Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Public Library of Science full text link Public Library of Science Free PMC article
Full text links

Actions

Share

.2014 Dec 22;9(12):e115590.
doi: 10.1371/journal.pone.0115590. eCollection 2014.

Enzymes from fungal and plant origin required for chemical diversification of insecticidal loline alkaloids in grass-Epichloë symbiota

Affiliations

Enzymes from fungal and plant origin required for chemical diversification of insecticidal loline alkaloids in grass-Epichloë symbiota

Juan Pan et al. PLoS One..

Abstract

The lolines are a class of bioprotective alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norloline), -NHCH3 (loline), -N(CH3)2 (N-methylloline), -N(CH3)Ac (N-acetylloline), -NHAc (N-acetylnorloline), and -N(CH3)CHO (N-formylloline). Other than the LolP cytochrome P450, which is required for conversion of N-methylloline to N-formylloline, the enzymatic steps for loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorloline is the first fully cyclized loline alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in loline alkaloid diversification. Two genes of the loline alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorloline, and complementation with the two wild-type genes restored production of N-formylloline and N-acetylloline. These results indicated that lolN and lolM are required in the steps from N-acetylnorloline to other lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norloline to loline, and of loline to N-methylloline. One of the more abundant lolines, N-acetylloline, was observed in some but not all plants with symbiotic Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline, suggesting that a plant acetyltransferase catalyzes N-acetylloline formation. We conclude that although most loline alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.

PubMed Disclaimer

Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Structures of common loline alkaloids.
Substitutions on the nitrogen at C1 differentiate the lolines.
Figure 2
Figure 2. Enrichment of deuteratedN-acetylnorloline (NANL) from application of tetradeuteratedexo-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
Figure 3. Replacement oflolN andlolM withhph maker gene.
(A) Schematic representation oflolN-lolM replacement by thehph marker gene via homologous recombination. Shown are maps of the wild-typelolN andlolM inEpichloë 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 representHindIII digestion sites. Colored arrowheads represent primers used to generate pKAES323 and to screen the transformants. (B) Southern-blot analysis ofE. festucae strains. Wild-type E2368 and transformants were probed with alolN fragment orlolM gene amplified from E2368 (old probe was stripped off the membrane before new hybridization). Lanes containedHindIII-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
Figure 4. GC-MS traces showing loline-alkaloid profiles of meadow fescue symbiotic with differentE. festucae strains.
(A) ThelolN-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 , in the biosynthetic pathway fromN-acetylnorloline (NANL) to the final product,N-formylloline (NFL).
Figure 5
Figure 5. Partial LolN amino-acid sequence alignment ofEpichloë coenophiala e4309 andN-formylloline (NFL) producers.
Red-framed sequences are three differentE. coenophiala isolates.Ecoe  = Epichloë coenophiala,Eaot  = Epichloë aotearoae,Efes  = Epichloë festucae,Echis  = Epichloë chisosa,Esig  = Epichloë siegelii,Eunci  = Epichloë uncinata.
Figure 6
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 andN-methylloline (NML) formation from norloline. AdoHcy  = S-adenosyl homocysteine.
Figure 7
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 ofN-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
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 oflolO, lolN, lolM, orlolP, or a plant acetyltransferase activity, determine which alkaloids accumulate in the symbiotic plant as the pathway end-products.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Schardl CL, Florea S, Pan J, Nagabhyru P, Bec S, et al. (2013) The epichloae: alkaloid diversity and roles in symbiosis with grasses. Curr Opin Plant Biol 16:480–488. - PMC - PubMed
    1. Schardl CL, Grossman RB, Nagabhyru P, Faulkner JR, Mallik UP (2007) Loline alkaloids: currencies of mutualism. Phytochemistry 68:980–996. - PubMed
    1. Moon CD, Craven KD, Leuchtmann A, Clement SL, Schardl CL (2004) Prevalence of interspecific hybrids amongst asexual fungal endophytes of grasses. Mol Ecol 13:1455–1467. - PubMed
    1. Ghimire SR, Rudgers JA, Charlton ND, Young C, Craven KD (2011) Prevalence of an intraspecific Neotyphodium hybrid in natural populations of stout wood reed (Cinna arundinacea L.) from eastern North America. Mycologia 103:75–84. - PubMed
    1. Kang Y, Ji Y, Zhu K, Wang H, Miao H, et al. (2011) A new Epichloë species with interspecific hybrid origins from Poa pratensis ssp. pratensis in Liyang, China. Mycologia 103:1341–1350. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Public Library of Science full text link Public Library of Science Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp