Abstract
Botcinic acid is a phytotoxic polyketide involved in the virulence of the gray mold fungus Botrytis cinerea. Here, we aimed to investigate the specific regulation of the cluster of Bcboa genes that is responsible for its biosynthesis. Our analysis showed that this cluster is located in a subtelomeric genomic region containing alternating G + C/A + T-balanced regions, and A + T-rich regions made from transposable elements that underwent RIP (Repeat-Induced Point mutation). Genetic analyses demonstrated that BcBoa13, a putative Zn2Cys6 transcription factor, is a nuclear protein with a major positive regulatory role on the expression of other Bcboa1-to-Bcboa12 genes, and botcinic acid production. In conclusion, the structure and the regulation of the botcinic acid gene cluster show similar features with the cluster responsible for the biosynthesis of the other known phytotoxin produced by B. cinerea, i.e., the sesquiterpene botrydial. Both clusters contain a gene encoding a pathway-specific Zn2Cys6 positive regulator, and both are surrounded by relics of transposons which raise some questions about the role of these repeated elements in the evolution and regulation of the secondary metabolism gene clusters in Botrytis.
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Acknowledgements
The authors would like to warmly thank Christopher Alford (Ludwig-Maximilians-Universität of Munich, Germany) for the English revision of this manuscript and Dr. Julia Schumacher (Bundesanstalt für Materialforschung und prüfung, Berlin, Germany) for providing cloning vectors. IGC thanks the financial support from MINECO-FEDER (AGL2015-65684-C2-1-R). MV thanks the Saclay Plant Sciences network. The authors declare no conflict of interest.
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Figure S1 Construction of the different strains. The primers used for the construction of the different strains are listed in Table S1. For more details, see “Materials and methods”. a For the generation of the ΔBcboa13, the whole ORF of the gene was replaced by a hygromycin-resistant cassette. b Southern blot analysis confirmed the integration of a single copy of the hygromycin-resistance cassette only in the ΔBcboa13 4.6.2. The used probe (P) is represented in A. cBcniaD locus was targeted for the reintroduction of the Bcboa13 ORF within the ΔBcboa13, as well as for the overexpression of Bcboa13 in the WT B05.10 strain. (PDF 211 KB)
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Figure S2 The deletion of Bcboa13 has no influence on B05.10 virulence on French bean leaves. The virulence of the WT strain B05.10 and the ΔBcboa13 4.6.2 was evaluated on detached leaves (Phaseolus vulgaris cv. Caruso grown under glasshouse conditions) inoculated with 3-day-old mycelium plugs a or conidia b. The inoculated plants were then incubated at 75% relative humidity with a photoperiod of 16 h of white light at 22 °C and 8 h of darkness at 18 °C. The results represent the mean of lesion diameter observed from at least 10 symptoms at 48, 72 and 96 h for a and 72, 96, and 120 h for b. Standard deviations are represented. Statistical (t test) analysis did not point out significant differences between the results regarding the WT B05.10 strain and the ΔBcboa13 4.6.2. (PDF 69 KB)
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Figure S3 Synteny of B. cinerea BOA genomic region with Sclerotinia sclerotiorum genome. Alignments between B. cinerea and S. sclerotiorum genomes were performed with blastn algorithm (-e < 1e-10). The synteny between BCIN01 genomic region 1-97500 and aligning S. sclerotiorum regions was visualized with Artemis ACT (Carver et al. 2005). Bcboa1 and Bcboa2 align with S. sclerotiorum chromosome 5 (CP017818), whereas the region containing Bcboa3-Bcboa13 genes align with S. sclerotiorum chromosome 15 (CP017828). Bcboa15–Bcboa17 do not align with S. sclerotiorum genome. The B. cinerea genomic region downstream the BOA gene cluster align with S. sclerotiorum chromosome 2 (CP017815). The S. sclerotiorum genes aligning with Bcboa1–Bcboa13 are, respectively, sscle_05g042570, sscle_05g042560, sscle_15g106540, sscle_15g106530, sscle_15g106520, sscle_15g106510, sscle_15g106500, sscle_15g106490, sscle_15g106480, sscle_15g106470, sscle_15g1064960, sscle_15g106450, and sscle_15g106440. (PDF 217 KB)
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Figure S4 Yeast One-Hybrid assays. The Yeast One-Hybrid technique was previously developed and successfully used in our lab for B. cinerea (Simon et al. 2013; Zhang et al. 2015). Here, we used the promoters of Bcboa6 and Bcboa9 as baits and BcBoa13 as a prey in direct interaction assays. As the pBcboa9 contains twice the candidate-binding motif, we focused on it and we designed and generated a series of constructs containing different parts of this promoter containing or not the candidate-binding motif fused upstream of the reporter gene HIS3 (pBcboa9 T1–T5) into the vector pINT-HIS3NB. In addition, we isolated the sequence of each of the candidate-binding motif versions and cloned them in a three-time repeat upstream of the HIS3 gene (pBcboa9 T6 and T7). This strategy has been successfully used before in B. cinerea (Zhang et al. 2015). As far as the Bcboa6 is concerned, we only cloned the full-length promoter (pBcboa6 T1) and the three-time repeat of the candidate motif 6 (pBcboa6 T2). All those promoter-based constructs were used to transform the Saccharomyces cerevisiae strain Y187. Regarding the prey generation, we cloned Bcboa13 in two different ways. The first consists of the classical full-length cloning fused in frame to the activation domain of yeast Gal4 into the plasmid pACTIIst. The second is restricted to the predicted binding domain of the protein. The interaction between BcBoa13 and promoters was assayed by transforming the Y187 mutants previously generated with the promoter constructs with the TF-containing pACTIIst. We could select co-transformants, but subcultures on selective histidine lacking media did not reveal any activation of the HIS3 gene. (PDF 254 KB)
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Porquier, A., Moraga, J., Morgant, G. et al. Botcinic acid biosynthesis in Botrytis cinerea relies on a subtelomeric gene cluster surrounded by relics of transposons and is regulated by the Zn2Cys6 transcription factor BcBoa13. Curr Genet 65, 965–980 (2019). https://doi.org/10.1007/s00294-019-00952-4
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DOI: https://doi.org/10.1007/s00294-019-00952-4