Fungal endophytes provide grasses with enhanced protection from herbivory, drought, and pathogens. The loline alkaloids (saturated 1-aminopyrrolizidines with an oxygen bridge) are fungal metabolites often present in grasses with fungal endophytes of the genera Epichloë or Neotyphodium. We conducted a Mendelian genetic analysis to test for activity of lolines produced in plants against aphids feeding on those plants. Though most loline-producing endophytes are asexual, we found that a recently described sexual endophyte, Epichloë festucae, had heritable variation for loline alkaloid expression (Lol+) or nonexpression (Lol-). By analyzing segregation of these phenotypes and of linked DNA polymorphisms in crosses, we identified a single genetic locus controlling loline alkaloid expression in those E. festucae parents. We then tested segregating Lol+ and Lol- full-sibling fungal progeny for their ability to protect host plants from two aphid species, and observed that alkaloid expression cosegregated with activity against these insects. The in planta loline alkaloid levels correlated with levels of anti-aphid activity. These results suggested a key role of the loline alkaloids in protection of host plants from certain aphids, and represent, to our knowledge, the first Mendelian analysis demonstrating how a fungal factor contributes protection to plant-fungus mutualism.

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Loline alkaloids are saturated pyrrolizidines with an oxygen bridge between carbon atoms C-2 and C-7 and an amino group on C-1. They are bioprotective alkaloids produced by Epichloë and Neotyphodium species, mutualistic fungal endophytes that are symbiotic with cool-season grasses. The sequence of bond formation in loline alkaloid biosynthesis was determined by synthesizing deuterated forms of potential intermediates and feeding them to cultures of the endophyte Neotyphodium uncinatum. These cultures incorporated deuterium from labeled N-(3-amino-3-carboxypropyl)proline and exo-1-aminopyrrolizidine into N-formylloline. The first result suggests that N-(3-amino-3-carboxypropyl)proline is the first committed intermediate in loline biosynthesis, and the second result demonstrates that the pyrrolizidine rings form before the ether bridge. The incorporation of these two compounds into lolines and the lack of incorporation of several related compounds clarify the order of bond formation in loline alkaloid biosynthesis.

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Loline alkaloids are saturated pyrrolizidines with a substituted 1-amino group and an oxygen bridge between C2 and C7, and are insecticidal metabolites of plant-symbiotic fungi (endophytes). Cultures of the endophyte, Neotyphodium uncinatum, incorporated labeled L-proline and L-homoserine into the 1-aminopyrrolizidine, N-formylloline. The A-ring carbons C1-C3 and the N1 were derived from L-homoserine; the B-ring carbons C5-C8 and the ring nitrogen were derived from L-proline. Incorporation of both deuterium atoms from L-[4,4-(2H2)]homoserine and feeding tests with labeled L-methionine indicated that L-homoserine incorporation was not achieved via aspartyl semialdehyde or S-adenosylmethionine, but probably involved a highly novel N--C bond-forming gamma-substitution reaction.