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.     

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.     

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.     

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.     

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.     

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.     

Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae.

Races of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, interact with cultivars of rice in a gene-for-gene specific manner. Multiple DNA fragments of various sizes from all strains of X. o. pv. oryzae hybridized with avrBs3, an avirulence gene from Xanthomonas campestris pv. vesicatoria, in Southern blots; this suggests the presence of several homologs and possibly a gene family. A genomic library of a race 2 strain of X. o. pv. oryzae, which is avirulent on rice cultivars carrying resistance genes xa-5, Xa-7, and Xa-10, was constructed. Six library clones, which hybridized to avrBs3, altered the interaction phenotype with rice cultivars carrying either xa-5, Xa-7, or Xa-10 when present in a virulent race 6 strain. Two avirulence genes, avrXa7 and avrXa10, which correspond to resistance genes Xa-7 and Xa-10, respectively, were identified and partially characterized from the hybridizing clones. On the basis of transposon insertion mutagenesis, sequence homology, restriction mapping, and the presence of a repeated sequence, both genes are homologs of avirulence genes from dicot xanthomonad pathogens. Two BamHI fragments that are homologous to avrBs3 and correspond to avrXa7 and avrXa10 contain a different number of copies of a 102-bp direct repeat. The DNA sequence of avrXa10 is nearly identical to avrBs3. We suggest that avrXa7 and avrXa10 are members of an avirulence gene family from xanthomonads that control the elicitation of resistance in mono- and dicotyledonous plants.     

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.     

Novel genomic locus with atypical G+C content that is required for extracellular polysaccharide production and virulence in Xanthomonas oryzae pv. oryzae.

Three exopolysaccharide (EPS)- and virulence-deficient mutants of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, were isolated by Tn5 mutagenesis. These insertions are not located within the gum gene cluster. A 40-kb cosmid clone that restored EPS production and virulence to all three mutants was isolated, and the three transposon insertions were localized to contiguous 4.3- and 3.5-kb EcoRI fragments that are included in this clone. Sequence data indicate that two of the transposon insertions are in genes that encode a putative sugar nucleotide epimerase and a putative glycosyl transferase, respectively; the third insertion is located between the glycosyl transferase gene and a novel open reading frame (ORF). A 5.5-kb genomic region in which these three ORFs are located has a G+C content of 5-1.7%, quite different from the G+C content of approximately 65.0% that is typical of X. oryzae pv. oryzae. Homologues of this locus have not yet been reported in any other xanthomonad.     

A novel two-component system PdeK/PdeR regulates c-di-GMP turnover and virulence of Xanthomonas oryzae pv. oryzae

Two-component systems (TCS) consisting of histidine kinases (HK) and response regulators (RR) play essential roles in bacteria to sense environmental signals and regulate cell functions. One type of RR is involved in metabolism of cyclic diguanylate (c-di-GMP), a ubiquitous bacterial second messenger. Although genomic studies predicted a large number of them existing in different bacteria, only a few have been studied. In this work, we characterized a novel TCS consisting of PdeK(PXO_01018)/PdeR(PXO_ 01019) from Xanthomonas oryzae pv. oryzae, which causes the bacterial leaf blight of rice. PdeR (containing GGDEF, EAL, and REC domains) was shown to have phosphodiesterase (PDE) activity in vitro by colorimetric assays and high-performance liquid chromatography analysis. The PDE activity of full-length PdeR needs to be triggered by HK PdeK. Deletion of pdeK or pdeR in X. oryzae pv. oryzae PXO99(A) had attenuated its virulence on rice. ΔpdeK and ΔpdeR secreted less exopolysaccharide than the wild type but there were no changes in terms of motility or extracellular cellulase activity, suggesting the activity of PdeK/PdeR might be specific.     

Controlled synthesis of the DSF cell-cell signal is required for biofilm formation and virulence in Xanthomonas campestris

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell-cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence.     

糖基转移酶基因rbfCxoo缺失突变导致水稻白叶枯病菌毒性表达增强

摘 要：【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,简称Xoo)基因组中推导的脂多糖O抗原合成蛋白基因rbfCxoo (XOO2599) 的结构和生物学功能。【方法】通过基因克隆、序列分析、缺失突变和表型测定,对rbfCxoo的分子特征及其功能进行了鉴定。【结果】 用特异性引物进行PCR扩增,从野生型菌株PXO99A基因组DNA中成功地获得了与己测序菌株KACC10331序列完全一致的全长基因序列; RbfCxoo序列N端和C端分别具有一个糖基转移酶的保守结构域(Glycos_transf_2)。用标记置换法获得了基因缺失突变体△rbfCxoo。与PXO99A相比,△rbfCxoo 脂多糖O抗原合成能力并未发生变化,但鞭毛素糖基化能力有所降低。此外,△rbfCxoo鞭毛运动性、生物膜形成和胞外纤维素酶和木聚糖酶活性都无明显改变,但对水稻的致病性显著增强,毒性相关基因的表达也有所增加。【结论】RbfCxoo可能与细菌鞭毛素糖基化修饰以及毒性表达有关。     

KdgR, an IClR family transcriptional regulator, inhibits virulence mainly by repression of hrp genes in Xanthomonas oryzae pv. oryzae

KdgR has been reported to negatively regulate the genes involved in degradation and metabolization of pectic acid and other extracellular enzymes in soft-rotting Erwinia spp. through direct binding to their promoters. The possible involvement of a KdgR orthologue in virulence by affecting the expression of extracellular enzymes in Xanthomonas oryzae pv. oryzae, the causal agent of rice blight disease, was examined by comparing virulence and regulation of extracellular enzymes between the wild type (WT) and a strain carrying a mutation in putative kdgR (ΔXoo0310 mutant). This putative kdgR mutant of X. oryzae pv. oryzae showed increased pathogenicity on rice without affecting the regulation of extracellular enzymes, such as amylase, cellulase, xylanase, and protease. However, the mutant carrying a mutation in an ortholog of xpsL, which encodes the functional secretion machinery for the extracellular enzymes, showed a dramatic decrease in pathogenicity on rice. Both mutants of kdgR and of xpsL orthologs showed higher expression of two major hrp regulatory genes, hrpG and hrpX, and the genes in the hrp operons when grown in hrp-inducing medium. Thus, both genes were shown to be involved in repression of hrp genes. The kdgR ortholog was thought to suppress virulence mainly by repressing the expression of hrp genes without affecting the expression of extracellular enzymes, unlike findings for the kdgR gene in soft-rotting Erwinia spp. On the other hand, xpsL was confirmed to be involved in virulence by promoting the secretion of extracellular enzymes in spite of repressing the expression of the hrp genes.