Comprehensive prediction of plant cytoplasmic and apoplastic effectors underlying Erwinia psidii pathogenicity

Erwinia psidii (Eps) is the causal agent of emerging diseases of eucalypt and guava; however, the mechanisms underlying its pathogenicity are not fully understood. Here, we predicted factors involved in the ability of Eps to cause disease on its host plants. For that, the genomes of four Eps strains exhibiting different virulence on eucalypt were sequenced, and hrp/hrc genes coding for the type III secretion system (T3SS), effectors injected into the plant cell cytoplasm through the T3SS (T3SEs) and their plant subcellular localizations, as well as proteins deployed to the host apoplast, were predicted. It was found that Eps possesses a complete hrp/hrc gene cluster based on comparison with Erwinia amylovora. A total of 18 T3SEs were predicted, 11 of which were shared among all strains, none were exclusive to any strain and seven were absent in at least one strain. No sequence variation among strains was found for five T3SE candidates whereas extensive variation was found for six, suggesting the latter may be determinants of virulence differences. The T3SE candidates are predicted to target the plant cell nucleus, cytoplasm, mitochondrion, chloroplast and peroxisome. The predicted apoplastic effector repertoire common to all four strains was over-represented in proteins of unknown functions or predicted to possess enzymatic activities, among which the most abundant were oxidoreductases and peptidases. Proteins with lytic transglycosylase activity were predicted in strain-specific apoplastic effector repertoires. These results provide an important framework for future research aimed at uncovering the factors underlying Eps pathogenicity.     

The tomato Pto gene confers resistance to Pseudomonas floridensis,an emergent plant pathogen with just nine type III effectors

A newly discovered bacterial species, Pseudomonas floridensis, has emerged as a pathogen of tomato in Florida. This study compares the virulence and other attributes of P. floridensis to Pseudomonas syringae pv. tomato, which causes bacterial speck disease of tomato. Pseudomonas floridensis reached lower population levels in leaves of tomato as compared to the P. syringae pv. tomato strains DC3000 and NYT1. Analysis of the genome sequence of the P. floridensis type strain GEV388 revealed that it has just nine type III effectors including AvrPtoB_GEV388, which is 66% identical to AvrPtoB in DC3000. Five of these effectors have been previously reported to be members of a ‘minimal effector repertoire’ required for full DC3000 virulence on Nicotiana benthamiana; however, GEV388 grew poorly on leaves of this plant species compared to the DC3000 minimal effector strain. The tomato Pto gene recognizes AvrPtoB in race 0 P. syringae pv. tomato strains, thereby conferring resistance to bacterial speck disease. Pto was also found to confer resistance to P. floridensis, indicating this gene will be useful in the protection of tomato against this newly emerged pathogen.     

一个推测的野油菜黄单胞菌Ⅲ型分泌效应子基因XopXccP对寄主植物的致病性分析

 野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv. campestris, Xcc)是一种在全球范围内引起十字花科植物黑腐病的重要病原细菌。Xcc中效应蛋白与植物互作机理的研究还不够全面, 在已经测序的菌株Xcc 8004中, Xc_2994基因预测编码一个III型分泌效应蛋白XopXccP, 其生物学功能尚不清楚。为了探索XopXccP的生物学功能, 我们构建了Xcc 8004菌株的XopXccP基因缺失突变体, 结果发现XopXccP基因缺失后, 病原菌在多个甘蓝、花椰菜以及模式植物拟南芥(Col-0)上的致病性显著下降, 甚至几乎不致病。同时, 采用农杆菌侵染拟南芥花序的转化方法, 获得了转XopXccP基因拟南芥纯系, 经过诱导效应蛋白XopXccP在拟南芥中表达, 发现转基因拟南芥出现类似病斑的细胞死亡。本研究结果初步证明, XopXccP是一种毒性蛋白, 是Xcc 8004对多数十字花科植物致病所必须的。     

Hypersensitive response suppression by type III effectors of plant pathogenic bacteria

The suppressor activity of four representative avirulence (avr) genes from the Pseudomonas syringae group against elicitors of a general hypersensitive response (HR) was examined in tobacco leaves inoculated with double transformants of Pseudomonas fluorescens containing both a cosmid plasmid (pHIR11) carrying the hrp gene cluster and a plasmid carrying each avr gene. The double transformants Pf (pHIR11) containing avrB, avrRps4, or avrPto failed to induce an HR, but that carrying avrRpt2 did induce an HR as Pf (pHIR11 + empty vector) did. Thus, some Avr proteins seem to have suppressor activity against a general HR and should promote aggressiveness of the pathogens.     

