A novel gene MGA1 is required for appressorium formation in Magnaporthe grisea

Insertional mutagenesis is an effective way to study the infection mechanism of fungal pathogens. In an attempt to identify the genes involved in appressorium formation from Magnaporthe grisea, we carried out Agrobacterium tumefaciens mediated transformation (ATMT) of the fungus. Analysis of the region flanking the T-DNA integration site in one of the appressorium mutants showed insertion in a gene coding a 78 amino acid protein (MGA1), showing no significant homology to any of the known proteins. The mutant mga1 caused neither foliar nor root infection. Complementation of the mutated gene with the full length wild type gene restored appressorium formation as well as rice infection demonstrating the involvement of this gene in pathogenicity of M. grisea. In an indirect immunolocalisation assay, the MGA1 expression was seen predominantly in germ tube and appressoria. The mutant was impaired in glycogen and lipid mobilization required for appressorium formation. The glycerol content in the mycelia of the mutant under hyperosmotic stress conditions was less as compared to wild type and was thus unable to tolerate the hyperosmotic stress induced by sorbitol. We hypothesize that MGA1 plays a crucial role in signal transduction leading to the metabolism of glycogen and lipids, which is a part of appressorium differentiation process.     

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.     

Serial Analysis of Gene Expression (SAGE) of<Emphasis Type="Italic"> Magnaporthe grisea</Emphasis>: genes involved in appressorium formation

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by E. Cerdá-Olmedo     

靛红衍生物的合成及其对稻瘟菌的生物活性

以靛红为原料合成了系列3-亚胺基/亚甲基-吲哚-2-酮化合物及其Mannich碱衍生物,研究了它们在抗稻瘟菌方面的活性,发现了这两种类型的若干化合物有较好的抑制稻瘟菌孢子萌发的活性,初步讨论了构效关系。认为1位的羟甲基和胺甲基、3位的亚甲基是药效团,芳基亚甲基苯环上对位取代基、羟基取代基和吸电子取代基不利于活性的提高,邻位的供电子取代基有利于活性的提高。     

稻瘟菌Ⅰ型烯醇化酶基因全长cDNA的电子克隆

利用电子克隆技术从稻瘟菌中克隆到一个新的Ⅰ型烯醇化酶全长cDNA,暂命为MgEno-1。MgEno-1全长1571核薯酸,其预测的ORF为1317核苷酸,共编码438个氨基酸。起始密码子ATG位于第53位,终止密码子TAA位于第1369位。序列分析表明该烯醇化酶与丝状真菌中已报道的其它烯醇化酶高度同源,且长度一致,这暗示烯醇化酶基因进化上高度保守,甚至有可能像18SrRNA一样可作为进化尺度。这将是第一个用电子克隆技术从稻瘟菌中克隆到的基因。     

Light-Enhanced Resistance to Magnaporthe grisea Infection in the Rice Sekiguchi Lesion Mutants

The rice sl mutant showed two types of responses to Magnaporthe grisea infection by light treatments. One was an sl -mutant-type response characterized by Sekiguchi lesion expression under light waves of 400–700 nm, and the other was a wild-type response characterized by blast and/or necrotic spot lesion expression in the dark or at wavelength between 290 and 330 nm. There was a large difference in the resistance to M. grisea infection between the mutant- and wild-type responses in the rice sl mutant. When the mutant-type response was induced in the rice sl mutant, the disease resistance was enhanced relative to that in the wild-type response. Enhanced resistance was demonstrated by two components: (a) the number of Sekiguchi lesions was reduced relative to that of blast or necrotic lesions; (b) sporulation of M. grisea was not induced in Sekiguchi lesions. The enhanced resistance was dependent on light of 400–700 nm.     

Calcium-regulated appressorium formation of the entomopathogenic fungusZoophthora radicans

