Inhibition of resistance gene-mediated defense in rice by Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv. oryzae and the closely related X. oryzae pv. oryzicola cause bacterial blight and bacterial leaf streak of rice, respectively. Although many rice resistance (R) genes and some corresponding avirulence (avr) genes have been characterized for bacterial blight, no endogenous avr/R gene interactions have been identified for leaf streak. Genes avrXa7 and avrXa10 from X. oryzae pv. oryzae failed to elicit the plant defense-associated hypersensitive reaction (HR) and failed to prevent development of leaf streak in rice cultivars with the corresponding R genes after introduction into X. oryzae pv. oryzicola despite the ability of this pathovar to deliver an AvrXa10:Cya fusion protein into rice cells. Furthermore, coinoculation of X. oryzae pv. oryzicola inhibited the HR of rice cultivar IRBB10 to X. oryzae pv. oryzae carrying avrXa10. Inhibition was quantitative and dependent on the type III secretion system of X. oryzae pv. oryzicola. The results suggest that one or more X. oryzae pv. oryzicola type III effectors interfere with avr/R gene-mediated recognition or signaling and subsequent defense response in the host. Inhibition of R gene-mediated defense by X. oryzae pv. oryzicola may explain, in part, the apparent lack of major gene resistance to leaf streak.     

Differentiation between Xanthomonas campestris pv. oryzae, Xanthomonas campestris pv. oryzicola and the bacterial 'brown blotch' pathogen on rice by numerical analysis of phenotypic features and protein gel electrophoregrams

Thirty-five Xanthomonas campestris pv. oryzae, fourteen X. campestris pv. oryzicola strains and six 'brown blotch' pathogens of rice, all of different geographical origin, were studied by numerical analysis of 133 phenotype features and gel electrophoregrams of soluble proteins, %G + C determinations and DNA:rRNA hybridizations. The following conclusions were drawn. (i) The Xanthomonas campestris pathovars oryzae and oryzicola display clearly distinct protein patterns on polyacrylamide gels and can be differentiated from each other by four phenotype tests. (ii) Both pathovars are indeed members of Xanthomonas which belongs to a separate rRNA branch of the second rRNA superfamily together with the rRNA branches of Pseudomonas fluorescens, Marinomonas, Azotobacter, Azomonas and Frateuria. (iii) 'Brown blotch' strains are considerably different from X. campestris pv. oryzae and oryzicola. They are not members of the genus Xanthomonas, but are more related to the generically misnamed. Flavobacterium capsulatum, Pseudomonas paucimobilis, Flavobacterium devorans and 'Pseudomonas azotocolligans' belonging in the fourth rRNA superfamily. (iv) No correlation was found between the virulence, pathogenic groups or geographical distribution of X. campestris pv. oryzae or oryzicola strains and any phenotypic or protein electrophoretic property or clustering.     

玉米细菌性条斑病非寄主抗性基因Rxo1转化水稻的研究

水稻细菌性条斑病是我国重要的水稻病害之一,但是在水稻种质资源中尚未发现抗细菌性条斑病单个主效基因。利用农杆菌介导的转化系统将从玉米中克隆的细菌性条斑病非寄主抗性基因Rxo1转入我国2个杂交稻恢复系和2个常规水稻品种。转基因植株的PCR和Southern分析结果表明Rxo1基因已整合到受体基因组中,Rxo1基因单拷贝整合的转化体在自交T1代呈现抗感3∶1分离。人工接种实验和病菌的生长曲线表明携带Rxo1的转基因植株对水稻细条病菌可以产生过敏性抗病反应。上述结果为利用非寄主抗性基因防治该病害提供了有用的信息。     

The avrRxo1 gene from the rice pathogen Xanthomonas oryzae pv. oryzicola confers a nonhost defense reaction on maize with resistance gene Rxo1

Maize lines that contain the single dominant gene Rxo1 exhibit a rapid hypersensitive response (HR) after infiltration with the rice bacterial streak pathogen Xanthomonas oryzae pv. oryzicola, but not with the rice bacterial blight pathogen X. oryzae pv. oryzae. The avirulence effector gene that corresponds to Rxo1, designated avrRxo1, was identified in an X. oryzae pv. oryzicola genomic library. When introduced into X. oryzae pv. oryzae, clones containing avrRxo1 induced an HR on maize with Rxo1, but not on maize without Rxo1. The avrRxo1 gene is 1,266 bp long and shows no significant homology to any database sequences. When expressed in an X. oryzae pv. oryzae hrpC mutant that is deficient in the type III secretion system, avrRxo1 did not elicit the HR, indicating that the avrRxo1-Rxo1 interaction is dependent on type III secretion. Transient expression of avrRxo1 in onion cells after biolistic delivery revealed that the protein product was associated with the plasma membrane. Transient expression in maize lines carrying Rxo1 resulted in cell death, suggesting that AvrRxo1 functions from inside maize cells to elicit Rxo1-dependent pathogen recognition.     

