AgSPA2 and AgBOI control landmarks of filamentous growth in the filamentous ascomycete Ashbya gossypii

The morphological requirements for the
development of a fungal mycelium include the
emergence of germ tubes, a permanent hyphal
tip extension, lateral and apical branching and
septation. The basis for each of these events,
also termed landmarks of fi lamentous growth,
is polarised growth. Thus polarised growth is
essential for hyphal and mycelial morphogenesis.
We wanted to investigate genes in the fi lamentous
ascomycete Ashbya gossypii that are involved
in polarised growth and control landmarks of
fi lamentous growth. In "Chapter 1" and "Chapter 2" I described the
identifi cation of two genes that are involved in
polarised growth and I highlighted their roles
in landmarks of fi lamentous growth. At the end
of the fi rst two chapters I draw a refi ned model
of the developmental pattern in A.gossypii with
the insight gained from these fi rst two chapters.
In "Chapter 3" and "Chapter 4" I described two
novel tools that were established for the functional
analysis of the genes investigated in the fi rst
two chapters. These two methods were the
construction of a versatile module for C-terminal
GFP fusion and an integration module based on
the AgADE2 marker. Chapter 1 We wanted to identify genes
in A.gossypii that are important for hyphal
morphogenesis. As polarised growth is crucial
for morphogenesis in A.gossypii and S.cerevisiae
we hypothesised that a similar set of genes is
required for polarised growth in A.gossypii and
S.cerevisiae; however, differences in orthologous
proteins might have an impact on the regulation
of polarised growth and thus guide the process
of fi lamentation in A.gossypii and budding in
S.cerevisiae. We screened for A.gossypii proteins
that have an orthologue in S.cerevisiae implicated
in polarised growth and that display a signifi cant
difference in the primary structure compared to the
S.cerevisiae orthologue. This revealed AgSpa2p,
a homologue of ScSpa2p. AgSpa2p is more than
twice as long as ScSpa2p due to an extended
internal domain without signifi cant homology to
ScSpa2p. AgSpa2p localises permanently to
hyphal tips and transiently to sites of septation.
We constructed two AgSPA2 mutants, a partial
deletion of the internal domain alone and a
complete deletion. We could show that these
mutant strains display alterations in the branching
frequency and in the hyphal tip growth speed
potential. The branching frequency and the hyphal
tip growth speed potential are two factors that have an impact on the hyphal tip growth speed.
The two mutants behave opposing in respect of
each of these factors and the two factors have a
competing effect on the hyphal tip growth speed.
We suggest that AgSpa2p is required to balance
these two factors to achieve an effi cient hyphal tip
growth speed and the extended internal domain in
AgSpa2p plays an important role in this process. Chapter 2 We screened an existing A.gossypii
knock out library for strains that show defects in
landmarks of fi lamentous growth. We identifi ed
a strain that frequently displayed spherically
enlarged hyphal tips. The deleted gene in that
strain was identifi ed as AgBOI. AgBoip represents
an orthologue of the redundant proteins in
S.cerevisiae ScBoi1p and ScBoi2p, which are
implicated in polarised growth. We could show
that AgBoip is required for the emergence
of germ tubes and for the initiation of lateral
branches. Moreover AgBoip is required for
permanent hyphal tip extension, as polarisation
of hyphal tips is not permanently maintained in
AgboiΔ strains. We could show that prior to a
spherical enlargement of hyphal tips in AgboiΔ
the polarisation marker AgSpa2p delocalises
from the tip. Tips can be repolarised which goes
in parallel with a relocalisation of AgSpa2p. In
spherically enlarged tips the actin cytoskeleton is
also depolarised. AgBoip itself localised to sites of
polarisation similar to cortical actin patches. We
suggest that AgBoip is required for establishment
of cell polarity to initiate germ tubes and lateral
branches and for maintenance of cell polarity to
allow a permanent hyphal tip extension. Chapter 3 The green fl uorescent protein (GFP)
is of extraordinary value in molecular biology. It
allows the visualisation of proteins in organisms
and together with advanced microscopy
techniques it enables dynamic studies in living
cells. We constructed a versatile module for PCR
based C-terminal GFP-fusion and established
microscopy techniques for dynamic GFP studies
in A.gossypii. The studies described in "Chapter
1" and "Chapter 2" would not have been possible
without the development of this module. Chapter 4 The functional analysis of genes often
requires the expression of truncated alleles, e.g.
reporter modules as the GFP, tagged proteins
for biochemical analyses, mutant alleles or
overexpression constructs. As it was of importance
in this work to express certain GFP fusion proteins
as second copies we constructed an integration module on the basis of the AgADE2 marker.
The mode of action of the integration module is
the reconstitution of a truncated AgADE2 ORF
thereby co-integrating the fragment of interest.