Elucidation and its pathological role of pectin metabolism in phytopathogenic fungus

本研究首次分离了柑橘黑腐病病原菌(Alternaria Citri)和柑橘褐斑病病原菌(A.ternata)编码内切多聚半乳糖醛酸酶(EndoplyGalacuronase，EndoPG)的基因，目的基因的缺失使得两个不同的突变株不能产生EndoPG。这两种病原菌都属于链格孢属，在形态上难以区分。这些真菌产生的内毒素具有相似的生化特性，基因高度相似(99.6%的核苷酸同源性)。然而，突变体的表型完全不同。柑橘内毒素突变体在引起柑橘黑腐病和马铃薯组织浸渍方面的能力显著降低，并且不能在柑橘果皮切片上定殖。相反，内毒素突变体在致病性上没有变化。这些结果表明，一种细胞壁降解酶在真菌病原菌的致病力中可能发挥不同的作用。细胞壁度…的作用更多的ADD酶取决于疾病的类型，而不是真菌的分类。将携带绿色荧光蛋白(GFP)基因的pTEFEGFP导入野生型柑橘及其内毒素干扰突变体M60中，研究柑橘内毒素对柑橘果实组织的侵染情况。野生型转化子G1和M60转化子GM4的孢子、芽管、附着体和侵染菌丝均有绿色荧光。G1菌丝垂直穿透果皮，GM4菌丝不垂直穿透果皮，但G1菌丝和GM4菌丝均匀分布在柑橘果实汁囊区。柑橘内切酶基因启动子控制下携带GFP基因的转化子EPG7的绿色荧光在侵染过程中被果胶在果皮中诱导，但在汁囊区被完全抑制，这可能是由于汁液中的糖对碳分解代谢的抑制。为了进一步确定这一机制，利用GFP基因作为报告基因，在包含Acpg1启动子的翻译起始点上游定义了813个碱基。这个上游序列包含五个推测的分解代谢抑制元件A(CREA)的结合序列，CREA是一种参与碳分解代谢抑制的顺式作用锌指抑制因子。我们构建了每个带有绿色荧光蛋白报告基因的CREA结合位点缺失的Acpg1启动子，并将它们转化到柑橘红曲霉中。从-401到-813缺失的PGPDL4具有底物诱导和分解代谢抑制作用，而从-1到-84缺失的PGPDL5包括一个可能的Cre-A结合位点，导致分解代谢抑制功能的丧失。果胶在果皮中诱导了PGPDL4的绿色荧光，但在柑橘果实的汁囊区完全被抑制。而PGPDL5在果皮区和汁囊区均能诱导出绿色荧光。这些证据还表明，Acpg1在汁囊区的抑制可能是通过碳分解代谢抑制实现的。较少

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