Pyricularia oryzae is a plant pathogenic fungus that severely affects rice production. Past studies, primarily using mutants generated by spontaneous mutations or artificial physical and chemical mutagenesis, have determined that melanin is required for appressorium turgor, penetration, and virulence of P. oryzae. However, these roles need to be verified by gene knockout and/or overexpression in different strains considering the potential differences in the level of virulence. Here, we confirmed the indispensable roles of melanin in the development and virulence of P. oryzae by knocking out and over-expressing three melanin synthesis genes (ALB1, RSY1, and BUF1) in two wild-type strains (Guy11 and 70-15). Deletion of ALB1, RSY1, or BUF1 led to loss of melanin and virulence in both strains. ALB1, RSY1 and BUF1 in Guy11, and BUF1 in 70-15 were required for conidiation, respectively. ALB1, RSY1, and BUF1 were required for conidial resistance to environmental stresses (UV exposure, oxidization, and freezing damage) in both strains. Guy11 cells had greater amounts of melanin and more transcripts of melanin synthesis genes than 70-15 cells. Paired culture experiments between the deletion or over-expression mutants of melanin synthesis genes suggested that the reaction catalyzed by Buf1, but not Alb1 and Rsy1, was likely a rate-limiting step in melanin biosynthesis in 70-15. These results expand our understanding on melanin and its synthesis genes in P. oryzae as well as its responses to biotic and abiotic environments.

稻瘟病菌是一种严重影响水稻生产的植物病原真菌。过去的研究主要使用自发突变或人工物理和化学诱变产生的突变体，已确定黑色素是稻瘟病菌的旺盛，穿透和毒力所必需的。但是，考虑到毒力水平的潜在差异，需要通过基因敲除和/或在不同菌株中的过表达来验证这些作用。在这里，我们通过敲除和过表达三个黑色素合成基因（ALB1，RSY1和BUF1），证实了黑色素在米曲霉的发育和毒性中不可或缺的作用。）在两个野生型菌株中（Guy11和70-15）。两种菌株中ALB1，RSY1或BUF1的缺失均导致黑色素损失和致病性。分别需要Guy11中的ALB1，RSY1和BUF1，以及70-15中的BUF1。ALB1，RSY1和BUF1在两个菌株中都需要分生孢子抵抗环境压力（紫外线暴露，氧化和冰冻破坏）。Guy11细胞比70-15细胞具有更多的黑色素和更多的黑色素合成基因转录物。在黑色素合成基因的缺失或过表达突变体之间的配对培养实验表明，由Buf1而不是Alb1和Rsy1催化的反应可能是70-15中黑色素生物合成的限速步骤。这些结果扩大了我们对米青假单胞菌中黑色素及其合成基因及其对生物和非生物环境的反应的理解。