Publications by authors named "Gabriel Scalliet"

22 Publications

  • Page 1 of 1

A world-wide analysis of reduced sensitivity to DMI fungicides in the banana pathogen Pseudocercospora fijiensis.

Pest Manag Sci 2021 Mar 25. Epub 2021 Mar 25.

Wageningen Research, Wageningen University and Research, Wageningen, The Netherlands.

Background: Pseudocercospora fijiensis is the causal agent of the black leaf streak disease (BLSD) of banana. Bananas are important global export commodities and a major staple food. Their susceptibility to BLSD pushes disease management towards excessive fungicide use, largely relying on multisite inhibitors and sterol demethylation inhibitors (DMIs). These fungicides are ubiquitous in plant disease control, targeting the CYP51 enzyme. We examined sensitivity to DMIs in P. fijiensis field isolates collected from various major banana production zones in Colombia, Costa Rica, Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon and determined the underlying genetic reasons for the observed phenotypes.

Results: We observed a continuous range of sensitivity towards the DMI fungicides difenoconazole, epoxiconazole and propiconazole with clear cross-sensitivity. Sequence analyses of PfCYP51 in 266 isolates showed 28 independent amino acid substitutions, nine of which correlated with reduced sensitivity to DMIs. In addition to the mutations, we observed up to six insertions in the Pfcyp51 promoter. Such promoter insertions contain repeated elements with a palindromic core and correlate with the enhanced expression of Pfcyp51 and hence with reduced DMI sensitivity. Wild-type isolates from unsprayed bananas fields did not contain any promoter insertions.

Conclusion: The presented data significantly contribute to understanding of the evolution and global distribution of DMI resistance mechanisms in P. fijiensis field populations and facilitate the prediction of different DMI efficacy. The overall reduced DMI sensitivity calls for the deployment of a wider range of solutions for sustainable control of this major banana disease.
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http://dx.doi.org/10.1002/ps.6372DOI Listing
March 2021

One Cut to Change Them All: CRISPR/Cas, a Groundbreaking Tool for Genome Editing in and Other Fungal Plant Pathogens.

Phytopathology 2021 Mar 29;111(3):474-477. Epub 2021 Jan 29.

Syngenta Crop Protection AG, Stein, Switzerland.

CRISPR/Cas is a genome editing technology that has opened new dimensions in functional biology. In a recent publication, we presented a highly efficient CRISPR/Cas technique for , which dramatically increases our options to mutagenize and modify single or multiple genes. In this Perspectives article, we describe the essential features of the method and demonstrate with several examples how it opens new avenues for unraveling the virulence mechanisms of and other plant pathogenic fungi and can accelerate research for the identification of new antifungal compounds.
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http://dx.doi.org/10.1094/PHYTO-09-20-0379-PERDOI Listing
March 2021

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea.

PLoS Pathog 2020 08 17;16(8):e1008326. Epub 2020 Aug 17.

University of Kaiserslautern, Department of Biology, Kaiserslautern, Germany.

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.
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http://dx.doi.org/10.1371/journal.ppat.1008326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451986PMC
August 2020

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

PLoS Pathog 2019 12 20;15(12):e1007780. Epub 2019 Dec 20.

Syngenta Crop Protection AG, Stein, Switzerland.

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.
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http://dx.doi.org/10.1371/journal.ppat.1007780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941823PMC
December 2019

sRNA Profiling Combined With Gene Function Analysis Reveals a Lack of Evidence for Cross-Kingdom RNAi in the Wheat - Pathosystem.

Front Plant Sci 2019 4;10:892. Epub 2019 Jul 4.

Biointeractions and Crop Protection, Rothamsted Research, Harpenden, United Kingdom.

Cross-kingdom small RNA (sRNA) silencing has recently emerged as a mechanism facilitating fungal colonization and disease development. Here we characterized RNAi pathways in , a major fungal pathogen of wheat, and assessed their contribution to pathogenesis. Computational analysis of fungal sRNA and host mRNA sequencing datasets was used to define the global sRNA populations in and predict their mRNA targets in wheat. 389 -induced sRNA loci were identified. sRNAs generated from some of these loci were predicted to target wheat mRNAs including those potentially involved in pathogen defense. However, molecular approaches failed to validate targeting of selected wheat mRNAs by fungal sRNAs. Mutant strains of carrying deletions of genes encoding key components of RNAi such as Dicer-like (DCL) and Argonaute (AGO) proteins were generated, and virulence bioassays suggested that these are dispensable for full infection of wheat. Nonetheless, our results did suggest the existence of non-canonical DCL-independent pathway(s) for sRNA biogenesis in . dsRNA targeting essential fungal genes applied or generated from an RNA virus vector in a procedure known as HIGS (Host-Induced Gene Silencing) was ineffective in preventing growth or disease. We also demonstrated that is incapable of dsRNA uptake. Collectively, our data suggest that RNAi approaches for gene function analyses in this fungal species and potentially also as a control measure may not be as effective as has been demonstrated for some other plant pathogenic fungi.
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http://dx.doi.org/10.3389/fpls.2019.00892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620828PMC
July 2019

Anilinopyrimidine Resistance in Is Linked to Mitochondrial Function.

