Molecular analysis of pathogenicity of Fusarium oxysporum by gene tagging

土传真菌尖孢镰刀菌（Fusarium oxysporum）通过直接穿透根并定殖维管组织而引起多种植物物种的维管枯萎。该物种内的单个致病菌株具有有限的宿主范围，并且相似或相同宿主范围的菌株被分配到种内组，称为特殊形式（f.sp.）。本研究从甜瓜枯萎病病原菌尖孢镰刀菌中分离到43个致病突变体。通过限制性内切酶介导的DNA整合诱变，获得了一株甜瓜新菌株。本研究通过分析突变体中标记位点的结构和功能，分离出致病性和症状发展所需的基因，并从5个突变体中鉴定出突变基因AGR 1、FOW 1、FOW 2、FOW 3和FOW 4。通过遗传互补和转化介导的靶向实验，证实了这些基因在该病原菌完全致病性中的重要作用。这些基因可能分别编码番茄琥珀酸裂解酶、线粒体载体蛋白、Zn（II）2Cys 6转录因子、Cys 2 His 2转录因子和磷酸烯酰丙酮酸羧激酶，是植物病原真菌新的致病基因; FOW 2和FOW 3编码不同的转录调节因子，可能调控直接参与致病的基因的表达。在本研究中，我们通过将野生型菌株的cDNA与fow 2突变体的cDNA相减，分离了Fow 2调控基因，命名为F2 R1。F2 R1编码一种类似于动物病原体新型隐球菌（Cryptococcus neoformans）的甘氨酸调节糖蛋白的蛋白质。f2 r1突变体对甜瓜的致病力略有降低，而fow 2突变体完全丧失致病力。这些结果表明，Fow 2调节多种致病基因的表达，包括F2 R1。

The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. Individual pathogenic strains within the species have a limited host range, and strains similar or identical host ranges are assigned to intraspecific groups, called formae speciales (f.sp.). We previously isolated 43 pathogenicity mutants of the melon wilt pathogen F.oxysporum f. sp. Melonis by restriction enzyme-mediated DNA integration mutagenesis. In this research, we analyzed the structures and functions of tagged sites in the mutants to isolate genes required for pathogenicity and symptom development by this pathogen.We identified the mutated genes, named AGR1, FOW1, FOW2, FOW3, and FOW4, from five mutants. Essential roles of these genes in full pathogenicity of this pathogen were confirmed by genetic complementation and transformation-mediated targeting experiments. These genes possibly encode argininosuccinate lyase, mitochondrial carrier protein, Zn(II)2Cys6 transcription factor, Cys2His2 transcription factor, and phosphoenoylpyruvate carboxykinase, respectively, and are novel pathogenicity genes of plant pathogenic fungi.FOW2 and FOW3 encode different transcription regulators, suggesting that they regulate expression of the genes directly involved in pathogenicity. In this research, we isolated a Fow2-regulated gene, named F2R1, by subtraction of cDNAs of the wild-type strain with those of the fow2 mutant. F2R1 encodes a protein similar to cytokine-regulated glycoprotein of the animal pathogen Cryptococcus neoformans. The f2r1 mutant showed slightly reduced virulence to melon plants, although the fow2 mutant completely lost pathogenicity. These results suggest that Fow2 regulates expression of multiple pathogenicity genes, including F2R1.