Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis

Abstract

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event

“…The AGL1 and LBA 4404 strains of A. tumefaciens which were used for transformation, and Escherichia coli strain DH5α (Thermo Fisher Scientific, Walthman, MA, USA), which was used during construction and maintenance of plasmid constructs, were grown at 28 and 37 • C, respectively, on either LB (Luria-Bertani) agar (5 g yeast extract, 10 g tryptone, 10 g NaCl in 1L of ddH 2 O) plates or in LB broth. PDA and LB media were supplemented with appropriate antibiotics as necessary [35].…”

“…Gene cassettes including the constitutive Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and the A. nidulans tryptophan biosynthesis terminator (trpCT), for cloning of the first reading frame A (RFA-A) cassette; the hygromycin B resistance gene (hph) under the control of the A. nidulans trpC promoter (trpCP) and terminator; and reporter genes including DsRed (DsRed-Express 736 bp, gb|DQ232603.1|), sgfp (gb|EF090408.1|), egfp (gb|HQ259114.1|) containing attB1 and attB2 sites were synthesised and obtained from GeneArt, Regensburg, Germany. The Gateway™-enabled destination vector (pEND0002) was constructed through modifications of the T-DNA region of pPZP200 [36] by cloning the 2.1 kb XbaI/KpnI fragment containing the hph expression cassette and 2.9 kb gpdP-(RFA-A)-trpCT cassette (Invitrogen, Thermo Fisher Scientific, Walthman, MA, USA) as previously described in [35].…”

“…The sensitivity of non-transformed endophyte mycelia to treatment with hygromycin B were determined by plating 400 µL of liquid culture onto PDA plates containing increasing concentrations of antibiotic (50,100,150,200,250, and 300 µg/mL) as previously described [35].…”

“…A. tumefaciens-mediated transformation of endophyte strains NEA12 and E1 was performed using A. tumefaciens cells (AGL1 and LBA 4404) as previously described in [35]. Control experiments were performed by co-cultivation of endophyte mycelia with strains of A. tumefaciens that had not been transformed with the plasmid vectors.…”

Novel bioassay to assess antibiotic effects of fungal endophytes on aphids

“…Developing endophyte strains that produce favourable quantities and types of alkaloids that remain harmless to grazing animals but retain the toxicity towards invertebrates has been the focus of recent research, particularly in Australia with Epichloë festucae var. lolii in perennial ryegrass [21][22][23].…”