丁香假单胞菌III型分泌系统组分HrpH酶学功能解析

 III型分泌系统(T3SS)是植物病原细菌的主要致病系统,负责分泌和转运效应蛋白。T3SS结构的形成需要跨越由肽聚糖包被的细菌细胞壁周质层。HrpH是丁香假单胞菌T3SS特化的裂解性转糖基酶。本研究通过原核表达纯化了HrpH及其相关结构域蛋白,发现HrpH具有结合肽聚糖的能力,其中SLT结构域是肽聚糖结合的关键功能域,第148位的谷氨酸是结合的关键位点。进一步研究发现HrpH能够抑制丁香假单胞菌番茄致病变种DC3000(Pst DC3000)的生长,透射电镜显示,HrpH蛋白处理Pst DC3000使其细胞壁的完整性受损。以上研究结果表明HrpH通过裂解细胞壁、结合肽聚糖从而协助T3SS穿透细菌细胞壁形成超分子复合体。     

具有III型分泌系统的Pseudomonas moraviensis FP1761植物益生功能及其全基因组分析

 本研究从节瓜根际分离到一株具有防病促生功能的荧光类假单胞菌FP1761。该菌株对部分植物病原真菌和细菌具有拮抗能力,能够解钾、解有机磷和无机磷,可产生氨、蛋白酶、嗜铁素、吲哚乙酸。生物测定表明菌株FP1761可显著促进小麦生长。生理生化、平均核苷酸相似度、16S rDNA和多基因分析将FP1761鉴定为摩拉维亚假单胞菌(Pseudomonas moraviensis)。菌株FP1761基因组草图全长6.12 Mb,(G+C)含量为59.9%,共编码5467个基因序列。将该菌株与种内3个代表性菌株进行泛基因组和核心基因组分析,共产生 4 357个共有基因,菌株FP1761特有基因327个。利用antiSMASH对菌株次生代谢基因簇进行预测,发现其含有8个潜在的次生代谢产物基因簇。其中两个基因簇与嗜铁素pyoverdine合成相关,未见聚酮类合成基因。基因组分析发现,该菌株具有与病原性假单胞菌相似的III型分泌系统,但丢失了效应蛋白调控因子hrpS和转运相关的hrpH和hrpK1基因。对全基因组扫描,菌株FP1761仅保留了病原性假单胞菌的保守效应蛋白AvrE和HopAA1-1。FP1761是目前已发现的唯一具有III型分泌系统的摩拉维亚假单胞菌。本研究表明摩拉维亚假单胞菌FP1761具有潜在的植物防病促生功能,但其III型分泌系统与植物益生互作机制有待进一步解析。     

Genetic diversity of Pseudomonas syringae pv. actinidiae,pathogen of kiwifruit bacterial canker

Bacterial canker disease of kiwifruit currently occurs in at least 15 countries, causing serious damage. The causative agent of the disease is Pseudomonas syringae pv. actinidiae (Psa), which is genetically diverse and is currently classified into five biovars, namely, biovars 1, 2, 3, 5 and 6. In Japan, four biovars except biovar 2 have been found so far. These biovars have been confirmed to have differences in the virulence and composition of pathogenicity-related genes, such as toxin biosynthesis and type III effector genes. Biovars 1 and 6 possess the tox island, a genomic island of approximately 38 kb, which contains phaseolotoxin biosynthesis genes (argK-tox cluster) and is confirmed to have been acquired from other bacteria through horizontal transfers. Also, on the megaplasmid possessed by biovar 6, there exist coronatine biosynthesis genes, and biovar 6 has the ability to produce two phytotoxins, phaseolotoxin and coronatine. In 2014, biovar 3, considered to be of foreign origin, was confirmed for the first time in Japan. Biovar 5, whose virulence is relatively weak, is distributed only in a limited area. In addition to the tox island and various plasmids, a large number of mobile genetic elements are confirmed to be present on the Psa genomes, which might have played a major role in helping Psa to acquire new features. In order to understand how Psa acquired the ability to infect kiwifruit systemically, it is important to make polyphasic comparisons with related pathovars, such as P. syringae pv. theae and pv. actinidifoliorum.     

