Victorin, the host-selective cyclic peptide toxin from the oat pathogen Cochliobolus victoriae, is ribosomally encoded

Abstract

The necrotrophic fungal pathogen Cochliobolus victoriae produces victorin, a host-selective toxin (HST) essential for pathogenicity to certain oat cultivars with resistance against crown rust. Victorin is a mixture of highly modified heterodetic cyclic hexapeptides, previously assumed to be synthesized by a nonribosomal peptide synthetase. Herein, we demonstrate that victorin is a member of the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Analysis of a newly generated long-read assembly of the C. victoriae genome revealed three copies of precursor peptide genes (vicA1-3) with variable numbers of "GLKLAF" core peptide repeats corresponding to the victorin peptide backbone. vicA1-3 are located in repeat-rich gene-sparse regions of the genome and are loosely clustered with putative victorin biosynthetic genes, which are supported by the discovery of compact gene clusters harboring corresponding homologs in two distantly related plant-associated Sordariomycete fungi. Deletion of at least one copy of vicA resulted in strongly diminished victorin production. Deletion of a gene encoding a DUF3328 protein (VicYb) abolished the production altogether, supporting its predicted role in oxidative cyclization of the core peptide. In addition, we uncovered a copper amine oxidase (CAO) encoded by vicK, in which its deletion led to the accumulation of new glycine-containing victorin derivatives. The role of VicK in oxidative deamination of the N-terminal glycyl moiety of the hexapeptides to the active glyoxylate forms was confirmed in vitro. This study finally unraveled the genetic and molecular bases for biosynthesis of one of the first discovered HSTs and expanded our understanding of underexplored fungal RiPPs.

Keywords: Cochliobolus; RiPP; copper amine oxidase; host-selective toxin; victorin.

Fig. 1.

Structures of known victorin derivatives (1–6) and proposed structures of related biosynthesis intermediates (7–10).

Fig. 2.

Analysis of victorin structure, biosynthesis, and genetic background. (A) Deduction of the unmodified peptide backbone sequence of victorin C from its structure. Red letters indicate the individual amino acids that make up the peptide repeat. (B) Precursor peptide amino acid sequence. The number of sequence repeats varies among the three gene copies (Note: not all repeats encoded in vicA3 are in frame, thus, the predicted numbers of core peptides in VicA3 vary with different intron–exon structure predictions). The signal peptide is underlined, the kexin protease recognition sites are shown in bold, and the core peptide is shown in red. (C) Putative victorin biosynthesis genes (Top) and putative biosynthetic gene clusters of vicA homologs in Apiospora montagnei and Colletotrichum eremochloae. Blue shade represents duplication of the highlighted gene or region. Numbers following a gene name indicate duplicates. Gene name identity between species does not necessarily imply homology (SI Appendix, Table S2). (D) Schematic of the genomic regions harboring the victorin precursor peptide genes (annotated here as A1–A3). Each chromosome is shown as a black line with the position in megabases (Mb) indicated. Gray blocks show locations where transposable elements have been annotated, and pink blocks show annotated FI3 genes. Blocks appearing above the chromosome line are encoded in the forward direction, and blocks below the line in the reverse. Other colors indicate the locations of genes with putative victorin biosynthetic genes with the same color code as in C. The red and blue ribbons connecting the two contigs show regions >2 kb with >80% identity between the two chromosomes. Red ribbons indicate alignments in the same direction, and blue ribbons indicate inversions. (E) Gene density heat map of the C. victoriae genome. Genes were binned based on the lengths of their 5′ and 3′ intergenic regions. Putative victorin biosynthesis genes are overlaid as white dots, and precursor peptide genes are overlaid as red dots.

Fig. 3.

Victorin production analysis in deletion mutants. (A) LC–MS extracted ion chromatograms (EICs) of victorin C standard and of culture filtrate extractions from different C. victoriae strains (Left) and spectra at indicated elution times (Right). m/z of 815.2 (Top) corresponds to victorin C (1), m/z of 798.2 (Bottom) corresponds to HV-toxin M (3). (B) Two replicates (Top/Bottom) of victorin toxicity bioassay on susceptible oat cultivar (Fulgrain) leaves with undiluted culture filtrate from different C. victoriae strains. Arrows indicate leaf wilting. Numbers in white indicate the independent transformants (SI Appendix, Table S5). Note only culture filtrate from WT strain FI3 caused the typical leaf wilting symptom in the first assay replicate (Top), whereas in the second assay replicate (Bottom), the leaf wilting symptom was also observed with culture filtrate from the ΔvicA* mutant strain.

Fig. 4.

Biosynthetic function of VicK. (A) Proposed reaction catalyzed by VicK. (B) MS¹ and MS² spectra of ions corresponding to masses of compounds 3, and 7–9 with proposed structures based on diagnostic fragment ions a–e in comparison to the corresponding glyoxylate analogs 1, 4, 2, and 5 (SI Appendix, Fig. S3). (C) In vitro oxidative deamination of 3 with cell-free lysate from A. nidulans expressing VicK. m/z of 798.2 corresponds to 3, m/z of 815.2 to 1. Chromatograms of samples containing cell-free lysates are from after 14 h of incubation with the substrate. Y axes of all seven chromatograms are scaled equally.