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.     

水稻条斑病细菌hrp基因诱导表达系统的建立

水稻条斑病细菌(Xanthomonas oryzae pv.oryzicola,Xooc)决定在非寄主植物上激发过敏反应(hypersensitive response)和在寄主水稻上具致病性(pathogenicity)的hrp基因簇是诱导表达的。为研究hrp基因的功能,利用hpa1和hrpX基因的启动子与gfp基因进行融合,构建了hrp基因诱导表达系统。绿色荧光蛋白表达揭示,Xooc的hrp基因在营养丰富的NB培养基上不能有效表达,在hrp诱导培养基XOM3上可有效表达。以hrpX和hrpG突变体为参照,RT-PCR研究结果提示,Xooc野生型菌株hpa1基因在NB上不能有效表达,在XOM3培养基上可有效表达。相应地,hrpX突变体中hpa1基因不能被诱导表达,而在hrpG突变体中hpa1基因转录表达水平低于野生菌。研究结果还证实,水稻悬浮细胞能高效诱导Xooc的hrp基因表达。Xooc hrp基因诱导表达系统的建立为研究hrp基因功能、发掘T3SS效应分子以及开展Xooc致病性研究奠定了基础。     

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.     

Gene involved in transcriptional activation of the hrp regulatory gene hrpG in Xanthomonas oryzae pv. oryzae

A novel regulatory gene, trh, which is involved in hrp gene expression, is identified in the plant pathogen Xanthomonas oryzae pv. oryzae. In the trh mutant, expression of HrpG, which is a key regulator for hrp gene expression, is reduced both under the in vitro hrp-inducing condition and in planta.     

Identification of two novel hrp-associated genes in the hrp gene cluster of Xanthomonas oryzae pv. oryzae

We have cloned a hrp gene cluster from Xanthomonas oryzae pv. oryzae. Bacteria with mutations in the hrp region have reduced growth in rice leaves and lose the ability to elicit a hypersensitive response (HR) on the appropriate resistant cultivars of rice and the nonhost plant tomato. A 12,165-bp portion of nucleotide sequence from the presumed left end and extending through the hrpB operon was determined. The region was most similar to hrp genes from Xanthomonas campestris pv. vesicatoria and Ralstonia solanacearum. Two new hrp-associated loci, named hpa1 and hpa2, were located beyond the hrpA operon. The hpa1 gene encoded a 13-kDa glycine-rich protein with a composition similar to those of harpins and PopA. The product of hpa2 was similar to lysozyme-like proteins. Perfect PIP boxes were present in the hrpB and hpa1 operons, while a variant PIP box was located upstream of hpa2. A strain with a deletion encompassing hpa1 and hpa2 had reduced pathogenicity and elicited a weak HR on nonhost and resistant host plants. Experiments using single mutations in hpa1 and hpa2 indicated that the loss of hpa1 was the principal cause of the reduced pathogenicity of the deletion strain. A 1,519-bp insertion element was located immediately downstream of hpa2. Hybridization with hpa2 indicated that the gene was present in all of the strains of Xanthomonas examined. Hybridization experiments with hpa1 and IS1114 indicated that these sequences were detectable in all strains of X. oryzae pv. oryzae and some other Xanthomonas species.     

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.     

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.     

Xanthomonas oryzae pv. oryzae avirulence genes contribute differently and specifically to pathogen aggressiveness

Genomic copies of three Xanthomonas oryzae pv. oryzae avirulence (avr) genes, avrXa7, avrXal0, and avrxa5, and four homologous genes, aB3.5, aB3.6, aB4.3, and aB4.5, were mutagenized individually or in combination to study the roles of avr genes in one component of pathogen fitness, i.e., aggressiveness or the amount of disease X. oryzae pv. oryzae causes in susceptible rice lines. These X. oryzae pv. oryzae genes are members of the highly related Xanthomonas avrBs3 gene family. Compared to the wild-type strain, X. oryzae pv. oryzae strains with mutations in avrXa7, avrxa5, and the four homologous genes caused shorter lesions on rice line IR24, which contains no resistance genes relevant to the wild-type strain. The contribution of each gene to lesion length varied, with avrXa7 contributing the most and avrXal0 showing no measurable effect on aggressiveness. The functional, plasmidborne copies of avrXa7, aB4.5, and avrxa5 restored aggressiveness only to strains with mutations in avrXa7, aB4.5, and avrxa5, respectively. Mutations in avrXa7 were not complemented by plasmids carrying any other avr gene family members. These data indicate that some, but not all, avr family members contribute to pathogen aggressiveness and that the contributions are quantitatively different. Furthermore, despite their sequence similarity, the aggressiveness functions of these gene family members are not interchangeable. The results suggest that selection and pyramiding resistance genes can be guided by the degree of fitness penalty that is empirically determined in avr gene mutations.     

