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A maize wall-associated kinase confers quantitative resistance to head smut

Abstract

Head smut is a systemic disease in maize caused by the soil-borne fungus Sporisorium reilianum that poses a grave threat to maize production worldwide. A major head smut quantitative resistance locus, qHSR1, has been detected on maize chromosome bin2.09. Here we report the map-based cloning of qHSR1 and the molecular mechanism of qHSR1-mediated resistance. Sequential fine mapping and transgenic complementation demonstrated that ZmWAK is the gene within qHSR1 conferring quantitative resistance to maize head smut. ZmWAK spans the plasma membrane, potentially serving as a receptor-like kinase to perceive and transduce extracellular signals. ZmWAK was highly expressed in the mesocotyl of seedlings where it arrested biotrophic growth of the endophytic S. reilianum. Impaired expression in the mesocotyl compromised ZmWAK-mediated resistance. Deletion of the ZmWAK locus appears to have occurred after domestication and spread among maize germplasm, and the ZmWAK kinase domain underwent functional constraints during maize evolution.

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Figure 1: Fine mapping of the resistance QTL qHSR1.
Figure 2: Transgenic validation of ZmWAK.
Figure 3: Subcellular localization and expression pattern of ZmWAK.
Figure 4: ZmWAK functions in mesocotyl to inhibit the upward growth of S. reilianum.

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NCBI Reference Sequence

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Acknowledgements

We thank J. Schirawski (RWTH Aachen University) for providing the mating-compatible S. reilianum isolates SRZ1 and SRZ2 and X. Yang (China Agricultural University) for providing the DNA for 522 maize inbred lines and 184 teosinte accessions. We also thank N. Dengler for her help in the identification of microscopy structures. This work was supported by the National High-Tech Research and Development Program of China (grant numbers 2012AA10A306 and 2012AA101104).

Author information

Authors and Affiliations

Authors

Contributions

W.Z. contributed the functional analysis of ZmWAK and preparation of the manuscript. N.Z. contributed the construction and transformation of the chimeric receptor. Q.C., J.Z., X.Z. and Y.C. contributed the fine mapping of qHSR1. B.Z. contributed the sequencing of the ZmWAK alleles. J. Ye contributed the microscopy of S. reiliamun. H.L. and J. Yan contributed the evolutionary analysis. Y.X. and G.T. contributed the field inoculation of head smut. B.L. and K.A.F. contributed the sequencing of the Mo17 and HZ4 BAC clones. J.L. contributed maize transformation. M.X. led the project and wrote the manuscript.

Corresponding author

Correspondence to Mingliang Xu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Fine mapping and genetic effect of qHSR1.

(ac) Sequential fine mapping of qHSR1. The empty and filled rectangles represent the homozygous HZ4/HZ4 and heterozygous Ji1037/HZ4 regions, respectively. In this way, the genomic architecture of each recombinant type is illustrated. The table to the right indicates the resistant percentages of progeny with or without the donor regions. “Total No. of progeny” indicates the sample sizes of recombinant-derived progeny with the same recombinant type. P values represent the significance of the difference between the homozygous and heterozygous genotypes. The phenotype of a given recombinant genotype was deemed resistant (R) if the P value was <0.05 or susceptible (S) if the P value was >0.05. Fine mapping of qHSR1 was performed by comparing the donor region to the deduced phenotype of the recombinants. (a) Fine mapping using 43 F1BC3 and F2BC2 recombinants narrowed resistance to the qHSR1 interval PHMI–STSN6AC. (b) Fine mapping using 54 F1BC4 and F2BC3 recombinants narrowed resistance to the qHSR1 interval PHMI–STS3M1. (c) Fine mapping using 43 F1BC6 and F2BC5 recombinants narrowed resistance to the qHSR1 interval STS1M3–STS3M1. Resistance percentages are given as mean ± standard error. (d) Genetic contribution of qHSR1 to head smut resistance. Light and dark gray filled bars correspond to the heterozygous Ji1037/HZ4 and homozygous HZ4/HZ4 genotypes at qHSR1, respectively. Values are means ± standard error.

Supplementary Figure 2 Confirmation of the mapped qHSR1 region with various test-cross progeny.

(a) Cross between recombinants with donor fragments proximal and distal to STS1M3. (b) Cross between recombinants with the smallest donor fragment and the donor fragment distal to STS3M1. (c) Cross between recombinants with the donor fragments distal to STS3M1 and proximal to CAPS3M3. (d) Cross between the recombinants with the donor fragment proximal to STS1M3. The black triangles represent the 147-kb insertion. Values are means ± standard error. On the right edge of each graph, significant differences (P < 0.05) are indicated by different lowercase letters. Differences that were not statistically significant (P > 0.05) are indicated by the same lowercase letters.

