TaRLK‐6A promotes Fusarium crown rot resistance in wheat

The plasma membrane-localized phytosulfokine receptor-like protein TaRLK-6A, interacting with TaSERK1, positively regulates the expression of defense-related genes in wheat, consequently promotes host resistance to Fusarium crown rot. F usarium crown rot (FCR), mainly caused by the soil-borne fungus Fusarium pseudograminearum, is a devastating disease of wheat (Triticum aestivum (Ta)). Fusarium crown rot causes substantial yield losses and generates mycotoxins in wheat grains that can cause serious health problems in humans and livestock (Powell et al., 2017). Identifying genes that provide resistance to F. pseudograminearum and characterizing the molecular mechanisms underlying such resistance are key to breeding wheat varieties that are resistant to this devastating fungus. To date, several genes have been reported to be associated with FCR resistance in wheat. A genome-wide association study of 435 wheat introgression lines, QTL analysis of 181 double haploid lines, and mutant-based functional analysis identified TaDIR-B1, which encodes a putative dirigent protein, as a negative regulator of FCR resistance (Yang et al., 2021). Other studies have shown that silencing of one wall-associated kinase gene TaWAK-6D or TaAACT1 (encoding acetoacetyl-CoA thiolase II) compromises wheat resistance to FCR (Qi et al., 2021; Xiong et al., 2023). However, none of these studies indicated whether overexpression of these genes would enhance FCR resistance in wheat. Here, we identified TaRLK-6A (TraesCS6A02G222000.1), a gene on chromosome 6A that contributes to FCR resistance in wheat. TaRLK-6A encodes a receptor-like kinase (RLK) and was upregulated in FCR-resistant near-isogenic lines (NILs) relative to susceptible NILs following inoculation with F. pseudograminearum (Figure S1, http://www.wheat-expression.com/; Ma et al., 2014). Real-time quantitative PCR (RT-qPCR) analysis showed that the transcript levels of TaRLK-6A in both the FCR-resistant wheat cultivar CI12633 and the susceptible wheat cultivar Yangmai 9 increased by F. pseudograminearum infection, peaking at 4 d post inoculation (dpi) (Figure S2). The transcript level of TaRLK-6A was higher in FCR-resistant wheat cultivars (Shanhongmai and CI12633) than in susceptible cultivars (Yangmai 18 and Yangmai 9) (Figure S3). Additionally, TaRLK-6A transcript levels were higher in roots and stems, but lower in leaves and spikes (Figure S4, http://www.wheat-expression.com/; International wheat genome sequencing consortium (IWGSC, 2014)), which is consistent with the plant organs where FCR usually occurs. Overall, these results suggested that TaRLK-6A might participate in the wheat resistance response to FCR. To identify a possible association of different TaRLK-6A variants with FCR resistance, we analyzed the genomic sequence of TaRLK-6A in 364 Chinese wheat accessions whose FCR resistance and susceptibility have been scored at the seedling stage (Yang et al., 2019). The presence or absence of three indels in the TaRLK-6A promoter sequences among these wheat accessions defined two haplotypes, with TaRLK-6A_Hap2 corresponding with significantly more resistance to FCR than TaRLK-6A_Hap1 (Figure S5). Accordingly, analyses of the putative cis-acting elements in the promoters of the two haplotypes of TaRLK-6A (Table S1), LUC-reporter-based analysis of promoter activities, and measurement of transcript levels of TaRLK-6A in typical cultivars of the two haplotypes (Figure S6) supported the likelihood that gene expression differed between the haplotype variants. To investigate the potential defensive functions of TaRLK-6A, we obtained TaRLK-6A-overexpressing transgenic wheat plants, wheat plants silenced for TaRLK-6A by virus-induced gene silencing (VIGS), and mutant wheat plants harboring a premature stop codon in TaRLK-6A. At 30 dpi with F. pseudograminearum, FCR lesion sizes on the stems of TaRLK-6A-overexpressing wheat plants were smaller than those on untransformed