Evaluating the effect of secreted ribonucleases from Ustilago maydis on the extracellular RNA profile/repertoire of Zea mays during corn smut disease. Evaluating the effect of secreted ribonucleases from Ustilago maydis on the extracellular RNA profile/repertoire of Zea mays during corn smut disease

The apoplast of host plants serves as a dynamic interface during invasion and subsequent establishment of disease by a microbial pathogen. In the recent past, host plant apoplasts have been demonstrated to harbour small membrane bound vesicles called extracellular vesicles (EVs) that contain both RNA and protein. EVs are considered to play a significant role in cell-cell communication between the host and the pathogen during infection. In this study the RNAs associated with EVs from the host plant Zea mays under infected and uninfected conditions were sequenced. Besides EV bound RNA, plant apoplasts also contain free RNA that are equally important for host pathogen interaction. We have also detected a pool of extracellular RNA (exRNA) in Z. mays located on leaf surfaces. In this experiment, we sequenced both the free apoplastic RNA and leaf surface RNA from Z. mays during U. maydis infection and under control uninfected conditions. The U. maydis genome codes for a number of secreted ribonucleases that are essential for the pathogenesis of the fungus. For three of these ribonucleases, we have also investigated their role in the regulation of cell:cell communication between Z. mays and U. maydis. Accordingly, this study includes comparative transcript profiling of all the three different forms of exRNA between Z. mays plants infected with either wild type U. maydis or U. maydis strains lacking one or more secreted ribonucleases. Overall design: Z. mays plants of the variety B73 were infected at 7 days seedling stage with U. maydis strains SG200 WT, SG200Δnuc1Δnuc2 or SG200Δnuc3. The infected leaf samples were harvested at 5 days post infection (5 dpi). For isolation of leaf surface RNA, the leaf surfaces were sprayed with vesicle isolation buffer (VIB) followed by RNA precipitation from the rinse buffer collected through centrifugation of the leaves. For isolating EV associated and free apoplastic RNAs, the harvested leaf samples were infiltrated with VIB followed by centrifugation to collect the apoplastic wash fluid (AWF). EVs were collected from the AWF by centrifugation at 40,000g for one hour and EV associated RNAs were isolated from the pellet. The supernatant was used for the isolation of the free apoplastic RNA. For uninfected samples, the seedlings were left uninfected and leaves were harvested at 12 days post sowing for RNA isolation. Thus, three different sets of RNAs were isolated and sequenced from four different conditions including one control uninfected and three infection conditions. Three biological replicates of the samples were sequenced.

宿主植物质外体（apoplast）在微生物病原菌入侵及后续病害定殖过程中，是一个动态互作界面。近期研究证实，宿主植物质外体中存在一类膜结合小型囊泡，即细胞外囊泡（extracellular vesicles, EVs），其内含RNA与蛋白质。目前认为，这类囊泡在侵染过程中宿主与病原菌的细胞间通讯环节发挥关键作用。本研究对感染与未感染状态下玉米（Zea mays）来源的细胞外囊泡结合RNA进行了测序。除囊泡结合RNA外，植物质外体中还存在游离RNA，其在宿主-病原菌互作中同样具有重要意义。我们还在玉米叶片表面检测到一类细胞外RNA（extracellular RNA, exRNA）池。本实验同时对玉米在玉米黑粉菌（U. maydis）侵染及未侵染对照条件下的质外体游离RNA与叶片表面RNA进行了测序。玉米黑粉菌基因组编码多种分泌型核糖核酸酶，这类酶对该真菌的致病过程不可或缺。针对其中3种核糖核酸酶，我们还探究了其在玉米与玉米黑粉菌之间细胞间通讯调控中的作用。据此，本研究针对三类不同形式的细胞外RNA，开展了转录谱比较分析，所用样本分别为接种野生型玉米黑粉菌、或缺失一种/多种分泌型核糖核酸酶的玉米黑粉菌菌株的玉米植株。实验设计概况：将B73品种的玉米幼苗于播种后7天（幼苗期）接种玉米黑粉菌菌株SG200 WT、SG200Δnuc1Δnuc2或SG200Δnuc3。接种后5天（5 dpi）收获受侵染的叶片样本。对于叶片表面RNA的提取，先使用囊泡分离缓冲液（vesicle isolation buffer, VIB）喷洒叶片表面，随后收集经叶片离心得到的冲洗液，通过沉淀法从中提取RNA。对于质外体囊泡结合RNA与游离质外体RNA的提取，先向收获的叶片样本中注入囊泡分离缓冲液，再经离心收集质外体冲洗液（apoplastic wash fluid, AWF）。通过40,000g离心1小时从质外体冲洗液中分离得到细胞外囊泡，从囊泡沉淀中提取囊泡结合RNA；上清液则用于提取游离质外体RNA。未侵染对照组的幼苗不做接种处理，于播种后12天收获叶片用于RNA提取。综上，本研究从4种不同条件下（1组未侵染对照、3组侵染处理）共分离得到3类不同的RNA并进行测序，每组样本设置3次生物学重复。