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.     

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。     

Productivity and biochemical properties of green tea in response to full-length and functional fragments of HpaG Xooc, a harpin protein from the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola

Harpin proteins from plant pathogenic bacteria can stimulate hypersensitive cell death (HCD), drought tolerance, defence responses against pathogens and insects in plants, as well as enhance plant growth. Recently, we identified nine functional fragments of HpaG;Xooc, a harpin protein from Xanthomonas oryzae pv.oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG;1-94'HpaG;10-42, and HpaG;62-138, which contain the HpaG;Xooc regions of the amino acid sequence as indicated by the number spans, exceed the parent protein in promoting growth, pathogen defence and HCD in plants. Here we report improved productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments tested in comparison with HpaG;Xooc and an inactive protein control. Field tests suggested that the four proteins markedly increased the growth and yield of green tea, and increased the leaf content of tea catechols, a group of compounds that have relevance in the prevention and treatment of human diseases. In particular, HpaG;1-94 was more active than HpaG;Xooc in expediting the growth of juvenile buds and leaves used as green tea material and increased the catechol content of processed teas. When tea shrubs were treated with HpaH;Xooc and HpaG;1-94 compared with a control, green tea yields were over 55% and 39% greater, and leaf catechols were increased by more than 64% and 72%, respectively. The expression of three homologues of the expansin genes, which regulate plant cell growth, and the CsCHS gene encoding a tea chalcone synthase, which critically regulates the biosynthesis of catechols, were induced in germinal leaves of tea plants following treatment with HpaG;1-94 or HpaG;Xooc. Higher levels of gene expression were induced by the application of HpaG;1-94 than HpaG;Xooc. Our results suggest that the harpin protein, especially the functional fragment HpaG;1-94, can be used to effectively increase the yield and improve the biochemical properties of green tea, a drink with medicinal properties.     

Transgenic elite Indica rice varieties,resistant to Xanthomonas oryzae pv. oryzae

The agronomically important Indica (group 1) rice varieties IR64, IR72, hybrid restorer line Minghui 63, and BG90-2 were co-transformed by microbombardment of embryogenic suspensions with plasmids that contain the Xa21 gene which confers resistance to Xanthomonas oryzae pv. oryzae and the hph gene for resistance to hygromycin B. Six of the 55 transgenic R0 plant lines containing the Xa21 gene displayed high levels of resistance to the pathogen, and no partial resistance was observed. The trait was stably inherited in subsequent generations, and transgenic plants are currently in field tests. The ability to transfer agronomically important genes into elite Indica rice varieties demonstrates the applicability of genetic engineering for the agronomic improvement of rice.     

水稻细菌性条斑病菌REC结构域蛋白基因vemRXoc的功能鉴定

水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)是水稻的主要病原细菌之一,该菌引起的水稻细菌性条斑病可导致水稻减产、品质下降.Xoc GX01菌株基因组中含有一个与十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)的致病相关基因vemR一致性高达95.27%的同源基因XOC2152,其编码蛋白中含有磷酸信号识别受体REC结构域.为了研究XOC2152基因在Xoc中的生物学功能,本研究通过基于自杀质粒pK18mob同源整合方法,构建了XOC2152的非极性突变体NK2152.该突变体的胞外多糖产量仅为野生菌株的27.33%,平板游动半径为野生菌株的40.96%,对铜离子耐受能力极显著降低.针刺接种水稻(日本晴品种) 10 d后,Xoc GX01处理的病斑平均长度为(3.86±1.19) cm,而突变体NK2152处理的病斑长度为(0.98±0.45) cm.带有该基因片段的pLAFR3可以互补突变体的表型和致病力变化.上述结果表明XOC2152基因具有与Xcc的vemR类似的功能,被命名为vemRXoc基因.本研究表明XOC2152基因(vemRXoc基因)与水稻细菌性条斑病菌的致病力、胞外多糖产量、运动能力以及对铜离子的耐性等相关.     

Functional interplay between two Xanthomonas oryzae pv,. oryzae secretion systems in modulating virulence on rice

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S-) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S- T3S- double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA- mutant or a ClsA- LipA- double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.     

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。