Project description:Hxt1 is a high affinity hexose transporter important for virulence of the phytopathogenic basidiomycete Ustilago maydis on its host plant maize. Hxt1 shows the highest similarities to the glucose sensors Rgt2 and Snf3 from S. cereviciae (42% and 39% identity on amino acid level, respectively). In these sensors, the substitution of a highly conserved arginine to lysine leads to a constitutive active signal, resulting in expression of several glucose induced genes. Introduction of an analogous mutation in Hxt1 leads to loss of transport activity in the resulting Hxt1(R164K) protein. Expression of Hxt1(R164K) in hxt1 deletion strains results in a completely avirulent phenotype. Pathogenic developement of M-bM-^HM-^Fhxt1 hxt1(R164K) strains is blocked immediately after plant penetration. Expression analysis of M-bM-^HM-^Fhxt1 hxt1(R164K) cells 24 hours post inoculation revealed downregulation of a set of genes involved in carbohydrate metabolism indicating a role of Hxt1 in carbohydrate signaling during initiation of the pathogenic stage. To analyze expression changes of SG200 and SG200M-bM-^HM-^Fhxt1 and SG200M-bM-^HM-^Fhxt1 hxt1(R164K) cells were harvested from the surface of maize leaves 24 hour post inoculation. For each strain three independent replicates were conducted.
Project description:The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. Strains in which the rep1 gene is inactivated (M-bM-^HM-^Frep1 strain) are affected in aerial hyphae formation. This makes these strains instrumental to assess changes in global gene expression as a consequence of aerial growth. Microarray analysis revealed that only 31 genes in the M-bM-^HM-^Frep1 SG200 strain had a fold change in expression of >= 2. Twenty-two of these genes are up-regulated and half of them encode small secreted proteins (SSPM-bM-^@M-^Ys) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the M-bM-^HM-^Frep1 SG200 strain encode secreted cysteine-rich proteins (SCRPM-bM-^@M-^Ys). Interestingly, most of the SCRPM-bM-^@M-^Ys are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the M-bM-^HM-^Frep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that only minor changes occur in the expression profile when U. maydis forms aerial structures. Key words: aerial hypha, repellent, hydrophobin-like protein, Ustilago maydis, SSP, SCRP, fungal pathogenicity. To analyze expression changes in aerial hyphae upon deletion of the rep1 gene, strains SG200 and SG200M-bM-^HM-^Frep1 were grown on charcoal nitrate minimal array media supplemented with vitamins and 1% glucose at 22 C for 48 h. For each experiment three biological replicates were performed.
Project description:Goals: Comparing the infection between Ustilago maydis SG200 with the wild-type strain FB1xFB2 previously published Methods: Comparative RNASeq analysis between U. maydis SG200 and U. maydis FB1xFB2 at three timepoints (axenic, 2dpi, 12dpi) Results: The RNASeq analysis in SG200 identifies differences in gene expression with FB1xFB2. These differences could be the result of a unequal contribution of each nuclei to transcription. Further analysis identified a set of differentially transcribed genes.