Christine R. Voisey

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

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.

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

SummaryWhite clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and β-glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.

Expression of the soybean (Kunitz) trypsin inhibitor in transgenic tobacco: Effects on larval development of Spodoptera litura

The coding region of the Tia allelic form of the soybean (Kunitz) trypsin inhibitor gene has been introduced, as a transcriptional fusion with the CAMV 35S promoter, into tobacco. Southern analysis of DNA extracted from progeny (Fl) plants confirmed that an intact copy (or copies) of the gene is integrated into the tobacco genome. Gel filtration column chromatography has been used to partially purify the inhibitor from leaves of transgenic tobacco and inhibition assays revealed that the protein can inhibit both bovine trypsin, and trypsin‐like (BApNA‐hydrolysing) activity extracted from Spodoptera litura digestive tracts. SDS‐PAGE and western blotting determined that the inhibitor accumulates as a protein of ca. 20 kD in transgenic leaf tissue. The protein has been purified to homogeneity using reverse‐phase column chromatography, and subsequent N‐terminal sequencing revealed that the inhibitor is processed in tobacco leaf tissue by the removal of the N‐terminal leader sequence. Insect feeding trials, using neonate larvae of S. litura, have been conducted with leaf tissue excised from transgenic progeny plants that either accumulated the inhibitor or from control (non‐transgenic) plants. These trials established that, when compared with insects fed non‐transformed leaf tissue, larvae fed transgenic leaf tissue demonstrated significantly greater mortality, and the survivors grew more slowly in terms of weight gain over time. These results are interpreted with respect to current opinion on the use of proteinase inhibitors as insect pest resistance factors in transgenic plants.

Prolific direct plant regeneration from cotyledons of white clover

SummaryA facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1-1 6-benzylaminopurine and 0.05 mg 1-1 α-napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.

Release of transgenic white clover plants expressing Bacillus thuringiensis genes: An ecological perspective

The destructive effects of indigenous insects on pasture plants are a significant cause of diminished pastoral productivity in New Zealand. Identification of white clover genotypes with natural immunity against native insects has not been successful, so farmers use chemical sprays to control pest populations. An alternative strategy to reduce insect damage to white clover is through development of a transformation system using Agrobacterium‐mediated transfer DNA. We have developed a rapid procedure, where transgenic white clover plants regenerate directly from the cotyledonary axil of germinated seedlings and can be transferred into the glasshouse within 42 days of culture initiation. This technology offers the opportunity to improve the sustainability of white clover in New Zealand pastures by introducing pest resistance genes directly into these plants. Insect feeding bioassays have identified several serotypes of Bacillus thuringiensis (Bt) with δ‐endotoxins active against larvae of the lepidopteran pe...

Mutualistic fungal endophytes in the Triticeae – survey and description

Grasses of the tribe Triticeae were screened to determine the presence of mutualistic epichloae fungal endophytes. Over 1500 accessions, from more than 250 species, encompassing 22 genera within the Triticeae were screened using immunodetection and direct staining/microscopy techniques. Only two genera, Elymus and Hordeum, were identified as harbouring epichloae endophytes with accessions native to a range of countries including Canada, China, Iran, Kazakhstan, Kyrgyzstan, Mongolia, Russia and the USA. Genetic analysis based on simple sequence repeat data revealed that the majority of endophytes cluster according to geographical regions rather than to host species; many strains isolated from Hordeum grouped with those derived from Elymus, and amongst the Elymus-derived strains, there was no clear correspondence between clustering topology and host species. This is the first detailed survey demonstrating the genetic diversity of epichloae endophytes within the Triticeae and highlights the importance of germplasm centres for not only preserving the genetic diversity of plant species but also the beneficial microorganisms they may contain.

Transgenic pest and disease resistant white clover plants.

Both insect pests and viral diseases have significant negative impacts on the yield and persistence of forage legumes, and often it is difficult to develop resistant germplasm using traditional plant breeding approaches. We illustrate some of the principles for the molecular breeding of forage legumes, by demonstrating how cloned resistance genes can be used to produce transgenic white clover plants, resistant to either an insect pest (Wiseana spp.) or white clover mosaic virus (WC1MV). Insect bioassays identified a Bacillus thuringiensis Cry1Ba toxin and the proteinase inhibitor, BPTI, as possible resistance factors for Wiseana larvae. Transgenic white clover plants expressing a modified crylBa gene, altered to give an increased proportion of G/C bases and mimic the codon use of plant genes, were toxic to feeding Wiseana larvae. While larvae feeding on white clover expressing the BPTI transgene survived, their growth rate was significantly reduced. Our strategy to develop resistance to WC1MV has been to express individual WC1MV genes in transgenic white clover plants. Expression of the coat protein gene of WC1MV in white clover gave low levels of resistance to the virus, but a substantial delay to systemic infection. Expression of a mutated form of the WC1MV 13K movement protein gene in white clover, resulted in plants with a high level of resistance and restricted systemic spread of the virus. Five of 22 transgenic white clover plants designed to express the WC1MV replicase gene showed immunity to WC1MV infection.

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.