云南省元阳县籼/粳型稻瘟病菌无毒基因Avr-Pia、Avr-Pita1和Avr-Pii多样性分析

为明确云南省元阳县籼/粳型稻瘟病菌中无毒基因的多样性及差异,采用接种鉴别品种方法和PCR技术对9株粳型菌株和21株籼型菌株中无毒基因Avr-Pia、Avr-Pita1和Avr-Pii的多样性进行分析。结果显示,30株稻瘟病菌菌株中无毒基因Avr-Pia、Avr-Pita1和Avr-Pii的平均出现频率分别为33.3%、76.7%和0,Avr-Pia、Avr-Pita1在籼型菌株和粳型菌株中的出现频率分别为4.8%和100.0%、66.7%和100.0%。Avr-Pia主要为存在/缺失的多样性,籼/粳型菌株对水稻品种Achiasahi(Pia)的致病率分别为100.0%和11.1%;Avr-Pita1可划分为3个类群,除菌株CH1195属于PO类群外,籼型菌株均属于J1类群,粳型菌株则属于J2/J3类群;籼/粳型菌株对水稻品种K1(Pita)的致病率分别为100.0%和0,且籼/粳型菌株Avr-Pita1蛋白序列在第83位和第192位氨基酸差异明显。Avr-Pii在30株稻瘟病菌菌株中均未检出,籼/粳型菌株对水稻品种Fujisaka No. 5(Pii)的致病率分别为100.0%和0。表明Avr-Pia和Avr-Pita1在粳型菌株中的出现频率高于籼型菌株且变异程度低,粳型菌株中可能存在其它无毒基因使其不能侵染Fujisaka No. 5。     

中国水稻白叶枯病菌小种C8菌株无毒基因的分离及功能鉴别

 白叶枯病菌与水稻间的互作符合基因对基因模式,不同小种中存在特异的avrBs3/pthA家族基因。对中国水稻白叶枯病菌小种C8菌株基因组DNA进行BamHⅠ酶切和Southern杂交,发现了其特有的约6.0 kb大小、含avrBs3/pthA家族基因的信号条带。经文库筛选、Southern杂交和限制性酶切分析,获得了C8小种avr/pthC8a基因。将该基因导入菲律宾小种6 的代表菌株PXO99A中,发现avr/pthC8a可使PXO99A丧失对水稻品种Asominori（携有成株期抗性基因Xa17）的毒性,暗示avr/pthC8a可能是与Xa17相应的无毒基因。序列分析发现,avr/pthC8a基因大小为3816 bp,编码1272个氨基酸,由102 bp编码的34个氨基酸重复单元在基因内的重复数为20.5个,其3′端的BamHⅠ酶切位点由GGATCC突变为AGATCC,其他特征均与avrBs3/pthA家族的成员相似,即C端存在1个亮氨酸拉链、3个核定位信号和1个酸性转录激活域。avr/pthC8a基因3′端的BamHⅠ酶切位点发生突变在avrBs3/pthA家族基因中鲜有报道。中国C8小种avr/pthC8a基因的发掘,为进一步了解水稻黄单胞菌毒性变异以及avrBs3/pthA家族基因的进化提供了理论依据。     

Transient expression of pepper Bs2 gene in Citrus limon as an approach to evaluate its utility for management of citrus canker disease

The pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains containing the avrBs2 avirulence gene in susceptible pepper and tomato. The avrBs2 gene is highly conserved in the Xanthomonas genus and when bacteria lack this gene their growth in a susceptible host is diminished, indicating that the avrBs2 gene product could confer an adaptive advantage to the pathogen. The avrBs2 of Xanthomonas citri subsp. citri (Xcc), cause of citrus canker, shares 96% homology with avrBs2 of Xcv. To evaluate if Bs2 could recognize avrBs2 of Xcc in citrus plants and thereby activate plant defence mechanisms to increase resistance to canker, transient expression experiments were conducted using Agrobacterium tumefaciens in lemon plants subsequently challenged with wildtype Xcc. The results showed that transient expression of Bs2 reduced canker formation in lemon and induced plant defence mechanisms, as shown by callose deposition and PR‐1 expression. Moreover, when an avrBs2 mutant of Xcc was used, no decrease in disease symptoms was observed. This work shows that the Bs2 gene from Solanaceae is functional in lemon, a member of the Rutaceae family. Therefore, Bs2 is a potential candidate gene for stable expression in transgenic citrus plants in order to improve resistance to canker disease.     

