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.     

水稻条斑病菌hrcC、hpa3和hrpE基因表达不依赖hrpG和hrpX基因调控

摘要：【目的】决定水稻条斑病菌（Xanthomonas oryzae pv. oryzicola）在非寄主植物上激发过敏反应（hypersensitive response, HR）和在寄主水稻上致病性（pathogenicity）的hrp基因簇是受hrpG和hrpX基因调控的,但还不清楚hrpG和hrpX基因是否共同决定着所有hrp基因的表达。【方法】本文通过基因敲除方式获得了水稻条斑病菌的hrpG和hrpX基因的双突变体。【结果】烟草和水稻上测定结果显示,双突变体与单突变体一样,均在烟草上失去HR激发能力和丧失在水稻上的致病性;相应地,功能互补后双突变体恢复至野生表型。细菌在水稻悬浮细胞、hrp诱导培养基XOM3和营养丰富的培养基NB中生长后的RT-PCR结果显示,NB中hrp基因低水平表达,XOM3和水稻细胞能够高水平诱导hrp基因表达。无论何种生长条件,hrpG单突变体中hrcC、hrcT、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcJ和hrpG基因不表达;hrpX单突变体中hpa2、hrcC、hpa3、hrpE和hrpG基因表达,而hpa1、hrcT、hpaB和hrcJ基因不表达;hrpG和hrpX双突变体中hrcC、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcT、hrcJ和hrpG基因不表达。【结论】这提示,水稻条斑病菌的hrcC、hrpE和hpa3基因不受hrpG和hrpX基因单独或同时调控,而hrcT基因受HrpG调控。由此推测,水稻条斑病菌III型分泌系统关键组份的表达有可能通过另外的信号途径进行调控,这为进一步分析III型分泌途经的形成提供了线索。     

Characterization of the hrpF pathogenicity peninsula of Xanthomonas oryzae pv. oryzae

The hrp gene cluster of Xanthomonas spp. contains genes for the assembly and function of a type III secretion system (TTSS). The hrpF genes reside in a region between hpaB and the right end of the hrp cluster. The region of the hrpF gene of Xanthomonas oryzae pv. oryzae is bounded by two IS elements and also contains a homolog of hpaF of X. campestris pv. vesicatoria and two newly identified genes, hpa3 and hpa4. A comparison of the hrp gene clusters of different species of Xanthomonas revealed that the hrpF region is a constant yet more variable peninsula of the hrp pathogenicity island. Mutations in hpaF, hpa3, and hpa4 had no effect on virulence, whereas hrpF mutants were severely reduced in virulence on susceptible rice cultivars. The hrpF genes from X. campestris pv. vesicatoria, X. campestris pv. campestris, and X. axonopodis pv. citri each were capable of restoring virulence to the hrpF mutant of X. oryzae pv. oryzae. Correspondingly, none of the Xanthomonas pathovars with hrpF from X. oryzae pv. oryzae elicited a hypersensitive reaction in their respective hosts. Therefore, no evidence was found for hrpF as a host-specialization factor. In contrast to the loss of Bs3-dependent reactions by hrpF mutants of X. campestris pv. vesicatoria, hrpF mutants of X. oryzae pv. oryzae with either avrXa10 or avrXa7 elicited hypersensitive reactions in rice cultivars with the corresponding R genes. A double hrpFxoo-hpa1 mutant also elicited an Xa10-dependent resistance reaction. Thus, loss of hrpF, hpal, or both may reduce delivery or effectiveness of type III effectors. However, the mutations did not completely prevent the delivery of effectors from X. oryzae pv. oryzae into the host cells.     

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。     

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.     

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.     

Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae

The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.     

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.     

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.     

Functional analysis of the aroC gene encoding chorismate synthase from Xanthomonas oryzae pathovar oryzae

Xanthomonas oryzae pv. oryzae causes bacterial blight in rice, and this bacterial blight has been widely found in the major rice-growing areas. We constructed a transposon mutagenesis library of X. oryzae pv. oryzae and identified a mutant strain (KXOM9) that is deficient for pigment production and virulence. Furthermore, the KXOM9 mutant was unable to grow in minimal medium lacking aromatic amino acids. Thermal asymmetric interlaced-PCR and sequence analysis of KXOM9 revealed that the transposon was inserted into the aroC gene, which encodes a chorismate synthase in various bacterial pathogens. In planta growth assays revealed that bacterial growth of the KXOM9 mutant in rice leaves was severely reduced. Genetic complementation of this mutant with a 7.9-kb fragment containing aroC restored virulence, pigmentation, and prototrophy. These results suggest that the aroC gene plays a crucial role in the growth, attenuation of virulence, and pigment production of X. oryzae pv. oryzae.     

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.     

Constitutive heterologous expression of avrXa27 in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains

The vascular pathogen Xanthomonas oryzae pv. oryzae ( Xoo ) and nonvascular pathogen Xanthomonas oryzae pv. oryzicola ( Xoc ) cause bacterial blight (BB) and bacterial leaf streak (BLS) diseases of rice, respectively. We have previously identified the avirulence gene avrXa27 from Xoo PXO99^A, which specifically induces the expression of the rice resistance gene Xa27 , ultimately leading to resistance against BB disease in rice. In this study, we have generated a transgenic rice line (L24) that expresses avrXa27 constitutively under the control of the PR1 promoter, and have examined its role in the host–pathogen interaction. L24 is not more susceptible to BB, indicating that avrXa27 does not contribute to virulence. AvrXa27 retains avirulence activity in L24 and, after crossing with a line containing Xa27 , progeny display phenotypic changes including inhibition of tillering, delay in flowering, stiff leaves, early leaf senescence and activation of pathogenesis-related ( PR ) genes. On challenge with a variety of compatible strains of Xoo and Xoc strain L8, lines with both avrXa27 and Xa27 also show enhanced resistance to bacterial infection. The induction of Xa27 and subsequent inhibition of Xoc growth in Xa27 plants are observed on inoculation with Xoc L8 harbouring avrXa27 . Our results indicate that the heterologous expression of avrXa27 in rice containing Xa27 triggers R gene-specific resistance and, at the same time, confers enhanced resistance to compatible strains of Xoo and Xoc . The expression of AvrXa27 and related proteins in plants has the potential to generate broad resistance in plants.     

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.     

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。