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Characterisation of novel secondary metabolism genes in plant-endophytic Epichloë fungi : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Manawatu, New Zealand
Plant-‐endophytic
fungi
of
the
genus
Epichloë
are
symbionts
of
cool
season
grasses.
Epichloë
infections
are
generally
asymptomatic,
with
endophyte
and
host
growth
synchronised
to
form
an
interconnected
hyphal
network
in
the
intercellular
spaces
separating
host
cells.
In
return
for
the
sustenance
received
from
the
host
apoplast,
Epichloë
spp.
produce
a
range
of
bioprotective
secondary
metabolites
(SM)
that
deter
both
vertebrate
and
invertebrate
herbivores.
Peramine
is
an
Epichloë-‐
derived
insect-‐deterring
SM
produced
by
the
two-‐module
non-‐ribosomal
peptide
synthetase
(NRPS)
PerA,
encoded
by
the
gene
perA.
The
perA
gene
is
widespread
across
Epichloë,
but
peramine
production
is
much
more
discontinuous.
Through
an
Epichloë-‐wide
survey
it
is
shown
that
the
peramine
chemotype
of
Epichloë
isolates
can
be
accurately
predicted
through
a
combination
of
PCR
and
sequencing
methods.
Furthermore,
the
protein
encoded
by
the
widespread
peramine-‐negative
perA-‐ΔR
allele
is
analysed
in
detail.
The
perA-‐ΔR
allele
has
a
transposable
element
inserted
into
the
3’
end
of
the
gene,
and
was
previously
assumed
to
be
non-‐functional.
Evidence
for
ongoing
perA-‐ΔR
functionality
is
presented,
with
the
PerA-‐ΔR
protein
shown
to
exhibit
altered
substrate
specificity
relative
to
PerA.
A
PerA/PerA-‐ΔR
domain
swap
is
shown
to
restore
peramine
production
to
PerA-‐ΔR,
providing
insight
into
the
functional
changes
that
separate
this
protein
from
PerA.
Another
major
project
investigated
the
role
SM
genes
may
play
in
Epichloë
sexual
development.
Many
Epichloë
spp.
are
transmitted
vertically
through
infection
of
the
host
seed.
However,
sexual
Epichloë
spp.
may
also
undergo
a
reproductive
cycle
that
involves
formation
of
pre-‐sexual
stromata
structures
over
developing
host
inflorescences.
This
sexual
cycle
culminates
in
horizontal
transfer
of
the
endophyte
to
endophyte-‐free
host
plants.
Deletion
of
the
symbiotically
regulated
endophyte
gene
irlA
induced
a
symbiosis-‐defective
phenotype
in
planta,
and
this
observation
led
to
the
identification
of
a
novel
Epichloë
SM
cluster.
Synteny
analysis
and
comparison
to
previously
characterised
fungal
SM
gene
clusters
defines
a
five-‐gene
cluster
centred
on
irlA
that
is
shown
to
be
upregulated
in
stromata.
A
model
is proposed
whereby
this
SM
cluster
controls
initiation
of
the
proliferative
hyphal
growth
associated
with
stromata
formation.