稻瘟菌无毒基因AvrPii编码蛋白分子特性及转基因水稻的构建

为了解稻瘟菌无毒基因AvrPii在水稻病原菌相关分子模式诱导的免疫反应(PTI)抗病分子信号调控中的功能,对AvrPii基因编码蛋白的分子结构特性进行分析,构建了AvrPii基因的植物表达载体幵进行遗传转化。结果表明:AvrPii蛋白由70个氨基酸组成,相对分子质量约为7 500,等电点为6.0,总平均亲水性值为–0.159;AvrPii蛋白包含1个信号肽、1个跨膜结构域和2个基序为LxAR和CxxCxxxxxxxxxxxH的保守结构域;利用农杆菌侵染转化法,获得AvrPii基因全长序列和不含信号肽AvrPiinsp的转基因株系13个,PCR扩增和实时荧光定量PCR鉴定结果表明,AvrPii和AvrPiinsp基因已成功转入受体品种日本晴幵得到稳定表达。     

Detection and Analysis of Magnaporthe oryzae Avirulence Genes AVR-Pib,AVR-Pik and AvrPiz-t in Heilongjiang Province

【Objective】The objective of this study is to investigate the distribution and variation mechanism of avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t in Magnaporthe oryzae strains from different regions and years in Heilongjiang Province, to understand the pathogenic phenotypes of different avirulence gene alleles, and to provide a reference for utilization and distribution of resistance cultivars in Heilongjiang Province.【Method】Based on the avirulence gene sequences published in NCBI, specific primers were designed to amplify full length and the coding sequence (CDS) regions of three genes, respectively. From 2016 to 2017, 335 M. oryzae strains in different regions of Heilongjiang Province were collected and isolated, and their DNA was PCR-amplified using avirulence genes primers and analyzed by agarose gel electrophoresis. The PCR products with different band patterns and from representative strains of different regions were selected for sequencing. The sequencing results were compared with the corresponding avirulence gene sequences for base and amino acid. The pathogenic phenotype of M. oryzae strains with different variant types was determined based on the rice resistance to single-gene lines.【Result】The specific bands of AVR-Pib, AVR-Pik and AvrPiz-t were detected in PCR detection and appeared in different distribution frequencies and mutation types, indicating that these 3 avirulence genes were all distributed in Heilongjiang Province. The average amplification frequency of the 3 avirulence genes was 75.52%, 87.16% and 85.67%, respectively. Among them, 4 types of band (bandless, high band, mid to high band and low band) of AVR-Pib were detected by electrophoresis analysis and 5 variant types AVR-Pib (1-1, 1-2, 2, 3, 3-1) were detected by PCR product sequencing. The genotypes AVR-Pib-1-1, AVR-Pib-1-2, AVR-Pib-2 and AVR-Pib-3-1 are newly discovered variant types, of which genotypes AVR-Pib-1-1 and AVR-Pib-1-2 are insertions of transposon Pot2 but with different insertion sites. The genotype AVR-Pib-2 has a small fragment insertion in the upstream of CDS region. The genotype AVR-Pib-3-1 base sequence has 4 differences from the original sequence, namely 32 (C/G) 35 (T/A) 36 (T/A) 38 (T/A), and the amino acid translation was terminated prematurely. Pathogenic analysis showed that except for the normal genotype AVR-Pib-3, the other alleles lost their avirulence functions. Seven AVR-Pik alleles (D, A, B, C, E, F, F2) were detected after PCR product sequencing, and the alterations in the nucleotide sequences of these alleles all resulted in amino acid missense mutations. The 7 AVR-Pik alleles have been reported previously. The avirulence gene AvrPiz-t was analyzed by electrophoresis and sequencing of PCR products, and 2 types of band (high band and normal band type) and 4 genotypes of AvrPiz-t (A, B, C, D) were revealed. Among them, AvrPiz-t-A is the original genotype, while AvrPiz-t-B has a base A insertion at position 191, causing premature termination of amino acid translation. Genotype AvrPiz-t-C is a newly discovered allelic type, characterized by the presence of a nucleotide variation at position 17 (T/C) and the insertion of base C at position 19 compared with type A, leading to the frameshift mutation and premature translation termination. The high band type avirulent genotype AvrPiz-t-D was sequenced and verified as having an insertion of the Pot3 transposon. Rice single-gene lines infection showed that the strains with AvrPiz-t-A were avirulent to Piz-t line due to Piz-t recognition, whereas the strains with AvrPiz-t (B, C, D) were virulent to Piz-t line due to lost the ability recognized by Piz-t.【Conclusion】The avirulence genes AVR-Pib, AVR-Pik and AvrPiz-t of M. oryzae in Heilongjiang Province are widely distributed and the types of variation are abundant. The results of this study can provide a reference for breeding and popularizing rice cultivars with corresponding disease-resistance genes.     

