水稻白叶枯病菌和水稻细菌性条斑病菌的实时荧光PCR快速检测鉴定

成功建立了水稻白叶枯菌与水稻细菌性条斑病菌快速检测鉴定的实时荧光PCR方法。根据含铁细胞接受子基因设计两菌的通用引物PSRGF/PSRGR（扩增一个152bpDNA片段）和特异性探针（Baiprobe和Tiaoprobe）,并对13种细菌和1种植原体进行实时荧光PCR。结果表明,两个特异性探针能分别特异性检测到目标病原菌产生荧光信号而其它参考菌不产生荧光信号。检测的绝对灵敏度是30.6fg/μL质粒DNA和103CFU/mL的菌悬浮液,相当于1个细菌细胞的基因,比常规PCR电泳检测高约100倍,相对灵敏度为105CFU/mL。整个检测过程只需2h,完全闭管,降低了污染的机会,无需PCR后处理。 用这两个特异性探针分别对自然感染白叶枯菌和条斑菌的叶片DNA提取液和种子浸泡液进行实时荧光PCR,结果均可特异性检测到目标菌的存在并完全可将两种病原细菌区分开来,且只需03g叶片和10g种子。     

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.     

炭样小单孢菌产抗生素对水稻细菌性条斑病菌的抗菌作用及其机理

【背景】水稻细菌性条斑病菌为水稻细菌性条斑病的病原菌,土壤中分离到的一株具有广谱抗菌活性的炭样小单孢菌JXNU-1,其发酵产物(即抗生素JX)对植物病原菌具有较强的抑菌活性。【目的】研究抗生素JX对水稻细菌性条斑病菌的抗菌作用及其机理。【方法】采用杯碟法测定抑菌圈大小,二倍稀释法测定最低抑菌浓度、最低杀菌浓度,并且从菌体形态观察、电导率变化、培养液大分子漏出、蛋白质合成、核酸合成和膜电位变化6个方面探究其作用机理。【结果】抗生素JX对水稻细菌性条斑病菌的抑菌圈直径达18.84±0.28mm,最低抑菌浓度和最低杀菌浓度分别为1.39μg/m L和2.78μg/mL,且杀菌速度很快,作用12 h的杀菌率达100%。在抗生素JX作用下,水稻细菌性条斑病菌的细胞形态发生改变,培养液电导率、膜电位和大分子漏出量均随抗生素浓度增加而增大,但菌体蛋白质含量随着抗生素浓度增加而降低,同时,通过实时荧光定量PCR方法检测发现ef-p表达量下调。【结论】抗生素JX对水稻细菌性条斑病菌具有较强的抗菌作用,推测其抑菌机理是通过抑制菌体蛋白质的生物合成和影响细胞膜完整性而起作用。     

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。     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

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。     

水稻细菌性条斑病菌RpfCxoc/RpfGxoc双组分系统的功能

【目的】旨在阐明双组分系统RpfCxoc/RpfGxoc在水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)DSF(diffusible signal factor)合成、致病性等相关方面的生物学功能。【方法】以Xoc野生型菌株Rs105为母体,利用自杀载体pK18mobsacB缺失突变rpfCxoc、rpfGxoc和rpfGCxoc(rpfCxoc和rpfGxoc双基因),测定突变体及其互补菌株的DSF合成水平、对水稻的致病性、胞外多糖(extracellular polysaccharide,EPS)产量、菌体形态及群体结构。【结果】从Rs105基因组中克隆了rpfCxoc和rpfGxoc基因,并获得了相应的单基因或双基因缺失突变体。与Rs105相比,ΔrpfCxoc和ΔrpfGCxoc过量合成DSF信号分子,但是ΔrpfGxoc合成DSF的能力显著下降;rpfCxoc和rpfGxoc单基因或双基因的缺失突变均导致Xoc的致病性丧失,EPS合成水平下降34.1%-48.5%,形成菌体高度聚集的生物膜结构。【结论】RpfCxoc/RpfGxoc双组分系统调控Xoc的DSF生物合成、EPS产生和生物膜的驱散,是Xoc保持致病性所必需的因子。     

Specific protein phosphorylation induced in Xanthomonas campestris pv. oryzae by bacteriophage Xp12

We have investigated the endogenous phosphorylation patterns of phosphorylated proteins of Xanthomonas campestris pv. oryzae induced by its bacteriophages. For bacteriophage Xp12-infected cells, at least three phosphoproteins with apparent molecular weights of 28, 28.5 and 45kDa were detected by in vitro labeling with [-³²P]-ATP. These Xp12-specific phosphoproteins only occurred with Xp12 infection, and were not shown in uninfected or Xp10-infected cells. The protein kinase(s) responsible could use either ATP or GTP as the nucleotide substrate with nearly the same efficiency. Magnesium was proved to be an essential factor for the phosphorylation. EGTA treatment excluding the possibility that the presumed protein kinase was calcium-dependent. Under our reaction conditions, the optimal phosphorylation occurred at pH 7 to 8, for 30 to 40 min at 25 to 37°C. The Xp12-specific protein phosphorylation hint the existence of a physiological regulation mechanism involved in the life cycle of bacteriophage Xp12. Furthermore, the presumed protein kinase was shown to be encoded by the genome of Xp12 rather than indirectly induced by Xp12 infection.     