Supplementary Figure 3 Predicted genes and genomic variation within the qHSR1 regions of three inbred lines.

Green arrows indicate the position and orientation of predicted genes. Blue and gray boxes indicate the locations of transposons and retrotransposons, respectively. Light-gray shading indicates collinear coding regions. The region between the red dotted lines is the mapped qHSR1 region, which is flanked by the markers STS1M2 and STS3M1 (highlighted in yellow).

Supplementary Figure 4 RNA expression of five candidate genes.

(a) Specificity test of the ZmWAK primers by using cDNA as the template. (b) Expression levels of five candidate genes in the newly emerged tissues of Ji1037 seedlings from mock- and S. reilianum–inoculated seedlings 3 DAS were assessed by qPCR. gDNA, genomic DNA. The ZmWAK primers span a large intron (~2.8 kb) and therefore produced no product with the genomic DNA template.

Supplementary Figure 5 Characterization of the qHSR1 region in 62 inbred lines.

(a) The predicted genes in the qHSR1 regions of Mo17 and HZ4 are depicted as arrows pointing in the direction of transcription. Exons are in orange, and introns are in yellow. (b) PCR amplification of the expressed genes from 62 inbred lines. Red indicates the same PCR product as that in Ji1037, green indicates the same PCR product as that in HZ4, yellow indicates mixed PCR products containing both Ji1037 and HZ4 sequences, gray indicates a PCR product that is different from both Ji1037 and HZ4, and blue indicates no amplification. R, resistant; S, susceptible; “number,” the number of lines sharing the same haplotype detected by each marker.

Supplementary Figure 6 MUSCLE alignment of the closest WAK proteins and phylogenetic analysis of the ZmWAK gene family.

(a) WAK proteins closely related to ZmWAK were aligned, including AtWAK1 and AtWAK2 (Arabidopsis), OsWAK104 (rice), BdWAK4L (B. distachyon), Sb05g027230.1 (Sorghum bicolor), SiWAK2L (S. italica) and TRIUR3_01549_P1 (T. urartu). The GUB, EGF_CA and kinase domains are highlighted with blue, green and purple lines, respectively. The kinase active site is outlined in red, and the mutated sites are outlined in yellow. The blue star indicates the arginine and non-arginine residues adjacent to the active site aspartic acid. Black shading indicates identical amino acids, and gray shading indicates similar amino acids to ZmWAK. (b) Phylogenetic analysis of the predicted WAK proteins (containing the GUB, EGF_CA and kinase domains) in maize and several other plants using the maximum-likelihood method. RD and non-RD WAKs clustered separately, except for two RD kinases (underlined in blue) that clustered in the non-RD clade and one WAK that contained the kinase domain but lacked the conserved active site aspartic acid (underlined in green).

Supplementary Figure 7 ZmWAK amino acid sequence variation and expression patterns of different ZmWAK alleles.

(a) The eight amino acid polymorphic sites detected in the eight ZmWAK proteins are marked by triangles. (b) Detailed description of the ZmWAK alleles from the Mo17, B73, Zheng58, 8902, 982, V022 and V4 inbred lines. R, resistant; S, susceptible; MR, moderately resistant. (c) PROVEAN analysis of the potential functional effects of the amino acid changes. (d) Basal expression of ZmWAK in the root, leaf and mesocotyl of resistant and susceptible inbred lines. Expression values are the mean ± s.d. of two independent experiments.

Supplementary Figure 8 Nucleotide diversity of ZmWAK.

The nucleotide diversity of maize (πM) and teosinte (πT) in the ZmWAK region shown in graphical (upper) and tabular (lower) form. Results of the Tajima’s D test and π ratios are shown. P1 and P2 indicate the promoter region upstream and downstream of the transposable element insertion, respectively.

Supplementary Figure 9 Genomic variation in the ZmWAK promoter region.

High sequence variation was detected at –462 bp in the ZmWAK promoter, where a number of residual transposable elements had been inserted. Type I and III transposable element arrangements were found in the majority of the inbred lines, such as Mo17 and B73, respectively. Type II and type IV arrangements were found mainly in teosinte entries and trophic maize lines.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9. (PDF 4722 kb)

Supplementary Table 1

Primers used in fine mapping and studies of molecular mechanism. (XLSX 14 kb)

Supplementary Table 2

ZmWAK information in 522 maize lines and 184 teosintes. (XLSX 37 kb)

Supplementary Table 3

Pedigree of ten inbred lines with HZ4 background. (XLSX 9 kb)

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Zuo, W., Chao, Q., Zhang, N. et al. A maize wall-associated kinase confers quantitative resistance to head smut. Nat Genet 47, 151–157 (2015). https://doi.org/10.1038/ng.3170

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