Analysis of a Series of Mutants Highlights Complex Regulation of Fusarium Head Blight Resistance Conferred by Fhb1 Locus in Wheat

Abstract

Fusarium head blight (FHB), which is mainly caused by Fusarium graminearum, is one of the devastating wheat diseases that threaten global wheat production (Rawat et al. 2016; Li et al. 2019; Su et al. 2019; Wang et al. 2020). Improvement of FHB resistance has become an urgent need for securing worldwide wheat production. Fhb1, a quantitative trait locus located on chromosome 3BS and exploited in Chinese variety cv Sumai 3 (SM3), provides the most stable and major effect on FHB resistance in wheat. Fhb1-mediated FHB resistance is reported to be conferred by either a pore-forming toxin-like (PFT) gene TaPFT (Rawat et al. 2016) or a putative histidine-rich calcium-binding protein (TaHRC or TaHis, hereafter referred as TaHRC) encoding gene (Li et al. 2019; Su et al. 2019). At the Fhb1 locus, TaPFT, which only has one homoeolog on chromosome 3BS, was suggested to confer wheat FHB resistance (Rawat et al. 2016). Later, TaHRC was identified to be the key determinant of resistance to FHB at the Fhb1 locus by two different laboratories (Li et al. 2019; Su et al. 2019). However, the two groups reached contrasting conclusions regarding this gene's function. This discrepancy may hinder further efforts in using the Fhb1 locus for improving FHB resistance and thus necessitates further investigation. Besides, given that common wheat is a hexaploid with three subgenomes (AABBDD), the roles of the other two homoeologs, TaHRC-3A and TaHRC-3D, in FHB resistance or susceptibility remain unclear. To clarify the roles of TaPFT and TaHRC at the Fhb1 locus in FHB resistance in the same genetic background of SM3, we first generated a series of mutant lines including Tapft, Tahrc-3B, Tapft/Tahrc-3B double mutant lines, and different mutant lines of three TaHRC homoeologs in SM3, respectively. TaPFT was a single-copy gene in SM3 (Rawat et al. 2016), and we designed a guide RNA (gRNA) targeting its first exon (Figure 1A). Furthermore, of the three TaHRC homoeologs in SM3, TaHRC-3B is 42-bp longer than TaHRC-3A and TaHRC-3D at the N-terminus of the encoding region due to a rare deletion spanning the start codon, which was supposed to result in either gain of function (Li et al. 2019) or loss of function of TaHRC (Su et al. 2019) and thus increased FHB resistance (Figure S1). Therefore, we designed a TaHRC-3B specific target sequence (Target 1) in this region, which was also used together with the target of TaPFT to simultaneously edit both genes. (Figure 1A,B; Figure S1). Moreover, we designed another gRNA (Target 2) targeting the conserved region for simultaneously editing of the three TaHRC homoeologs (Figure 1A; Figure S1; Table S1). We successfully obtained two Tapft, two Tahrc-3B, and two Tapft/Tahrc-3B mutant lines, and seven null mutant lines of TaHRC homoeologs including single, double, or triple mutant lines in SM3, respectively (Table S2). Following segregation, we generated a series of transgene-free homozygous mutants in T1 progenies derived from the T0 mutant lines (Table S3). The editing profiles of the representative lines were indicated in Figure 1C. To evaluate the FHB resistance, the transgene-free homozygous lines were grown in two different environments, with the wheat spikes inoculated by Fusarium and examined for pathogen spread at 21 days post inoculation (dpi). The evaluations in both the greenhouse and the field showed that all the Tapft, Tahrc-3B, and Tapft/Tahrc-3B null mutant lines exhibited high FHB resistance similar to the wild type (WT) SM3 (Figure 1D and S2), indicating that TaPFT at the Fhb1 locus is not necessary for FHB resistance, consistent with the recent report by Shi et al. (2025). Furthermore, the seven mutant lines of TaHRC homoeologs, including one aaBBDD, two AAbbDD, two aabbDD, and two aabbdd lines generated by Target 2 (Figure 1C; Table S3), also showed no significant differences in FHB resistance compared to WT SM3 (Figure 1D; Figure S2), implying that the loss of function of TaHRC-3B due to a naturally occurred causal mutation may account for increased FHB resistance in SM3 (Su et al. 2019). To investigate whether genome editing of TaHRC may be employed to enhance the FHB resistance in FHB-susceptible wheat varieties, we generated a series of mutant lines of three TaHRC homoeologs in three varieties including Zhengmai 7698 (ZM7698), Aikang 58 (AK58) and Jimai 22 (JM22), respectively, by designing another common gRNA (Target 3) (Figure 1E; Figure S1). In total, we identified six, four and five independent T0 Tahrc mutant