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.