Natasha Forester

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand’s agricultural economy by conferring biotic advantages to the host grass. Epichloë endophyte strain AR37 provides ryegrass with improved agronomic performance, insect protection and plant persistence. Indole diterpenes are a class of well characterised secondary metabolites produced by Epichloë endophytes, with the AR37 strain expressing epoxyjanthitrems, a class of decorated indole diterpenes. These have been associated with the observed effects of AR37 on livestock and insect pests. Here we used gene inactivation by CRISPR-Cas9 to deconvolute the genetic basis for epoxyjanthitrem biosynthesis and manipulate this secondary metabolite pathway to reduce or remove endophyte-induced mammalian toxicity whilst retaining activity against some important agricultural insect pests. We also show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) and that these gene edits can be classified as site-directed nuclease 1 (SDN-1), which are not regulated as genetically modified organisms in selected jurisdictions, including Australia. These SDN-1 technology edits have been successfully grown out of containment in the field in Australia for seed multiplication, followed by agronomy trials, which are now being evaluated. This has the potential to provide a step change in the future use of animal safe Epichloë strains in New Zealand pastures which will significantly reduce chemical inputs and increase animal welfare.

Observations taken over decades of seed production, and examination of anther tissues from Epichloë infected grasses suggest that Epichloë endophytes are vertically transmitted through seed and are not horizontally transferred by pollen to new host plants. However, no study has quantified the potential for Epichloë transmission via pollen. We studied the incidence of Epichloë DNA detection in pooled pollen samples collected from subsets of 1680 Lolium perenne plants undergoing seed multiplications in a containment glasshouse. Most plants were infected with one of several Epichloë strains, representing different species of Epichloë. These data were compared with the incidence of Epichloë DNA in anthers, pseudostem and seed with known infection status. Sensitive qualitative and quantification assays, high resolution melting analysis and digital droplet PCR were employed to detect and/or quantify either Epichloë or Lolium DNA within plant-host tissues. Epichloë DNA sequences were not detected in any pollen samples tested. Epichloë is therefore unlikely to be spread between plants via pollen. In countries such as New Zealand, where genetically modified organisms are regulated, risks of potential spread into the environment associated with maternal compared to paternal transmission may affect regulatory decisions regarding the use of such Epichloë-grass symbioses outside of physical containment facilities.

Facial eczema (FE) in grazing ruminants is a debilitating liver syndrome induced by ingestion of sporidesmin, a toxin belonging to the epipolythiodioxopiperazine class of compounds. Sporidesmin is produced in spores of the fungus Pseudopithomyces chartarum , a microbe which colonises leaf litter in pastures. New Zealand has a high occurrence of FE in comparison to other countries as animals are fed predominantly on ryegrass, a species that supports high levels of Pse. chartarum spores. The climate is also particularly conducive for Pse. chartarum growth. Here, we present the genome of Pse. chartarum and identify the putative sporidesmin gene cluster. The Pse. chartarum genome was sequenced using single molecule real-time sequencing (PacBio) and gene models identified. Loci containing genes with homology to the aspirochlorine, sirodesmin PL and gliotoxin cluster genes of Aspergillus oryzae, Leptosphaeria maculans and Aspergillus fumigatus , respectively, were identified by tBLASTn. We identified and annotated an epipolythiodioxopiperazine cluster at a single locus with all the functionality required to synthesise sporidesmin. Highlights: The whole genome of Pseudopithomyces chartarum has been sequenced and assembled. The genome is 39.13 Mb, 99% complete, and contains 11,711 protein coding genes. A putative sporidesmin A toxin (cause of facial eczema) gene cluster is described. The genomes of Pse. chartarum and the Leptosphaerulina chartarum teleomorph differ. Comparative genomics is required to further resolve the Pseudopithomyces clade.

In ascomycetes and basidiomycetes, iron-responsive GATA-type transcriptional repressors are involved in regulating iron homeostasis, notably to prevent iron toxicity through control of iron uptake. To date, it has been unknown whether this iron regulator contributes toward mutualistic endosymbiosis of microbes with plants, a system where the endophyte must function within the constraints of an in-host existence, including a dependency on the host for nutrient acquisition. Functional characterization of one such protein, SreA from Epichloë festucae, a fungal endosymbiont of cool-season grasses, indicates that regulation of iron homeostasis processes is important for symbiotic maintenance. The deletion of the sreA gene (ΔsreA) led to iron-dependent aberrant hyphal growth and the gradual loss of endophyte hyphae from perennial ryegrass. SreA negatively regulates the siderophore biosynthesis and high-affinity iron uptake systems of E. festucae, similar to other fungi, resulting in iron accumulation in mutants. Our evidence suggests that SreA is involved in the processes that moderate Epichloë iron acquisition from the plant apoplast, because overharvesting of iron in ΔsreA mutants was detected as premature chlorosis of the host using a hydroponic plant growth assay. E. festucae appears to have a tightly regulated iron management system, involving SreA that balances endophyte growth with its survival and prevents overcompetition with the host for iron in the intercellular niche, thus promoting mutualistic associations. Mutations that interfere with Epichloë iron management negatively affect iron-dependent fungal growth and destabilize mutualistic Epichloë –ryegrass associations.