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稻瘟病菌(Magnaporth oryzae)引起的稻瘟病是水稻上一种毁灭性病害,每年给全球水稻生产造成10%~30%的产量损失,严重威胁着全球的粮食生产安全。从分子水平探究该病菌的致病机制,对于挖掘潜在的控制稻瘟病的药剂靶标具有重要的理论和实践意义。本研究在稻瘟病菌中鉴定到2个分别与酿酒酵母亚甲基四氢叶酸还原酶Met12和Met13同源的蛋白,命名为Mo Met12和Mo Met13。Mo Met12和Mo Met13分别含有690和631个氨基酸,两者的一致性为32%。对这2个蛋白编码基因分别进行敲除突变,发现ΔMomet12和ΔMomet13突变体在CM营养丰富培养基上生长减慢、气生菌丝减少。ΔMomet13在MM基本培养基上不能生长,在SDC和OM营养饥饿培养基上生长显著减慢,气生菌丝稀薄。ΔMomet12在M M、SDC和OM培养基上生长均显著减慢;对突变体进行产孢和致病性测定发现,ΔMomet13突变体在CM和SDC培养基上均丧失了产孢能力,而ΔMomet12在CM培养基上产孢量显著下降,在SDC上产孢能力丧失;ΔMomet13突变体侵染菌丝扩展和致病力显著下降,ΔMomet12突变体致病力与野生型比较无明显变化。加入外源的甲硫氨酸可恢复ΔMomet12和ΔMomet13突变体在不同培养基上的生长和产孢缺陷,且能部分恢复ΔMomet13突变体的致病能力。上述结果表明,Mo Met12和Mo Met13通过参与甲硫氨酸的生物合成,从而控制稻瘟病菌的生长和无性繁殖。Mo Met13同时是一个重要的致病相关因子,参与病菌侵染菌丝的生长和致病过程。
Rice blast caused by Magnaport oryzae is a devastating disease on rice, which causes 10% -30% loss of global rice production every year, which seriously threatens the global food production safety. To explore the pathogenic mechanism of this bacterium at the molecular level has important theoretical and practical significance for exploring potential drug targets for controlling rice blast. In this study, two proteins homologous to Met12 and Met13 of Saccharomyces cerevisiae were identified in M. grisea, named as Mo Met12 and Mo Met13. Mo Met12 and Mo Met13 contain 690 and 631 amino acids, respectively, with a consistency of 32%. Knock-out mutagenesis of these two protein-coding genes respectively showed that ΔMomet12 and ΔMomet13 mutants slowed down on CM nutrient-rich medium and reduced the number of aerial mycelia. ΔMomet13 can not grow on MM minimal medium, grows slowly on SDC and OM nutrient starvation medium, and thinly on aerial mycelium. ΔMomet12 growth on MM, SDC and OM medium were significantly slowed; sporulation and pathogenicity of mutants were determined, ΔMomet13 mutant in CM and SDC medium lost sporulation ability, and ΔMomet12 in CM The amount of sporulation in the medium decreased significantly and the spore forming ability was lost in SDC. The mycelial expansion and virulence of ΔMomet13 mutant significantly decreased. The pathogenicity of ΔMomet12 mutant showed no significant change compared with wild type. Addition of exogenous methionine restored the growth and sporulation defects of ΔMomet12 and ΔMomet13 mutants on different media and partially restored the virulence of ΔMomet13 mutants. The above results show that Mo Met12 and Mo Met13 can control the growth and clonal reproduction of Magnaporthe grisea by participating in the biosynthesis of methionine. Mo Met13 is also an important pathogenicity-related factor involved in the growth and pathogenicity of mycotoxins infecting mycelia.