All authors except Gert HJ Kema were employees of Syngenta Crop Protection or affiliates during the course of the research project. the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.

34Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a 35 broad range of fungal diseases. This has been the most rapidly expanding fungicide group in 36 terms of new molecules discovered and introduced for agricultural use over the past fifteen 37 years. A particular pattern of differential sensitivity (resistance) to a subclass of chemically-38 related SDHIs (SHA-SDHIs) was observed in naïve Zymoseptoria tritici populations. Class 39 specific SHA-SDHI resistance was confirmed at the enzyme level but did not correlate with 40 the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and 41 characterization of the genetic factor responsible for standing SHA-SDHI resistance in natural 42 field isolates identified a gene (alt-SDHC) encoding a paralog of the C subunit of succinate 43 dehydrogenase. This paralog was not present within our sensitive reference isolates and found 44 at variable frequencies within Z. tritici populations. Using reverse genetics, we showed that 45 alt-SDHC associates with the three other SDH subunits leading to a fully functional enzyme 46 and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. 47 Enzymatic assays, computational modelling and docking simulations for the two types of 48 SQR enzymes (alt-SDHC, SDHC) enabled us to describe protein-inhibitor interactions at an 49 atomistic level and to propose rational explanations for differential potency and resistance 50 across SHA-SDHIs. European Z. tritici populations displayed a presence (20-30%) / absence 51 polymorphism of alt-SDHC, as well as differences in alt-SDHC expression levels and splicing 52 efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. 53Characterization of the alt-SDHC promoter in European Z. tritici populations suggest that 54 transposon insertions are associated with the strongest resistance phenotypes. These results 55 establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in 56 natural populations of Z. tritici. This study paves the way to an increased awareness of the 3 57 role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount 58 importance of population genomics in fungicide discovery. 59 Author Summary 60Zymoseptoria tritici is the causal agent of Septoria tritici leaf blotch (STB) of wheat, the most 61 devastating disease for cereal production in Europe. Multiple succinate dehydrogenase 62 inhibitor (SDHI) fungicides have been developed and introduced for the control of STB. We 63 report the discovery and detailed characterization of a paralog of the C subunit of the SDH 64 enzyme conferring standing resistance towards a particular chemical subclass of the SDHIs. 65The resistance gene is characterized by its presence/absence, expression and splicing 66 polymorphisms which in turn affect resistance levels. The identified mechanism influenced 67 the chemical optimization phase which led to the discovery of pydiflu...

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