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Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight

Abstract

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of wheat and barley that leads to reduced yield and mycotoxin contamination of grain, making it unfit for human consumption. FHB is a global problem, with outbreaks in the United States, Canada, Europe, Asia and South America. In the United States alone, total direct and secondary economic losses from 1993 to 2001 owing to FHB were estimated at $7.67 billion1. Fhb1 is the most consistently reported quantitative trait locus (QTL) for FHB resistance breeding. Here we report the map-based cloning of Fhb1 from a Chinese wheat cultivar Sumai 3. By mutation analysis, gene silencing and transgenic overexpression, we show that a pore-forming toxin-like (PFT) gene at Fhb1 confers FHB resistance. PFT is predicted to encode a chimeric lectin with two agglutinin domains and an ETX/MTX2 toxin domain. Our discovery identifies a new type of durable plant resistance gene conferring quantitative disease resistance to plants against Fusarium species.

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Figure 1: Fhb1 phenotype and mapping.
Figure 2: Gene structure and mutant analysis of PFT.
Figure 3: Functional validation of PFT with RNAi-induced gene silencing.

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Acknowledgements

This work was supported by US Wheat and Barley Scab Initiative (USDA-ARS grant 59-0206-2-088) and US National Science Foundation grant contract (IIP-1338897). We thank G. Bai for (USDA-ARS, Manhattan, Kansas) providing seeds of wheat cultivars and landraces for association studies and S. McCormick (USDA-ARS, Peoria, Illinois) for providing purified DON for inoculation experiments. We thank A. Akhunova, R. Matniyazov, S. Sehgal, S. Simsek, K. Benson, B. Friebe, D. Wilson, J.W. Raupp, N. Tyagi, U.M. Quraishi and L. Jiarui for technical support. We acknowledge help from student workers C. McDaniel, K. Madden and R. Clay-Pettis. This is contribution 16-147-J from the Kansas Agricultural Experiment Station.

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Authors and Affiliations

Authors

Contributions

N.R. and M.O.P. performed most of the experimental work. S.L. developed the near-isogenic lines and markers for the initial part of the work. X.Z. carried out DON and D3G analyses, and additional phenotyping of the mutants. V.K.T. made the RNAi crosses and did their phenotyping. K.A. isolated some of the TILLING mutants. H.N.T. generated RNAi plants. W.W.B. produced inocula for all the phenotyping work. E.A. developed the BAC library and provided useful discussions. N.R., J.A.A. and B.S.G. conceived the research and co-wrote the manuscript.

Corresponding author

Correspondence to Bikram S Gill.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Quantitative RT–PCR analysis of the genes of the Fhb1 region in spikes of R-NIL and S-NIL.

Gene expression changes were calculated as fold change in Fusarium graminearum inoculations relative to water inoculations (expression = 1). Error bars denote the standard deviation. No significant differences (P < 0.05, Student’s t-test) were found between R-NIL and S-NIL expression in genes except for PFT and NBA, which were expressed only in the R-NIL.

Supplementary Figure 2 Expression profile of PFT in R-NIL based on quantitative RT–PCR.

(a) Average expression of PFT in R-NIL spikes at various intervals after inoculation with water and FHB. PFT was expressed constitutively and the expression declined progressively in both the treatments. (b) Spikes at various stages of heading showed maximum expression of PFT in pre-emergence spikes (shown by the green column, next to the green y-axis on left), after which the PFT transcript levels started declining drastically. A blue secondary axis on the right shows 10X magnified expression values at stages after pre-anthesis (shown by blue columns). (c) Average expression of PFT in various aerial parts of R-NIL plants at pre-anthesis stage. The expression was highest in the spikes, followed by leaf sheaths. Error bars in the expression charts represent the standard deviation. Asterisks denote statistically significant differences (P < 0.05, Student’s t-test).

Supplementary Figure 3 Mis-splicing in susceptible cultivars of haplotype S3 and TILLING mutant pft528.

(a) Multiple sequence alignment of the susceptible cultivars, TILLING mutant pft528, Sumai 3, resistant TILLING parent HR58, and BAC383G12 showing intronic region having a G>A SNP (highlighted in red) 4 bp upstream of the start of exon 2. (b) Mis-splicing in mRNA of representative susceptible cultivars from haplotype S3: Nanda2419 and Funo and, TILLING mutant pft528 as compared to resistant cultivars Sumai 3 and WSB, and homozygous resistant TILLING parent HR58. No reverse transcription (NRT) controls for Sumai 3 and Nanda2419 rule out any genomic DNA contamination in the mRNA samples. No template control is an overall negative control for the experiment, ruling out any other contamination. (c) Position of primers used for testing aberrant splicing shown by blue arrows.

Supplementary Figure 4 Overexpression of PFT in susceptible cultivars Bobwhite and Fielder.

Transcript levels of PFT in 11 putative transgenic T0 plants. Quantitative RT-PCR showed PFT expression in three T0 plants (#7879, #7993, and #7636) out of five events in Bobwhite, whereas in Fielder only one event (#7978) showed expression of the gene. Untransformed cultivars Bobwhite and Fielder do not have the gene. The level of expression was different in all the independent events.

Supplementary Figure 5 FHB-infected spikes of T1 family plants from event #7978 in Fielder overexpressing PFT after 21 d of inoculation.

The plants that showed PFT expression had spikes with either very slow or no progress of disease in the spikelets down from the point of inoculation (white arrows), whereas the plants that lacked the PFT expression showed spikes with severe bleaching like the untransformed Fielder control. The ‘+’ and ‘–’ symbols denote presence and absence of the gene, respectively, in the plants.

Supplementary Figure 6 DON treatment on the spikes of R-NIL, S-NIL, PFT truncation mutants and resistant parent HR58.

(a) Only the S-NIL spikes showed bleaching upon DON inoculation, whereas the R-NIL, wild type HR58 and mutant spikes did not have any bleaching, indicating that PFT does not play a role in DON detoxification. (b) DON and D3G content (in ppm) in the middle spikelets after 1, 3, 7,14 and 21 days after inoculation (dai) with purified DON. The values are given as average of 3 replications ± their standard deviations. D3G content of S-NIL spikelets was significantly lower than the others till 7 dai, after that the ratios were similar in all the plants (P < 0.05, Student’s t-test).

Supplementary Figure 7 Neighbor-joining tree of proteins similar to pore-forming toxin-like protein

Sequences were aligned with MUSCLE. MEGA6 was used to generate the phylogenetic tree using Bootstrap confidence values based on 100 iterations, indicated in the nodes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. Accession numbers of all the proteins used are provided in Supplementary Table 8. Evolutionary analyses were conducted in MEGA6.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1, 2 and 4–7. (PDF 1760 kb)

Supplementary Table 3

SNPs found in the Fhb1 region in a collection of 41 wheat cultivars and landraces known for their Fhb1-mediated resistance to Fusarium head blight. (XLSX 44 kb)

Supplementary Table 8

GenBank accession numbers for all genes, nucleotides and proteins used in the study. (XLSX 12 kb)

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Rawat, N., Pumphrey, M., Liu, S. et al. Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight. Nat Genet 48, 1576–1580 (2016). https://doi.org/10.1038/ng.3706

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