A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici.

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to the stretched heterocycle amide SDHIs (SHA-SDHIs), a subclass of chemically-related SDHIs, was observed in naïve Zymoseptoria tritici populations not previously exposed to these chemicals. Subclass-specific resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the molecular mechanisms responsible for standing SHA-SDHI resistance in natural field isolates identified a gene paralog of SDHC, termed ZtSDHC3, which encodes for an alternative C subunit of succinate dehydrogenase, named alt-SDHC. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits, leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two SQR enzymes (altC-SQR, WT_SQR) enabled us to describe enzyme-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. European Z. tritici populations displayed a presence (20-30%) / absence polymorphism of ZtSDHC3, as well as differences in ZtSDHC3 expression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of the ZtSDHC3 promoter in European Z. tritici populations suggests that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations of Z. tritici. This study paves 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.

Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola.

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.

The salt-responsive transcriptome of chickpea roots and nodules via deepSuperSAGE.

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: This 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 enriched the information about the salt stress dynamics of more than 9,000 chickpea ESTs, and enlarged their pool of alternative transcripts isoforms. As an example for the high resolution of the employed technology that we coin deepSuperSAGE, we demonstrate that ROS-scavenging and -generating pathways undergo strong global transcriptome changes in chickpea roots and nodules already 2 hours after onset of moderate salt stress (25 mM NaCl). Additionally, a set of more than 15 candidate transcripts are proposed to be potential components of the salt overly sensitive (SOS) pathway in chickpea. Newly identified transcript isoforms are potential targets for breeding novel cultivars with high salinity tolerance. We demonstrate that these targets can be integrated into breeding schemes by micro-arrays and RT-PCR assays downstream of the generation of 26 bp tags by SuperSAGE.