基于晚疫病菌效应子识别策略挖掘马铃薯潜在抗病基因资源

为探索利用晚疫病菌效应子识别策略快速挖掘具有潜在抗晚疫病基因的马铃薯资源,挑选了晚疫病菌侵染马铃薯时早期上调表达的68个RXLR类效应子基因,将它们分别克隆到PVX病毒植物表达载体pGR106上,采用农杆菌牙签穿刺方法,在55份不同基因型的马铃薯叶片上进行瞬时表达,根据过敏反应（Hypersensitive response,HR）发生与否来推断马铃薯中是否存在潜在抗病基因。结果表明,10个效应子基因,包括无毒基因AVR2家族成员PITG_23008,细胞死亡激发子PexRD2、无毒基因PexRD39（AVRblb2）,以及其它未知功能效应子基因Pex147-2、PexRD8、PexRD49、PITG_10232、PITG_11484、PITG_07555和PITG_22724,能够在10份马铃薯材料上诱导HR,预示这些马铃薯材料中具有潜在抗病基因。另外,离体叶片晚疫病菌接种鉴定表明,识别6个晚疫病菌效应子的马铃薯材料IVP196-2比识别3个效应子的马铃薯材料HJT349-3抗病性更强。     

Sequence variation in the putative effector gene SIX8 facilitates molecular differentiation of Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc), causal agent of fusarium wilt of banana, is among the most destructive pathogens of banana and plantain. The development of a molecular diagnostic capable of reliably distinguishing between the various races of the pathogen is of key importance to disease management. However, attempts to distinguish isolates using the standard molecular loci typically used for fungal phylogenetics have been complicated by a poor correlation between phylogeny and pathogenicity. Among the available alternative loci are several putative effector genes, known as SIX genes, which have been successfully used to differentiate the three races of F. oxysporum f. sp. lycopersici. In this study, an international collection of Foc isolates was screened for the presence of the putative effector SIX8. Using a PCR and sequencing approach, variation in Foc‐SIX8 was identified which allowed race 4 to be differentiated from race 1 and 2 isolates, and tropical and subtropical race 4 isolates to be distinguished from one another.     

稻瘟病菌无毒基因研究进展

水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。无毒基因是病原物遗传因子,能诱发寄主植物产生抗病性,是稻瘟菌与水稻互作最重要的激发子。为寻求稻瘟病防治的新策略,本研究概述了研究稻瘟病菌无毒基因的意义,归纳了抗性基因与无毒基因互作的3种模式,总结了国内外稻瘟病菌无毒基因的克隆及功能研究,探讨了无毒基因在生产上病害流行预测中的应用,指出利用抗性基因与无毒基因的关系对稻瘟菌分类将会更为科学和实用,应进一步加快稻瘟病菌无毒基因的克隆。     

利用酵母双杂交系统筛选感病番茄中蔬四号与无毒蛋白AvrPto、AvrPtoB互作的蛋白

 【目的】利用酵母双杂交系统在感病番茄中蔬四号中筛选分别与AvrPto、AvrPtoB互作的蛋白,研究AvrPto、AvrPtoB作为毒性因子在感病番茄中的作用通路。【方法】提取感病番茄中蔬四号的总RNA,合成中蔬四号cDNA文库,分别构建AvrPto和AvrPtoB的诱饵载体,从中蔬四号cDNA文库中筛选分别与AvrPto、AvrPtoB互作的蛋白,并对阳性克隆进行分析和鉴定。【结果】最终筛选到7个与AvrPto互作的蛋白,2个与AvrPtoB互作的蛋白,核酮糖-1,5-二磷酸（RuBP）羧化酶/加氧酶小亚基和AvrPto、AvrPtoB都能够互作,说明AvrPto、AvrPtoB参与破坏植物的光合作用;另外与AvrPto互作的蛋白质还包括叶绿体原卟啉原氧化酶、叶绿体醛缩酶、叶绿体延伸因子TufA、翻译延伸因子等参与植物光合作用蛋白。【结论】分析筛选到的蛋白主要是参与植物光合作用的蛋白,推测AvrPto、AvrPtoB通过与植物光合作用有关的蛋白互作,或者影响卡尔文循环,或者影响叶绿素和相关蛋白质的合成,从而破坏植物光合作用,促进侵袭位点叶肉细胞衰老、死亡,进而引起坏死症状。     

基于毒性相关基因序列的稻瘟病菌群体遗传多样性分析

 选用16对毒性相关基因特异性引物对四川和重庆9个县(市)分离到的200个稻瘟病菌单孢菌株进行PCR扩增,并采用最长距离法进行聚类分析,结果显示各引物均能扩增出其目的条带,多态位点百分率(P)高达93.75%,扩增频率差异较大;200个菌株可归为70个不同的单元型,其中单元型SCH13为优势单元型;在0.86遗传相似水平上,200个菌株可划分为27个遗传宗谱,包括1个优势宗谱,3个亚优势宗谱,14个次要宗谱,9个小宗谱,层次丰富;在群体平均水平上,病菌群体具有丰富的遗传多样性(H=0.324 4,I=0.484 2),且群体间差异较大;9个种群在遗传距离为0.05水平上可分为4个类群,种群遗传谱系与地理区域分布呈一定相关性。同时,该地区的群体存在一定的遗传分化(HT=0.320 0),群体内多样性大于群体间多样性(Hs=0.179 6,Dst=0.140 4),总遗传变异的56.13%存在于群体内(Gst=0.438 7),群体间基因流动性较小(Nm=0.639 6)。本研究揭示了四川和重庆部分区域稻瘟病菌群体遗传结构、遗传多样性及其与地理分布之间的关系,为抗病育种和品种布局奠定了基础。