An In Planta -Expressed Polyketide Synthase Produces ( R )-Mellein in the Wheat Pathogen Parastagonospora nodorum

Abstract

dParastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN477), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as (R)-mellein and (R)-O-methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression system, we successfully demonstrated that SN477 is the only enzyme required for the production of (R)-mellein. This is the first identification of a fungal PKS that is responsible for the synthesis of (R)-mellein. The P. nodorum ⌬SN477 mutant did not show any significant difference from the wild-type strain in its virulence against wheat. However, (R)-mellein at 200 g/ml inhibited the germination of wheat (Triticum aestivum) and barrel medic (Medicago truncatula) seeds. Comparative sequence analysis identified the presence of mellein synthase (MLNS) homologues in several Dothideomycetes and two sodariomycete genera. Phylogenetic analysis suggests that the MLNSs in fungi and bacteria evolved convergently from fungal and bacterial 6-methylsalicylic acid synthases.

SnPKS19 Encodes the Polyketide Synthase for Alternariol Mycotoxin Biosynthesis in the Wheat Pathogen Parastagonospora nodorum

“…The capacity of AOH to affect seed germination was also assessed. Unlike what we had observed for (R)-mellein in a previous study (39), AOH did not inhibit the germination of wheat and barrel medic seeds (data not shown). We have also tested the antimicrobial activity of AOH against Escherichia coli, Saccharomyces cerevisiae, Zymoseptoria tritici, and three environmental bacterial strains isolated from field wheat samples (Bacillus cereus, a Flavobacterium sp., and Sphingobacterium multivorum) (see the methods in the supplemental material).…”

“…Using both reverse genetics and heterologous expression, we recently identified the P. nodorum PKS gene responsible for production of R-(Ϫ)-mellein, which exhibited antigerminative activity (39). Here, using a similar approach, we unequivocally demonstrated that SnPKS19, which is highly similar to the A. alternata pksI product (86% protein identity), is solely responsible for the biosynthesis of AOH in P. nodorum.…”

“…The metabolite profile analyses were performed on an Agilent 1200 LC system coupled to a diode array detector (DAD) and an Agilent 6120 quadrupole MS with an ESI source. Chromatographic separation and conditions were exactly as described previously (39). The authenticity of AOH was confirmed by comparing the m/z ratio, UV spectrum, and retention time with those of the corresponding commercial standards (LGC standards).…”

Functional genomics‐guided discovery of a light‐activated phytotoxin in the wheat pathogen Parastagonospora nodorum via pathway activation

“…The metabolite products encoded by these gene clusters are mostly unknown. Recently, we characterized the PKS genes in P. nodorum that encode an anti‐germinative compound ( R )‐mellein (Chooi et al ., ) and the mycotoxin alternariol (Chooi et al ., ). As part of our ongoing effort to understand the chemistry and pathobiology of P. nodorum , we focus on a P. nodorum polyketide synthase (PKS) gene cluster ( SNOG_08608 ‐ SNOG_08616 ) in this study.…”

Saccharomyces cerevisiae as a tool for mining, studying and engineering fungal polyketide synthases

“…When SN477 , was cloned from cDNA under the ADH2 promoter and transformed into S. cerevisiae BJ5464-NpgA, ( R )-mellein was produced by the yeast host, revealing that SN477 is the only enzyme required for ( R )-mellein synthesis. Though ( R )-mellein showed no relevance to the virulence against wheat, it was able inhibit the germination of wheat seeds (Chooi et al, 2015). …”