MoSfl1 Is Important for Virulence and Heat Tolerance in Magnaporthe oryzae

The formation of appressoria, specialized plant penetration structures of Magnaporthe oryzae, is regulated by the MST11-MST7-PMK1 MAP kinase cascade. One of its downstream transcription factor, MST12, is important for penetration and invasive growth but dispensable for appressorium formation. To identify additional downstream targets that are regulated by Pmk1, in this study we performed phosphorylation assays with a protein microarray composed of 573 M. oryzae transcription factor (TF) genes. Three of the TF genes phosphorylated by Pmk1 in vitro were further analyzed by coimmunoprecipitation assays. One of them, MoSFL1, was found to interact with Pmk1 in vivo. Like other Sfl1 orthologs, the MoSfl1 protein has the HSF-like domain. When expressed in yeast, MoSFL1 functionally complemented the flocculation defects of the sfl1 mutant. In M. oryzae, deletion of MoSFl1 resulted in a significant reduction in virulence on rice and barley seedlings. Consistent with this observation, the Mosfl1 mutant was defective in invasive growth in penetration assays with rice leaf sheaths. In comparison with that of vegetative hyphae, the expression level of MoSFL1 was increased in appressoria and infected rice leaves. The Mosfl1 mutant also had increased sensitivity to elevated temperatures. In CM cultures of the Mosfl1 and pmk1 mutants grown at 30°C, the production of aerial hyphae and melanization were reduced but their growth rate was not altered. When assayed by qRT-PCR, the transcription levels of the MoHSP30 and MoHSP98 genes were reduced 10- and 3-fold, respectively, in the Mosfl1 mutant. SFL1 orthologs are conserved in filamentous ascomycetes but none of them have been functionally characterized in non-Saccharomycetales fungi. MoSfl1 has one putative MAPK docking site and three putative MAPK phosphorylation sites. Therefore, it may be functionally related to Pmk1 in the regulation of invasive growth and stress responses in M. oryzae.稻瘟病菌（Magnaporthe oryzae）特有的植物侵入结构——附着胞（appressoria）的形成，受MST11-MST7-PMK1丝裂原活化蛋白激酶级联反应（MAP kinase cascade）调控。其下游转录因子之一MST12，对病菌的侵入与侵染生长至关重要，但对于附着胞形成并非必需。为鉴定Pmk1调控的其他下游靶标，本研究利用包含573个稻瘟病菌转录因子（transcription factor，TF）基因的蛋白质微阵列开展磷酸化检测。经体外实验证实可被Pmk1磷酸化的3个转录因子基因，进一步通过免疫共沉淀（coimmunoprecipitation）实验进行验证。其中一个名为MoSFL1的基因，在体内可与Pmk1发生相互作用。与其他Sfl1同源蛋白类似，MoSfl1蛋白含有HSF样结构域。当在酵母中表达时，MoSFL1可功能性互补sfl1突变体的絮凝缺陷。在稻瘟病菌中，敲除MoSFL1会导致水稻与大麦幼苗的致病力显著下降。与此结果一致的是，在水稻叶鞘穿透实验中，Mosfl1突变体的侵染生长能力存在缺陷。与营养菌丝相比，MoSFL1在附着胞以及侵染的水稻叶片中的表达水平有所升高。Mosfl1突变体对高温环境的敏感性也有所增强。在30℃培养的CM培养基中，Mosfl1与pmk1突变体的气生菌丝产生与黑色素化程度均有所降低，但生长速率未发生改变。通过实时定量反转录聚合酶链反应（qRT-PCR）检测发现，Mosfl1突变体中MoHSP30与MoHSP98基因的转录水平分别下调了10倍与3倍。SFL1同源蛋白在丝状子囊菌中保守存在，但目前尚未在非酿酒酵母目（Saccharomycetales）真菌中对其功能进行表征。MoSfl1含有1个推定的MAPK结合位点与3个推定的MAPK磷酸化位点。因此，MoSfl1可能与Pmk1在稻瘟病菌的侵染生长与胁迫响应调控中发挥功能关联。