Chitinase induction in Phaseolus vulgaris during race-specific interactions with Pseudomonas syringae pv. phaseolicola

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The biology of the endophyte/grass partnership

The association of Epichloë and Neotyphodium endophytes with host grasses is one of balance between both partners. The host plants are symptomless with the exception of stromata that can form on reproductive tillers of grasses infected with Epichloë spp. The hyphae of the endophytes, spread systemically throughout the above ground parts of plants and in leaves, are aligned parallel with the longitudinal leaf axis and are seldom branched. In this review, the reader is guided through the interaction of host grass and endophytes as revealed by microscopy and then given a model to explain the unique association that these endophytic fungi have with host grasses. This model proposes that hyphae within meristematic tissue branch profusely, providing hyphal strands that extend by intercalary growth amongst enlarging plant cells. Hyphae cease branching and elongating when amongst stationary cells but remain metabolically active, producing a range of bioactive products that enhance the persistence of host grasses. By this process the synchronisation of endophyte and plant growth is achieved, providing a generally mutualistic relationship. Keywords: Neotyphodium, Epichloë, intercalary hyphal extension</jats:p

Insights into the molecular biology of epichloë endophyte alkaloid biosynthesis

The production of secondary metabolite alkaloids by epichloë endophytes of grasses is of great interest to agriculture due to the opposing effects of pasture protection against insect herbivory and concomitant toxicity to stock. Recent molecular genetic analysis, that has greatly enhanced our understanding of secondary metabolite biosynthesis, is reviewed and potential outcomes for pastoral agriculture are discussed. Keywords: endophyte, Epichloë, Neotyphodium, alkaloid, secondary metabolite, gene cluster</jats:p

Identification of NRPS gene families from Neotyphodium/Epichloe endophytic fungi that form mutualistic associations with cool season grasses

Neotyphodium and Epichloë species are closely related asexual and sexual endophytic fungi, respectively, that form symbiotic associations with cool season grasses of the sub family Pooideae, including several important forage and turf grass species. The endophytes confer a number of advantages to their hosts, but also can cause animal toxicosis. These positive and negative effects are, in many cases, due to the production of fungal secondary metabolites. In filamentous fungi secondary metabolite genes are commonly clustered and are well documented to often contain non-ribosomal peptide synthetases (NRPSs) at their core. Members of this gene family encode large multifunctional proteins that synthesise a diverse range of bioactive compounds, many of which have been shown to serve as pathogenicity or virulence factors, in addition to suggested roles in niche adaptation. We have used a degenerate PCR approach to identify members of the NRPS gene family from Neotyphodium and Epichloë species, and have shown that at least 13 NRPS genes exist among these genomes. The distribution of these genes among different Neotyphodium/Epichloë lineages suggests that a common ancestor contributed most of the complement of NRPS genes, which have been either retained or lost during the evolution of these fungi. Keywords: Neotyphodium, Epichloë, non-ribosomal peptide synthetase, NRPS</jats:p

Characterisation of a novel endophyte NRPS gene and its role in endophyte-grass symbioses

Symbiotic grass associations with fungal endophytes (genera Neotyphodium and Epichloë) display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. Perennial ryegrass, an important agronomic grass, is typically associated with the N. lolii endophyte. The endophyte lives within the intercellular spaces without inducing any symptoms in the plant. The aim of this study is to elucidate the biosynthetic function of fungal secondary metabolite gene clusters. Non-ribosomal peptide synthetase genes (NRPSs) of unknown function were targeted, as these genes are commonly associated with the production of bioactive peptides some of which are ecologically important. Some novel endophyte NRPS genes have been identified using a degenerate PCR screen; one of these, NRPS5 will be discussed here. Clones were obtained by screening a fosmid Epichloë festucae genomic DNA library and we are currently determining gene function by using targeted gene replacement followed by an assessment in vitro and in planta using metabolomics and appropriate bioassay screens. Keywords: endophyte, NRPS, secondary metabolism</jats:p

Heterologous synthesis of the fungal alkaloid peramine

Endophytic fungi of temperate grasses form stable symbiotic associations that provide mutual benefits for both species.</jats:p

Biosynthesis of an extracellular siderophore is essential for maintenance of mutualistic endophyte-grass symbioses

We have identified a novel siderophore from the mutualistic grass symbiont Epichloë festucae. Targeted gene replacement of a non-ribosomal peptide synthetase (termed sidF) from E. festucae eliminated biosynthesis of this extracellular siderophore. Structural characterisation, based on extensive mass spectrometry showed similarities to fusarinine-type siderophores. Plants inoculated with strains carrying a targeted deletion in sidF have a dramatic phenotype. Siderophore loss changed a mutualistic symbiont into an antagonistic fungus, causing de-regulation and proliferation of fungal hyphae, plant stunting and sometimes even tiller death. We hypothesise that loss of the biosynthesis of the siderophore disrupts iron homeostasis within the whole symbiotum, which is a critical factor for maintenance of mutualistic endophyte-grass symbioses. Keywords: siderophore, iron homeostasis, NRPS</jats:p

Dual Affymetrix GeneChip analysis of the perennial ryegrass-endophyte symbiosis

Grass associations with Epichloë/Neotyphodium endophytes display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. To gain a comprehensive understanding of the complex biological interactions that occur between a plant host and fungal symbiont, a transcriptomics approach using custom designed Affymetrix GeneChip®s was employed. We are currently comparing and analysing symbiotic interactions of perennial ryegrass (Lolium perenne) with endophytes N. lolii and E. festucae as well as comparing it against endophyte-free perennial ryegrass. Both N. lolii and E. festucae have been grown in culture in order to compare in planta versus in vitro gene expression. Additionally, targeted gene replacements in E. festucae have been performed and a comparative analysis of the knock-outs with wild-type E. festucae infected plants is in progress. These comparative analyses have revealed changes in gene expression which may lead to the identification of gene pathways/networks and the roles of these genes in symbiosis. Keywords: Neotyphodium, Epichloë, transcriptomics, symbiosis</jats:p