7种检疫性植物病原黄单胞菌III型分泌系统和效应蛋白编码基因的差异性分析

 γ-变形菌纲(γ-Proteobacteria)的黄单胞菌属(Xanthomonas)的大多数种类可引起植物病害,多数是我国检疫对象。与其他革兰氏阴性植物病原细菌一样,植物病原黄单胞菌可通过高度保守的III型分泌系统(type-III secretion system, T3SS)分泌效应蛋白(T3SS-secreted effectors, T3SEs)进入植物细胞,在非寄主植物和抗病寄主植物上产生过敏反应(hypersensitive response, HR)以及在感病寄主植物上具有致病性。尚不清楚哪些种类的黄单胞菌具有T3SS和缺少哪些T3SE是否可作为检疫的依据。搜集7种检疫性植物病原黄单胞菌,通过PCR和Southern杂交试验结果发现:香蕉细菌性青枯病菌(X. campestris pv. musacearum)的ICMP287和ATCC49084菌株、甘蔗流胶病菌(X. axonopodis pv. vasculorum)ATCC13901菌株、洋葱细菌性叶枯病菌(X. axonopodis pv. allii)的LMG576和LMG578菌株中不含有tale基因,并且ATCC13901菌株既不含有T3SS基因也不含有hpa1和xopQ基因;菜豆细菌性疫病菌(X. campestris pv. phaseoli)ATCC49119菌株不含有hpa1基因。相应地,推测含有2~12个tale基因的黄单胞菌有:大豆斑疹病菌(X. axonopodis pv. glycines)ICMP5732和ATCC43911菌株、豌豆细菌性疫病菌(X. axonopodis pv. vignicola)ATCC11648菌株、棉花细菌性角斑病菌(X. campestris pv. malvacearum)ATCC12131和(X. campestris pv. phaseoli)ATCC49119菌株。大豆细菌性斑疹病菌ATCC43911菌株尽管含有hpa1、xopQ和hrcC基因,但在非寄主烟草上不能激发HR反应;而甘蔗流胶病菌ATCC13901菌株不含有hpa1、xopQ和hrcC基因,却激发烟草产生HR反应。这些结果对于分析比较不同植物病原黄单胞菌的致病性因子和设计特定的植物检疫靶点提供了科学线索。     

Genetic structure analysis of strains causing citrus canker in Iran reveals the presence of two different lineages of Xanthomonas citri pv. citri pathotype A*

West Asia has been recognized as a major centre for the diversification of Xanthomonas citri pv. citri, a citrus quarantine pathogen of considerable economic importance. However, little genotyping data is available mainly due to the paucity of microbial resources in this region. Using a comprehensive strain collection, several genotyping techniques and a pathogenicity assay, the status of strains causing Asiatic citrus canker in Iran, an internationally significant citrus‐producing country, was clarified. All strains were genetically related to X. citri pv. citri pathotype A* (i.e. strains with a host range restricted to Mexican lime and related species) but not to pathotype A (i.e. strains with a wide host range among rutaceous species). The findings were based on discriminant analysis of the principal components of MLVA‐31 data and were further confirmed by pathogenicity data. Two genetically, geographically and pathologically separate groups of strains in Iran were identified. One of the groups had never been previously reported anywhere in the world. A very strong genetic structure was found (R_ST = 0·938), consistent with their geographical isolation. Strains from these two groups also differed in terms of their type III effector repertoire. The atypical host range of one of these groups could explain why some Iranian strains had previously been mistakenly identified as pathotype A. This study suggests the absence of invasive pathotype A strains in Iran (known as DAPC 1), which account for most of the economically important outbreaks internationally.     

