利用酵母双杂交筛选与致病疫霉菌效应蛋白PITG_07586互作的寄主蛋白

 致病疫霉是引起马铃薯晚疫病的病原菌,其分泌的效应蛋白是侵染寄主的重要毒力因子。本研究在致病疫霉转录组中筛选到一个侵染早期表达的效应蛋白基因PITG_07586(GenBank:XM_002904522.1)。该基因全长447 bp,其编码蛋白包含1个信号肽,1个核定位信号序列(RRRRKRRKKKK)。在本氏烟草中,瞬时表达该基因显著促进病原菌侵染。亚细胞定位表明GFP-PITG_07586定位在细胞核中。利用酵母双杂交技术并以PITG_07586为诱饵筛选马铃薯cDNA文库,最终获得3个靶标蛋白。经序列比对分析,这3个靶标蛋白分别是马铃薯POM30蛋白、电压依赖阴离子通道蛋白VDAC以及SRC2类蛋白。研究结果为致病疫霉菌效应蛋白PITG_07586及其靶标蛋白如何调控植物免疫提供重要依据。     

辣椒疫霉菌PiAvr3a-like基因的鉴定及功能分析

疫霉菌分泌的RxLR效应子能干扰寄主植物细胞的正常生理代谢和功能。我们通过同源序列搜索在辣椒疫霉菌中鉴定到9个PiAvr3a的同源基因(PiAvr3a-like),同时发现PiAvr3a-like在大豆疫霉菌中也有分布,表明该类基因可能在疫霉菌致病过程中发挥重要作用。从辣椒疫霉菌中成功克隆了5个PiAvr3a-like基因,表达模式分析发现PcAvh128/132/134这3个基因在疫霉菌侵染早期诱导表达,暗示它们可能在侵染前期发挥功能。接种实验表明在本氏烟中瞬时表达其中的PcAvh128可以显著促进辣椒疫霉菌的侵染,而PcAvh132活性相反,其他3个基因没有明显活性。这5个基因均不能抑制INF1和效应因子诱导的细胞死亡,也不能诱导可见的过敏反应。上述结果表明PiAvr3a-like基因不同成员之间功能存在分化,这为疫霉菌和寄主的分子互作研究提供了材料和参考。     

效应因子PsCRN77基因在本氏烟中的表达降低其对寄生疫霉的抗性

CRN(Crinkler)是一类疫霉菌胞内效应因子,绝大多数CRN的功能和作用机制尚不明确。本研究利用农杆菌介导的瞬时表达技术,发现大豆疫霉效应因子PsC RN77可以在本氏烟中抑制激发子诱导的细胞死亡。接种分析表明在本氏烟中表达该效应因子基因可以促进寄生疫霉的侵染。定量RT-PCR结果显示在本氏烟中表达PsCRN77可以负调控水杨酸信号途径标记基因PRb-1b的表达,正调控茉莉酸信号途径标记基因LOX的表达。进一步通过DAB染色表明,与表达GFP对照相比,表达PsCRN77可以降低疫霉菌侵染后本氏烟中的活性氧积累。以上结果表明PsCRN77是一个毒性效应因子,可以通过影响激素抗性信号途径和活性氧积累干扰植物的防卫反应,促进病原菌的侵染。该研究为认识疫霉菌的致病机制提供了线索。     

RXLR类效应分子PITG_21645.2的基因序列分析及功能验证

 马铃薯晚疫病黑龙江菌株效应分子PITG_21645.2在本氏烟(Nicotiana benthamiana)上可以抑制由INF1引起的过敏性细胞坏死(Hypersensitive response, HR)。为验证其为致病疫霉致病的重要效应分子,克隆了10个致病疫霉菌株以及3个同属卵菌菌株中的PITG_21645.2同源基因,它们在氨基酸序列上相似度为93%~100%。与INF1共表达的结果显示,仅有PITG_21645.2^MP903丧失了抑制HR的功能。其中PITG_21645.2^MP903的第31位氨基酸存在特异性,编码丝氨酸,其余同源基因在该位点均编码天冬酰胺。PITG_21645.2^MP903回复突变体(S31N)能够恢复该基因抑制INF1引起HR的功能,说明第31位氨基酸是影响抑制功能的关键位点。另外,PITG_21645.2还能抑制BAX、大豆疫霉PsojNIP和效应分子Avh238在本氏烟上激发的HR,而PITG_21645.2^MP903都丧失抑制功能。本氏烟上表达PITG_21645.2能够促进致病疫霉在寄主上的定殖,从而提示PITG_21645.2在致病疫霉的侵染中发挥致病性功能。     

