Project description:Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host Zea mays. The biotrophic interaction is initiated upon host penetration, and involves expansion of the host plasma membrane around hyphae, which is thought to facilitate the exchange of nutrients and virulence factors. Transcriptional regulators involved in the establishment of an infectious dikaryon and penetration into the host have been identified, however, regulators involved in the post-penetration stages remained to be elucidated. In the study we report the identification of an Ustilago maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumour development in planta. Microarray analyses of Δfox1-infected plant tissue identified Fox1 as a transcriptional activator, involved in the expression of secreted effectors required for virulence. Maize plants were infected with a mixture of either FB1 and FB2 (wild-type), or FB1∆fox1 and FB2∆fox1 crossings, to measure the impact of fox1 on pathogenic development. Early Golden Bantam maize plants were grown in a phytochamber in a 15h/9h light-dark cycle; light period started/ended with 1h ramping of light intensity. Maize plants were kept at 28°C (light) and 20° (dark). Plantlets were individually sown in pots with potting soil (Fruhstorfer Pikiererde) and infected 7 days after sowing, 1 h before end of the light period. Infected leaf tumor material from at least 10 plants was collected 5 days post infection, 1 h before the end of the light period and directly frozen in liquid nitrogen for RNA-extraction. RNA samples were extracted from infected leaf tissue 5 days after infection.
Project description:Gene expression of FB2 wt in minimal medium with 2 percent arabinose or glucose after 24h of growth was compared with the conditional promoter regulated strain Pcrg1:grx4 (#55). Glucose is a repressor of this promoter while arabinose acts as inducer. Grx4 was initially pre-depleted during growth on MM with glucose over night. Afterwards cells were transferred to MM with either glucose or arabinose and cells were collected after 24h. 3 biological repeats were performed for each strain and each condition. Cells were flash frozen and used to isolate total RNA. RNA sequencing was done by Genewiz. DEG’s between wt and mutant for the different conditions were obtained. Iron regulated genes such as the high affinity uptake system and the siderophore system of Ustilago maydis were repressed in the Pcrg1:grx4 (#55) strain when grown in glucose. Expression of these genes were similar for wt and the conditional strain when grown in arabinose. Grx4 was identified as an essential gene in Ustilago maydis which regulates the iron regulon and related genes.
Project description:The basidiomycete fungus Ustilago maydis causes smut disease in maize and has become an important model for elucidating the strategies used for host colonization by biotrophic fungi. In this study, we performed an in-depth transcriptional profiling of the plant-associated development of a cross between U. maydis FB1 and FB2 wildtype strains. The analysis of eight different stages, including the development on the leaf surface, early colonization, tumor induction and spore maturation, offers an unprecedented view of the changes in the fungal transcriptome associated with the passage through the entirely biotrophic life cycle. In our analysis, we focus on fungal metabolism, nutritional strategies, secreted effectors and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to the plant surface, establishment of biotrophy and tumor formation, respectively. These modules are likely the key determinants for U. maydis virulence. 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 show that oligopeptide transporters likely involved in nitrogen supply during infection are important virulence determinants. By measuring the intramodular connectivity of transcription factors, we identified potential drivers for the virulence modules. While known components of the b-cascade served 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. We demonstrate a crucial role in effector gene expression and tumor formation for one of these transcription factors.