Elucidation and its pathological role of pectin metabolism in phytopathogenic fungus

本研究首先从柑橘黑腐病病原菌（Alternaria citri）和柑橘褐斑病病原菌（A. alternata）。这两种病原体都属于链格孢属，它们在形态上难以区分。这些真菌产生的endoPG具有相似的生化特性，基因高度相似（99.6%核苷酸同一性）。然而，突变体的表型完全不同。A. Citri在其引起柑橘黑腐病症状的能力以及在马铃薯组织的浸渍中的能力显著降低，并且不能定殖于柑橘皮段。相反，A. alternata的致病力没有变化。结果表明，细胞壁降解酶在真菌病原体的致病性中可以发挥不同的作用。细胞壁的作用 ...更多信息 酶的活性取决于疾病的类型，而不是真菌的分类。为了研究柑橘炭疽病菌在柑橘果实组织中的定殖。citri，然后将携带绿色荧光蛋白（GFP）基因的pTEFEGFP导入野生型A。Citri及其endoPG破坏突变体M60。在转化体G1（来自野生型）和GM 4（来自M60）的孢子、芽管、附着胞和感染菌丝中观察到绿色荧光。G1菌丝垂直穿透果皮，而GM 4菌丝不垂直穿透果皮，但G1和GM 4菌丝在柑橘汁囊区均匀分布。A.的绿色荧光。柑橘EPG 7携带在A.在感染阶段，柑橘的果皮中的果胶诱导了柑橘的感染，但在汁囊区域完全被抑制，这可能是由于果汁中的糖对碳分解代谢物的抑制。为了进一步确定该机制，使用GFP基因作为报告基因，以限定包含Acpg 1启动子的翻译起始位点上游的813个碱基。该上游序列包含5个分解代谢产物阻遏元件A（CreA）的推定结合序列，CreA是一种参与碳分解代谢产物阻遏的顺式作用锌指阻遏物。我们构建了每个带有GFP报告基因的CreA结合位点缺失的Acpg 1启动子，并将它们转化到A.柑橘。从-401到-813缺失的构建体PGPDL 4显示底物诱导和分解代谢物阻遏，而PGPDL 5另外从-1到-84缺失，包括一个推定的Cre-A结合位点，导致分解代谢物阻遏功能的丧失。果胶在柑橘果皮中诱导PGPDL 4发出绿色荧光，但在汁囊区完全被抑制。而PGPDL 5在果皮和汁囊区均被诱导产生绿色荧光。这些证据也表明，Acpg 1在汁囊区的阻遏可能是通过碳分解代谢物阻遏。少

In this project, the genes encoding endopolygalacturonase (endoPG) were first isolated, and a target-gene disruption made two distinct mutants lacking the endoPG production from citrus black rot pathogen (Alternaria citri) and from citrus brown spot pathogen (A. alternata). Both pathogens belonging to the genera of Alternaria and they are morphologically indistinguishable. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degr … More ading enzyme depends upon the type of disease but not the taxonomy of the fungus. In order to investigate colonization of citrus fruit tissues by A. citri, pTEFEGFP carrying a green fluorescent protein (GFP) gene was then introduced in to wild type A. citri and its endoPG-disrupted mutant M60. Green fluorescence was observed in spores, germ tubes, appressoria, and infection hyphae of transformants G1 (derived from wild type) and GM4 (derived from M60). Hyphae of G1 but not GM4 vertically penetrated the peel, but the hyphae of both G1 and GM4 spread equally in the juice sac area of citrus fruit. Green fluorescence of A. citri transformant EPG7 carrying a GFP gene under control of the endoPG gene promoter of A. citri was induced by pectin in the peel during the infection stage, but repressed completely in the juice sac area, likely by carbon catabolite repression by sugars in the juice. To make sure the mechanisms further, a GFP gene was employed as a reporter gene to define 813 bases upstream of the translation start site comprising the Acpg1 promoter. This upstream sequence contains five putative binding sequences of catabolite repressive element A (CreA), a cis-acting zinc finger repressor involved in carbon catabolite repression. We constructed each CreA-biding site-deleted Acpg1 promoters with GFP reporter gene and transformed them into A. citri. The construct PGPDL4 deleted from -401 to - 813 showed both substrate induction and catabolite repression, while PGPDL5 additionally deleted from -1 to -84, including one putative Cre-A-binding site, resulted in a loss of catabolite repression function. Green fluorescence of PGPDL4 was induced by pectin in the peel but repressed completed in the juice sac area of citrus fruit. However, green fluorescence of PGPDL5 was induced both in the peel and juice sac area. These evidences also indicated that the repression of Acpg1 in the juice sac area is likely by carbon catabolite repression. Less