Brian A. Tapper

A symbiosis expressed non‐ribosomal peptide synthetase from a mutualistic fungal endophyte of perennial ryegrass confers protection to the symbiotum from insect herbivory

While much is known about the biosynthesis of secondary metabolites by filamentous fungi their biological role is often less clear. The assumption is these pathways have adaptive value to the organism but often the evidence to support this role is lacking. We provide the first genetic evidence that the fungal produced secondary metabolite, peramine, protects a host plant from insect herbivory. Peramine is a potent insect feeding deterrent synthesized by Epichloë/Neotyphodium mutualistic endophytes in association with their grass hosts. The structure of peramine, a pyrrolopyrazine, suggests that it is the product of a reaction catalysed by a two‐module non‐ribosomal peptide synthetase (NRPS). Candidate sequences for a peramine synthetase were amplified by reverse transcription polymerase chain reaction. Four unique NRPS products were identified, two of which were preferentially expressed in planta. One of these hybridized to known peramine producing strains. This clone was used to isolate an Epichloë festucae cosmid that contained a two‐module NRPS, designated perA. Nine additional genes, which show striking conservation of microsynteny with Fusarium graminearum and other fungal genomes, were identified on the perA‐containing cosmid. Associations between perennial ryegrass and an E. festucae mutant deleted for perA lack detectable levels of peramine. A wild‐type copy of perA complemented the deletion mutant, confirming that perA is a NRPS required for peramine biosynthesis. In a choice bioassay, plant material containing the perA mutant was as susceptible to Argentine stem weevil (ASW) (Listronotus bonariensis) feeding damage as endophyte‐free plants confirming that peramine is the E. festucae metabolite responsible for ASW feeding deterrent activity.

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.

The exploitation of epichloae endophytes for agricultural benefit

Epichloae endophytes of family Clavicipitaceae (comprising genera Epichloë and Neotyphodium) are fungal symbionts of Pooideae grasses. The associations formed, range from mutually beneficial to antagonistic and the nature of this relationship is dependent upon the importance of vertical (via host seeds) versus horizontal (ascospore mediated) transmission of the fungus. These endophytes can enhance their hosts’ survival through protection from abiotic and biotic stresses and can thus be utilized in an agricultural context. Animal-safe grass-endophyte associations that confer bio-protective properties for increased pasture persistence and productivity have been developed and commercialized. One of the crucial drivers underpinning the selection of epichloae strains for commercial development is endophyte derived bioactivity. The potential of next generation endophytes is determined by testing a number of attributes such as agronomic fitness, animal and food safety as well as compatibility with host plants of interest. Strategic research supports these activities by focusing on elucidating mechanisms of compatibility between host and fungal symbiont, as well as investigating other molecular drivers of symbiosis such as siderophore mediated iron-uptake, fungal signalling, fungal growth in host plants and fungal secondary metabolism. This review weaves together the different strands of multidisciplinary research aimed at ultimately exploiting epichloae endophytes for increased pasture performance.

Indole-Diterpene Biosynthetic Capability of Epichloë Endophytes as Predicted by ltm Gene Analysis

ABSTRACT Bioprotective alkaloids produced by Epichloë and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloë festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloë endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.

Effect of Growth Conditions on Alkaloid Concentrations in Perennial Ryegrass Naturally Infected with Endophyte

A preliminary study of the effect of water stress on alkaloid concentrations in endophyte-infected perennial ryegrass (Lolium perenne) focused on artificial infections with selected endophyte strains (Barker et al., 1993). This paper reports the results of studies of the impact of environmental factors (nitrogen, water, temperature) on the concentration of alkaloids in “Nui” perennial ryegrass naturally infected with Neotyphodium lolii, a more typical component of New Zealand pastoral forage.

Functional analysis of an indole‐diterpene gene cluster for lolitrem B biosynthesis in the grass endosymbiont Epichloë festucae

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole‐diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub‐telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole‐diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.

The effect of endophyte on the performance of irrigated perennial ryegrasses in subtropical Australia

