水稻白叶枯病菌HD-GYP结构域蛋白PXO_03945的功能鉴定

为了揭示水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,简称Xoo)环二鸟苷酸(c-di-GMP)信号相关蛋白PXO_03945的生物学功能,本研究通过基因同源重组和无标记交换,对PXO_03945基因进行了缺失突变,对野生型、突变体和互补菌株进行表型测定,分析该基因缺失突变对病菌胞内c-di-GMP水平、致病性、运动性、胞外多糖产生和生物膜形成的影响。结果表明,PXO_03945蛋白具有参与c-di-GMP降解的磷酸二酯酶(PDE)的HD-GYP结构域和功能未知的DUF3391结构域。全长基因缺失可导致体内c-di-GMP浓度明显上升。与野生型相比,ΔPXO_03945对水稻品种日本晴的致病性显著下降,而游动性增强,胞外多糖产生和生物膜形成增加。基因互补可以使之恢复。因此,HD-GYP结构域蛋白PXO_03945可能通过降解胞内c-di-GMP,正向调控了致病性,负向调控了运动性、EPS产生和生物膜形成能力。     

水稻白叶枯病菌鞭毛基体基因fliExoo缺失突变分析

 为了阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,简称Xoo)鞭毛基体组分蛋白基因fliExoo的生物学功能,本研究用Gm^R抗性基因标记交换法成功构建了基因缺失突变体△fliExoo。与野生型菌株PXO99^A相比,△fliExoo鞭毛缺失,菌体易沉降,在0.3%半固体培养基上运动能力明显降低;尽管生长速率和胞外纤维素酶活性无明显变化,但胞外多糖产生和生物膜形成能力明显下降;对水稻品种日本晴的致病性和对非寄主烟草的致敏性反应明显减弱。基因互补可以使上述突变表型恢复。因此,鞭毛基因fliExoo突变不仅影响了细菌鞭毛依赖的运动性,而且对病原菌毒性相关的表型也产生了影响。     

The filamentous phage ϕRSS1 enhances virulence of phytopathogenic Ralstonia solanacearum on tomato

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ?RSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ?RSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ?RSS1, resulting in early wilting. Tomato plants inoculated with ?RSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ?RSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ?RSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ?RSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.     

hrp基因关键调控因子HrpG在水稻白叶枯病菌和条斑病菌中的交叉互补性研究

 水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)和条斑病菌(X. oryzae pv. oryzicola, Xoc)在非寄主烟草上产生过敏反应(HR)和在寄主水稻上具有致病性,是由hrp致病岛决定的,其中HrpG为关键调控因子。Xoo和Xoc侵染水稻途径和在水稻上产生病害症状的不同,是否与hrpG基因有关,还不清楚。本研究利用反向遗传学方法获得了Xoo和Xoc的hrpG突变体PΔhrpG和RΔhrpG,并用hrpG_Xoo基因和hrpG_Xoc基因分别互补上述2个突变体,获得了相应的互补菌株。致病性测定结果显示,PΔhrpG和RΔhrpG突变体丧失了在水稻上的致病性和在非寄主烟草上产生HR的能力,而hrpG_Xoo基因和hrpG_Xoc基因可分别互补上述突变体至野生型水平。利用GUS报告基因检测基因启动子活性发现,hrpG_Xoo和hrpG_Xoc的启动子活性没有显著差别;RT-PCR和Western杂交结果显示,hrpG基因交叉互补菌株中HrpG调控的下游基因hrpX、hpaR、hrcT、hpa2和hrpD6的表达没有差异,且III型分泌系统分泌蛋白Hpa2的分泌性没有受到影响。这些结果表明,稻黄单胞菌hrpG基因可在Xoo和Xoc中交叉互置,位于其上游和下游的调控途径可能相似,而决定Xoo和Xoc在水稻上的侵染途径以及所致病害症状差异可能与hrpG基因位点无关。     

Growth Complementation of hrpXo Mutants of Xanthomonas oryzae pv. oryzae by Virulent Strains in Rice Cultivars Resistant and Susceptible to the Parental Strain

Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 is virulent on rice cultivar IR24 and avirulent on IR-BB2. From recent reports, some virulence and avirulence factors of plant pathogenic bacteria are transferred to plant cells through the hrp-dependent type III secretion system. In this study, we investigated the involvement of hrp genes in the compatible and the incompatible interactions between rice and X. o. pv. oryzae after co-inoculation with hrpXo mutants derived from T7174 and virulent strains. Growth of the mutants, named 74ΔHrpXo and 76ΔHrpXo, was repressed in IR24 when the mutants were applied alone. However, growth of the mutants was complemented by co-inoculation with virulent strains. Growth of bioluminescent hrpXo mutant 76ΔHrpXo in IR24 and its growth in IR-BB2 after co-inoculation with T7133, which is virulent on both cultivars, was equally complemented, as detected by bioluminescence from the mutant. On the other hand, only partial complementation of growth of T7174L76, which is a bioluminescent and pathogenic derivative of T7174, by T7133 was observed in IR-BB2. Thus, growth of the hrpXo mutant of X. o. pv. oryzae was complemented by virulent strains in both susceptible and resistant rice leaves with the parental strain. Received 21 July 2000/ Accepted in revised form 26 October 2000     

磷酸甘油转移酶基因在水稻白叶枯病菌致病力中的作用研究

稻黄单胞菌稻致病变种(Xanthomonas oryzae pv.oryzae,Xoo)引起的水稻白叶枯病,是水稻上的重要致灾病害之一。前期基因组学分析发现,Xoo菌株PXO99^A的PXO＿04062基因可能编码一个磷酸甘油转移酶,推测与病原菌的致病力有关。本研究采用自杀质粒pK18mobsacB介导的方法,构建了该基因的缺失突变体,并对突变体进行了反式互补。水稻上致病性测定发现,与野生型菌株相比,PXO＿04062基因的缺失突变体DM04062在寄主水稻日本晴上的致病力显著下降,并且其胞外多糖产量、运动性、生物膜形成等均明显降低。进一步采用RNA-seq方法比较突变体转录组的变化,发现突变体中的差异表达基因共有533个,其中包括多个致病相关基因,如胞外多糖合成和细胞运动性等相关基因。与野生型相比,这些基因在突变体中的表达均显著下调。这些结果说明,白叶枯病菌的磷酸甘油转移酶基因是通过影响多种致病力相关因子的合成,来影响病菌在水稻上的致病力。     

Characterization of pilP, a gene required for twitching motility, pathogenicity, and biofilm formation of Acidovorax avenae subsp. avenae RS-1

The Gram-negative bacterium Acidovorax avenae subsp. avenae is the causal agent of bacterial brown stripe (BBS), which can cause severe diseases in many plants, including rice, with huge economic importance. Type IV pili (TFP) are hair-like appendages involved in several bacterial activities such as bacterial surface motility, surface adherence, colonization, biofilm formation, and virulence. The aim of our study is to characterize the association of A. avenae subsp. avenae TFP with BBS in rice. We generated a transposon (Tn5) mutant library. Then, an insertional mutagenesis on the background of this bacterium was identified as reduced pathogenicity. The confirmed inserted genetic region was into gene pilP, which encodes a TFP assembly protein. The pilP-deficient mutant strain seriously affected the motility twitching ability, biofilm formation and virulence. Collectively, our results clearly indicated that the pilP gene and TFP in A. avenae subsp. avenae play a key role in plant pathogenicity, twitching motility, and biofilm formation.     

A single amino acid substitution in the SdhB protein of succinate dehydrogenase determines resistance to amicarthiazol in Xanthomonas oryzae pv. oryzae

BACKGROUND: Xanthomonas oryzae pv. Oryzae Ishiyama, a causal agent of rice bacterial leaf blight, was found to be sensitive in vitro to the systemic fungicide amicarthiazol (2‐amino‐4‐methylthiazole ‐5‐carboxanilide), which is a potent inhibitor of succinate dehydrogenase (SDH, EC 1.3.99.1). This paper aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzae to amicarthiazol. RESULTS: UV‐induced resistant mutants of X. oryzae pv. oryzae to amicarthiazol were isolated. The activity of SDH in wild‐type X. oryzae pv. oryzae was strongly inhibited by amicarthiazol, while that in resistant mutants was insensitive, although their SDH activity was decreased compared with the wild‐type sensitive strain without amicarthiazol. A mutation of Histidine₂₂₉ (CAC) to Tyrosine₂₂₉ (TAC) was identified in sdhB, which encoded the iron–sulfur protein subunit of SDH. The sdhB from the mutant was ligated into a cosmid, pUFR034, to generate pUFR034RAX, which conferred resistance to amicarthiazol when transformed into the wild‐type sensitive strain. CONCLUSION: A mutation of His₂₂₉ (CAC) to Tyr₂₂₉ (TAC) in SdhB was responsible for determining amicarthiazol resistance. Copyright © 2010 Society of Chemical Industry     

