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

Abstract

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 beta-tubulin (beta -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.

Reactive Oxygen Species Play a Role in Regulating a Fungus–Perennial Ryegrass Mutualistic Interaction

“…This experiment showed that the level of b-tubulin transcript in planta was ;1/ 80th the level in mycelia ( Figure 6A), a result consistent with estimates of endophyte biomass in perennial ryegrass of between 0.4 and 1.9% (Panaccione et al, 2001;Young et al, 2005). Semiquantitative RT-PCR expression analysis showed that noxA is weakly expressed in axenic culture but strongly expressed in planta ( Figure 6A).…”

“…Real-time quantitative PCR determination of endophyte DNA biomass in perennial ryegrass associations infected with Fl1 wild type or the A44 DnoxA mutant was performed using primers endo1 and endo2, as described previously (Young et al, 2005).…”

“…Protoplasts of E. festucae were prepared as described previously (Young et al, 1998(Young et al, , 2005. Protoplasts were transformed with 5 mg of either circular (pPN74 and pPN82) or linear PCR-amplified product (primers M13F and M13R for pPN75, pII99-2 and pII99-3 for pPN78) or cotransformed with 5 mg of circular pII99 and 10 mg of pPN83 using the method described previously (Itoh et al, 1994).…”

“…Protection of the symbiotum from insect and small animal herbivory through endophyte synthesis of bioprotective secondary metabolites (alkaloids) is the best-studied benefit of this association (Bush et al, 1997;Lane et al, 2000). Genes for the biosynthesis of the four main classes of bioprotective alkaloids have now been cloned, including genes for the biosynthesis of ergot alkaloids (Panaccione et al, 2001;Wang et al, 2004), lolines (Spiering et al, 2002(Spiering et al, , 2005, peramine (Tanaka et al, 2005), and indole-diterpenes (Young et al, 2005). Expression studies have shown that the genes for all four pathways are preferentially and highly expressed in planta (Spiering et al, 2002;Tanaka et al, 2005;Young et al, 2005), suggesting that host-specific signaling is required for the induction of these pathways.…”

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

“…Initially, employing Penicillium paxilli as a model experimental system to dissect the biochemistry of indole-diterpene biosynthesis ) (an obvious parallel can be drawn with the ergots and their model synthesizer: Claviceps purpurea) allowed for the isolation of three orthologous genes from the grass endophytes, Neotyphodium lolii and Epichloe¨festucae, and subsequent confirmation of their contiguous clustering (Young et al 2005). While further attempts to clone additional linked ltm genes in the endophytes by such approaches as inverse PCR were thwarted by the presence of large blocks of highly repetitive retrotransposon sequences flanking both sides of the cluster, an alternative strategy combining suppression subtractive hybrydization (a technique applied successfully in case of LOL characterization) with chromosome walking was taken up.…”