lines in ZM7698, AK58 and JM22, respectively (Table S2). Following segregation in T1 progenies, we obtained different types of transgene-free Tahrc mutant lines, including two AAbbDD, two AABBdd, one aaBBdd and two aabbdd lines in ZM7698, and one AAbbDD, one AABBdd and two aabbdd lines in AK58, and two AAbbDD, one AABBdd and two aabbdd lines in JM22, respectively (Figure 1F; Figure S3; Table S3). We then evaluated the performances of these Tahrc mutant lines of different varieties in FHB resistance in different environments. For the Tahrc mutant lines of ZM7698, albeit not high FHB resistance as observed in SM3, the numbers of diseased spikelets of the two single mutant lines, AAbbDD and AABBdd, were significantly decreased compared to that of the ZM7698 WT across three environments, indicating that both AAbbDD and AABBdd lines exhibited improved FHB resistance (Figure 1G,H). Notably, the AABBdd lines showed further decline of diseased spikelets compared to AAbbDD lines (Figure 1G,H). However, we also observed that the double (aaBBdd) and triple (aabbdd) mutant lines of ZM7698 did not show significant differences in the number of diseased spikelets compared to WT ZM7698 (Figure 1G,H).Similar to the above results, the AAbbDD and AABBdd lines of AK58 also exhibited significant decreases in diseased spikelets relative to WT control, and the AABBdd line showed superior FHB resistance over the AAbbDD line across two environments (Figure 1G,H). These results reinforce that knocking out TaHRC-3D could more potently enhance FHB resistance. It is worth noting that the expression levels of TaHRC-3B and TaHRC-3D, especially TaHRC-3D, in ZM7698 and AK58, are much higher than those of TaHRC-3A in wheat spikelets at different time points after inoculation with F. graminearum, respectively, suggesting that both of them, especially TaHRC-3D, may play more crucial roles in FHB susceptibility (Figure S4). Similar to ZM7698, simultaneous knockout of all three TaHRC homoeologs in AK58 did not give rise to FHB resistance either (Figure 1G,H). Therefore, we speculate that the regulation of FHB resistance by TaHRC is very complex and only certain null mutations of specific homoeologs in some genetic backgrounds, such as Tahrc-3B in SM3, the Tahrc-3B and Tahrc-3D mutants in ZM7698 and AK58 backgrounds demonstrated in this study, as well as Tahrc-3B in wheat variety cv Bobwhite in previous studies (Su et al. 2019; Chen et al. 2022), could confer improved FHB resistance. In addition, the mutations of TaHRC homeologs had no negative effects on major agronomic traits in ZM7698 and AK58 (Figures S5–S8). Unexpectedly, we also observed that the Tahrc mutant lines of JM22, including AAbbDD, AABBdd and aabbdd, did not exhibit significant differences in the number of diseased spikelets compared to WT JM22 (Figure S9), implying that different genes/networks may be involved in wheat FHB susceptibility or the existence of inhibitors in different genetic backgrounds (Zheng et al. 2022). This adds another layer of complexity to the regulation of FHB resistance conferred by the Fhb1 locus in wheat. In summary, through systematically and rigorously evaluating a relatively large number of edited null mutants, we here demonstrate that Tahrc-3B rather than TaPFT is responsible for Fhb1-mediated resistance to FHB in SM3. Compared to Tahrc-3B, Tahrc-3D increases FHB resistance in the tested two elite varieties, providing a practical and valuable strategy for bolstering FHB resistance in some elite wheat cultivars through genome editing. Overall, our work uncovers high complexity underlying the regulation of FHB resistance conferred by Fhb1 locus, whose exact nature and mode of action thus need to be explored in future, and may benefit from the different sets of null mutant lines generated in our work. L.X. conceived the project. L.Y., J.T., J.L., S.L., C.Z., and Y.H. performed the experiments. L.Y. and J.T. wrote the manuscript. L.X. revised the manuscript. All the authors read the final version of this manuscript. We thank Professor Lingrang Kong from Shandong Agricultural University and Professor Wenjing Hu from Lixiahe Institute of Agricultural Sciences for their help in the evaluations of FHB resistance. This work is partly funded by The Shennong Laboratory (Grant No. SN01-2022-01 to L.X.) and National Natural Science Foundation of China (Grant No. 32188102 to L.X.). The data that supports the findings of this study are available in the Supporting Information of this article. Data S1: pbi70318-sup-0001-DataS1.docx. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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