Front Microbiol 2017 30;8:2361. Epub 2017 Nov 30.

Syngenta Crop Protection AG, Stein, Switzerland.

Crop protection anilinopyrimidine (AP) fungicides were introduced more than 20 years ago for the control of a range of diseases caused by ascomycete plant pathogens, and in particular for the control of gray mold caused by . Although early mode of action studies suggested an inhibition of methionine biosynthesis, the molecular target of this class of fungicides was never fully clarified. Despite AP-specific resistance having been described in . field isolates and in multiple other targeted species, the underlying resistance mechanisms were unknown. It was therefore expected that the genetic characterization of resistance mechanisms would permit the identification of the molecular target of these fungicides. In order to explore the widest range of possible resistance mechanisms, AP-resistant . UV laboratory mutants were generated and the mutations conferring resistance were determined by combining whole-genome sequencing and reverse genetics. Genetic mapping from a cross between a resistant field isolate and a sensitive reference isolate was used in parallel and led to the identification of an additional molecular determinant not found from the characterized UV mutant collection. Together, these two approaches enabled the characterization of an unrivaled diversity of resistance mechanisms. In total, we report the elucidation of resistance-conferring mutations within nine individual genes, two of which are responsible for almost all instances of AP resistance in the field. All identified resistance-conferring genes encode proteins that are involved in mitochondrial processes, suggesting that APs primarily target the mitochondria. The functions of these genes and their possible interactions are discussed in the context of the potential mode of action for this important class of fungicides.
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http://dx.doi.org/10.3389/fmicb.2017.02361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714876PMC
November 2017

A new mechanism for reduced sensitivity to demethylation-inhibitor fungicides in the fungal banana black Sigatoka pathogen Pseudocercospora fijiensis.

Mol Plant Pathol 2018 06 13;19(6):1491-1503. Epub 2018 Feb 13.

Wageningen University and Research, Wageningen Plant Research, 6700 AA Wageningen, the Netherlands.

The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.
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http://dx.doi.org/10.1111/mpp.12637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637983PMC
June 2018

Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control.

PLoS Genet 2016 08 11;12(8):e1005876. Epub 2016 Aug 11.

Plant Research International, Wageningen University and Research, Wageningen, The Netherlands.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.
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http://dx.doi.org/10.1371/journal.pgen.1005876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981457PMC
August 2016

A gapless genome sequence of the fungus Botrytis cinerea.

Mol Plant Pathol 2017 01 9;18(1):75-89. Epub 2016 Jun 9.

Syngenta Crop Protection Münchwilen AG, Crop Protection Research, CH-4332, Stein, Switzerland.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.
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http://dx.doi.org/10.1111/mpp.12384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638203PMC
January 2017

Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans.

Mol Plant Pathol 2014 Sep 14;15(7):664-76. Epub 2014 Apr 14.

Cell and Molecular Sciences, James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.

Phenylamide fungicides have been widely used for the control of oomycete-incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi-dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the 'insensitive' allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.
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http://dx.doi.org/10.1111/mpp.12124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638662PMC
September 2014

A review of current knowledge of resistance aspects for the next-generation succinate dehydrogenase inhibitor fungicides.

Phytopathology 2013 Sep;103(9):880-7

Syngenta Crop Protection, Research Biology, Schaffhauserstrasse, Stein, Switzerland.

