Fig 1.
Baseline cross-resistance of Z. tritici populations to SDHI fungicides.
Sensitivity towards different SDHIs was determined in liquid culture assays for a collection of 97 Z. tritici strains sampled for fungicide resistance monitoring in 2009 in Europe (plain circles). Two strains 06STD024 (red triangle) and 07STGB009 (green square), were considered fluopyram-resistant in monitoring performed in 2006 and 2007 respectively. 09STF011 (blue circle), belongs to the collection of 97 isolates sampled in 2009 and is the isolate with lowest sensitivity towards fluopyram in this set. Panels (A), (B) and (C) represent liquid culture cross-resistance plots with SHA-SDHI fluopyram on the y axis and non-SHA SDHIs benzovindiflupyr, fluxapyroxad or boscalid on the x-axis respectively. Panels (D), (E) and (F) correspond to cross resistance plots of non-SHA SDHIs fluxapyroxad, benzovindiflupyr and boscalid, compared as pairs. 06STD024, 07STGB009 and 09STF011 are circled in red.
Fig 2.
Fine mapping of fluopyram resistance factor using 06STD024 x IPO323 progeny.
(A) Agar plate growth assay used for characterizing progeny isolates for resistance or sensitivity to fluopyram. 2 μl of 2.106 cells.ml-1 were spotted onto AE agar supplemented or not with 10 mg.L-1 fluopyram and incubated at 20°C. Pictures were taken either 7 days (control) or 14 days (fluopyram) after inoculation. (B) IPO323 mapping intervals determined by BSA using 60 progeny isolates (i) and by CAPS markers (ii) on the full mapping population (234 progeny isolates). (C) 16 kb mapping interval of IPO323 chromosome 3. Structural variations at this locus between IPO323 and 06STD024 were determined using long range PCRs. Insert 1 was fully sequenced, only borders of insert 2 were sequenced. Insert 1 and insert 2 positions are based on the IPO323 genome. (D) Gene content of insert 1 region. Predicted genes and their orientation are visualized with arrows, green: putative genes, red: ZtSDHC3, blue: transposable element. Diagonally striped rectangles represent regions of high similarity (>90% identity) to other fully assembled Z. tritici genomes, corresponding chromosomal coordinates are indicated.
Fig 3.
Z. tritici SDHC proteins alignment.
Z. tritici ZtSDHC3 (alt-SDHC, NCBI MK067274, isolate 06STD024), ZtSDHC1 (SDHC, Uniprot F9XH52, isolate IPO323) and ZtSDHC2 (SDHC2, NCBI SMR59342, isolate IPO323) proteins were aligned with AlignX (Blosum62). Asterisk (*) is located above the predicted cleavage sites of the pre-proteins indicated by a red line. Red arrow highlights the Qp-site amino-acid residue likely involved in differential SDHI sensitivity pattern.
Fig 4.
The role of alt-SDHC_I78 residue in conferring SHA-SDHIs-specific resistance in vivo and in planta.
(A) Agar growth phenotypes of IPO323 mutants (left panel) and 06STD024 mutants (right panel). Left panel: Flu21 is an IPO323 SDHC_A84I UV mutant, pTet::SDHC3: IPO323 transformants carrying the ZtSDHC3 gene under the control of a tetracycline-repressible promoter, pTet::SDHC3_I78A IPO323 transformants carry a similar construct but contain a mutated version of SDHC3 gene encoding an alt-SDHC_I78A variant. Right panel: 06STD024 and individual deletion mutants of either the core ZtSDHC1 (KO_SDHC1) or of the dispensable ZtSDHC3 (KO_SDHC3). Pictures were taken at 6DPI, + Dox indicates the addition of doxycycline (30 mg.L-1) to the medium. (B) Liquid culture sensitivity of IPO323 and 06STD024 mutants towards SDHIs. The set of characterized IPO323 (white bars) and 06STD024 (grey bars) mutants was similar to panel A. EC50s (nM) were determined in duplicate in at least 3 biological replicates (see S5 Table). Values obtained for a broader range of marketed and research SDHIs are presented in S5 Table. (C) In planta SDHI-sensitivity assays. The presented graphs are derived from a single biological experiment, each value / data point represents the mean disease control value of 12 individual plants. The sensitivity curves were obtained by non-linear regression of the data using GraphPad Prism software. (D) In planta EC50s (g.ha-1) of reference strain (705) and 06STD024 mutants for commercial SDHIs, benzovindiflupyr, isopyrazam (non SHA-SDHIs) and pydiflumetofen (SHA-SDHI). Values are derived from four biological replicates of 12 technical replicates each (EC50 +/- 95% confidence interval).
Table 1.
