Figure 1.
Structures of common loline alkaloids.
Substitutions on the nitrogen at C1 differentiate the lolines.
Table 1.
Fungal isolates in this study.
Figure 2.
Enrichment of deuterated N-acetylnorloline (NANL) from application of tetradeuterated exo-1-acetemidopyrrolizidine ([2H4]AcAP) to loline alkaloid producing culture.
Shown are (A) GC-MS total ion chromatogram, (B) mass spectrum at retention time 13.948 and (C) 13.921 min, and (D) proposed scheme of [2H4]NANL formation from [2H4]AcAP.
Figure 3.
Replacement of lolN and lolM with hph maker gene.
(A) Schematic representation of lolN-lolM replacement by the hph marker gene via homologous recombination. Shown are maps of the wild-type lolN and lolM in Epichloë festucae E2368 (WT), targeting vector (pKAES323), and the locus after homologous recombination (KO). Black bars represent DNA sequence, and filled arrows represent genes. Bent blue lines on the bars represent HindIII digestion sites. Colored arrowheads represent primers used to generate pKAES323 and to screen the transformants. (B) Southern-blot analysis of E. festucae strains. Wild-type E2368 and transformants were probed with a lolN fragment or lolM gene amplified from E2368 (old probe was stripped off the membrane before new hybridization). Lanes contained HindIII-digested genomic DNA from E2368 (WT), lolN-lolM knockout transformant (KO), ectopic transformant of E2368 with pKAES323 (Ect), and E2368 transformed with the empty vector pKAES173 (WT+vec).
Figure 4.
GC-MS traces showing loline-alkaloid profiles of meadow fescue symbiotic with different E. festucae strains.
(A) The lolN-lolM knockout (KO), (B) an empty-vector control transformant (WT+vec), and (C and D) complementation strains (KO+lolN+lolM). The numbers after complementation strains represent different meadow fescue plants inoculated with independent transformants. (E) Proposed roles of LolN and LolM (this work), and reported role of LolP [14], in the biosynthetic pathway from N-acetylnorloline (NANL) to the final product, N-formylloline (NFL).
Table 2.
Loline alkaloid profiles and LOL-gene screening results for endophyte isolates. a
Figure 5.
Partial LolN amino-acid sequence alignment of Epichloë coenophiala e4309 and N-formylloline (NFL) producers.
Red-framed sequences are three different E. coenophiala isolates. Ecoe = Epichloë coenophiala, Eaot = Epichloë aotearoae, Efes = Epichloë festucae, Echis = Epichloë chisosa, Esig = Epichloë siegelii, Eunci = Epichloë uncinata.
Figure 6.
Assay of LolM methyltransferase activity.
(A) Chromatogram of loline alkaloids from incubation of norloline and AdoMet with protein extract of yeast transformed with empty vector. (B) Chromatogram of loline alkaloids from incubation of norloline and AdoMet with crude protein extract from yeast that expresses LolM. (C) Chromatogram of loline alkaloids from incubation of loline and AdoMet with protein extract of yeast transformed with empty vector. (D) Chromatogram of loline alkaloids from incubation of loline and AdoMet with crude protein extract from yeast that expresses LolM. (E) Proposed scheme of loline and N-methylloline (NML) formation from norloline. AdoHcy = S-adenosyl homocysteine.
Figure 7.
GC-MS chromatogram of loline alkaloids after application of loline to asymbiotic plants.
Shown are loline alkaloids extracted from loline applications to (A and B) endophyte-free (E-) meadow fescue (MF), and (C and D) E- perennial ryegrass (PRG), (E) mass spectrum of N-acetylloline (NAL) from application of loline to E- meadow fescue, and (F) proposed scheme of NAL formation from loline. Quinoline was added as internal standard (istd). Unlabeled peaks are non-loline alkaloid compounds. Numbers after MF or PRG indicate independent trials.
Figure 8.
Summarized loline-alkaloid biosynthetic pathway.
Labeled arrows are for steps that contribute to diversity of the lolines. Presence or absence of functional copies of lolO, lolN, lolM, or lolP, or a plant acetyltransferase activity, determine which alkaloids accumulate in the symbiotic plant as the pathway end-products.