Gramiketides, Novel Polyketide Derivatives of Fusarium graminearum, Are Produced during the Infection of Wheat

Abstract

The plant pathogen Fusarium graminearum is a proficient producer of mycotoxins and other in part still unknown secondary metabolites, some of which might act as virulence factors on wheat. The PKS15 gene is expressed only in planta, so far hampering the identification of an associated metabolite. Here we combined the activation of silent gene clusters by chromatin manipulation (kmt6) with blocking the metabolic flow into the competing biosynthesis of the two major mycotoxins deoxynivalenol and zearalenone. Using an untargeted metabolomics approach, two closely related metabolites were found in triple mutants (kmt6 tri5 pks4,13) deficient in production of the major mycotoxins deoxynivalenol and zearalenone, but not in strains with an additional deletion in PKS15 (kmt6 tri5 pks4,13 pks15). Characterization of the metabolites, by LC-HRMS/MS in combination with a stable isotope-assisted tracer approach, revealed that they are likely hybrid polyketides comprising a polyketide part consisting of malonate-derived acetate units and a structurally deviating part. We propose the names gramiketide A and B for the two metabolites. In a biological experiment, both gramiketides were formed during infection of wheat ears with wild-type but not with pks15 mutants. The formation of the two gramiketides during infection correlated with that of the well-known virulence factor deoxynivalenol, suggesting that they might play a role in virulence.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones

“…MS 2 mass fragments of the FDDP-D [M + H]+ (RT5.70_m/z 503.3004) observed in our study do not match those reported by Seidl et al [27] for gramiketide B ([M + H] + m/z 503.3003). Rather, the MS 2 [M + H] + mass fragments reported for gramiketide B [27] closely match the MS 2 [M + H] + mass fragments of the C16 BGC unknown mass feature observed in our study as RT5.51_m/z 503.3004 from in planta wheat head challenge (Table S7). A potential mass feature corresponding to Seidl et al's proposed gramiketide A (m/z 521.3109) was also detected in our wild-type in planta extracts (m/z ∆ppm < ±5), albeit at detection intensities at the limit of detection of our instrument when searching using extraction ion chromatograms-the detected amounts were insufficient to carry out MS 2 comparison for further validation.…”

“…For example, microarray expression profiles of F. graminearum C16 BGC genes significantly increase after 72 h postinoculation on barley and 96 h postinoculation on wheat followed by a subsequent decrease [8]. This result was recently corroborated using LC-HRMS profiling of F. graminearum-infected wheat heads, where postulated C16 BGC products along with DON were observed 72 h postinoculation, with concentrations increasing over time [27]. Transcriptomics experiments have also linked the co-expression of the C16 BGC with that of the fusaoctaxin BGC (C64) in F. graminearum, where transcripts of both BGC genes were observed to peak 64-96 h after infection in infected barley and wheat kernels followed by a subsequent decrease [7].…”

“…In a recent research study using multi-gene deletion strains to identify secondary metabolites associated with the F. graminearum C16 BGC, Seidl et al [27] discerned two metabolite products (having an observed [M + H] + m/z of 521.3109 ± 3ppm and an [M + H] + m/z of 503.3003 ± 3ppm) that were differentially expressed under in vitro cultivation and in planta challenge in wheat heads between a "triple mutant" strain (∆tri5, ∆pks4,13, and ∆kmt6) and a pks15 gene disruption in the triple mutant background (∆tri5, ∆pks4,13, ∆kmt6, and ∆pks15). Scaled-up cultivation yielded insufficient material for a full structural characterization of the molecules; however, based on MS 2 experiments of culture extracts derived using C 12 -and C 13 -labeled substrates, Seidl et al [27] proposed the candidate names gramiketides A and B (for m/z 521.3109 and m/z 503.3003, respectively) as products of the F. graminearum C16 BGC. MS 2 mass fragments of the FDDP-D [M + H]+ (RT5.70_m/z 503.3004) observed in our study do not match those reported by Seidl et al [27] for gramiketide B ([M + H] + m/z 503.3003).…”

“…Scaled-up cultivation yielded insufficient material for a full structural characterization of the molecules; however, based on MS 2 experiments of culture extracts derived using C 12 -and C 13 -labeled substrates, Seidl et al [27] proposed the candidate names gramiketides A and B (for m/z 521.3109 and m/z 503.3003, respectively) as products of the F. graminearum C16 BGC. MS 2 mass fragments of the FDDP-D [M + H]+ (RT5.70_m/z 503.3004) observed in our study do not match those reported by Seidl et al [27] for gramiketide B ([M + H] + m/z 503.3003). Rather, the MS 2 [M + H] + mass fragments reported for gramiketide B [27] closely match the MS 2 [M + H] + mass fragments of the C16 BGC unknown mass feature observed in our study as RT5.51_m/z 503.3004 from in planta wheat head challenge (Table S7).…”

“…Structural characterization enables dereplication efforts and prevents duplicity of names assigned to molecular analogs derived from the same BGC of a given organism-which appears to be the case in the products characterized for the F. graminearum C16 BGC. As a surrogate C16 BGC was heterologously expressed in A. oryzae, and the resulting products were structurally characterized in full by Tsukada et al [12] two years before the proposition of the gramiketide naming scheme by Seidl et al [27], retention of the FDDP naming convention should have been given precedence by Seidl et al to prevent naming duplicity.…”