Methionine Biosynthesis is Essential for Infection in the Rice Blast Fungus <i>Magnaporthe oryzae</i>

Methionine is a sulfur amino acid standing at the crossroads of several biosynthetic pathways. In fungi, the last step of methionine biosynthesis is catalyzed by a cobalamine-independent methionine synthase (Met6, EC 2.1.1.14). In the present work, we studied the role of Met6 in the infection process of the rice blast fungus, Magnaporthe oryzae. To this end MET6 null mutants were obtained by targeted gene replacement. On minimum medium, MET6 null mutants were auxotrophic for methionine. Even when grown in presence of excess methionine, these mutants displayed developmental defects, such as reduced mycelium pigmentation, aerial hypha formation and sporulation. They also displayed characteristic metabolic signatures such as increased levels of cysteine, cystathionine, homocysteine, S-adenosylmethionine, S-adenosylhomocysteine while methionine and glutathione levels remained unchanged. These metabolic perturbations were associated with the over-expression of MgCBS1 involved in the reversed transsulfuration pathway that metabolizes homocysteine into cysteine and MgSAM1 and MgSAHH1 involved in the methyl cycle. This suggests a physiological adaptation of M. oryzae to metabolic defects induced by the loss of Met6, in particular an increase in homocysteine levels. Pathogenicity assays showed that MET6 null mutants were non-pathogenic on both barley and rice leaves. These mutants were defective in appressorium-mediated penetration and invasive infectious growth. These pathogenicity defects were rescued by addition of exogenous methionine and S-methylmethionine. These results show that M. oryzae cannot assimilate sufficient methionine from plant tissues and must synthesize this amino acid de novo to fulfill its sulfur amino acid requirement during infection.甲硫氨酸（methionine）是一种处于多条生物合成途径交叉节点的含硫氨基酸（sulfur amino acid）。在真菌体内，甲硫氨酸生物合成的最终步骤由不依赖钴胺素的甲硫氨酸合酶（cobalamine-independent methionine synthase，Met6，EC 2.1.1.14）催化完成。本研究针对Met6在稻瘟病菌（Magnaporthe oryzae）侵染过程中的功能展开探究。为此，我们通过靶向基因替换（targeted gene replacement）技术构建了MET6基因敲除突变体。在基础培养基中，MET6敲除突变体表现为甲硫氨酸营养缺陷型。即便在添加过量甲硫氨酸的培养基中培养，此类突变体仍显现出多项发育缺陷：菌丝色素沉着减弱、气生菌丝形成受阻以及产孢能力下降。同时，这些突变体呈现出特征性的代谢谱改变：半胱氨酸（cysteine）、胱硫醚（cystathionine）、同型半胱氨酸（homocysteine）、S-腺苷甲硫氨酸（S-adenosylmethionine）、S-腺苷同型半胱氨酸（S-adenosylhomocysteine）的水平显著升高，而甲硫氨酸与谷胱甘肽（glutathione）的水平则保持稳定。上述代谢紊乱与两个基因的过表达相关：一是参与反向转硫化途径（reversed transsulfuration pathway，可将同型半胱氨酸代谢为半胱氨酸）的MgCBS1，二是参与甲基循环（methyl cycle）的MgSAM1与MgSAHH1。这一结果提示，稻瘟病菌会对Met6缺失引发的代谢缺陷产生生理适应性响应，尤其是针对同型半胱氨酸水平升高的状况。致病性检测结果显示，MET6敲除突变体在大麦与水稻叶片上均丧失了致病性。此类突变体存在附着胞（appressorium）介导的侵入缺陷以及侵入后侵染生长缺陷。外源添加甲硫氨酸与S-甲基甲硫氨酸（S-methylmethionine）可修复上述致病性缺陷。本研究结果表明，稻瘟病菌无法从植物组织中获取足量的甲硫氨酸，必须在侵染过程中从头合成该氨基酸，才能满足自身对含硫氨基酸的需求。