Summary The fungusZoophthora radicans (Zygomycetes: Entomophthorales) requires external Ca²⁺ for appressorium formation but not for conidial germination. The number of appressoria formed depends on the Ca²⁺ concentration of the medium. At low [Ca²⁺] (100 pM) nuclear division and germ tube growth are significantly reduced compared to higher Ca²⁺ concentrations (10 and 1,000 M). By contrast, neither external K⁺ nor external Cl^– is needed for germination or appressorium formation. Treatment of conidia with a Ca²⁺-antagonist, Nd³⁺, and a Ca²⁺-channel blocker, nifedipine, inhibits appressorium formation, showing that a Ca²⁺ influx is required for appressorium formation. Furthermore, the partial yet saturating inhibition by nifedipine and complete inhibition by Nd³⁺ indicates that at least two kinds of Ca²⁺ channels are involved in appressorium formation. A contribution of intracellular Ca²⁺ to the signal transduction chain for the formation of appressoria is demonstrated by the inhibitory effect of the intracellular Ca²⁺ antagonist TMB-8. The calmodulin antagonists R24571, TFP, W-7, and W-5 inhibit appressorium formation at concentrations which have no effect on germination. The data presented in this paper are consistent with the hypothesis that a Ca²⁺/calmodulin system is involved in regulating appressorium formation. However, since the direct effects of the drugs were not specifically tested on their proposed binding sites, we leave room for alternative hypotheses that have yet to be formulated.Abbreviations A-9-C 9-anthracenecarboxylic acid - DAPI 4,6 diamino-2-phenylindole - EGTA ethylene glycol bis(-aminoethylether)-N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - H-7 N-(2-methylamino)ethyl-5-isoquino-linesulphonamide dihydrochloride - IC₅₀ concentration of inhibitor that causes 50% inhibiton - R24571 (calmidazolium) 1-[bis-(4-chlorophenyl)methyl]-3-[2,4-dichloro--(2,4-dichlorobenzyloxy)phenethyl]-imidazolium chloride - TEA tetraethylammonium - TFP (trifluoperazine) 10-[3-(4-methylpiperazine-1-yl)-propyl]-2-trifluomethylphenothiazine - TMB-8 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride - W-5 N-(6-aminohexyl)-1-naphthalene-sulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide     

Disruption of a Magnaporthe grisea cutinase gene.

Summary Using a one-step strategy to disrupt CUT1, a gene for cutinase, cut1^– mutants were generated in two strains of Magnaporthe grisea. One strain, pathogenic on weeping lovegrass and barley and containing the arg3–12 mutation, was transformed with a disruption vector in which the Aspergillus nidulans ArgB⁺ gene was inserted into CUT1. Prototrophic transformants were screened by Southern hybridization, and 3 of 53 tested contained a disrupted CUT1 gene (cut1 : : ArgB⁺). A second strain, pathogenic on rice, was transformed with a disruption vector in which a gene for hyg B resistance was inserted into CUT1. Two of the 57 transformants screened by Southern hybridization contained a disrupted CUT1 gene (cut1:. Hyg). CUT1 mRNA was not detectable in transformants that contained a disrupted gene. Transformants with a disrupted CUT1 gene failed to produce a cutin-inducible esterase that is normally detected by activity staining on non-denaturing polyacrylamide gels. Enzyme activity, measured either with tritiated cutin or with p-nitrophenyl butyrate as a substrate, was reduced but not eliminated in strains with a disrupted CUT1 gene. The infection efficiency of the cut1^– disruption transformants was equal to that of the parent strains on all three host plants. Lesions produced by these mutants had an appearance and a sporulation rate similar to those produced by the parent strains. We conclude that the M. grisea CUT1 gene is not required for pathogenicity.     

Increased tryptophan decarboxylase and monoamine oxidase activities induce Sekiguchi lesion formation in rice infected with Magnaporthe grisea

Sekiguchi lesion (sl)-mutant rice infected with Magnaporthe grisea showed increased light-dependent tryptophan decarboxylase (TDC) and monoamine oxidase (MAO) activities. TDC and MAO activities were observed before the penetration of M. grisea to rice cells and maintained high levels even after Sekiguchi lesion formation. Light-dependent expression of TDC gene was observed in leaves inoculated with M. grisea before Sekiguchi lesion formation. Spore germination fluid (SGF) of M. grisea also induced Sekiguchi lesion formation accompanied by increased enzymes activities and tryptamine accumulation. Sekiguchi lesion was also induced by treatments with tryptamine and beta-phenylethylamine, which are substrates for MAO, but was not induced by non-substrates such as indole-3-propionic acid, (+/-)-phenylethylamine and tryptophan under light. Light-dependent induction of Sekiguchi lesion by tryptamine was significantly inhibited in the presence of MAO inhibitors, metalaxyl and semicarbazide, and H2O2-scavengers, ascorbic acid and catalase. H2O2 in M. grisea-infected leaves with and without Sekiguchi lesions was demonstrated directly in situ by strong 3,3'-diaminobenzidine (DAB) staining. On the other hand, H2O2 induced Sekiguchi lesions on leaves of cv. Sekiguchi-asahi under light, but not in darkness. This difference was associated with the decrease of catalase activity in infected leaves under light and the absence of decrease in darkness. We hypothesize that the H2O2-induced breakdown of cellular organelles such as chloroplasts and mitochondria in mesophyll cells may cause high TDC and MAO activities and the development of Sekiguchi lesion, and that the sl gene products in wild-type rice may function as a suppressor of organelle breakdown caused by chemical or environmental stress.     