Succinate-quinone SDHIs sensitivity assays on purified mitochondria of field isolates and transformants of Z. tritici.
Fig 5.
Comparison of Z. tritici 3D models of the two SQR paralogs and putative binding modes for SHA SDHIs.
(A) Homology model of Z. tritici WT-SQR (blue) superimposed on the homology model of Z. tritici altC-SQR (salmon). (B) Putative binding mode of pydiflumetofen in Z. tritici WT-SQR. (C) 2D depiction of SDH inhibitors used for docking comparisons and discussed in the text. (D) Putative binding mode of isofetamid in Z. tritici WT-SQR. E. superposition of energy minimum conformations for pydiflumetofen and compound 3.
Table 2.
Occurrence of the ZtSDHC3 gene in European Z. tritici monitoring populations and the pangenome.
Fig 6.
Resistance to SHA-SDHIs in Z. tritici EU populations.
(A) Box and whisker plot presenting EC50 sensitivity data of 93 Z. tritici isolates sampled in the EU in 2009. Sensitivity data are sorted by genotype according to the presence of the ZtSDHC3 gene. ** p value of 0.0029 in Welch’s corrected unpaired t-test. (B) Solid agar growth of a collection of 93 Z. tritici isolates sampled in 2009 (same set as above). Individual strains from this collection are boxed and numbered 1 to 93. Boxes A and B correspond to reference resistant strains 06STD024 and 07STGB009 respectively. Box C corresponds to IPO323 reference sensitive isolate. The yellow framed boxes correspond to strains carrying the ZtSDHC3 gene. Yellow arrow designates strain 09STIR20.1 (number 78) carrying a non-functional alt-SDHC (frameshift, S6 Table). Each individual strain was spotted onto AE agar plates (approx. 700 cells per spot) supplemented or not with isofetamid 5 mg.L-1, fluopyram 5 mg.L-1 and pydiflumetofen 0.1 mg.L-1. Plates were left to grow at 20°C in the dark and imaged at 10 DPI (DMSO control and isofetamid) or 18 DPI (fluopyram and pydiflumetofen).
Fig 7.
Fungicide sensitivity, gene expression and mRNA splicing in alt-SDHC-containing field isolates.
(A) Gel electrophoresis of RT-PCR products of ZtSDHC1 and ZtSDHC3 (5’ regions encompassing 2 introns each). gDNA of strain 06STD024 was used as control. Two control strains (IPO323 and IPO94269) lack the ZtSDHC3 gene whereas the other eight isolates (09STIR20.3, 09STD053, 09STD041, 09STF037, 09STF011, 09STF112, 07STGB009 and 06STD024) all carry the gene. (B) Absolute quantification by hydrolysis probe RT-qPCR of total ZtSDHC1 mRNA, and of total and unspliced ZtSDHC3 mRNAs. Error bars correspond to standard deviation (± SD) corresponding to eight individual determinations. (C) Plot of total ZtSDHC3 mRNA for each isolate versus calculated percentage of spliced ZtSDHC3 mRNA. Results for strain 09STIR20.3 not displayed (calculation leading to negative value, S5 Dataset). (D) Plot of spliced ZtSDHC3 mRNA against fluopyram sensitivity in liquid culture (EC50 in nM). Vertical error bars correspond to the standard error of the mean (± SEM) corresponding to at least five individual determinations of fluopyram EC50 (S5 Table). Results for strain 09STIR20.3 not displayed.
Table 3.
Overview of genotyping, phenotyping and protein assays for a panel of Z. tritici field isolates.
Fig 8.
Structural variation at the ZtSDHC3 locus in European Z. tritici field isolates and populations.
(A) Structural overview of ZtSDHC3 locus variations in a set of sequenced Z. tritici isolates. Mutations are lined up to 3D7 genomic structure, only mutations located within the region between ZT3D7_G4529 start codon to the stop codon of the ZtSDHC3 gene are shown. Positions are numbered according to the ZtSDHC3 start codon (+1). Sequences have been deposited at NCBI under references MK067275-MK067282. (B) Insertion of transposable elements in the promoter of ZtSDHC3 of highly resistant 06STD024 and 07STGB009 field isolates. Target site duplications of 9 bp are flanking each transposon insertion. (C) European map with pie charts representing the 4 genotypes detected in Zymoseptoria tritici isolates collected in 2016. Green: ZtSDHC3 gene absent, grey: ZtSDHC3 gene present and no promoter amplification product, yellow: ZtSDHC3 gene present and promoter of classical size, red: ZtSDHC3 gene present and promoter of larger size. The total count of isolates for each sampling location is presented in white boxes. Right panel: Bar chart showing the total count of isolates of each type (similar color code) listed by countries.