Die frühe Infektionsphase von Ustilago maydis

The fungus Ustilago maydis is the causative agent of corn smut disease. Prerequisite for successful infection is the fusion of two compatible haploid sporidia and formation of a dikaryon. This process is controlled by the two mating type loci a and b. The a-locus encodes a biallelic pheromone/pheromone receptor system which is responsible for cell-cell recognition and fusion of the sporidia. The subsequent pathogenic development is controlled by the multiallelic b-locus, encoding two homeodomain proteins, bW and bE. After fusion of haploid sporidia, bW and bE proteins from different alleles can form heterodimers, triggering downstream cascades of regulatory processes. These control filamentous growth, penetration of the plant surface, growth inside the plant and induction of galls. The aim of this work was the isolation of genes regulated by the bW/bE heterodimer that play a role during early pathogenic development. For this U. maydis strains were constructed in which bW and bE genes from different alleles can be turned on and off in a regulated fashion. These strains were used in a non-radioactive RNA-fingerprint analysis to identify differences in the transcriptome in response to the formation of the bW/bE heterodimer. The screen covered more than half of all genes transcribed in U. maydis, and 348 amplicons appeared to originate from differentially expressed genes. Further analysis of 48 chosen amplicons led to the identification of 12 previously unknown b-regulated genes. Of those, 7 are upregulated, 5 are downregulated. Therefore the bW/bE heterodimer is a central regulator, triggering a vast change in the transcriptome. For one of the upregulated genes, frb52 encoding a DNA polymerase X homolog, direct regulation by the bW/bE heterodimer could be shown. Deletion of this gene has no effect on mating and pathogenicity, providing no hint as to the role of the protein in these processes. Another upregulated gene encodes a MAP kinase homolog, named Kpp6. Kpp6 has an unusual amino terminal extension of 150 amino acids that exhibits no similarity to any known protein sequence and has no apparent function. kpp6 is not only induced after b-induction, but also shows a different shorter transcript in response to pheromone. Present data indicate that both transcripts encode for the same polypeptide. Deletion of kpp6 leads to a greatly reduced pathogenicity. In infections of corn plants with deletion mutants, no anthocyanin production of the plant is visible, an early defense reaction normally seen in infections with wild-type cells. Therefore Kpp6 has a crucial role in the early infection process and mediates most likely the communication between fungus and plant. Analysis of three of the downregulated genes led to the identification of a cluster of six genes (the cab locus) that are upregulated during phereomone stimulation and shut off after successful cell fusion. Unlike all the other genes known to be upregulated upon pheromone response, this regulation is independent of the transcription factor Prf1. The regulatory role of Prf1 seems to be restricted to the mating type genes, prf1 itself, and genes necessary for pheromone processing or transport. The expression of the cab locus genes is negatively regulated by the pheromone MAP kinase cascade and positively regulated by the cAMP signalling cascade. This antagonistic control points to a differential interaction between the two signalling modules and the processes regulated by the bW/bE heterodimer during different stages of the early infection process.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2001

Author dc:creator

• Brachmann, Andreas

Contributors dc:contributor

• Kahmann, Regine

Repository record source_url

https://edoc.ub.uni-muenchen.de/28/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:28