| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
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
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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
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