RNAseq profiling of U. maydis during biotrophic stages of development
We have performed an in depth transcriptional profiling of the entire plant-associated development initiated by U. maydis wild type strains by RNAseq analysis between 12 hours and 12 days post infection. In this analysis we focused mainly on fungal metabolism, nutritional strategies, secreted effectors and regulatory networks. We uncovered 14 discrete expression modules to which U. maydis genes could be assigned. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We showed that oligopeptide transporters likely involved in nitrogen assimilation are important virulence determinants. Secreted proteins are expressed in characteristic expression waves, suggesting that they are only transiently needed (Figure 4). We also show that the previously observed downregulation of many effector genes late in infection is associated with expression heterogeneity: in hyphal aggregates which are embedded in the polysaccharide matrix, downregulation is observed in the center of these structures while cells lying in the periphery of the aggregates continue to express the respective effectors. Secreted proteins were enriched in three of the 14 expression modules corresponding to stages on the plant surface, establishment of biotrophy and induction of tumors. These modules are likely the key determinants for U. maydis virulence. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. While mutants lacking one of these transcription factors (Nlt1) showed normal proliferation in leaf tissue and induced anthocyanin formation, they were unable to induce leaf tumors. A comparison of effector gene expression in wild type and mutant infected tissue revealed that effector gene expression was specifically abrogated in the nlt1 mutant. This makes it likely that one or several of the Nlt1-regulated effectors will be responsible for the induction of leaf tumors.