Participation of Xanthomonas axonopodis pv. citri hrp cluster in citrus canker and nonhost plant responses

The participation of the Xanthomonas axonopodis pv. citri hypersensitive response and pathogenicity ( hrp ) cluster in interactions with host and nonhost plants was characterized in pathogenicity and avirulence models. The hrp cluster encodes the type III secretion system indispensable for trafficking of proteins to the plant cell. Mutations in operons hrpB and hrpD and the hrpF gene failed to produce canker in citrus plants or hypersensitive response in cotton plants. The interaction of the phytopathogen with various nonhost plants has been characterized . The results showed that the hypersensitive response is activated in leaves of cotton, bean, tobacco, tomato, pepper and Nicotiana benthamiana , and that genes present in operons hrpB and hrpD and the hrpF gene are required for pathogenicity in hosts and induction of the hypersensitive response in nonhost plants.     

Influence of amylovoran production on virulence of Erwinia amylovora and a different amylovoran structure in E. amylovora isolates from Rubus

The amylovoran structures of five Erwinia amylovora isolates from Malaceae sp. and four isolates from Rubus sp. host plants were fully established, mainly by NMR. The structural data on one E. amylovora isolate from a Malaceae sp. host, which had been previously suggested by mass and NMR (Nimtz et al., 1996), were completed. E. amylovora strains infective on Malaceae sp. host plants had an amylovoran composed of pentasaccharide and 30–40% hexasaccharide repeating-substructures, whereas amylovoran from E. amylovora isolates from Rubus sp. host plants had only the pentasaccharide substructures. On the other hand, the exopolysaccharide (EPS) production differed in wild-type E. amylovora strains. Data on in vitro amylovoran production per cell could account for the differences in aggressiveness found in E. amylovora strains, as deduced from a pilot test with highly, moderately, and weakly aggressive strains. This correlation was confirmed with several other wild-type E. amylovora strains from different origin.     

Comprehensive diversity assessment of walnut-associated xanthomonads reveal the occurrence of distinct Xanthomonas arboricola lineages and of a new species (Xanthomonas euroxanthea) within the same tree

Xanthomonas arboricola pv. juglandis (Xaj) is the aetiological agent of walnut diseases causing economic losses on walnut production worldwide. This phytopathogen is spread around the world where walnuts are produced and has a considerable genetic diversity. Using a comprehensive sampling methodology, focusing on factors that could influence the diversity of walnut-colonizing Xaj in Portugal, this work provides new insights on xanthomonad populations on walnut. Genetic diversity was assessed by multilocus sequence analysis (MLSA) and dot blot hybridization patterns on 131 Xanthomonas isolates obtained from 64 walnut trees considering epidemiological metadata such as year of isolation, distinct bioclimatic regions, production regimes, and host-related features. The results showed that the majority of isolates were split into 17 lineages of Xaj, while the other isolates clustered in four MLSA groups that did not include Xaj strains. These four groups were represented by three lineages of X. arboricola, and 11 lineages of Xanthomonas spp., including strains assigned to the recently proposed new species Xanthomonas euroxanthea. Furthermore, distinct Xaj, X. arboricola, and Xanthomonas spp. were isolated from the same walnut tree, suggesting possible genetic admixture within the same host. Phylogenetic analysis through geoBurst revealed the high diversity of these Xanthomonas spp. populations. Assessment of type III effector genes gave the indication that some Xanthomonas spp. strains were nonpathogenic on walnut, with the exception for X. euroxanthea CPBF 424. Altogether, these findings add to the thorough characterization of walnut-associated xanthomonads in Portugal, providing a comprehensive snapshot of the current diversity that could contribute to risk assessment analysis and improve phytosanitary control.     

