A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness.We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field.

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.

BackgroundThe combination of high-throughput transcript profiling and next-generation sequencing technologies is a prerequisite for genome-wide comprehensive transcriptome analysis. Our recent innovation of deepSuperSAGE is based on an advanced SuperSAGE protocol and its combination with massively parallel pyrosequencing on Roche's 454 sequencing platform. As a demonstration of the power of this combination, we have chosen the salt stress transcriptomes of roots and nodules of the third most important legume crop chickpea (Cicer arietinum L.). While our report is more technology-oriented, it nevertheless addresses a major world-wide problem for crops generally: high salinity. Together with low temperatures and water stress, high salinity is responsible for crop losses of millions of tons of various legume (and other) crops. Continuously deteriorating environmental conditions will combine with salinity stress to further compromise crop yields. As a good example for such stress-exposed crop plants, we started to characterize salt stress responses of chickpeas on the transcriptome level.ResultsWe used deepSuperSAGE to detect early global transcriptome changes in salt-stressed chickpea. The salt stress responses of 86,919 transcripts representing 17,918 unique 26 bp deepSuperSAGE tags (UniTags) from roots of the salt-tolerant variety INRAT-93 two hours after treatment with 25 mM NaCl were characterized. Additionally, the expression of 57,281 transcripts representing 13,115 UniTags was monitored in nodules of the same plants. From a total of 144,200 analyzed 26 bp tags in roots and nodules together, 21,401 unique transcripts were identified. Of these, only 363 and 106 specific transcripts, respectively, were commonly up- or down-regulated (>3.0-fold) under salt stress in both organs, witnessing a differential organ-specific response to stress.Profiting from recent pioneer works on massive cDNA sequencing in chickpea, more than 9,400 UniTags were able to be linked to UniProt entries. Additionally, gene ontology (GO) categories over-representation analysis enabled to filter out enriched biological processes among the differentially expressed UniTags. Subsequently, the gathered information was further cross-checked with stress-related pathways.From several filtered pathways, here we focus exemplarily on transcripts associated with the generation and scavenging of reactive oxygen species (ROS), as well as on transcripts involved in Na+ homeostasis. Although both processes are already very well characterized in other plants, the information generated in the present work is of high value. Information on expression profiles and sequence similarity for several hundreds of transcripts of potential interest is now available.ConclusionsThis report demonstrates, that the combination of the high-throughput transcriptome profiling technology SuperSAGE with one of the next-generation sequencing platforms allows deep insights into the first molecular reactions of a plant exposed to salinity. Cross validation with recent reports enric...

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...