Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.     

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

两个稻瘟病菌假想蛋白的纯化和特性分析

稻瘟病菌(Magnaporthe Grisea)引起的水稻稻瘟病是世界水稻生产最具毁灭性的病害之一,也是研究植物与病原物相互作用分子机理的模式系统之一.真菌分泌蛋白由于其本身所具有的分泌特性极有可能成为被宿主植物识别的作用病菌分泌蛋白,该研究到目前为止还比较有限. 文章证明了稻瘟病菌分泌蛋白的存在并且检验了其中几个蛋白在其宿主植物水稻中的诱导因子作用. 通过同源基因的时空表达技术,表明分泌蛋白在稻瘟病菌中大量表达.其中两个在稻瘟病菌中的未知蛋白被测试出具有诱导因子作用.     

Construction of an electronic physical map of Magnaporthe oryzae using genomic position-ready SSR markers

Magnaporthe oryzae is a model for plant pathogenic filamentous fungi. We have assembled a simple sequence repeat (SSR)-based physical map of the species, using in silico sequence data. A set of 120 SSR markers was developed from the genomic sequence of the reference isolate 70-15. These markers were readily amplified from the genomic DNA of other isolates, and high levels of allelic variation characterised the parental isolates of the two crosses tested. All the markers were locatable to one of the seven M. oryzae chromosomes. An SSR-based physical in silico map was constructed, and pre-existing SSR and RFLP loci were integrated into the map, along with 23 Avr (avirulence) genes and two other genes of importance to the plant/pathogen interaction. This map provides a platform for population genetics and functional genomics studies in the model pathogen, and even in other evolu- tionally related pathogens.     

用抑制差减杂交技术分离烯丙异噻唑诱导水稻特异表达的基因

烯丙异噻唑(PBZ)处理水稻根能使其产生对稻瘟病的系统获得性抗性,因此在东南亚稻区被广泛用于防治稻瘟病,然而关于其作用的分子机理还知之甚少．运用抑制差减杂交技术,试图通过分离鉴定受PBZ诱导调控的关键基因,探索其作用的分子机理．以PBZ处理后的水稻叶片cDNA为目标群体(tester),以未处理水稻叶片cDNA为对照群体(driver),用经过对照cDNA差减的、烯丙异噻唑处理的cDNA群体构建了一个含260个重组子的差减文库．通过差示筛选鉴定出了26个。PBZ诱导水稻特异表达和增强表达的候选克隆．对26个cDNA克隆进行了双向测序和同源性比较,发现其中3个克隆：rJAB1,rTAB2和蛋白磷酸酯酶2Aδ调节亚基同型物基因,位于抗病相关信号转导途径上,它们与哺乳动物和人类免疫途径上的信号因子有明显相似之处,因此推断可能与诱导抗性有关．另外8个克隆与已知基因同源性为70％～99％．经Northern杂交分析,其中rJAB1(编码c-jun激活区结合蛋白1)受烯丙异噻唑和稻瘟菌诱导表达;膜糖蛋白同源基因及肌动蛋白(actin)α1受烯丙异噻唑诱导表达,部分克隆为低丰度转录本．     

迷迭香抗植物病原菌活性研究

用滤纸片扩散法和菌丝生长速率法实验研究了迷迭香乙醇提取物对6种植物病原真菌菌丝生长的抑制作用。结果表明迷迭香乙醇提取物对水稻稻瘟病菌、番茄霜霉病菌、黄瓜枯萎病菌有较好的抑制活性，浓度在5mg／mL以上时，抑菌率达到100％。     

Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.     

稻瘟菌REMI突变体库的构建

以稻瘟病菌菌株P131和Y34为材料，利用REMI技术构建了含有8000个转化体的突变体库．对REMI转化的部分条件进行了优化，比较了不同限制性内切酶(XhoI、ApaI和EcoRV)之间REMI转化效率的差异．结果表明，稻瘟菌分生孢子在摇培32h和Drisdase消化菌丝2h或3h时较适宜REMI转化；XhoI和ApaI之间没有明显差异，而两者的转化率高于EcoRV．