The new broad-spectrum fungicides from the succinate dehydrogenase inhibitor (SDHI) class have been quickly adopted by the market, which may lead to a high selection pressure on various pathogens. Cases of resistance have been observed in 14 fungal pathogens to date and are caused by different mutations in genes encoding the molecular target of SDHIs, which is the mitochondrial succinate dehydrogenase (SDH) enzyme. All of the 17 marketed SDHI fungicides bind to the same ubiquinone binding site of the SDH enzyme. Their primary biochemical mode of action is the blockage of the TCA cycle at the level of succinate to fumarate oxidation, leading to an inhibition of respiration. Homology models and docking simulations explain binding behaviors and some peculiarities of the cross-resistance profiles displayed by different members of this class of fungicides. Furthermore, cross-resistance patterns among SDHIs is complex because many mutations confer full cross resistance while others do not. The nature of the mutations found in pathogen populations varies with species and the selection compound used but cross resistance between all SDHIs has to be assumed at the population level. In most of the cases where resistance has been reported, the frequency is still too low to impact field performance. However, the Fungicide Resistance Action Committee has developed resistance management recommendations for pathogens of different crops in order to reduce the risk for resistance development to this class of fungicides. These recommendations include preventative usage, mixture with partner fungicides active against the current pathogen population, alternation in the mode of action of products used in a spray program, and limitations in the total number of applications per season or per crop.
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http://dx.doi.org/10.1094/PHYTO-01-13-0009-RVWDOI Listing
September 2013

Gray mold populations in german strawberry fields are resistant to multiple fungicides and dominated by a novel clade closely related to Botrytis cinerea.

Appl Environ Microbiol 2013 Jan 19;79(1):159-67. Epub 2012 Oct 19.

Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany.

The gray mold fungus Botrytis cinerea is a major threat to fruit and vegetable production. Strawberry fields usually receive several fungicide treatments against Botrytis per season. Gray mold isolates from several German strawberry-growing regions were analyzed to determine their sensitivity against botryticides. Fungicide resistance was commonly observed, with many isolates possessing resistance to multiple (up to six) fungicides. A stronger variant of the previously described multidrug resistance (MDR) phenotype MDR1, called MDR1h, was found to be widely distributed, conferring increased partial resistance to two important botryticides, cyprodinil and fludioxonil. A 3-bp deletion mutation in a transcription factor-encoding gene, mrr1, was found to be correlated with MDR1h. All MDR1h isolates and the majority of isolates with resistance to multiple fungicides were found to be genetically distinct. Multiple-gene sequencing confirmed that they belong to a novel clade, called Botrytis group S, which is closely related to B. cinerea and the host-specific species B. fabae. Isolates of Botrytis group S genotypes were found to be widespread in all German strawberry-growing regions but almost absent from vineyards. Our data indicate a clear subdivision of gray mold populations, which are differentially distributed according to their host preference and adaptation to chemical treatments.
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http://dx.doi.org/10.1128/AEM.02655-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536109PMC
January 2013

Biological activity of sedaxane---a novel broad-spectrum fungicide for seed treatment.

Pest Manag Sci 2013 Apr 9;69(4):527-34. Epub 2012 Oct 9.

Syngenta AG, Crop Protection Research, Biology, Stein, Switzerland.

Background: Sedaxane is a new broad-spectrum seed treatment fungicide developed by Syngenta Crop Protection for control of seed- and soil-borne diseases in a broad range of crops. Its physicochemical properties and activity spectrum have been optimised for use as a seed treatment providing both local and systemic protection of the seed and roots of target crops.

Results: Sedaxane inhibits respiration by binding to the succinate dehydrogenase complex in the fungal mitochondrium. Its activity spectrum covers seed-borne fungi such as Ustilago nuda, Tilletia caries, Monographella nivalis and Pyrenophora graminea, as well as the soil-borne fungi Rhizoctonia solani, R. cerealis and Typhula incarnata. Under greenhouse conditions, sedaxane showed high levels and consistent protection against U. nuda, P. graminea and Rhizoctonia spp. Under field conditions, efficacy against Rhizoctonia spp. resulted in increased yield compared with the untreated check. Efficacy against snow mould has been shown under very high disease pressure conditions. The combination of sedaxane plus fludioxonil against snow mould can provide resistance management for sustainable use.

Conclusions: The broad spectrum and high level of activity in combination with excellent crop tolerance allow the use of sedaxane as a seed treatment in a wide variety of crops. It is a potential tool for precautionary resistance management when combined with other fungicides, especially against pathogens showing a potential for resistance development, such as M. nivalis.
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http://dx.doi.org/10.1002/ps.3405DOI Listing
April 2013

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

PLoS One 2012 19;7(4):e35429. Epub 2012 Apr 19.

Syngenta Crop Protection Münchwilen AG, Stein, Switzerland.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035429PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334918PMC
August 2012

New capabilities for Mycosphaerella graminicola research.

Mol Plant Pathol 2010 Sep;11(5):691-704

Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire, UK.