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.     

Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques

A polymerase chain reaction (PCR) technique was developed for detecting the presence of Xanthomonas oryzae pv. oryzae, the bacterial leaf blight (BLB) pathogen in rice seed and for studying the transmission of this bacterium from seed to plant. Primers TXT and TXT4R from an insertion sequence (IS1113) of the pathogen were used to amplify a 964-bp DNA fragment. A combined biological and enzymatic amplification (BIO-PCR) technique was used to detect the pathogen in naturally infected seed. The level of detection of TXT and TXT4R primers was 55 fg DNA of X. o. pv. oryzae, which is roughly the equivalent of seven cells (and four cells in pure culture suspension) of X. o. pv. oryzae. Hybridization of IS1113 with the amplified DNA fragment in Southern blot analysis confirmed that the 964-bp DNA fragment was amplified from X. o. pv. oryzae. The presence of the IS1113 element in strains of X. o. pv. oryzae from 16 rice-growing countries was confirmed by DNA dot blot analysis. X. o. pv. oryzae was detected from the seed washes and DNA extracted from the seed washes of naturally infected seeds of cvs Jaya and TN1. When stored at 4 degrees C, the pathogen was recovered up to 4 months and 9 months from naturally infected seeds of cvs Jaya and TN1, respectively. The BLB bacterium was also detected in seedlings, mature plants and seeds collected from plants raised from naturally infected seeds.     

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.     

Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious rice diseases worldwide. A rice gene, Xa26, conferring resistance against Xoo at both seedling and adult stages was isolated by map-based cloning strategies from the rice cultivar Minghui 63. Xa26 belongs to a multigene family consisting of four members. It encodes a leucine-rich repeat (LRR) receptor kinase-like protein and is constitutively expressed. Sequence analysis revealed that IRBB3 and Zhachanglong lines that are resistant to a broad range of Xoo strains, also carry Xa26. However, significant difference in lesion length was observed among these lines after inoculation with a set of Xoo strains. Moreover, transgenic plants carrying Xa26 showed enhanced resistance compared with the donor line of the gene in both seedling and adult stages. These results suggest that the resistance conferred by Xa26 is influenced by the genetic background.     

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。     

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.     

Rapid inhibition of protein histidine phosphorylation by UV-irradiation in Xanthomonas oryzae pv. oryzae

Abstract Exposure of Xanthomonas oryzae pv. oryzae cells to 254 nm UV radiation resulted in an alteration of protein phosphorylation. Labelling of the phosphohistidine-containing proteins with molecular masses of 81 and 32 kDa, named p81 and p32, was rapidly reduced following UV irradiation in the early exponential cells, but the decrease was not detected in mid-exponential cells. Mitomycin C, a DNA replication inhibitor, and rifampicin, a drug generally used to inhibit RNA synthesis and DNA replication, were also found to reduce the histidyl phosphorylation. However, this alteration of protein phosphorylation was not hindered by chloramphenicol treatment. A possible role for these histidyl phosphopfoteins in sensing UV light is proposed.     

TAL effector-dependent Bax gene expression in transgenic rice confers disease resistance to Xanthomonas oryzae pv. oryzae

The hypersensitive response (HR) is a form of programmed cell death of plant cells occurring in the local region surrounding pathogen infection site to prevent the spread of infection by pathogens. Bax, a mammalian pro-apoptotic member of Bcl-2 family, triggers HR-like cell death when expressed in plants. However, constitutive expression of the Bax gene negatively affects plant growth and development. The Xa10 gene in rice (Oryza sativa) is an executor resistance (R) gene that confers race-specific disease resistance to Xanthomonas oryzae pv. oryzae strains harboring TAL effector gene AvrXa10. In this study, the Xa10 promoter was used to regulate heterologous expression of the Bax gene from mouse (Mus musculus) in Nicotiana benthamiana and rice. Cell death was induced in N. benthamiana after co-infiltration with the P_Xa10:Bax:T_Xa10 gene and the P_PR1:AvrXa10:T_Nos gene. Transgenic rice plants carrying the P_Xa10:Bax:T_Xa10 gene conferred specific disease resistance to Xa10-incompatible X. oryzae pv. oryzae strain PXO99^A(pHM1AvrXa10), but not to the Xa10-compatible strain PXO99^A(pHM1). The resistance specificity was confirmed by the AvrXa10-dependent induction of the P_Xa10:Bax:T_Xa10 gene in transgenic rice. Our results demonstrated that the inducible expression of the Bax gene in transgenic rice was achieved through the control of the executor R gene promoter and the heterologous expression of the pro-apoptosis regulator gene in rice conferred disease resistance to X. oryzae pv. oryzae.

     

Novel Candidate Virulence Factors in Rice Pathogen Xanthomonas oryzae pv. oryzicola as Revealed by Mutational Analysis

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice. Transposon-mediated mutational analysis of the pathogen with a quantitative assay revealed candidate virulence factors including genes involved in the pathogenesis of other phytopathogenic bacteria, virulence factors of animal pathogens, and genes not previously associated with virulence.