中国水稻白叶枯病菌毒性变异研究

 利用13个含已知抗病基因的近等基因系材料,在水稻孕穗期采用剪叶接种法对我国285个水稻白叶枯菌株(其中108个采集于1970~1992年,177个采集于2003~2004年)的致病力变异进行研究。结果表明,病菌的致病力呈现多样性。通过试验系统比较了不同时期病菌的毒性和小种组成变化。在此基础上对病菌群体毒性变化的原因进行了分析。     

katExoo基因缺失突变对水稻白叶枯病菌H2O2抗性和致病性的影响

 为了揭示过氧化氢酶基因katExoo在水稻白叶枯病菌(Xanthomonasoryzaepv.oryzae,Xoo)过氧化氢(H₂O₂)抗性和致病性中的功能,本研究构建了基因缺失突变体ΔkatExoo,测定了突变体的H₂O₂抗性、过氧化氢酶(CAT)活性、在离体培养条件下的生长速率以及对水稻的致病性。用标记基因置换法获得了ΔkatExoo突变体,其保守的CAT结构域(GATase1_catalase和catalase_clade_2)被Gm^R片段所替换。katExoo基因缺失突变并不导致病菌的H₂O₂抗性和CAT活性降低或丧失,反而在一定程度上使之增强和升高。ΔkatExoo离体生长量显著降低,水稻接种叶片病斑明显缩短、在叶组织内的种群量下降。表明基因缺失突变显著地影响了病菌的生长、定殖和致病性。本研究结果为“KatExoo可能是Xoo的一个毒性因子”的假设提供了遗传学证据。     

Pathogenesis-related gene expression in rice is correlated with developmentally controlled Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae

Disease resistance mediated by the resistance gene Xa21 is developmentally controlled in rice. We examined the relationship between Pathogenesis Related (PR) defense gene expression and Xa21-mediated developmental disease resistance induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1a, OsPR1b, and OsPR1c genes were cloned and their induction was analyzed, in addition to the OsPR10a gene, at the juvenile and adult stages in response to a wildtype Xoo strain that induces a resistance response (incompatible interaction) and an isogenic mutant Xoo strain that does not (compatible interaction). We found that the adult stage leaves are more competent to express these OsPR1 genes and that the Xa21 locus is required for the highest levels of induction.     

Isolation and Sequence of a Gene, celD Xo Involved in Cellobiose Utilization in Xanthomonas oryzae pv. oryzae

A genomic library of Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 was screened for 4-methylumbelliferyl β-D-glucoside-hydrolyzing (MUGase) activity. In subcloning of one of the MUGase-positive clones, an approximately 4.2-kb SacI-SphI fragment conferred not only MUGase activity but also 4-methylumbelliferyl β-D-cellobioside-hydrolyzing (MUCase) activity. Sequence analysis showed that the fragment contained an ORF of 2951 bp. The conceptual ORF product was significantly homologous with 1,4-β-D-glucan glucohydrolase D (CELD) from Pseudomonas fluorescens subsp. cellulosa, and was named CELD_Xo. Cell fractionation experiments suggested that CELD_Xo is localized in the cell-envelope fraction. We constructed a CELD_Xo-deficient mutant (74ΔCELD) from X. o. pv. oryzae. Little MUCase activity was detected in the cell-envelope fraction prepared from the mutant. The mutant 74ΔCELD did not grow in synthetic medium containing cellobiose as the sole sugar source. On the other hand, growth in rice leaves and pathogenicity of the mutant and the parental strain did not differ. These results suggested that CELD_Xo is involved in cellobiose utilization of X. o. pv. oryzae but that the gene is not required for bacterial growth in rice leaves. Received 16 February 2001/ Accepted in revised form 11 April 2001     

Baseline sensitivity of natural populations and resistance of mutants of Xanthomonas oryzae pv. oryzae to a novel bactericide,zinc thiazole

