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, SG200?nuc1?nuc2 or SG200?nuc3. 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.