Mycosphaerella graminicola is a major pathogen of wheat worldwide, causing Septoria leaf blotch disease. Targeted gene disruption in M. graminicola, by Agrobacterium tumefaciens-mediated transformation, has become an established functional genomics tool for M. graminicola research in recent years. However, in order to advance research into this economically important pathogen, further functional genomics tools need to be developed. Here, we report three new capabilities for M. graminicola research: (i) two selectable markers have been shown to work robustly in M. graminicola, namely G418 and the fungicide carboxin; (ii) the generation of a strain of M. graminicola in which the KU70 (MUS-51) homologue has been disrupted; in this strain, homologous recombination efficiencies increased to more than 95%, whilst maintaining wild-type growth in vitro and full pathogenicity on wheat leaves; (iii) the ability to efficiently target and generate precise mutations of specific genes in the genomic context in M. graminicola. In addition, the insertion of the E198A mutation into the beta-tubulin gene (MgTUB1), conferring resistance to the fungicide benomyl, suggests that this mutant allele may provide an additional selectable marker. The collective use of these tools will permit further advancements in our knowledge of the biology and pathogenicity of this important plant pathogen.
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http://dx.doi.org/10.1111/j.1364-3703.2010.00629.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640411PMC
September 2010

Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, Phytophthora infestans.

Mol Plant Pathol 2010 Mar;11(2):227-43

Syngenta Crop Protection AG, CH-4332 Stein, Switzerland.

Oomycete plant pathogens cause a wide variety of economically and environmentally important plant diseases. Mandipropamid (MPD) is a carboxylic acid amide (CAA) effective against downy mildews, such as Plasmopara viticola on grapes and potato late blight caused by Phytophthora infestans. Historically, the identification of the mode of action of oomycete-specific control agents has been problematic. Here, we describe how a combination of biochemical and genetic techniques has been utilized to identify the molecular target of MPD in P. infestans. Phytophthora infestans germinating cysts treated with MPD produced swelling symptoms typical of cell wall synthesis inhibitors, and these effects were reversible after washing with H(2)O. Uptake studies with (14)C-labelled MPD showed that this oomycete control agent acts on the cell wall and does not enter the cell. Furthermore, (14)C glucose incorporation into cellulose was perturbed in the presence of MPD which, taken together, suggests that the inhibition of cellulose synthesis is the primary effect of MPD. Laboratory mutants, insensitive to MPD, were raised by ethyl methane sulphonate (EMS) mutagenesis, and gene sequence analysis of cellulose synthase genes in these mutants revealed two point mutations in the PiCesA3 gene, known to be involved in cellulose synthesis. Both mutations in the PiCesA3 gene result in a change to the same amino acid (glycine-1105) in the protein. The transformation and expression of a mutated PiCesA3 allele was carried out in a sensitive wild-type isolate to demonstrate that the mutations in PiCesA3 were responsible for the MPD insensitivity phenotype.
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http://dx.doi.org/10.1111/j.1364-3703.2009.00604.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640402PMC
March 2010

Scent evolution in Chinese roses.

Proc Natl Acad Sci U S A 2008 Apr 14;105(15):5927-32. Epub 2008 Apr 14.

Institut Fédératif de Recherche 128, Unité Mixte de Recherche 5667, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Université Lyon 1, Ecole Normale Supérieure de Lyon, France.

The phenolic methyl ether 3,5-dimethoxytoluene (DMT) is a major scent compound of many modern rose varieties, and its fragrance participates in the characteristic "tea scent" that gave their name to Tea and Hybrid Tea roses. Among wild roses, phenolic methyl ether (PME) biosynthesis is restricted to Chinese rose species, but the progenitors of modern roses included both European and Chinese species (e.g., Rosa chinensis cv Old Blush), so this trait was transmitted to their hybrid progeny. The last steps of the biosynthetic pathways leading to DMT involve two methylation reactions catalyzed by the highly similar orcinol O-methyltransferases (OOMT) 1 and 2. OOMT1 and OOMT2 enzymes exhibit different substrate specificities that are consistent with their operating sequentially in DMT biosynthesis. Here, we show that these different substrate specificities are mostly due to a single amino acid polymorphism in the phenolic substrate binding site of OOMTs. An analysis of the OOMT gene family in 18 species representing the diversity of the genus Rosa indicated that only Chinese roses possess both the OOMT2 and the OOMT1 genes. In addition, we provide evidence that the Chinese-rose-specific OOMT1 genes most probably evolved from an OOMT2-like gene that has homologues in the genomes of all extant roses. We propose that the emergence of the OOMT1 gene may have been a critical step in the evolution of scent production in Chinese roses.
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http://dx.doi.org/10.1073/pnas.0711551105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2311369PMC
April 2008

Production and emission of volatile compounds by petal cells.

