Research ArticlePLANT PATHOLOGY

The hijacking of a receptor kinase–driven pathway by a wheat fungal pathogen leads to disease

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Science Advances  26 Oct 2016:
Vol. 2, no. 10, e1600822
DOI: 10.1126/sciadv.1600822
  • Fig. 1 Map-based cloning of the Snn1 gene.

    (A) The genomic region containing the Snn1 gene on the short arm of chromosome 1B is shown in red. (B) The genetic linkage map of the Snn1 region. Markers in blue are from Reddy et al. (12), markers in red are from previous unpublished work, and markers in black were developed in this research. (C) BAC-based physical map of the Snn1 region anchored to the genetic linkage map. The four BACs in yellow represent the Snn1 candidate gene region. (D) Genetic linkage mapping of the seven candidate genes identified in the four BACs from the candidate gene region in (C). The green oval represents TaWAK, which cosegregated with Snn1.

  • Fig. 2 Functional validation of the TaWAK gene by mutagenesis and transgenesis.

    (A) Gene structure of the TaWAK (Snn1) gene with exons in yellow and UTRs in gray. Red arrowheads indicate mutation sites in EMS-induced mutants, all of which were insensitive to SnTox1. PKC, protein kinase C. (B) Infiltration and inoculation reactions on leaves of wild-type CS and the EMS mutant CSems6125 are shown as an example. (C) Reactions to SnTox1 infiltrations of CS (Snn1+), untransformed Bobwhite (BW; Snn1−), and sensitive and insensitive T1 transgenic plants both derived from the same event (BW5240). (D) Transgenic plants that were sensitive to SnTox1 were also susceptible to disease caused by spores of an SnTox1-producing fungal isolate. (E) All SnTox1-sensitive T1 plants derived from event BW5240 had the TaWAK transgene, and all insensitive plants lacked the transgene. (F) Similarly, all SnTox1-sensitive BW5240 T1 plants expressed TaWAK, whereas the insensitive plants did not.

  • Fig. 3 Transcriptional expression of Snn1.

    (A) Snn1 expression survey in CS by reverse transcription polymerase chain reaction (RT-PCR) with GAPDH as an endogenous control. The absence of an amplicon in CS 1BS-18 indicates that Snn1 transcription is unique to 1B. (B) Snn1 expression levels in 2-week-old plants entrained with a 12-hour light/dark cycle evaluated every 3 hours over a 72-hour period (blue) and in plants subjected to continuous darkness for the same time points (orange) using relative quantitative PCR (RQ-PCR). (C) RQ-PCR evaluation of Snn1 expression in SnTox1-challenged plants (green bars) and YPD mediuminfiltrated plants (blue bars). The dotted lines are shown to compare expression of Snn1 at 11:00 a.m. over 3 days. Note that the expression of Snn1 in the SnTox1-infiltrated plants becomes reduced over time compared to the control.

  • Fig. 4 Overview of the Snn1-SnTox1 and Tsn1-SnToxA interactions and known downstream events that result in NETS in the wheat–P. nodorum pathosystem.

    The SnTox1 and SnToxA proteins are secreted by the fungus. SnToxA is internalized into the cell (23), but SnTox1 is not (11). Upon recognition of SnTox1 and SnToxA by the Snn1 and Tsn1 proteins, respectively, signaling leading to up-regulation of defense response pathways and events resulting in programmed cell death (9, 11) ultimately provide a means for the pathogen to gain nutrients and reproduce. Plants with either Tsn1 or Snn1 are susceptible, and plants with both genes experience even higher levels of disease (24). Elimination of both genes renders the plant resistant.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/10/e1600822/DC1

    fig. S1. Conserved domains and active sites identified in the deduced Snn1 protein.

    fig. S2. An unrooted phylogenetic tree showing relationships between Snn1 and other plant wall–associated receptor kinase (WAK) proteins.

    fig. S3. Deduced amino acid sequence alignment of mutants and informative lines.

    fig. S4. Transcription analysis of the splice site mutant CSems-6141.

    fig. S5. DNA blot analysis.

    fig. S6. DNA alignment of Snn1 from chromosome 1B and its putative homoeoallele from chromosome 1D.

    fig. S7. Phylogenetic tree of 24 genotypes based on deduced amino acid sequences of the Snn1 gene.

    fig. S8. Transcription analysis of Snn1 in the durum wheat variety Lebsock.

    fig. S9. TaMAPK3 transcription analysis after SnTox1 spray inoculation.

    fig. S10. Y2H assays showing that Snn1 interacts with SnTox1 in a sequence-specific manner.

    table S1. PCR-based molecular markers used to anchor the CS chromosome 1BS BAC contig to the genetic linkage map containing the Snn1 gene.

    table S2. PCR-based molecular markers developed for the seven candidate genes.

    table S3. Descriptions of induced mutations identified within the Snn1 gene-coding region.

    table S4. Top five BLASTP hits in the NCBI nr database using the Snn1 deduced amino acid sequence as a query.

    table S5. The 826 Triticum accessions and 123 Ae. speltoides accessions evaluated for the presence of the Snn1 DNA sequence and/or for reaction to SnTox1.

    table S6. Primers used for sequencing of Snn1 from genomic DNA.

    table S7. PCR primers used to amplify the Snn1 cDNA fragments for sequencing.

    table S8. PCR primers used to amplify the Snn1 cDNA 5′ and 3′ ends.

    table S9. PCR primers used for RQ-PCR analysis.

    table S10. PCR primers used for Y2H analysis.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Conserved domains and active sites identified in the deduced Snn1 protein.
    • fig. S2. An unrooted phylogenetic tree showing relationships between Snn1 and other plant wall–associated receptor kinase (WAK) proteins.
    • fig. S3. Deduced amino acid sequence alignment of mutants and informative lines.
    • fig. S4. Transcription analysis of the splice site mutant CSems-6141.
    • fig. S5. DNA blot analysis.
    • fig. S6. DNA alignment of Snn1 from chromosome 1B and its putative homoeoallele from chromosome 1D.
    • fig. S7. Phylogenetic tree of 24 genotypes based on deduced amino acid sequences of the Snn1 gene.
    • fig. S8. Transcription analysis of Snn1 in the durum wheat variety Lebsock.
    • fig. S9. TaMAPK3 transcription analysis after SnTox1 spray inoculation.
    • fig. S10. Y2H assays showing that Snn1 interacts with SnTox1 in a sequence-specific manner.
    • table S1. PCR-based molecular markers used to anchor the CS chromosome 1BS BAC contig to the genetic linkage map containing the Snn1 gene.
    • table S2. PCR-based molecular markers developed for the seven candidate genes.
    • table S3. Descriptions of induced mutations identified within the Snn1 gene-coding region.
    • table S4. Top five BLASTP hits in the NCBI nr database using the Snn1 deduced amino acid sequence as a query.
    • table S5. The 826 Triticum accessions and 123 Ae. speltoides accessions evaluated for the presence of the Snn1 DNA sequence and/or for reaction to SnTox1.
    • table S6. Primers used for sequencing of Snn1 from genomic DNA.
    • table S7. PCR primers used to amplify the Snn1 cDNA fragments for sequencing.
    • table S8. PCR primers used to amplify the Snn1 cDNA 5′ and 3′ ends.
    • table S9. PCR primers used for RQ-PCR analysis.
    • table S10. PCR primers used for Y2H analysis.

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