Investigation of signalling involved in maintaining the mutually beneficial association between Epichloe festucae and perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand
In the mutually beneficial association between the fungal endophyte Epichloë festucae and
perennial ryegrass, fungal growth is highly regulated and coordinated with that of the
host. This implies there must be signalling between the fungus and its host to maintain
this close association. Recent work has shown a novel role for reactive oxygen species
(ROS) in this symbiotic maintenance, with multiple components of the superoxideproducing
NADPH oxidase (Nox) complex being essential for normal association.
However, the mechanism by which the Nox complex is regulated is unclear.
To identify potential regulators of the E. festucae Nox complex, comparisons were made
with well-characterised mammalian systems. This search identified three candidate
regulators: a stress activated MAP kinase, sakA, and the p21-activated kinases, pakA and
pakB. To investigate if these genes were involved in symbiotic maintenance, replacement
mutants were generated by homologous recombination. In culture analysis revealed that
the ?sakA mutant was hypersensitive to a range of stresses, whereas the pak mutants
were hypersensitive to cell wall stress-inducing agents and displayed altered growth and
morphology. Examination of perennial ryegrass infected with these mutants revealed
drastically altered plant interaction phenotypes for the ?sakA and ?pakA mutants in
comparison to the wild-type strain. ?sakA-infected plants were stunted and displayed
striking changes in development, with the base of tillers showing loss of anthocyanin
pigmentation and disorganisation of host cells below the meristem, resulting in swollen
bases. Plants infected with the ?pakA mutant were severely stunted, had no more than
two tillers and senesced soon after planting. In contrast, plants infected with the ?pakB
mutant were similar to wild-type, with only slight deregulation of growth in planta.
Examination of ROS in culture revealed that ?sakA and ?pakA displayed elevated levels
of both superoxide and hydrogen peroxide. ROS levels were also elevated around ?sakA
hyphae in planta. These results support roles for SakA and PakA in Nox regulation. This
work highlights the fine balance between mutualism and antagonism, and provides
insight into the molecular basis for mutualism.
Content removed from thesis due to copyright restrictions:
Eaton, C. J., I. Jourdain, et al. (2008). "Functional analysis of a fungal endophyte stress-activated MAP kinase." Current Genetics 53(3): 163-174.
Scott, B. and C. J. Eaton (2008). "Role of reactive oxygen species in fungal cellular differentiations." Current Opinion in Microbiology 11(6): 488-493.