During 2009–2010, a total of 323 isolates of Xanthomonas oryzae pv. oryzae were obtained from rice with symptoms of bacterial leaf blight (BLB) in four provinces (Zhejiang, Jiangsu, Anhui and Hubei) in China. These isolates were tested for baseline sensitivity to zinc thiazole, a novel bactericide with strong antibacterial activity against Xanthomonas. The sampled pathogenic population had similar sensitivity to zinc thiazole (0·1–16·8 mg L^?1) in all four regions and over the whole two‐year study period. The baseline sensitivity was distributed as a unimodal curve with a mean EC₅₀ value of 6·79 ± 1·61 mg L^?1. The risk of mutation to resistance of zinc thiazole in X. oryzae pv. oryzae was further evaluated in vitro and in vivo. Twelve zinc thiazole‐resistant mutants were obtained through ultraviolet (UV) irradiation, culturing on zinc thiazole‐amended nutrient agar (NA) plates, and culturing on zinc thiazole‐treated rice plants. These zinc thiazole‐resistant mutants had resistance factors (RF = EC₅₀ value of a mutant / EC₅₀ value of the wildtype parent of this mutant) of 12·4 to 186·1 with a mean RF value of 44·1. Mutants obtained via UV irradiation, culturing on NA plates and culturing on rice plants had mean RF values of 51·8, 24·5 and 14·4, respectively. All mutants showed decreases in resistance to zinc thiazole after 20 successive transfers on bactericide‐free media or 10 successive inoculation–reisolations on bactericide‐free rice plants. No significant difference was found in bacterial growth and sensitivity to bismerthiazol between zinc thiazole‐resistant mutants and their parents. However, a significant decrease was observed in the pathogenicity of zinc thiazole‐resistant mutants compared with their parents, especially for mutants obtained via UV irradiation.     

水稻白叶枯病菌在离体培养条件下生物膜形成的检测

分析了不同培养和测试条件(塑料和玻璃表面、培养时间、培养温度和培养基)对水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae,Xoo)生物膜形成的影响,并测定了不同地区来源的Xoo野生型菌株、基因缺失突变体的生物膜形成。结果表明,在聚苯乙烯表面生长、用M210培养基在23 ℃下静止培养24 h是Xoo生物膜形成比较适宜的条件;不同地区来源的菌株生物膜形成能力不同;鞭毛相关基因fleQxoo、fliExoo和rbfCxoo以及H2O2降解调控基因oxyRxoo的缺失突变均影响生物膜形成。     

Pathogenicity and population structure analysis of Pyricularia oryzae in different districts of Jiangsu province,China

Rice blast caused by Pyricularia oryzae is one of the most destructive rice diseases worldwide. In this study, 224 isolates were isolated from neck blast samples from nine districts in Jiangsu. We analysed the resistance frequency of 24 resistance (R) genes using 32 monogenic rice lines from the International Rice Research Institute (IRRI), including Pii, Pik-h, Pi5, Piz-5, and Piz, which exhibit high resistance frequencies. PAC (pathogenicity association coefficients) and VAC (virulence association coefficients) analyses identified three combinations of R genes, Piz/Pii, Piz/Piz-5, and Piz/Pi5, as being suitable for use in Jiangsu. Mating-type analysis of P. oryzae isolates indicated that sexual reproduction occurred less frequently in northern Jiangsu than in other areas, which may affect genetic diversity and dissemination. Pot2-TIR analysis indicated that the genetic diversity of P. oryzae in Xuzhou was mainly due to the insertion of transposable elements, while that of Nanjing was due to both the insertion of transposable elements and sexual recombination. Therefore, some R genes or gene combinations were suitable for resistance breeding in Jiangsu, and repetitive-PCR (rep-PCR) is a cost-effective tool for genetically differentiating distinct cultivar-specific populations or lineages with well-defined virulence patterns, because of the close correspondence between rep-PCR based clusters and pathotypes of inbred lines.     

Virulence factors analysis of native isolates of Xanthomonas albilineans and Xanthomonas sacchari from Tucumán,Argentina, reveals differences in pathogenic strategies

Xanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan-like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species.     

Phosphohexose mutase of Xanthomonas oryzae pv. oryzicola is negatively regulated by HrpG and HrpX,and required for the full virulence in rice