葡萄座腔菌候选效应子抑制了Bax诱导的PCD反应并促进烟草疫霉的侵染

 轮纹病是苹果生产中发生严重又难以防治的病害。为探究致病机理,本研究选择葡萄座腔菌的3个候选效应子进行研究。农杆菌介导的烟草瞬时表达显示,Bdo_01520、Bdo_11198、Bdo_11545都能够完全抑制Bax诱导的烟草PCD反应。酵母分泌系统测试表明3个候选效应子的信号肽都具有外泌活性。qRT-PCR测定候选效应子在葡萄座腔菌ZY7有伤接种苹果果实后的相对表达量,结果显示3个基因在病菌侵染36~72 h上调表达。在本生烟中瞬时表达Bdo_11198显著促进了烟草疫霉的侵染,并降低了疫霉菌侵染后本生烟中活性氧的积累。结果表明,葡萄座腔菌候选效应子可以通过影响植物的PCD反应和过氧化氢积累促进病原菌的侵染,研究结果为进一步研究葡萄座腔菌的致病机制提供了基础。     

辣椒疫霉CRN编码基因的克隆、转录特征及在与寄主互作中的作用

 CRN (crinkling and necrosis-inducing protein)蛋白是一类卵菌特有的效应分子,然而目前对其功能和分子机制知之甚少。为分析CRN效应分子在辣椒疫霉(Phytophthora capsici)与植物互作中的作用,本研究利用前期转录组测序结果,对辣椒疫霉基因组数据库进行序列比对分析,获得了7个CRN编码基因的全长序列;采用RT-PCR方法分析其在辣椒疫霉生长发育阶段(菌丝、游动孢子囊、游动孢子、萌发的休止孢)和侵染寄主阶段(本氏烟(Nicotiana benthamiana)灌根1.5、3、6、12、24、36和72 h后)的转录水平,发现3个基因在辣椒疫霉生长发育阶段和侵染阶段上调表达;将这3个基因克隆测序,发现其编码蛋白均含有保守的LFLAK和HVLVVVP基序;在本氏烟上的基因瞬时表达结果表明,Pc559084和Pc570403能够抑制由致病疫霉elicitin蛋白INF1或老鼠促凋亡蛋白BAX诱发的植物程序性细胞死亡,并且Pc559084能够促进辣椒疫霉侵染植物。这些研究结果为理解CRN效应分子在辣椒疫霉致病过程中的作用提供了重要的实验数据。     

本氏烟NbWRKY40亚家族转录因子抗病相关功能研究

 植物WRKY类转录因子的IIa亚类广泛参与调控植物的生物胁迫和非生物胁迫过程。本研究根据拟南芥和普通烟中具有抗病和抗胁迫功能的IIa亚类WRKY40转录因子的序列,利用基因同源克隆法,获得本氏烟中NbWRKY40基因。序列分析表明在本氏烟中有5个属于IIa亚类的基因,其序列之间具有高度相似性。qRT-PCR检测发现NbWRKY40基因是受寄生疫霉侵染诱导上调表达的基因,通过VIGS技术研究该类基因在本氏烟中的抗病相关功能,用半活体营养型寄生疫霉进行抗病性试验,结果表明NbWRKY40基因的沉默降低了本氏烟对寄生疫霉的抗性,且在侵染点的细胞坏死程度增强,过氧化氢积累和胼胝质沉积都明显减少。NbWRKY40基因沉默同样降低了本氏烟对死体营养型灰霉菌的抗性。检测NbWRKY40基因沉默后4个不同抗病信号通路下游基因的表达,发现基因沉默导致参与抗病和SA途径的PR1b、PR2b基因受疫霉侵染诱导表达的水平明显下降。综上,推测NbWRKY40基因可能依靠SA介导的信号通路参与调控本氏烟抗病性。     

番茄抗晚疫病基因Ph-3的分子标记开发及应用

正番茄晚疫病由卵菌疫霉属的致病疫霉菌(Phytophthora infestans)侵染引起。病菌对番茄致病性强,侵染寄主后通过气流散发孢子,多次重复侵染植株,引起晚疫病爆发,对番茄生产造成严重危害[1]。利用抗晚疫病资源,培育番茄抗病品种是降低晚疫病危害的有效途径。在野生番茄中发现     

