Studies on morphogenesis of infection structure and signal network in plant pathogenic fungi - regulatory mechanisms of environmental response and gene expression

1)侵染结构和信号网络形态发生相关基因的分离与表征。酿酒酵母酵母（Saccharomyces cerevisiae） FUS3/KSS1 MAP激酶基因同源的lagenarium GMK1基因参与了分生孢子萌发、附着胞形成和侵袭性生长的调控。在酿酒链球菌中，已知Ste12p是Fus3p/Kss1p MAP激酶下游的转录因子。本研究为研究CMK1 MAP激酶通路，分离并鉴定了lagenarium中Ste12同源基因CST1。Cst1Δ菌株在完整寄主叶片上无致病性，但接种在受伤叶片上可形成病变。cst1Δ菌株的分生孢子在寄主叶片表面和人造纤维素膜上都能萌发并形成黑化的附着胞，但不能从附着胞中产生感染性菌丝，说明CST1对病菌渗透附着胞至关重要。此外，与野生型菌株相比，cst1Δ菌株成熟后的附着胞脂滴含量极低。cst1Δ菌株分生孢子中脂滴丰富，但在附着胞形成过程中脂滴迅速消失。这种错误的脂质降解可能与cst1Δ菌株的附着胞渗透失败有关。本研究将农杆菌介导的转化（Agrobacterium tummefociens -mediated transformation, AtMT）应用于lagenarium炭疽病（Colletotrichum lagenarium）进行随机插入诱变。以携带耐潮霉素基因盒的双载体pBIG2RHPH2为载体，引入a . tumefaciens C58C1，将野生型104-T与pBIG2RHPH2共培养，获得耐潮霉素转化体。利用热不对称交叉聚合酶链式反应（TAIL-PCR），可以从几种致病性或形态发生缺陷突变体中鉴定出T-DNA侧翼的真菌基因组DNA片段。其中一个突变体的序列与我们之前描述过的lagenarium的黑色素生物合成基因PKS1相同。这些结果表明，AtMT可以作为植物病原菌C. lagenarium致病性和形态发生相关基因标记的有效工具。少

1) Isolation and characterization of genes involved in morphogenesis of infection structure and signal networkThe C. lagenarium GMK1 gene, a homologue of the Saccharomyces cerevisiae FUS3/KSS1 MAP kinase genes, was shown to regulate conidial germination, appressorium formation, and invasive growth. In S. cerevisiae, Ste12p is known to be a transcriptional factor downstream of Fus3p/Kss1p MAP kinases. In this research, to evaluate the CMK1 MAP kinase pathway, the Ste12 homologue CST1 gene from C. lagenarium was isolated and characterized. cst1Δ strains were nonpathogenic on intact host leaves, but could form lesions when inoculated on wounded leaves. Conidia of the cst1Δ strains could germinate and form melanized appressoria on both host leaf surface and artificial cellulose membrane, but could not produce infectious hyphae from appressoria, suggesting that CST1 is essential for appressorium penetration in C. lagenarium. In addition, matured appressoria of the cst1Δ strains contained ex … More tremely low level of lipid droplets, compared with that of the wild type strain. Lipid droplets were abundant in conidia of the cst1Δ strains, but rapidly disappeared during appressorium formation. This misscheduled lipid degradation might be related to the failure of appressorium penetration in the cst1Δ strain.2) Establishment of Agrobacterium tumefaciens-mediated transformation as an experimental tool for random insertional mutagenesis in Colletotrichum lagenariumIn this research, Agrobacterium tumefociens-mediated transformation (AtMT) was applied to Colletotrichum lagenarium for random insertional mutagenesis. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2, a binary vector carrying hygromycin-resistant gene cassette, introduced A. tumefaciens C58C1 led to good production of hygromycin-resistant transformants. The fungal genomic DNA segments flanking T-DNA could be identified from several pathogenicity or morphogenesis deficient mutants by thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR). Sequence from one of the mutants was identical with melanin biosynthesis gene PKS1 of C. lagenarium that we have previously characterized. These results indicated that AtMT could be an effective tool for tagging genes relevant to pathogenicity and morphogenesis in the plant pathogenic fungus C. lagenarium. Less