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A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat


As there are numerous pathogen species that cause disease and limit yields of crops, such as wheat (Triticum aestivum), single genes that provide resistance to multiple pathogens are valuable in crop improvement1,2. The mechanistic basis of multi-pathogen resistance is largely unknown. Here we use comparative genomics, mutagenesis and transformation to isolate the wheat Lr67 gene, which confers partial resistance to all three wheat rust pathogen species and powdery mildew. The Lr67 resistance gene encodes a predicted hexose transporter (LR67res) that differs from the susceptible form of the same protein (LR67sus) by two amino acids that are conserved in orthologous hexose transporters. Sugar uptake assays show that LR67sus, and related proteins encoded by homeoalleles, function as high-affinity glucose transporters. LR67res exerts a dominant-negative effect through heterodimerization with these functional transporters to reduce glucose uptake. Alterations in hexose transport in infected leaves may explain its ability to reduce the growth of multiple biotrophic pathogen species.

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Figure 1: Mutational and comparative genomic and analysis of the Lr67 region.
Figure 2: Location of amino acid changes resulting from mutagenesis in the LR67 protein.
Figure 3: Characterization of the LR67sus hexose transporter.
Figure 4: Characterization of LR67 homeologs and interaction analysis.

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This work was supported by funds provided through Grains Research and Development Corporation (CSP00161, CSP00164), Australia, and the Office of the Chief Executive (OCE) Post Doctoral Fellowship of the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia. We are grateful to S. Chandramohan, V. Calvo-Salazar, T. Richardson, H. Miah and X. Xia for technical assistance. We thank A. Ashton, I. Dry, P. Ryan and A. Rae for helpful discussions.

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Authors and Affiliations



S.H.-F., C.L., W. Schnippenkoetter, L.V., J.H.-E., M.L., S.P., R.M., X.K., H.B., R.S. and E.L. conducted pathological studies, molecular genetic analysis of populations, development and analysis of gamma and EMS mutants. J.W.M., J.W.P., P.D. and E.L. planned the transporter functional studies. J.W.M. conducted the transporter functional experiments. W. Schnippenkoetter, M.A. and E.L. conducted transgenic studies in wheat and barley. W. Spielmeyer provided DNA samples of azide mutants. J.W.M. and M.T. conducted the microscopy studies. E.L., J.W.M., M.A., P.D. and J.W.P. wrote the manuscript, and all coauthors commented on the draft.

Corresponding author

Correspondence to Evans Lagudah.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Quantification of fungal biomass (relative fluorescence units, RFU) in Lr67 mutants using a wheat germ agglutinin fluorophore binding to chitin.

(a) EMS mutants. (b) Gamma mutants. Resistant (R) and susceptible (S) sibs from flag leaves at a field site infected with Pst in Cobbitty, Australia 2013. Samples are n = 4, and error bars are s.d.

Supplementary Figure 2 Transgenic wheat and barley plants with Lr67res genomic construct expressing rust resistance.

(a) Field plot of Lr67 transgenic and non-transgenic wheat (Fielder) infected with stripe rust (Pst). (b) Genomic blot of NdeI restricted DNA probed with DNA fragment from Lr67 promoter region. The first lane is from Fielder sib line without the transgene, and the remaining lanes from Lr67res transgenic lines showing DNA fragments inherited from copies of the transgene (shown by asterisks). (c) Gene construct used in wheat transformation. The Lr67res genomic fragment (7,133 bp) was inserted in the NotI site (N) of pVecNeo and consists of the native promoter (5’ region), three exons, two introns and the 3’ regulatory region. The probe used to determine copy number is shown as a blue box in the 5’ region, and left and right borders of the T-DNA are marked as LB and RB, respectively. (d) Leaves of Lr67 transgenic and non-transgenic sib lines of barley (Golden Promise) infected with barley leaf rust (P. hordeii) 14 days post infection. (e,f) UV fluorescence microscopic images of barley leaf rust infected Golden Promise and transgenic Golden Promise stained with WGA-FITC at 12 days post infection. Well developed uredia are observed in the non-transgenic sib line without Lr67 (f) in contrast to restricted fungal growth in the Lr67 transgenic line (e).

Supplementary Figure 3 Phylogeny of LR67 relative to other sugar transporter proteins from the STP family.

At, Arabidopsis thaliana; Bradi, Brachypodium distachyon; Hv, Hordeum vulgare; Le, Lycopersicon esculentum; Mt, Medicago truncatula; Os, Oryza sativa; Sb, Sorghum bicolor; Si, Setaria italica; Vv, Vitis vinifera; Zm, Zea mays. Phylogenetic analysis was carried out using MUSCLE alignment, Gblocks curation followed by PhyML phylogeny (Dereeper, A. et al., robust phylogenetic analysis for the non-specialist, Nucleic Acids Res. 36, W465–W469, 2008).

Supplementary Figure 4 Expression of the Lr67 homoeologs from the AA, BB and DD genomes of Tc (Thatcher) and Tc67 (RL6077) seedlings by qRT-PCR.

Mock-inoculated plants are labeled as Mock 24 h and Mock 72 h, and plants inoculated with Pt are at 24 h and 72 h post inoculation (mean ± s.e.m., n = 3). Statistical significance levels, depicted by lowercase letters, are from comparisons within homoeologs for each genotype separately.

Supplementary Figure 5 Functional characterization of LR67.

(a) Glucose transport by LR67 allelic variants. Time course of EBY.VW4000 expressing LR67 resistant and susceptibility alleles following exposure to 50 μM D-[U-14C] glucose in pH 5 phosphate buffer. Samples are taken at 2 min junctures and an empty p426ADH1 provided background scintillation counter readings (mean ± s.e.m., n = 3). (b,c) Substrate binding specificity of LR67sus. EBY.VW4000 expressing LR67sus was incubated with 50 μM D-[U-14C] glucose and 500 μM of the specified competing sugar or glucose analog. Data are represented as percentage of the transport rate obtained for LR67sus supplied with 50 μM D-[U-14C] glucose only (mean ± s.e.m., n = 3). (d) Effect of pharmacological membrane modifiers and metabolic inhibitors on LR67sus carrier function. EBY.VW4000 expressing LR67sus was incubated with 50 μM D-[U-14C] glucose and the membrane modifier/metabolic inhibitor at the specified concentration. Data are represented as percentage of the transport rate obtained for LR67sussupplied with 50 μM D-[U-14C] glucose only (mean ± s.e.m., n = 3). Phlorizin inhibition is suggestive of a sugar transporter; PCMBS and NEM are sulfhydryl reagents indicating presence of such groups in the protein that contribute to its transport ability. NEM is membrane permeant while PCMBS is not. The latter finding points to a plasma membrane localization of the transporter. DEPC binds to a histidine residue in the binding site of the transporter. (e) LR67sus glucose transport function is pH dependent. EBY.VW4000 expressing LR67sus was incubated with 50 μM D-[U-14C] glucose in 25 mM NaHPO4/NaH2PO4 buffered to the specified pH (mean ± s.e.m., n = 3). (f) Co-expression of HXT and LR67. Glucose uptake of EBY.VW4000 individually expressing ScHXT5 in p426ADH1 and co-expressed with LR67res in pJM67 (mean ± s.e.m., n = 3).

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Moore, J., Herrera-Foessel, S., Lan, C. et al. A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat Genet 47, 1494–1498 (2015).

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