The effect of fungal endophyte (Neotyphodium lolii) infection on the performance of perennial ryegrass (Lolium perenne) growing under irrigation in a subtropical environment was investigated. Seed of 4 cultivars, infected with standard (common toxic or wild-type) endophyte or the novel endophyte AR1, or free of endophyte (Nil), was sown in pure swards, which were fertilised with 50 kg N/ha.month. Seasonal and total yield, persistence, and rust susceptibility were assessed over 3 years, along with details of the presence of endophyte and alkaloids in plant shoots. Endophyte occurrence in tillers in both the standard and AR1 treatments was above 95% for Bronsyn and Impact throughout and rose to that level in Samson by the end of the second year. Meridian AR1 only reached 93% while, in the standard treatment, the endophyte had mostly died before sowing. Nil Zendophyte treatments carried an average of ?0.6% infection throughout. Infection of the standard endophyte was associated with increased dry matter (DM) yields in all 3 years compared with no endophyte. AR1 also significantly increased yields in the second and third years. Over the full 3 years, standard and AR1 increased yields by 18% and 11%, respectively. Infection with both endophytes was associated with increased yields in all 4 seasons, the effects increasing in intensity over time. There was 27% better persistence in standard infected plants compared with Nil at the end of the first year, increasing to 198% by the end of the experiment, while for AR1 the improvements were 20 and 134%, respectively. The effect of endophyte on crown rust (Puccinia coronata) infection was inconsistent, with endophyte increasing rust damage on one occasion and reducing it on another. Cultivar differences in rust infection were greater than endophyte effects. Plants infected with the AR1 endophyte had no detectable ergovaline or lolitrem B in leaf, pseudostem, or dead tissue. In standard infected plants, ergovaline and lolitrem B were highest in pseudostem and considerably lower in leaf. Dead tissue had very low or no detectable ergovaline but high lolitrem B concentrations. Peramine concentration was high and at similar levels in leaf and pseudostem, but not detectable in dead material. Concentration was similar in both AR1 and standard infected plants. Endophyte presence appeared to have a similar effect in the subtropics as has been demonstrated in temperate areas, in terms of improving yields and persistence and increasing tolerance of plants to stress factors.

Effects of natural reseeding and establishment method on contamination of a novel endophyte-infected perennial ryegrass dairy pasture with other ryegrass/endophyte associations

Abstract The contamination of a perennial ryegrass (Lolium perenne) dairy pasture infected with a novel endophyte with other ryegrass/endophyte associations was studied over 2 years in Hamilton, New Zealand. Five management treatments (turnip crop, usual rotational grazing, usual rotational grazing followed by topping, silage making, hay making) were applied to produce a range of natural reseeding levels, and then three establishment methods (spray/cultivation, double‐spray/fallow, hard‐grazing) were used before drilling AR1 endophyte‐infected perennial ryegrass (no lolitrem B or ergovaline production). Contamination from wild endophyte (Neotyphodium lolii)‐infected perennial ryegrass, endophyte‐free perennial ryegrass, wild endophyte‐infected hybrid (L. boucheanum syn. L. hybridum)/Italian ryegrass (L. multiflorum), N. occultans endophyte‐infected hybrid/Italian ryegrass and endophyte‐free hybrid/Italian ryegrass tillers was determined. Contamination was assessed in the two summers following the autumn (May 2000) sowing by measuring lolitrem B concentration in bulk ryegrass samples and by sampling individual ryegrass tillers for endophyte identification in late summer of Year 1. Within the hard‐grazed establishment treatment, the percentage of ryegrass tillers infected with AR1 in late summer of Year 1 was highest (73%) following the turnip crop pre‐sowing treatment and lowest (4%) after hay making (SED = 5.3%, P < 0.001). The percentages of AR1‐in‐fected perennial ryegrass tillers in pastures established following spray/cultivation and double‐spray/ fallow treatments were 86 and 76%, respectively, compared with only 15% in the hard‐grazed treatment (SED = 2.5, P < 0.001) (turnips excluded). The spray/cultivation and double‐spray/fallow establishment treatments consistently had lower concentrations of lolitrem B in bulk ryegrass samples than the hard‐grazed treatment, for example in early autumn of Year 1 (0.3, 0.5, 1.1 mg kg−1 DM, P < 0.001) and Year 2 (0.1, 0.2, 1.0 mg kg−1 DM, P < 0.001). Pasture production from August 2000 to August 2001 was similar following the spray/cultivation, double‐spray/fallow and hard‐grazed establishment treatments, averaging 14.9t DM ha−1. It is recommended that ryegrass pastures containing a novel endophyte be sown after a summer crop, or following a spray/ cultivation or double‐spray/fallow establishment method, instead of under‐sowing into existing hard‐grazed pasture. Methodology for assessing pasture contamination with different ryegrass/endophyte associations is also discussed.

Occurrence of Extreme Alkaloid Levels in Endophyte-Infected Perennial Ryegrass, Tall Fescue, and Meadow Fescue

In the course of measurements of alkaloid concentrations in several thousand samples of endophyte-infected perennial ryegrass, tall fescue, and meadow fescue plants for diverse studies, we have encountered a number of samples with alkaloid concentrations which greatly exceed the highest levels reported in previous studies (e.g. Welty et al., 1994).

Deletion and gene expression analyses define the paxilline biosynthetic gene cluster in Penicillium paxilli.

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.