甘肃省辣椒疫霉菌的交配型分布及其致病力差异

采用单孢菌落直接配对法和游动孢子悬浮液土壤接种法对2003—2006年采自甘肃省辣椒主产区的辣椒疫霉菌菌株Phytophthora capsici进行交配型和致病力测定。结果表明,甘肃省辣椒疫霉菌存在A1、A2和A0三种交配型,以A1和A0占优势,在各产区均有分布,发生频率分别达57.9%和31.6%;A2交配型仅2株,分别出现在酒泉和庆阳。不同来源菌株对5个辣椒品种的致病力存在显著差异,平均病情指数为2.2～56.4,强、中、弱致病性菌株分别占10.6%、52.6%和36.8%,不同地区的菌株致病力有强弱之分,以中、弱致病性菌株为主,同一地区中不同菌株的致病力也存在一定的差异性。     

鳞翅目昆虫迁飞相关基因的鉴定及进化分析

为明确迁飞性昆虫迁飞行为的分子调控机制,选取12种鳞翅目昆虫为研究对象,将其分为迁飞组和非迁飞昆虫组,采用PAML软件分析单拷贝直系同源基因在2个分组中的进化速率,从基因组水平对涉及生物钟调节、神经调节和内分泌调节3类迁飞相关基因进行注释和比较。结果表明,部分基因在迁飞昆虫和非迁飞昆虫中的进化速率存在显著差异,且这些基因富集在肌肉结构发育、复眼及光感细胞发育、能量代谢、腺体发育、醇酮类及脂蛋白合成代谢、磷酸化调节等生物过程中;与神经调节相关的基因在迁飞昆虫中有不同程度的显著扩增或收缩,而涉及生物钟调节的基因中仅ebony在迁飞组昆虫中的拷贝数显著多于非迁飞组的拷贝数,与内分泌调节相关的法尼醇脱氢酶编码基因在迁飞组和非迁飞组中呈现显著差异。表明迁飞和非迁飞2组昆虫在基因组水平上存在部分基因拷贝数增加或减少的规律,可能是迁飞昆虫或非迁飞昆虫的共同特征。     

内蒙古自治区向日葵小核盘菌的菌丝亲和组类型及其菌株的生物学特性、致病力和交配型

为明确内蒙古自治区阴山北麓地区向日葵小核盘菌Sclerotinia minor的遗传变异,对自内蒙古自治区乌兰察布市、包头市和呼和浩特市向日葵上分离纯化的110株向日葵小核盘菌菌株进行菌丝亲和组(mycelium compatibility group,MCG)划分,并对5个主要MCG组间和组内各菌株的生物学特性、致病力和交配型进行测定。结果表明,供试的110株菌株被划分为14个亲合组,其中MCG1为主要类型,包含32株菌株,占总菌株的29.1%;MCG2包含来自7个地点的25株菌株;5个MCG仅包含1株菌株;在这14个MCG中,MCG1～MCG5包含92株菌株,占总菌株数的83.6%。MCG1～MCG5组间各菌株在菌落直径和草酸分泌量上存在差异,但在菌核形成量、多聚半乳糖醛酸酶活性和致病力上无显著差异;而MCG1～MCG5组内各菌株在菌落直径、草酸分泌量、菌核形成量、多聚半乳糖醛酸酶活性和致病力上均有一定差异。在MCG1～MCG5各菌株的交配型中,除MCG2中菌株的负反转型与正反转型比例接近1∶1外,其它4个MCG中菌株的负反转型与正反转型比例均偏离1∶1,表明内蒙古自治区向日葵小核盘菌具有较高的遗传变异程度。     

瓜类褪绿黄化病毒自然侵染南瓜及分子进化分析

瓜类褪绿黄化病毒(cucurbit chlorotic yellows virus,CCYV)可侵染多种瓜类,主要危害叶片,影响其光合作用,导致减产.采集湖南省南瓜疑似病毒病样品经siRNA测序发现CCYV,进一步RT-PCR验证CCYV可自然侵染南瓜,检出率为5.78％,这是我国内地首次发现CCYV可自然侵染南瓜.持...