Gregory T. Bryan

Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass

Lolitrems are potent tremorgenic mycotoxins that are synthesised by clavicipitaceous fungal endophytes of the Epichloë/Neotyphodium group in association with grasses. These indole–diterpenes confer major ecological benefits on the grass–endophyte symbiotum. A molecular signature for diterpene biosynthesis is the presence of two geranylgeranyl diphosphate (GGPP) synthases. Using degenerate primers for conserved domains of fungal GGPP synthases, we cloned two such genes, ltmG and ggsA, from Neotyphodium lolii. Adjacent to ltmG are two genes, ltmM and ltmK, that are predicted to encode an FAD-dependent monooxygenase and a cytochrome P450 monooxygenase, respectively. The cluster of ltm genes is flanked by AT-rich retrotransposon DNA that appears to have undergone extensive repeat induced point (RIP) mutation. Epichloë festucae, the sexual ancestor of N. lolii, contains an identical ltm gene cluster, but lacks the retrotransposon “platform’‘ on the right flank. Associations established between perennial ryegrass and an E. festucae mutant deleted for ltmM lack detectable levels of lolitrems. A wild-type copy of ltmM complemented this phenotype, as did paxM from Penicillium paxilli. Northern hybridization and RT-PCR analysis showed that all three genes are weakly expressed in culture but strongly induced in planta. The relative endophyte biomass in these associations was estimated by real-time PCR to be between 0.3 and 1.9%. Taking this difference into account, the steady-state levels of the ltm transcripts are about 100-fold greater than the levels of the endogenous ryegrass β-tubulin (β -Tub1) and actin (Act1) RNAs. Based on these results we propose that ltmG, ltmM and ltmK are members of a set of genes required for lolitrem biosynthesis in E. festucae and N. lolii.

An Extracellular Siderophore Is Required to Maintain the Mutualistic Interaction of Epichloë festucae with Lolium perenne

We have identified from the mutualistic grass endophyte Epichloë festucae a non-ribosomal peptide synthetase gene (sidN) encoding a siderophore synthetase. The enzymatic product of SidN is shown to be a novel extracellular siderophore designated as epichloënin A, related to ferrirubin from the ferrichrome family. Targeted gene disruption of sidN eliminated biosynthesis of epichloënin A in vitro and in planta. During iron-depleted axenic growth, ΔsidN mutants accumulated the pathway intermediate N5-trans-anhydromevalonyl-N5-hydroxyornithine (trans-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of Lolium perenne (perennial ryegrass) with ΔsidN mutants resulted in perturbations of the endophyte-grass symbioses. Deviations from the characteristic tightly regulated synchronous growth of the fungus with its plant partner were observed and infected plants were stunted. Analysis of these plants by light and transmission electron microscopy revealed abnormalities in the distribution and localization of ΔsidN mutant hyphae as well as deformities in hyphal ultrastructure. We hypothesize that lack of epichloënin A alters iron homeostasis of the symbiotum, changing it from mutually beneficial to antagonistic. Iron itself or epichloënin A may serve as an important molecular/cellular signal for controlling fungal growth and hence the symbiotic interaction.

A novel family of cyclic oligopeptides derived from ribosomal peptide synthesis of an in planta-induced gene, gigA, in Epichloë endophytes of grasses.

Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum.

Gene expression profiling of the endophytic fungus Neotyphodium lolii in association with its host plant perennial ryegrass

Endophytes of the Neotyphodium/Epichloë complex are filamentous fungi that typically form mutualistic associations with temperate grasses. The endophytes systemically colonise the intercellular spaces of their grass hosts and confer several biotic and abiotic attributes, but can also cause mammalian toxicoses. These symbioses are therefore of significant agricultural importance, and furthermore, the symbioses represent models to understand how such symbioses are established and maintained. To gain a greater understanding of the Neotyphodium lolii-perennial ryegrass (Lolium perenne) symbiosis, we have generated, sequenced and analysed four in planta expressed sequence tag libraries, enriched for genes differentially expressed during symbiosis via suppression subtractive hybridisation. Subtracted libraries were largely comprised of perennial ryegrass sequences, and comparative functional profiling of endophyte-infected ryegrass libraries with endophyte-free ryegrass libraries revealed downregulation of carbohydrate metabolism and photosynthesis during symbiosis, the latter confirming observations made in previous studies. Functional categories up regulated in the plant host in association with endophyte infection included cellular protein transport and protein synthesis and turn over.We also identified 24 N. lolii transcripts expressed during symbiosis, some of which were homologous to demonstrated pathogenicity/virulence genes, and others with proven roles in endophyte secondary metabolism. This study offers insights into biological processes underlying the N. lolii-perennial ryegrass symbiosis, and provides a list of novel candidate genes from both symbionts, which will form the basis for future investigations.

Proteomic analysis of a filamentous fungal endophyte using EST datasets

Proteomic analysis of many species of fungi, particularly filamentous fungi, is difficult due to the lack of publicly available genome sequence data and the problems associated with cross‐species comparisons. Furthermore, the detection of fungal proteins in biological systems where there are a greater number of proteins present from other eukaryote species provides additional challenges. We present an EST‐based approach for identifying proteins from a fungal endophyte of temperate grasses and demonstrate that this method is well suited for fungi with minimal sequence data.

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.

Identification of a gene involved in the regulation of hyphal growth of Epichloë festucae during symbiosis

Secreted proteins, those involved in cell wall biogenesis, are likely to play a role in communication in the symbiotic interaction between the fungal endophyte Epichloë festucae with perennial ryegrass (Lolium perenne), particularly given the close association between fungal hyphae and the plant cell wall. Our hypothesis was that secreted proteins are likely to be responsible for establishing and maintaining a normal symbiotic relationship. We analyzed an endophyte EST database for genes with predicted signal peptide sequences. Here, we report the identification and characterization of rhgA; a gene involved in the regulation of hyphal growth in planta In planta analysis of ΔrhgA mutants showed that disruption of rhgA resulted in extensive unregulated hyphal growth. This phenotype was fully complemented by insertion of the rhgA gene and suggests that rhgA is important for maintaining normal hyphal growth during symbiosis.

Fluorescence chromosome banding and FISH mapping in perennial ryegrass, Lolium perenne L.

BackgroundThe unambiguous identification of individual chromosomes is a key part of the genomic characterization of any species. In this respect, the development and application of chromosome banding techniques has revolutionised mammalian and especially, human genomics. However, partly because of the traditional use of chromosome squash preparations, consistent fluorescence banding has rarely been achieved in plants. Here, successful fluorescence chromosome banding has been achieved for the first time in perennial ryegrass (Lolium perenne), a forage and turf grass with a large genome and a symmetrical karyotype with chromosomes that are difficult to distinguish.ResultsBased on flame-dried chromosome preparations instead of squashes, a simple fluorescence Q-banding technique using quinacrine mustard, unambiguously identified each chromosome and enabled the development of a banded karyotype and ideogram of the species. This Q-banding technique was also shown to be compatible with sequential FISH mapping enabling labelled genes and molecular markers to be precisely assigned to specific cytogenetic bands. A technique for DAPI-banding, which gave a similar pattern to Q-banding, was also introduced. This was compatible with FISH mapping and was used to anchor a single copy gene from an earlier mapped linkage group of L. perenne, thus providing a step towards integration of the genetic and cytogenetic maps.ConclusionsBy enabling the allocation of genes mapped by other methods to physically identified chromosome positions, this work will contribute to a better understanding of genomic structures and functions in grasses.