Epichloë seed transmission efficiency is influenced by plant defense response mechanisms.

Asexual Epichloë are endophytic fungi that form mutualistic symbioses with cool-season grasses, conferring to their hosts protection against biotic and abiotic stresses. Symbioses are maintained between grass generations as hyphae are vertically transmitted from parent to progeny plants through seed. However, endophyte transmission to the seed is an imperfect process where not all seeds become infected. The mechanisms underpinning the varying efficiencies of seed transmission are poorly understood. Host gene expression in response to Epichloë sp. LpTG-3 strain AR37 was examined within inflorescence primordia and ovaries of high and low endophyte transmission genotypes within a single population of perennial ryegrass. A genome-wide association study was conducted to identify population-level single nucleotide polymorphisms (SNPs) and associated genes correlated with vertical transmission efficiency. For low transmitters of AR37, upregulation of perennial ryegrass receptor-like kinases and resistance genes, typically associated with phytopathogen detection, comprised the largest group of differentially expressed genes (DEGs) in both inflorescence primordia and ovaries. DEGs involved in signaling and plant defense responses, such as cell wall modification, secondary metabolism, and reactive oxygen activities were also abundant. Transmission-associated SNPs were associated with genes for which gene ontology analysis identified "response to fungus" as the most significantly enriched term. Moreover, endophyte biomass as measured by quantitative PCR of Epichloë non-ribosomal peptide synthetase genes, was significantly lower in reproductive tissues of low-transmission hosts compared to high-transmission hosts. Endophyte seed-transmission efficiency appears to be influenced primarily by plant defense responses which reduce endophyte colonization of host reproductive tissues.

Seed Transmission of Epichloë Endophytes in Lolium perenne Is Heavily Influenced by Host Genetics.

Vertical transmission of symbiotic Epichloë endophytes from host grasses into progeny seed is the primary mechanism by which the next generation of plants is colonized. This process is often imperfect, resulting in endophyte-free seedlings which may have poor ecological fitness if the endophyte confers protective benefits to its host. In this study, we investigated the influence of host genetics and environment on the vertical transmission of Epichloë festucae var. lolii strain AR37 in the temperate forage grass Lolium perenne. The efficiency of AR37 transmission into the seed of over 500 plant genotypes from five genetically diverse breeding populations was determined. In Populations I-III, which had undergone previous selection for high seed infection by AR37, mean transmission was 88, 93, and 92%, respectively. However, in Populations IV and V, which had not undergone previous selection, mean transmission was 69 and 70%, respectively. The transmission values, together with single-nucleotide polymorphism data obtained using genotyping-by-sequencing for each host, was used to develop a genomic prediction model for AR37 seed transmission. The predictive ability of the model was estimated at r = 0.54. While host genotype contributed greatly to differences in AR37 seed transmission, undefined environmental variables also contributed significantly to seed transmission across different years and geographic locations. There was evidence for a small host genotype-by-environment effect; however this was less pronounced than genotype or environment alone. Analysis of endophyte infection levels in parent plants within Populations I and IV revealed a loss of endophyte infection over time in Population IV only. This population also had lower average tiller infection frequencies than Population I, suggesting that AR37 failed to colonize all the daughter tillers and therefore seeds. However, we also observed that infection of seed by AR37 may fail during or after initiation of floral development from plants where all tillers remained endophyte-infected over time. While the effects of environment and host genotype on fungal endophyte transmission have been evaluated previously, this is the first study that quantifies the relative impacts of host genetics and environment on endophyte vertical transmission.

cAMP Signaling Regulates Synchronised Growth of Symbiotic Epichloë Fungi with the Host Grass Lolium perenne.

The seed-transmitted fungal symbiont, Epichloë festucae, colonizes grasses by infecting host tissues as they form on the shoot apical meristem (SAM) of the seedling. How this fungus accommodates the complexities of plant development to successfully colonize the leaves and inflorescences is unclear. Since adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent signaling is often essential for host colonization by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA). Consistent with deletions of this gene in other fungi, acyA mutants had a slow radial growth rate in culture, and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium (NBT) staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesize superoxide, indicating that cAMP signaling regulates accumulation of reactive oxygen species (ROS). Despite significant defects in hyphal growth and ROS production, E. festucae ΔacyA mutants were infectious and capable of forming symbiotic associations with grasses. Plants infected with E. festucae ΔacyA were marginally less robust than the wild-type (WT), however hyphae were hyper-branched, and leaf tissues heavily colonized, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires functional cAMP signaling.