antibodies identified a 110 kDa outer membrane protein that was sensitive to protease treatment of whole cells. XadA expression is induced in minimal medium. Homology modelling suggests that XadA adopts a beta-helix conformation-like pertactin, a non-fimbrial adhesin of Bordetella pertussis. This work is the first characterization of a non-fimbrial adhesin-like molecule in a plant pathogenic bacterium. It extends our knowledge about the repertoire of homologous virulence factors that are deployed by animal and plant pathogenic bacteria to include functions potentially involved in adhesion.     

PhyA, a secreted protein of Xanthomonas oryzae pv. oryzae, is required for optimum virulence and growth on phytic acid as a sole phosphate source

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a novel virulence deficient mutant (BXO1691) of X. oryzae pv. oryzae that has a Tn5 insertion in an open reading frame (phyA; putative phytase A) encoding a 373-amino acid (aa) protein containing a 28-aa predicted signal peptide. Extracellular protein profiles revealed that a 38-kDa band is absent in phyA mutants as compared with phyA+ strains. A BLAST search with phyA and its deduced polypeptide sequence indicated significant similarity with conserved hypothetical proteins in Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and limited homology to secreted phytases of Bacillus species. Homology modeling with a Bacillus phytase as the template suggests that the PhyA protein has a similar six-bladed beta-propeller architecture and exhibits conservation of certain critical active site residues. Phytases are enzymes that are involved in degradation of phytic acid (inositol hexaphosphate), a stored form of phosphate in plants. The phyA mutants exhibit a growth deficiency in media containing phytic acid as a sole phosphate source. Exogenous phosphate supplementation promotes migration of phyA X. oryzae pv. oryzae mutants in rice leaves. These results suggest that the virulence deficiency of phyA mutants is, at least in part, due to inability to use host phytic acid as a source of phosphate. phyA-like genes have not been previously reported to be involved in the virulence of any plant pathogenic bacterium.     

rpfF mutants of Xanthomonas oryzae pv. oryzae are deficient for virulence and growth under low iron conditions

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. In the related bacterium Xanthomonas campestris pv. campestris, the rpfF gene is involved in production of a diffusible extracellular factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes. Transposon insertions in the rpfF homolog of X. oryzae pv. oryzae are deficient for virulence and production of a DSF but are proficient for EPS and extracellular enzyme production. The rpfF X. oryzae pv. oryzae mutants exhibit an unusual tetracycline susceptibility phenotype in which exogenous iron supplementation is required for phenotypic expression of a tetracycline resistance determinant that is encoded on an introduced plasmid. The rpfF X. oryzae pv. oryzae mutants also overproduce one or more siderophores and exhibit a growth deficiency under low iron conditions as well as in the presence of reducing agents that are expected to promote the conversion of Fe+3 to Fe+2. Exogenous iron supplementation promotes migration of rpfF X. oryzae pv. oryzae mutants in rice leaves. The results suggest that rpfF may be involved in controlling an iron-uptake system of X. oryzae pv. oryzae and that an inability to cope with the conditions of low iron availability in the host may be the reason for the virulence deficiency of the rpfF X. oryzae pv. oryzae mutants.