Plant Signal Behav 2007 Nov;2(6):525-6

Laboratoire de Biotechnologies Végétales; Plantes Aromatiques et Médicinales; Université Jean Monnet; Saint-Etienne, France.

We localized the tissues and cells that contribute to scent biosynthesis in scented and non-scented Rosa x hybrida cultivars as part of a detailed cytological analysis of the rose petal. Adaxial petal epidermal cells have a typical conical, papillate shape whereas abaxial petal epidermal cells are flat. Using two different techniques, solid/liquid phase extraction and headspace collection of volatiles, we showed that, in roses, both epidermal layers are capable of producing and emitting scent volatiles, despite the different morphologies of the cells of these two tissues. Moreover, OOMT, an enzyme involved in scent molecule biosynthesis, was localized in both epidermal layers. These results are discussed in view of results found in others species such as Antirrhinum majus, where it has been shown that the adaxial epidermis is the preferential site of scent production and emission.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634358PMC
http://dx.doi.org/10.4161/psb.2.6.4659DOI Listing
November 2007

Both the adaxial and abaxial epidermal layers of the rose petal emit volatile scent compounds.

Planta 2007 Sep 23;226(4):853-66. Epub 2007 May 23.

Laboratoire de Biotechnologies Végétales, Plantes Aromatiques et Médicinales, EA 3061, Université Jean Monnet, 23, rue du Dr. Michelon, 42023 Saint-Etienne Cedex 2, France.

The localization and timing of production and emission of scent was studied in different Rosa x hybrida cultivars, focusing on three particular topics. First, it was found that petals represent the major source of scent in R. x hybrida. In heavily scented cultivars, the spectrum and levels of volatiles emitted by the flower broadly correlated with the spectrum and levels of volatiles contained within the petal, throughout petal development. Secondly, analysis of rose cultivars that lacked a detectable scent indicated that the absence of fragrance was due to a reduction in both the biosynthesis and emission of scent volatiles. A cytological study, conducted on scented and non-scented rose cultivars showed that no major difference was visible in the anatomy of the petals either at small magnification in optical sections or in ultrathin sections observed by TEM. In particular, the cuticle of epidermal cells was not thicker in scentless cultivars. Thirdly, using two different techniques, solid/liquid phase extraction and headspace collection of volatiles, we showed that in roses, both epidermal layers are capable of producing and emitting scent volatiles, despite the different morphologies of the cells of these two tissues. Moreover, OOMT, an enzyme involved in scent molecule biosynthesis was localized in both epidermal layers.
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http://dx.doi.org/10.1007/s00425-007-0531-1DOI Listing
September 2007

Role of petal-specific orcinol O-methyltransferases in the evolution of rose scent.

Plant Physiol 2006 Jan 16;140(1):18-29. Epub 2005 Dec 16.

Laboratoire Reproduction et Développement des Plantes, Unité Mixte de Recherche 5667 Centre National de la Recherche Scientifique, IFR128 Biosciences Lyon-Gerland, France.

Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominantly in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells' secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses.
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http://dx.doi.org/10.1104/pp.105.070961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1326028PMC
January 2006

Biosynthesis of the major scent components 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by novel rose O-methyltransferases.

FEBS Lett 2002 Jul;523(1-3):113-8

Reproduction et Développement des Plantes Laboratory, Ecole Normale Supérieure de Lyon, UMR 5667 CNRS-INRA-ENSL-UCBL, 46 allée d'Italie, 69364 Cedex 07, Lyon, France.

In Chinese rose species and in many modern varieties, two methylated phenolic derivatives, 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene, are major scent components. We show that cell-free extracts of rose petals catalyse the synthesis of 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene by methylation of precursor molecules. An expressed sequence tag approach was used to identify four highly similar O-methyltransferase sequences expressed specifically in petals and anthers. Thin layer chromatography analysis showed that the activities of these enzymes with different substrates and the proportions of reaction products produced closely mimicked those observed using cell-free petal extracts, indicating that orcinol O-methyltransferases are responsible for the biosynthesis of 3,5-dimethoxytoluene and 1,3,5-trimethoxybenzene from un-methylated precursors in this organ.
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http://dx.doi.org/10.1016/s0014-5793(02)02956-3DOI Listing
July 2002

Analysis of gene expression in rose petals using expressed sequence tags.

FEBS Lett 2002 Mar;515(1-3):35-8

Reproduction et Développement des Plantes, UMR 5667 CNRS-INRA-ENSL, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Cedex 07, Lyon, France.

Single-pass sequences were obtained from the 5'-ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent.
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http://dx.doi.org/10.1016/s0014-5793(02)02413-4DOI Listing
March 2002