Elucidation of the hrp clusters of Xanthomonas oryzae pv. oryzicola that control the hypersensitive response in nonhost tobacco and pathogenicity in susceptible host rice

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.     

Characteristics of a PCR-Based Assay for In Planta Detection of Xanthomonas campestris pv. pelargonii

Polymerase chain reaction (PCR) amplification was carried out with a primer pair targeting a sequence in the genome of Xanthomonas campestris pv. pelargonii , the causative agent of bacterial blight in geraniums. PCR amplification with the primer pair XcpMl/XcpM2 using total nucleic acid preparations from 22 geographicallydiverse isolates of X. campestris pv. pelargonii generated a major 197 bp DNA product. In contrast, no major amplification products were consistently generated from 12 other pathovars of X. campestris or from 19 isolates representing 10 different plant pathogenic bacteria, including two other bacterial pathogens of geraniums, Corynebacterium fascians and Pseudomonas cichorii . After PCR using this primer pair, between 1380 and 13800 copies of the X, campestris pv. pelargonii bacterial DNA target as template were detected by ethidium bromide staining of agarose gels, and between 13.8 and 138 copies by blot hybridization to a pathovar-specific biotinylated probe. Similarly, between 630 and 6300 colonyforming units (CFU) of X. campestris pv. pelargonii could be detected after ethidium bromide staining of agarose gels, and between 63 and 630 CFU after blot hybridization. The PCR-based assay was used to identify X. campestris pv. pelargonii in diseased geraniums; whereas discrete amplification products were not obtained with healthy plants.     

Exopolysaccharides of Xanthomonas pathovar strains that infect rice and wheat crops

In order to understand the mode of action of the taxonomically related pathogens Xanthomonas campestris pv. translucens, Xanthomonas oryzae pv. oryzae, and Xanthomonas oryzae pv. oryzicola, which attack wheat and rice crops, we examined the compositional differences of their exopolysaccharides (EPSs). Maximum production of polysaccharide in shake cultures of these pathogens was observed between 24 and 72 h. X. campestris pv. translucens, the leaf streak pathogen of wheat, produced a higher amount of polysaccharide (46.97 microg/ml) at 72 h compared to X. oryzae pv. oryzae (42.02 microg/ml), the bacterial blight pathogen of rice, and X. oryzae pv. oryzicola (41.91 microg/ml), the bacterial leaf streak pathogen of rice. Infrared (FTIR) spectra suggested that the polysaccharides of all three Xanthomonas pathovar strains have an -OH group with intermolecular hydrogen bonding, a C-H group of methyl alkanes, an aldehyde (RCHO) group, a C=C or C=O group, and a C-O group. FTIR spectra also revealed the presence of an acid anhydride group in X. oryzae pv. oryzae, a secondary aromatic or aliphatic amine group in X. campestris pv. translucens, and a primary aromatic or aliphatic amine group in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Nuclear magnetic resonance (NMR) spectra revealed the presence of unsubstituted sugars, an acetyl amine of hexose or pentose, and a beta-anomeric carbon of hexose or pentose in the polysaccharides of all bacteria. NMR spectra also identified the alpha-anomeric carbon of hexose or pentose in all strains, and a branching at the fourth carbon of the sugar only in X. campestris pv. translucens; the presence of an uronic acid molecule (acid anhydride group) in X. oryzae pv. oryzae; and a deoxy sugar, rhamnose, in X. oryzae pv. oryzicola.     

水稻条斑病菌gacA基因的克隆及功能初析

根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacAXooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacAXooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。     

水稻条斑病菌gacA基因的克隆及功能初析

根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacA_Xooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacA_Xooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。     

Molecular detection of plant pathogenic bacteria using polymerase chain reaction single-strand conformation polymorphism

The application of polymerase chain reaction (PCR) technology to molecular diagnostics holds great promise for the early identification of agriculturally important plant pathogens. Ralstonia solanacearum, Xanthomoans axonopodis pv. vesicatoria, and Xanthomonas oryzae pv. oryzae are phytopathogenic bacteria, which can infect vegetables, cause severe yield loss. PCR-single-strand conformation polymorphism (PCR-SSCP) is a simple and powerful technique for identifying sequence changes in amplified DNA. The technique of PCR-SSCP is being exploited so far, only to detect and diagnose human bacterial pathogens in addition to plant pathogenic fungi. Selective media and serology are the commonly used methods for the detection of plant pathogens in infected plant materials. In this study, we developed PCR-SSCP technique to identify phytopathogenic bacteria. The PCR product was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids. We tested over 56 isolates of R. solanacearum, 44 isolates of X. axonopodis pv. vesicatoria, and 20 isolates of X. oryzae pv. oryzae. With the use of universal primer 16S rRNA, we could discriminate such species at the genus and species levels. Species-specific patterns were obtained for bacteria R. solanacearum, X. axonopodis pv. vesicatoria, and X. oryzae pv. oryzae. The potential use of PCR-SSCP technique for the detection and diagnosis of phytobacterial pathogens is discussed in the present paper.     

Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.     

非寄主抗病基因Rxo1介导的水稻对Xanthomonas oryzae pv.oryzicola的抗病反应

通过表型鉴定、反转录PCR和实时定量PCR方法,利用转基因和非转基因水稻植株,研究由Rxol基因介导的,水稻对细菌性条斑病菌的抗性反应。结果观察到3个涉及过敏性反应的基因由Rxol基因诱导表达,并对其进行了分析。这3个基因参与编码病程相关蛋白,在转基因水稻植株中呈上调表达,表明水杨酸信号转导途径在抗性反应中发挥重要的作用。     

Manipulating broad-spectrum disease resistance by suppressing pathogen-induced auxin accumulation in rice

Breeding crops with the quality of broad-spectrum disease resistance using genetic resources is one of the principal goals of crop improvement. However, the molecular mechanism of broad-spectrum resistance remains largely unknown. Here, we show that GH3-2, encoding an indole-3-acetic acid (IAA)-amido synthetase, mediates a broad-spectrum resistance to bacterial Xanthomonas oryzae pv oryzae and Xanthomonas oryzae pv oryzicola and fungal Magnaporthe grisea in rice (Oryza sativa). IAA, the major form of auxin in rice, results in rice more vulnerable to the invasion of different types of pathogens, which is at least partly due to IAA-induced loosening of the cell wall, the natural protective barrier of plant cells to invaders. X. oryzae pv oryzae, X. oryzae pv oryzicola, and M. grisea secrete IAA, which, in turn, may induce rice to synthesize its own IAA at the infection site. IAA induces the production of expansins, the cell wall-loosening proteins, and makes rice vulnerable to pathogens. GH3-2 is likely contributing to a minor quantitative trait locus for broad-spectrum resistance. Activation of GH3-2 inactivates IAA by catalyzing the formation of an IAA-amino acid conjugate, which results in the suppression of expansin genes. Thus, GH3-2 mediates basal resistance by suppressing pathogen-induced IAA accumulation. It is expected that, regulated by a pathogen-induced strong promoter, GH3-2 alone may be used for breeding rice with a broad-spectrum disease resistance.     

Virulence deficiency caused by a transposon insertion in the purH gene of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a Tn5-induced virulence-deficient mutant (BXO1704) of X. oryzae pv. oryzae. The BXO1704 mutant exhibited growth deficiency in minimal medium but was proficient in inducing a hypersensitive response in a non-host tomato plant. Sequence analysis of the chromosomal DNA flanking the Tn5 insertion indicated that the Tn5 insertion is in the purH gene, which is highly homologous to purH genes of other closely related plant pathogenic bacteria Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris. Purine supplementation reversed the growth deficiency of BXO1704 in minimal medium. These results suggest that the virulence deficiency of BXO1704 may be due to the inability to use sufficient purine in the host.     

水稻对白叶枯病和细菌性条斑病的抗性遗传研究进展

水稻白叶枯病和水稻细菌性条斑病是由稻黄单胞细菌(Xanthomonas oryzae)不同致病变种引起的两种最重要的水稻细菌性病害。发掘和利用抗性基因,培育抗病品种是防治这两种病害的最有效手段之一。本文分别综述了这两种高度相关的病害的抗性遗传研究进展,包括已发掘和利用的主效抗性基因特点及目前国内外对这两种病害的抗性QTL定位研究进展,为水稻抗白叶枯病和细菌性条斑病育种研究提供有用信息。     

Genetic locus encoding functions involved in biosynthesis and outer membrane localization of xanthomonadin in Xanthomonas oryzae pv. oryzae

Xanthomonadins are membrane-bound, brominated, aryl-polyene pigments specific to the genus Xanthomonas. We have characterized a genetic locus (pig) from Xanthomonas oryzae pv. oryzae which contains four open reading frames (ORFs) that are essential for xanthomonadin production. Three of these ORFs are homologous to acyl carrier proteins, dehydratases, and acyl transferases, suggesting a type II polyketide synthase pathway for xanthomonadin biosynthesis. The fourth ORF has no homologue in the database. For the first time, we report that a putative cytoplasmic membrane protein encoded in the pig locus is required for outer membrane localization of xanthomonadin in X. oryzae pv. oryzae. We also report the identification of a novel 145-bp palindromic Xanthomonas repetitive intergenic consensus element that is present in two places in the pig locus. We estimate that more than 100 copies of this element might be present in the genome of X. oryzae pv. oryzae and other xanthomonads.