Towards take-all control: a C-21β oxidase required for acylation of triterpene defence compounds in oat

Abstract

Oats produce avenacins, antifungal triterpenes that are synthesized in the roots and provide protection against take-all and other soilborne diseases. Avenacins are acylated at the carbon-21 position of the triterpene scaffold, a modification critical for antifungal activity. We have previously characterized several steps in the avenacin pathway, including those required for acylation. However, transfer of the acyl group to the scaffold requires the C-21β position to be oxidized first, by an as yet uncharacterized enzyme. We mined oat transcriptome data to identify candidate cytochrome P450 enzymes that may catalyse C-21β oxidation. Candidates were screened for activity by transient expression in Nicotiana benthamiana. We identified a cytochrome P450 enzyme AsCYP72A475 as a triterpene C-21β hydroxylase, and showed that expression of this enzyme together with early pathway steps yields C-21β oxidized avenacin intermediates. We further demonstrate that AsCYP72A475 is synonymous with Sad6, a previously uncharacterized locus required for avenacin biosynthesis. sad6 mutants are compromised in avenacin acylation and have enhanced disease susceptibility. The discovery of AsCYP72A475 represents an important advance in the understanding of triterpene biosynthesis and paves the way for engineering the avenacin pathway into wheat and other cereals for control of take-all and other diseases.

Keywords: Avena strigosa; avenacins; cytochromes P450; disease resistance; metabolic engineering; natural products; triterpenes.

Fig. 1

Identification of C-21β oxidase-deficient (sad6) oat mutants. (a) Structures of the four avenacins. Avenacin A-1 is the major avenacin found in oat roots. Avenacins A and B are differentiated by the presence (A) or absence (B) of a hydroxyl group at C-23 (R₁). Avenacins A-1 and B-1 are acylated with N-methyl anthranilate at the C-21 position, and avenacins A-2 and B-2 have benzoate at this position (R₂). AsHMGR, Avena strigosa HMG-CoA reductase. (b) Current understanding of the pathway for the biosynthesis of avenacin A-1. The triterpene scaffold originates from the isoprenoid pathway (left), and the N-methyl anthranilate acyl group from the shikimate pathway (right). (c) LC-MS analysis of root extracts of seedlings of the wild-type oat accession and sad6 and sad7 mutant lines. The major avenacin peaks (A-1, A-2 and B-2, 15.5–19.6 min) observed in the wild-type are absent in sad6 and sad7 mutants (the least abundant, B-2, was below the detection limit in these analyses). The sad7 mutants accumulate nonacylated avenacins (des-acyl-avenacins A and B) as previously reported (Mugford et al., 2009), owing to lack of the acyltransferase, AsSCPL1/SAD7. The sad6 mutants #825 and #1243 accumulate a form of nonacylated avenacin, putatively designated des-acyl, des-21-hydroxy-avenacin A (Supporting Information Fig. S1).

Fig. 2

Candidate CYPs identified by transcriptome mining. (a) Root-expressed candidate CYPs identified in Avena strigosa. The corresponding scaffold names from the oat root transcriptome database (Kemen et al., 2014) are shown. (b) RT-PCR analysis of the transcript abundances of candidate CYP genes in different tissues from 3-d-old oat seedlings. Two previously characterized avenacin biosynthetic genes (AsbAS1/Sad1 and AsCYP51H10/Sad2) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) are included as controls.

Fig. 3

Production of EpdiHβA by transient expression of AsCYP72A475 in Nicotiana benthamiana. (a) GC-MS total ion chromatograms (TIC) of extracts from leaves expressing AstHMGR/AsbAS1/AsCYP51H10 alone or with AsCYP72A475. Mass spectra for 12,13β-epoxy-16β-hydroxy-β-amyrin (EpHβA, 12.4 min) and the novel peak putatively identified as 12,13β-epoxy, 16β,21β-dihydroxy-β-amyrin (EpdiHβA, 14.9 min) are shown on the right. Spectra are provided in Supporting Information Fig. S3. (b) Proposed pathway for biosynthesis of EpdiHβA from β-amyrin in N. benthamiana. (c) LC-charged aerosol detection chromatograms of extracts from leaves transiently expressing AstHMGR, AsbAS1 and AsCYP51H10 with different combinations of AsCYP72A475 and the C-3 arabinosyltransferase, AsAAT1. Upon coexpression of all enzymes, a novel peak was observed at 6.2 min (marked with an asterisk). (d) Structure of the purified compound with retention time 6.2 min (c) as determined by nuclear magnetic resonance (further details provided in Fig. S4). AsHMGR and AstHMGR, Avena strigosa wild-type and feedback-insensitive HMG-CoA reductase, respectively (Reed etal., 2017).

