The role of AgRax1p, AgRax2p, AgBud7p and AgBud10p in mycelial development of the filamentous fungus "Ashbya gossypii"

Polarized growth is essential for hyphal and
mycelial morphogenesis. The diversity of fungal morphology
and development raises many questions
considering the mechanism involved in selection of
new polar growth sites. Landmark events of fungal
growth include the emergence of germ tubes from a
germinated spore, sustained hyphal tip extensions,
lateral and apical branching and septation. The basis
for each of these events is the polarized growth machinery
and in contrast to the well studied polarized
growth pattern in the budding yeast S.cerevisiae, the
molecular requirements for the development of a fungal
mycelium are not well known.
The fi lamentous fungus Ashbya gossypii and the
budding yeast S. cerevisiae have different life styles
despite very similar gene contents and conserved
domain compositions of gene products.
In this work, I was investigating the role of several
homologues of S.cerevisiae genes involved in
the budding pattern in the fi lamentous ascomycete
A.gossypii. Since A.gossypii does not grow by budding,
it was interesting to search for the function of
S.cerevisiae BUD gene homologues. It was hypothesized
that genes controlling the budding pattern of
this yeast could be landmarks involved in polarized
growth control, branching or the stabilization of growth
axis in the fi lamentous fungus A.gossypii.
The goal of Chapter one was to describe the
role of AgRax1p and AgRax2p, homologues of the
S.cerevisiae genes ScRAX1 and ScRAX2. AgRax1p
and AgRax2p are implicated in maintenance of cell
polarity. They play important roles in emergence of
germ tubes and lateral branches as well as in maintenance
of permanent hyphal tip extension during
tip branching. A related role was also suggested for
ScRax2p in S.cerevisiae. Yeast Rax2p was implicated
in the maintenance of the bipolar budding pattern
but not in its establishment, in diploid S.cerevisiae
cells (Chen, et al. 2000). AgSpa2-GFP, a polarity marker
was maintained at the hyphal tip in Agrax1Δ and
Agrax2Δ strain during polarized growth as described
before for the wild type. Thus AgRax1p and AgRax2p
might not be permanently required during polarized
growth but only in response to distinct events. Such
events might be the initiation of a second germ tube
or a lateral branch, which cause the hyphal tip growth
speed to temporary slow down even though polarization
at the tip is maintained. Thus, AgRax1p and
AgRax2p might be required for reinforcement of polarization
in response to branching in order to maintain
permanent hyphal tip extension.
I show here that AgRax2p is involved in the temporal
regulation of branch emergence by maintenance
of polarity at selected branch sites. Furthermore, we
prove that AgRax1p and AgRax2p signal peptide
are essential for the proper localization of AgRax2p.
AgRax2p might be placed at the tips in response to
a lateral branching or septation event. Additionally, I
demonstrate that AgRax2p has a role in the septation
process where it may persist to direct future branching
events. The fi rst chapter also contains information
about the probable role of AgBud10p in polarity
maintenance.
In the second Chapter, I describe a possible
role of AgBud7p in the maintenance of sustained
polar growth and for the sporulation process. The
fact that the AgBUD7 gene has two homologues in
S.cerevisiae gave this work a more interesting dimension.
I verifi ed the role of both homologues in
S.cerevisiae and performed in addition experiments
with a number of double deletions. The phenotypes
obtained helped to analyse the role of AgBud7p in
fi lamentous growth. GFP fl uorescence of AgBud7p-
GFP transformants was highly enriched in small organelles,
which were constantly oscillating with about
the same amplitude in young and in old mycelium.
Moreover, I present evidence that the observed Ag-
Bud7-GFP movements rather depend on the fl ow of
cytoplasm than on actin-based structures. Co-localization
studies indicated that a small number of Ag-
Bud7p-vesicles co-localize with Spindle Pole Bodies.
Studies done in S.cerevisiae indicated a colocalization
of ScBud7p with late Golgi structures. Presently, late
Golgi structures cannot be visualized in A.gossypii to
allow a comparison with the S.cerevisiae data. The
40% decrease in maximal radial growth speed determined
from Agbud7Δ colonies and signifi cant deviations
from the growth axis suggest that AgBud7p
could be required for the maintenance of hyphal tip
shape by delivering certain substances to the cortical
membrane. In the absence of AgBud7p an insuffi cient
amount of building materials is transported to the
growing tips leading to a decrease in hyphal diameter
and to changes in growth direction.
The last chapter presents the results of heterologous
complementation experiments done
in S.cerevisiae. The goal was to analyze whether
A.gossypii proteins involved in diverse polar growth
events can complement S.cerevisiae deletions of
homologous genes. In these cases the A.gossypii
and the S.cerevisiae gene diverged from a common
ancestral gene over 100 million years ago. In all of
the cases tested full or at least partial complementation
was found. This chapter again shows the power
of knowledge about A.gossypii genomics in understanding
the degree of evolutionary conservation of
protein functions and of gene promoters.
These and other results highlight common
themes for the genetic regulation of growth guidance
in eukaryotic cells and make fi lamentous fungi powerful
model systems to elucidate the molecular mechanisms
that regulate these processes.