A wheat cysteine-rich receptor-like kinase confers broad-spectrum resistance against Septoria tritici blotch

Abstract

The poverty of disease resistance gene reservoirs limits the breeding of crops for durable resistance against evolutionary dynamic pathogens. Zymoseptoria tritici which causes Septoria tritici blotch (STB), represents one of the most genetically diverse and devastating wheat pathogens worldwide. No fully virulent Z. tritici isolates against synthetic wheats carrying the major resistant gene Stb16q have been identified. Here, we use comparative genomics, mutagenesis and complementation to identify Stb16q, which confers broad-spectrum resistance against Z. tritici. The Stb16q gene encodes a plasma membrane cysteine-rich receptor-like kinase that was recently introduced into cultivated wheat and which considerably slows penetration and intercellular growth of the pathogen.

Fig. 1. Map-based cloning of the Stb16q gene.

a Scaffold1066 and Scaffold45305 from the Ae. tauschii physical map were identified as co-segregating with Stb16q in the DH population derived from TA4152-19 × ND495. Polymorphic SSR markers and SNP markers identified from gene/pseudogene fragments of these two scaffolds were genetically mapped based on 1980 F₂ individuals (distances in centiMorgans). This delimits Stb16q to a 0.07 cM interval between markers cfd335 and cfn80033. Screening a TA4152-19 BAC library and the wheat reference sequence from Chinese Spring (RefSeq v1.0) identified a 272 kb interval with two candidate genes (scale bar = 50 kb). Unk1 and Crk6 are depicted as orange and blue arrows, respectively. A magnification of the Crk6 structure reveals seven exons indicated as light gray boxes (scale bar = 0.5 kb). Yellow, dark gray, and green boxes represent the predicted DUF26 domains, the transmembrane domain, and the serine/threonine protein kinase domains, respectively. Mutation identified in the EMS-derived mutant family 236 is represented on the 5^th exon. b Identification of the susceptible mutant family 236 derived from an EMS treatment of TA4152-19 seeds and carrying the deleterious mutation S508F. Phenotypes of leaf 1 (L1) and leaf 2 (L2) of three individual M₄ plants (M₄_13, M₄_14, and M₄_15) from family 236 are represented 21 days after inoculation with Z. tritici isolate IPO88018. Mutation genotyping with marker cfn80052 reveals that susceptible M₄ plants carry the mutation at the homozygous state while the resistant M₄ plants present the wild-type (wt) allele. c The native sequence of the Crk6 gene including promoter and terminator (12.9 kb) was introduced via biolistic into the susceptible French wheat cultivar Courtot. T₂ plants from family Ct_4.1.1.1 carrying the Crk6 transgene and its sister line Ct_4.1.1.3 lacking Crk6 present resistant and susceptible phenotypes, respectively, on leaf 2 against three Z. tritici isolates (IPO9699, IPO92006, and IPO9699) at 21 dpi.

Fig. 2. Stb16q genetic diversity and origin.

a Schematic representation of the 12 Stb16q exon1 haplotypes (H1 from H12) identified from re-sequencing 156 wheat accessions (including the 76SHWs, Supplementary Data 4). Stb16q structure reveals seven exons indicated as light gray boxes. Yellow, dark gray, and green boxes represent the predicted DUF26 domains, the transmembrane domain, and the serine/threonine protein kinase, interleukin-1 receptor associated kinase (STKc_IRAK) domains, respectively. Vertical black, white, and blue bars represent SNP, deletions, and insertions, respectively, compared to haplotype 1. On the left of each haplotype are indicated in brackets the most relevant representative and the number of accessions identified for each haplotype. On the right of haplotypes are indicated the number of SNP, number of deletions (their size in bp), and number of insertions (their size in bp). Asterisks highlight the presence of a stop codon. Physical position of diagnostic markers cfn80044 and cfn80045 are indicated at the bottom. All haplotypes have been identified in the SHWs collection except haplotype H12. b Topographic distribution of Ae. tauschii accessions carrying the Stb16q gene (green star) or not (blue square) and undetermined allele (gray round) according to diagnostic markers cfn80044 and cfn80045 (Supplementary Data 6). The map was created using the GPS Visualizer software (https://www.gpsvisualizer.com/about.html).

Fig. 3. Zymoseptoria tritici infection during compatible and incompatible interactions using wheat near isogenic lines carrying the Stb16q gene.

Leaf surfaces and leaf cross sections of Chinese Spring carrying or not Stb16q were observed using a confocal at three time points after infection (7, 10, and 13 dpi) with the Stb16q avirulent GFP-labeled Z. tritici isolate 3D7. Epiphyllous and penetrating hyphae, shown in green or in purple when they were stained by propidium iodide, are marked by full and empty white triangles, respectively. At 7 dpi, in the incompatible reaction (CS with Stb16q), the fungus is observed only at the leaf surface and no instance of penetration was recorded (a). At 10 dpi, some instances of penetration were observed (d) and by 13 dpi, hyphae start growing inside the substomatal cavity (f). In the compatible interaction (CS without Stb16q), by 7 dpi the fungus hyphae colonized the mesophyll (b). By 10 and 13 dpi, it formed a dense network and began to fill the substomatal cavities and to form pycnidia (e, g). Images a–g are maximum projections. The corresponding orthogonal views (yz) are shown in the narrow panels to the right of each main image. Scale bars represent 20 µm. The autofluorescence of the chloroplasts is shown in purple and the cell walls stained with propidium iodide in pink. The experiment was performed twice (n = 2). In the first replicate, infected leaves were collected at 6 and 9 dpi while they were collected at 7, 10, and 13 dpi in the second replicate. Both replicates show the same result.