A Nucleolar Protein, MoRRP8 Is Required for Development and Pathogenicity in the Rice Blast Fungus

The nucleolus is the largest, membrane-less organelle within the nucleus of eukaryotic cell that plays a critical role in rRNA transcription and assembly of ribosomes. Recently, the nucleolus has been shown to be implicated in an array of processes including the formation of signal recognition particles and response to cellular stress. Such diverse functions of nucleolus are mediated by nucleolar proteins. In this study, we characterized a gene coding a putative protein containing a nucleolar localization sequence (NoLS) in the rice blast fungus, <i>Magnaporthe oryzae</i>. Phylogenetic and domain analysis suggested that the protein is orthologous to Rrp8 in <i>Saccharomyces cerevisiae</i>. MoRRP8-GFP (translational fusion of MoRRP8 with green fluorescence protein) co-localizes with a nucleolar marker protein, MoNOP1 fused to red fluorescence protein (RFP), indicating that MoRRP8 is a nucleolar protein. Deletion of the <i>MoRRP8</i> gene caused a reduction in vegetative growth and impinged largely on asexual sporulation. Although the asexual spores of Δ<i>Morrp8</i> were morphologically indistinguishable from those of wild-type, they showed delay in germination and reduction in appressorium formation. Our pathogenicity assay revealed that the <i>MoRRP8</i> is required for full virulence and growth within host plants. Taken together, these results suggest that nucleolar processes mediated by MoRRP8 is pivotal for fungal development and pathogenesis.

核仁是真核细胞核内体积最大的无膜细胞器，在核糖体RNA（rRNA）转录与核糖体组装过程中发挥关键作用。近年来研究证实，核仁还参与一系列生物学进程，包括信号识别颗粒（signal recognition particle）的形成以及细胞应激响应。核仁的多样功能均由核仁蛋白介导。本研究针对稻瘟病菌（*Magnaporthe oryzae*）中一个编码含核仁定位序列（nucleolar localization sequence, NoLS）的推定蛋白的基因展开功能鉴定。系统发育与结构域分析显示，该蛋白与酿酒酵母（*Saccharomyces cerevisiae*）中的Rrp8蛋白为直系同源物。将MoRRP8与绿色荧光蛋白（GFP）融合得到的MoRRP8-GFP，与融合了红色荧光蛋白（RFP）的核仁标记蛋白MoNOP1发生共定位，证实MoRRP8为核仁蛋白。敲除*MoRRP8*基因会导致菌丝营养生长速率下降，并显著抑制无性产孢过程。尽管Δ*Morrp8*突变体的无性孢子在形态上与野生型菌株无明显差异，但二者的孢子萌发速率存在延迟，且附着胞形成率降低。致病性测定结果显示，*MoRRP8*基因对于病原菌实现完全毒力以及在寄主植物体内的增殖均不可或缺。综上，本研究结果表明，由MoRRP8介导的核仁相关过程对于稻瘟病菌的生长发育与致病过程至关重要。