NbRbohB基因在本氏烟与疫霉菌互作中的功能分析

活性氧(active oxygen species, AOS)在植物抗病中发挥着重要作用,主要由NADPH氧化酶(nicotinamide adenine dinucleotide phosphate oxidase)系统产生.为明确NADPH氧化酶NbRbohB基因在本氏烟与疫霉菌亲和与非亲和性互作中的功能,采用荧光定量PCR技术以及病毒诱导的基因沉默方法探究了NbRbohB基因在本氏烟中对2种疫霉菌抗性中的作用,并利用NADPH氧化酶抑制剂对辣椒疫霉的抗性进行了检测.结果发现:2种疫霉菌均能诱导本氏烟发生氧迸发,且NbRbohB基因可能参与了疫霉菌诱导本氏烟发生的氧迸发过程.该基因沉默后降低了本氏烟对亲和互作辣椒疫霉菌的抗性,但对非亲和互作疫霉菌的抗性没有肉眼可见的影响;NADPH氧化酶抑制剂处理本氏烟后也能降低其对辣椒疫霉的抗性.表明该基因通过介导AOS产生,参与植物对亲和性与非亲和性互作疫霉的抗病反应,在亲和互作中尤为重要.     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

Development of a species-specific and sensitive detection assay for Phytophthora infestans and its application for monitoring of inoculum in tubers and soil

A specific and sensitive PCR assay for the detection of Phytophthora infestans , the cause of late blight of potato, in soil and plant tissues was developed. A P. infestans -specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of rDNA internal transcribed spacer regions of most of the known Phytophthora species. PCR amplification of P. infestans DNA with primers INF FW2 and INF REV generated a 613 bp product, and species specificity was demonstrated against DNA from nine other Phytophthora species and seven potato-blemish pathogens. In a single-round PCR assay, 0·5 pg pure P. infestans DNA was detectable. Sensitivity was increased to 5 fg DNA in a nested PCR assay using Peronsporales-specific-primers in the first round. As few as two sporangia or four zoospores of P. infestans could be detected using the nested assay. Procedures are described for detection of P. infestans in leaves, stem and seed potato tubers before expression of symptoms. A soil assay in which 10 oospores per 0·5 g soil were detectable was developed and validated using samples of field soil. The PCR assay was used to examine the long-term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of P. infestans in leaf material buried in a replicated experiment under natural field conditions. Oospores were consistently detected using the PCR assay up to 24 months (total length of the study) after burial in soil, whereas the sporangial inoculum was detected for only 12 months after burial. Sporangial inoculum was shown to be nonviable using a baiting assay, whereas leaf material containing oospores remained viable up to 24 months after burial.     

Loss of Production of the Elicitor Protein INF1 in the Clonal Lineage US-1 of Phytophthora infestans

ABSTRACT The extracellular protein INF1 of Phytophthora infestans is a member of the elicitin family of protein elicitors known to induce a hypersensitive response on some solanaceous and cruciferous plants. The presence of INF1 elicitin in culture filtrates of 102 P. infestans isolates from 15 countries was examined. All tested isolates produced INF1 except five isolates collected in 1976 and 1977 from infected potatoes in East Germany (the former German Democratic Republic). Based on hybridization to the multi-locus DNA fingerprint probe RG57, all the INF1-nonproducing isolates were shown to belong to the clonal lineage US-1 that dominated world populations until the 1980s. Phylogenetic analysis of a set of European US-1 isolates using amplified fragment length polymorphism fingerprint data indicated that loss of INF1 production evolved independently in separate lineages within US-1. DNA and RNA blot hybridizations showed that INF1-nonproducing isolates still retain a copy of the inf1 gene, whereas little inf1 mRNA could be detected. Hypothetical interpretations of the evolution in a restricted geographic area of P. infestans lineages deficient in the production of a specific elicitor protein are discussed.     

Synthesis and fungicidal activity of ethaboxam against Oomycetes

This study describes the chemical synthesis and intrinsic fungicidal activity of ethaboxam [(RS)-N-(alpha-cyano-2-thenyl)-4-ethyl-2-(ethylamino)-1,3-thiazole-5-carboxamide], a new Oomycetes fungicide. In in vitro tests, ethaboxam showed inhibitory activity against isolates of Phytophthora and some Pythium spp, with MIC values ranging from 0.1 to 0.5 mg litre(-1) for nine isolates of Phytophthora infestans (Montagne) de Bary and from 1.0 to 5.0 mg litre(-1) for eight isolates of Phytophthora capsici Leonian. In tests to determine time and concentration for complete inactivation of each pathogen (five isolates of P infestans and five isolates of P capsici), ethaboxam inactivated all isolates of P infestans within 48h at 10 mg litre(-1) and those of P capsici within 96 h at 10 mg litre(-1). Ethaboxam effectively suppressed development of tomato late blight caused by P infestans and pepper Phytophthora blight caused by P capsici in the studies conducted to determine its preventive, curative, persistent and systemic activity. These results show that ethaboxam has desirable fungicidal characteristics as an Oomycetes fungicide.     