稻瘟菌糖蛋白激发子(CSBI)的纯化及其鉴定

稻瘟菌（Magnaporthe grisea）ZC1l3菌株97-151a菌丝经离心、超滤、Sephacryl S-100凝胶柱、DEAE-Sepharose FF阴离子交换柱层析,纯化获得糖蛋白激发子CSBI。CSBI经SDS-PAGE后银染显示单一条带,糖,蛋白比例约为9．32。CSBI对非亲和性互作水稻叶片中过氧化物酶的诱导显著高于亲和性互作水稻（P〈0．05）。经N端氨基酸同源序列比对表明,CSBI与MG07877．4推测蛋白的同源性最高。经基质辅助激光解析电离飞行时间质谱鉴定也表明CSBI是该推测蛋白。     

Transformation of the rice blast fungus Magnaporthe grisea to benomyl resistance

A transformation method based on a dominant selectable marker (benomyl resistance) was developed for the rice blast fungus Magnaporthe grisea. The heterologous gene for -tubulin from Neurospora crassa (pBT3) was used to obtain benomyl-resistant M. grisea transformants at a frequency of 20 to 30/g of DNA. Control transformations carried out with a plasmid conferring hygromycin resistance or a derivative of pBT3 containing a repetitive DNA sequence, yielded the same frequency of transformation as that of pBT3. Molecular analysis of the transformants indicated multiple integration of the vector DNA.     

稻瘟菌侵染诱导水稻凝集素基因的表达

利用mRNA差异显示技术(DDRT-PCR),从非亲和性稻瘟菌生理小种131侵染的水稻品种爱知旭(Oryza sati-va L. cv.Aichi-asahi)叶片中分离了8个诱导差异表达的cDNA片段.对这8个差示片段进行了回收、重扩增和克隆,以其中一个长度为321碱基并与甘露糖结合水稻凝集素和水稻盐诱导蛋白基因高度同源的差示片段为探针,筛选水稻非亲和性cDNA文库,获得12个阳性克隆.序列测定和数据库查询表明该基因的cDNA与水稻凝集素基因的cDNA及盐诱导蛋白基因的cDNA核苷酸同源性高达96%,推定的氨基酸序列与甘露糖结合水稻凝集素的氨基酸序列一致,与水稻盐诱导蛋白仅相差2个氨基酸.Southern杂交显示该基因在水稻基因组中有两个同源拷贝数,Northern杂交表明非亲和性稻瘟菌侵染可强烈诱导该基因表达.因此推测该基因参与了水稻对稻瘟菌侵染的防御反应.     

Fatty Acids and Their Derivatives as Modulators of Appressorium Formation in Magnaporthe grisea

Appressorium formation in germinating conidia of Magnaporthe grisea was inhibited on inductive and on noninductive surfaces by monounsaturated fatty acids with chain lengths of 16, 18, or 20 carbon atoms. On a noninductive surface, the inhibition was only observed upon stimulation with 1,16-hexadecanediol or oleyl alcohol, but not upon stimulation with 8-(4-chlorophenylthio)-adenosine-3′,5′-monophosphate. In the C₁₈-series, fatty acids with a double bond in position 9 were the most active ones. At 1 μg/ml of oleic or elaidic acid, less than 30% of the germinated conidia formed appressoria. The mode of inhibition was competitive to the inducing agent. On an inductive surface, compared to a noninductive surface the concentrations of oleic and elaidic acid needed for inhibition of appressorium formation were one order of magnitude higher. Methyl esters of inhibitory fatty acids and acids with two double bonds were not active. Like oleyl alcohol, elaidyl alcohol and petroselinyl alcohol stimulated infection structure formation on the noninductive surface.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。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。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library. Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves. mac1- transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.