Academic journal article Genetics

Gene Clusters for Insecticidal Loline Alkaloids in the Grass-Endophytic Fungus Neotyphodium Uncinatum

Academic journal article Genetics

Gene Clusters for Insecticidal Loline Alkaloids in the Grass-Endophytic Fungus Neotyphodium Uncinatum

Article excerpt

ABSTRACT

Loline alkaloids are produced by mutualistic fungi symbiotic with grasses, and they protect the host plants from insects. Here we identify in the fungal symbiont, Neotyphodium uncinatum, two homologous gene clusters (LOL-1 and LOL-2) associated with loline-alkaloid production. Nine genes were identified in a 25-kb region of LOL-1 and designated (in order) lolF-1, lolC-1, lolD-1, lolO-1, lolA-1, lolU-1, lolP-1, lolT-1, and lolE-1. LOL-2 contained the homologs lolC-2 through lolE-2 in the same order and orientation. Also identified was lolF-2, but its possible linkage with either cluster was undetermined. Most lol genes were regulated in N. uncinatum and N. coenophialum, and all were expressed concomitantly with loline-alkaloid biosynthesis. A lolC-2 RNA-interference (RNAi) construct was introduced into N. uncinatum, and in two independent transformants, RNAi significantly decreased lolC expression (P < 0.01) and loline-alkaloid accumulation in culture (P < 0.001) compared to vector-only controls, indicating involvement of lolC in biosynthesis of lolines. The predicted LolU protein has a DNA-binding site signature, and the relationships of other lol-gene products indicate that the pathway has evolved from various different primary and secondary biosynthesis pathways.

SEED-BORNE endophytic fungi-specifically the Epichloë species (asexual states, Neotyphodium species)-in symbiosis with cool-season grasses (Poaceae subfam. Poöideae) can impart to those grasses a variety of fitness enhancements including resistance to vertebrate and invertebrate herbivores, resistance to pathogens and parasites, enhanced phosphate uptake and nitrogen utilization, and increased tolerance of drought and heat (BUSH et al. 1997; MALINOWSKI and BELESKY 2000; SCHARDL et al 2004). Loline alkaloids produced by several Neotyphodium and Epichloë species are potent, broad-spectrum insecticides (RIEDELL et al. 1991; DOUGHERTY et al. 1998; WILKINSON et al. 2000), have little or no antimammalian activities (JACKSON et al. 1996), and sometimes accumulate in the plant to levels up to 20 mg g~! plant dry weight (DW; CRAVEN et al. 2001). These alkaloids have an unusual structure, comprising a saturated 1-aminopyrrolizidine-ring system, with a highly strained ether bridge between C-2 and C-7 (PETROSKI et al. 1989). Lolines are almost exclusively found in these grass-endophyte symbioses (HARTMANN and WITTE 1995; BUSH etal. 1997); outside of the grasses they have been identified in only a few plant species in the families Fabaceae and Convolvulaceae (HARTMANN and WITTE 1995; TOFERN et al. 1999). Genetic tests have confirmed linkage between capability of endophytes to produce lolines in planta and their bioprotective effects against aphids (WiLKiNSON et al. 2000). Improved survival under drought and/or competition of grass-endophyte symbiota possessing lolines has raised the possibility of additional roles of these alkaloids in host plant fitness and persistence (MALINOWSKI and BELESKY 2000; SCHARDL et al. 2004).

The biochemical pathway for the lolines is so far unknown. On the basis of certain structural similarities of lolines with plant pyrrolizidines (HARTMANN and WITTE 1995), BUSH et al. (1993) proposed that lolines are produced by a pathway involving polyamines such as spermidine. However, results of precursor feeding studies have now ruled out this possibility and suggest that lolines are formed by a novel biosynthetic pathway from the amino acids L-proline and L-homoserine (BLANKENSHIP et al. 2005).

We have previously identified two genes, lolA and lolC, showing strong upregulation in loline-alkaloid-producing Neotyphodium uncinatum cultures and strict association with in symbio loline-alkaloid-production phenotypes among isolates of different endophyte species (SPIERING et al. 2002). In this study we show that two gene clusters in the genome of N. uncinatum contain homologs of lolA and lolC, along with several additional genes whose predicted products showed relationships to enzymes typical of primary or secondary metabolic pathways. …

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