可可毛色二孢效应子LT_397基因的克隆及转录分析

可可毛色二孢Lasiodiplodia theobromae是引起葡萄溃疡病的主要致病菌之一。病原菌会分泌许多效应子抑制植物的防御反应。本试验对可可毛色二孢特有的效应子LT_397基因进行克隆,开放阅读框(open reading frame,ORF)全长1140 bp,含有11个半胱氨酸。生物信息学软件预测显示:效应子LT_397信号肽为位于N端的1~16个氨基酸。qRT-PCR结果表明:LT_397受可可毛色二孢诱导下,在侵染24 h的基因相对转录水平最高;在逆境胁迫处理方面:LT_397在营养、氧化、离子、酸碱和UV胁迫下基因的相对转录水平都上升。烟草瞬时表达表明,效应子LT_397可抑制水稻细菌性谷枯病菌Burkholderia glumae引发的过敏反应(hypersensitive response,HR)。以上结果为进一步分析效应子对寄主的致病机制奠定了基础。     

Commercial Fungicide Formulations Induce In Vitro Oospore Formation and Phenotypic Change in Mating Type in Phytophthora infestans

ABSTRACT A wide range of commercially formulated fungicides cause in vitro effects on mating behavior in specific isolates of Phytophthora infestans, the causal agent of late blight of potato and tomato. Four isolates of P. infestans representing each of the four common US genotypes, US-1, US-6, US-7, and US-8 and varying in their sensitivity to metalaxyl, were exposed to a variety of fungicides used to control late blight in petri dish assays at concentrations ranging from 1 to 100 mug a.i./ml. Exposure of each of these normally heterothallic single mating type isolates of P. infestans to 9 of the 11 commercial fungicide formulations tested resulted in the formation of oospores after 2 to 4 weeks. The highest numbers of oospores were formed on media amended with Ridomil 2E (metalaxyl) and Ridomil Gold EC (mefenoxam) at 0.1 to 10 mug a.i./ml, averaging as many as 471 and 450 oospores per petri dish, respectively. Several other fungicides including Maneb, Manzate (Mancozeb), Curzate (cymoxanil + mancozeb), and Acrobat MZ (dimethomorph + mancozeb) also induced oospore formation, producing from 0 to 200 oospores per plate at fungicide concentrations from 0.1 to 10 mug a.i./ml. The metalaxyl resistant isolates formed oospores in response to the fungicides more often than the metalaxyl sensitive isolates. No oospores were formed on media amended with Bravo (chlorothalonil) or Tattoo C (chlorothalonil + propamocarb HCl) and these compounds completely suppressed growth of the isolates at 0.1 and 1 mug a.i./ml. Three metalaxyl resistant A2 isolates mated with both A1 and A2 isolates after exposure to the fungicides Ridomil 2E and Ridomil Gold EC. Alterations in mating type expression were also observed in a metalaxyl sensitive A1 isolate after exposure to Benlate (benomyl). Copious amounts of chemicals are applied annually to potato and tomato production areas to control late blight. Our results indicate that a wide range of chemically diverse fungicides can induce normally heterothallic metalaxyl resistant isolates of P. infestans to form oospores in vitro after short exposures to the fungicides.     

Epidemiological importance of Solanum sisymbriifolium, S. nigrum and S. dulcamara as alternative hosts for Phytophthora infestans

Lesions of Phytophthora infestans were found on woody nightshade ( Solanum dulcamara ), black nightshade ( S. nigrum ) and S. sisymbriifolium during a nationwide late blight survey in the Netherlands in 1999 and 2000. Pathogenicity and spore production of P. infestans isolates collected from potato ( S. tuberosum ), S. nigrum , S. dulcamara and S. sisymbriifolium were determined on several host plant species, and oospore formation in naturally infected and inoculated foliage of hosts was quantified. The present population of P. infestans in the Netherlands is pathogenic on S. nigrum , S. dulcamara and S. sisymbriifolium . Oospores were produced in leaves of S. nigrum , S. dulcamara and S. sisymbriifolium following infection with A1 and A2 isolates. Therefore these plant species should be regarded as alternative hosts for the late blight pathogen. In the case of S. nigrum and S. dulcamara infection was a relatively rare event, suggesting that diseased plants do not significantly contribute to the overall late blight disease pressure present in potato-production areas. Oospore production in ageing S. nigrum and S. dulcamara plants in autumn, however, may generate a considerable source of (auto) infections in following years. Considerable numbers of sporangia and oospores were produced on S. sisymbriifolium following infection with P. infestans . Additional field infection data are needed to evaluate the epidemiological consequences of a commercial introduction of S. sisymbriifolium as a potato cyst nematode trap crop.