The genome of Xanthomonas oryzae pv. oryzicola annotates one uncharacterized gene, XOC_3841, only one ORF in this strain is annotated to encode Phosphohexose mutase (XanA), which reversibly converts glucose 1-phosphate to glucose 6-phosphate that implicates in the carbon metabolism pathways. However, it is unclear whether the XanA-coding gene is involved in the full virulence of X. oryzae pv. oryzicola. In this report, we showed that the mutagenesis in unique xanA, led the pathogen effectively to unable to utilize glucose and galactose for growth. The expression of xanA was strongly induced by glucose, sucrose, fructose, mannose or galactose at least 3 times higher than that by non-sugar NY medium. Intriguingly, xanA promoter region contains an imperfect PIP-box (plant-inducible promoter) (TTCGC-N16-TTCGA), and the expression of xanA was inducible in rice suspension cells rather than in a nutrient-rich (NB) medium and negatively regulated by a key hrp regulatory HrpG and HrpX cascade. More importantly, mutation in xanA resulted in impairment of bacterial growth and virulence in planta, and reduced bacterial cell motility and extracellular polysaccharides (EPS) production in media. In addition, the lost properties mentioned above in RΔxanA were completely restored to the wild-type level by the presence of xanA in trans. All these results suggest that xanA is required for EPS production, cell motility and the full virulence of X. oryzae pv. oryzicola.     

Selection and mutation of the avirulence gene AVR-Pii of the rice blast fungus Magnaporthe oryzae

Magnaporthe oryzae avirulence (AVR) genes are predicted to be involved in pathogen invasion and their virulence functions are restricted by the presence of the cognate resistance (R) genes. In this study, the distribution and variation of the avirulence (AVR-Pii) gene of M. oryzae in Yunnan province, China were analysed to understand haplotype diversity of AVR-Pii under field conditions. The presence of AVR-Pii in 454 field isolates of M. oryzae collected in Yunnan province was examined using gene-specific PCR markers. The results showed that 82 M. oryzae isolates carried AVR-Pii. Among them, 39 (35.5%), 5 (15.2%), 4 (14.3%), 2 (13.3%), 25 (12.8%) and 7 (9.7%) of the M. oryzae isolates carried AVR-Pii from central, southeastern, southwestern, northwestern, western and northeastern Yunnan province, respectively. Of these isolates, 55 were sequenced, while the remaining 27 isolates were not suitable for PCR-based sequencing and were not used for further analysis. Moreover, three AVR-Pii haplotypes were identified among the 55 isolates, in which H1 was identical with a previous sequence in GenBank (accession no. AB498874 ) and H2 and H3 were novel variants. All DNA sequence variations were found to occur in the protein-coding region resulting in amino acid substitutions. One virulent haplotype of AVR-Pii to Pii was identified among 55 field isolates, suggesting that the AVR-Pii gene has lost avirulence function through base substitution. These findings suggest that AVR-Pii is under positive selection and AVR-Pii mutations are responsible for overcoming race-specific resistance in nature.     

云南高原粳稻白叶枯病菌毒素及其与致病性关系

 为了探寻云南高原粳稻白叶枯病菌间毒素的差异及毒素与病菌致病性的关系,本研究选用致病性差异较大的3个云南高原粳稻白叶枯病菌株,采用乙酸乙酯法提取其毒素,用水稻幼苗浸根法和种子发芽抑制法测定毒素粗提物的生物活性,并用TLC法分析毒素组分及组分含量的差异。结果表明3个菌株单细胞产毒素量与菌株的致病性强弱成正相关;供试的3个菌株,无论其致病力强弱,其产生的毒素只要有足够的量,都能抑制水稻种子发芽,也能使水稻幼苗萎蔫,且毒素浓度越高,作用越明显;菌株间毒素组分和组分含量存在差异,一些特异的组分是否与其致病性有关,需要深入研究。     

水稻条斑病菌异柠檬酸脱氢酶在致病性中的功能分析

 异柠檬酸脱氢酶(isocitrate dehydrogenase,IcdH)催化异柠檬酸转化成α-酮戊二酸,参与碳代谢途径末端的三羧酸(tricarboxylic acid,TCA)循环。然而,编码异柠檬酸脱氢酶基因是否参与水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的致病性,我们并不清楚。为了阐明IcdH的作用,通过同源重组技术获得了Xoc的icdH基因缺失突变体(RΔicdH),并对该突变体进行了相关功能研究。研究表明:该突变体不能利用苹果酸、丙酮酸和柠檬酸,在寄主水稻上的生长能力和致病力相对于野生型均显著降低,其游动性也显著减弱; 功能互补子恢复RΔicdH的上述表型至野生型水平; Real-time PCR结果显示,六碳单糖、蔗糖、苹果酸、丙酮酸与柠檬酸能显著诱导icdH基因的转录表达;与水稻细胞互作时icdH基因受诱导表达,并受HrpX和HrpG负调控。这些结果说明:icdH基因是Xoc获取碳源和在寄主水稻上具有致病性所需的。