Growth, Differentiation and Sexuality

382 M. Feldbrügge et al.
for unbiased genetic approaches to pathogenesis
(Bölker et al. 1995).
Initialapproachestoisolategenesregulated
through the bE/bW heterodimer relied on differential
methods in which the b-induced filamentous
form was compared with the yeast-like form. With
the construction of U. maydis strains which harbour
an inducible combination of bE1/bW2 genes,
it became possible to time-resolve the b-dependent
regulatory cascade (Brachmann et al. 2001). Using
such b-inducible strains, a large number of bregulated
genes have been identified by RNA fingerprinting.
So far, only three of the b-dependent
genes have been shown to possess binding sites
for the bE/bW heterodimer in their respective promoter
regions, implicating a direct regulation by
the bE/bW heterodimer: lga2, a gene located in the
a2 mating type locus which appears to be involved
in mitochondrial fusion, polX, encodingaprotein
with weak similarities to DNA polymerase X, and
dik6, a putative membrane protein without similarity
to known proteins (Bohlmann et al. 1994;
Romeis et al. 2000; Brachmann et al. 2003; Bortfeld
et al. 2004). The finding that the majority of bcontrolled
genes are indirectly regulated suggests
that the bE/bW heterodimer triggers a regulatory
cascade with only a limited number of genes being
direct targets (termed class 1 genes, Fig. 18.5). It follows
that within this class there must be genes with
regulatory function which are in turn required for
the induction or repression of the indirect bE/bW
targets (termed class 2 genes, Fig. 18.5). Deletion
analysis revealed that neither the three directly regulated
genes, nor any of the other, indirectly regulated
genes affected pathogenicity, with the exception
of the MAP kinase kpp6 (see above). One
explanation is that b-regulated genes required for
cell wall structure, like the repellent Rep1 (Wösten
et al. 1996), the hydrophobin Hum2 (Bohlmann
1996), cell wall-modifying enzymes like the potential
exochitinase Exc1 (Brachmann et al. 2001) and
the endoglucanase Egl1 (Schauwecker et al. 1995),
are members of gene families with redundant functions.
In this scenario, single gene knockouts will
be without effect. However, class 1 genes with a regulatory
function are expected to play crucial roles
for pathogenic development, as they would presumably
co-regulate larger subsets of b-regulated
genes.
With the availability of the genomic sequence of
U. maydis (http://www.broad.mit.edu/annotation/
fungi/ustilago_maydis/), it became possible to
design genome-wide DNA arrays (J. Kämper and
R. Kahmann, unpublished data). The currently
available Affymetrix high-density arrays represent
approximately 93% of all U. maydis genes.These
arrays were used to monitor changes in gene
expression after b induction over a 12-h time
course leading to the identification of about 250
b-responsive genes, of which about 50% are either
up-regulated or down-regulated (M. Scherer and
J. Kämper, unpublished data). The functional
classification of these genes allowed to visualize
that cellular processes like the restructuring of
the cell wall as well as lipid metabolism are
controlled by the b mating type locus. Another
large number of b-regulated genes are involved
in the cell cycle, mitosis and DNA replication,
which is consistent with the observation that
b induction leads to cell cycle arrest until the
fungus has infected the plant. Systematic analysis
of b-regulated genes with potential regulatory
function has already allowed identification of new
pathogenicity factors. Of particular interest is
a b dependently expressed transcription factor
required for both filamentous growth as well as
pathogenic development. Microarray experiments
revealed that this transcription factor indeed
controls expression of a subset of class 2 genes
(M. Scherer and J. Kämper, unpublished data).
Insight into b-dependent processes was also
possible by comparing sequences from cDNA
clones obtained from two different developmental
stages, namely, germinating teliospores and
a filamentous diploid strain heterozygous for both
mating type loci. From differences in expression
in these stages, it can be inferred, for instance,
that the filament is involved in nutrient acquisition,
and that germinating teliospores may be
utilizing stored metabolites. Similarly, more ESTs
corresponding to ABC transporters were found in
the filament-specific library, which could indicate
that some of these proteins may be required to
eliminate toxic compounds produced by the host
(Sacadura and Saville 2003; Nugent et al. 2004).
Independent approaches to identify
pathogenicity factors have relied on the identification
of fungal genes differentially regulated
during fungal growth in planta. This has led to
the discovery of chromosomal loci containing coexpressed
genes. The so-called pig (plant-induced
gene) locus, identified by an enhancer-trapping
mutagenesis approach, consists of 11 genes of
which five are differentially regulated in planta
(Aichinger et al. 2003). pig4 and pig6 encode proteins
with similarities to a membrane transporter