Fig. 4

Phylogenetic and comparative analysis. (a) Neighbour-joining phylogenetic tree of 16 functionally characterized angiosperm CYP72A enzymes (Irmler et al., 2000; Seki et al., 2011; Fukushima et al., 2013; Itkin et al., 2013; Miettinen et al., 2014; Saika et al., 2014; Biazzi et al., 2015; Umemoto et al., 2016; Han et al., 2018; Yano et al., 2017; this work). Branch lengths represent number of amino acid substitutions per site (scale bar shown at bottom). Bootstrap support is given as percentages (1000 replicates) next to the branches. The tree is rooted using the (nonCYP72) Arabidopsis thaliana CYP734A1 enzyme, as previously described (Prall et al., 2016). Monocot sequences are indicated in bold. The eudicot CYP72A family members known to oxidize b-amyrin (or derivatives of β-amyrin) are marked with asterisks and the carbon position that these enzymes modify are showed on the right. As, Avena strigosa; At, A. thaliana; Cr, Catharanthus roseus; Gm, Glycine max; Gu, Glycyrrhiza uralensis; Kp, Kalopanax septemlobus; Mt, Medicago truncatula; Os, Oryza sativa; Sl, Solanum lycopersicum. (b) The soybean GmCYP72A69 has previously been reported to oxidize the β-amyrin-derived scaffold soyasapogenol B at the C-21β position to produce soyasapogenol A (Yano et al., 2017). (c) Coexpression of soybean GmCYP72A69 with AstHMGR and AsbAS1 results in a new peak (12.37 min), which was putatively identified as 21β-hydroxy-β-amyrin. A representative mass spectrum for the putative 21β-hydroxy-β-amyrin is given on the right. The mass spectrum for a minor coeluting product (labelled * in the controls) is shown in Supporting Information Fig. S5. (d) Coexpression of GmCYP72A69 with AstHMGR, AsbAS1 and AsCYP51H10 results in accumulation of a novel product (14.92 min) with a matching retention time and mass spectrum to EpdiHβA, as produced by coexpression of AstHMGR, AsbAS1, AsCYP51H10 and AsCYP72A475. AstHMGR, feedback-insensitive A. strigosa HMG-CoA reductase (Reed et al., 2017).

Fig. 5

Analysis of Ascyp72a475 (sad6) mutants. (a) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of transcript abundances of AsbAS1/Sad1, AsCYP51H10/Sad2 and AsCYP72A475 in RNA extracted from 3-d-old Avena strigosa seedlings. (b) qRT-PCR analysis of AsCYP72A475 transcript abundances in RNA extracted from roots of wild-type and sad6 mutant lines. Expression levels are shown relative to the wildtype. All qPCR data transcript abundances were normalized to those for the elongation factor 1 (EF1-α) housekeeping gene, using the ΔΔCq method (Livak & Schmittgen, 2001). Values are means ± SE (three technical replicates). (c) Representative roots of wild-type, sad1 and sad6 A. strigosa lines following inoculation with the take-all fungus (Gaeumannomyces graminis var. tritici strain T5). Roots were scored based on presence and extent of lesions (arrows) as previously described (Papadopoulou et al., 1999). (d) Disease scores for a suite of sad6 mutant lines (Supporting Information Fig. S7a) compared with those of the wild-type (S75) and a susceptible sad1 mutant line (#109). The bars represent mean disease scores (21–25 seedlings per line). Error bars represent the SE of the mean.