wheat Yangmai 18 (wild-type (WT)) plants (Figures S7, 1A). Compared with Yangmai18 plants, both the average disease indexes (DIs) of TaRLK-6A overexpression wheat lines in the T1 generation (Figure 1B) and their corresponding infection types (ITs) were significantly lower (Table S2). These trends were also seen in three TaRLK-6A overexpression wheat lines in the T2 generation (Table S2), suggesting that TaRLK-6A overexpression significantly enhanced wheat resistance to FCR. Functional characterization of TaRLK-6A in defense to Fusarium crown rot in wheat (A, B) Typical Fusarium crown rot (FCR) symptoms and disease indexes (DIs) of FCR in three TaRLK-6A-overexpressing lines and the wild-type (WT) Yangmai 18 line at 30 dpi with F. pseudograminearum (Student's t-test: **P < 0.01). (C, D) FCR symptoms and DIs of WT Cadenza plants, plus Tarlk-6A1 and Tarlk-6A2 (premature-stop mutants of TaRLK-6A) plants, at 30 dpi (t-test: **P < 0.01). (E) Yeast-two-hybrid (Y2H) assays show that TaRLK-6A interacted with TaSERK1-2D. (F) Pull-down assay showing a direct interaction between His-TFTaRLK-6A and GST-TaSERK1-2D in vitro. (G) Bimolecular fluorescence complementation (BiFC) assays show that TaRLK-6A interacted with TaSERK1-2D in Nicotiana benthamiana leaves. Bar, 50 μm. (H) Y2H assays show that TaRLK-6A interacted with TaSERK1-2D via their LRR domains. (I, J) FCR symptoms and DI of WT Cadenza plants, plus Taserk1-2A, Taserk1-2B, and Taserk1-2C (premature-stop mutants of TaSERK1-2A, TaSERK1-2B, and TaSERK1-2D) plants, at 30 dpi (t-test: **P < 0.01, *P < 0.05). In contrast, the stems of TaRLK-6A-silenced CI12633 plants exhibited more serious FCR symptoms, with larger necrotic areas and higher ITs and average DIs (Figures S8, S9), than did BSMV:GFP-infected (control) CI12633 seedlings. Transcript levels of TaRLK-6A and TaRLK-6B (but not TaRLK-6D) were also significantly decreased in the TaRLK-6A-silenced plants relative to control plants (Figure S10). Further, we selected mutants of TaRLK-6A and TaRLK-6B from the EMS-mutagenized libraries of hexaploid spring wheat cultivar Cadenza (Krasileva et al., 2017) for further assessment. Two TaRLK-6A premature-stop mutant lines also showed significantly higher disease severity than WT Cadenza plants (Figures 1C, D, S11A). Also, a premature-stop mutant of TaRLK-6B had reduced wheat resistance to FCR (Figure S11B–D). These results suggested that TaRLK-6A and TaRLK-6B are both required for wheat resistance to FCR. Sequence and phylogenetic-tree analyses indicated that TaRLK-6A, which belongs to the phytosulfokine receptor (PSKR) family (Matsubayashi et al., 2002; Figure S12), contains a signal peptide, an extracellular LRR-repeat-containing domain (residues 29–689), an island domain, a transmembrane domain, and a cytoplasmic kinase domain. Subcellular localization results showed that TaRLK-6A-GFP was co-localized with the plasma membrane marker AtPAI2A-mCherry in wheat protoplasts (Figure S13), suggesting that TaRLK-6A-GFP was localized at the plasma membrane. Because some RLKs can interact with other RLKs to co-regulate downstream signals in certain biological processes, including plant immunity (Wang et al., 2020), we searched for interacting partners to identify possible regulatory mechanisms for TaRLK-6A. In a yeast-two-hybrid (Y2H) screen, we identified a somatic embryogenesis receptor kinase, TaSERK1-2D (TraesCS2D02G321400), that interacted with TaRLK-6A (Table S4). As expected, TaSERK1-2D also localized at the plasma membrane (Figure S14). Yeast-two-hybrid, bimolecular fluorescence complementation (BiFC), and pull-down assays indicated that TaRLK-6A indeed interacted with TaSERK1-2D both in vitro and in Nicotiana benthamiana leaves (Figure 1E–G). Further Y2H assays using defined regions of these proteins showed that their extracellular domains were required for interaction, whereas the intracellular kinase domains did not interact (Figure 1H). To determine whether TaSERK1 was involved in the resistant response to FCR, we analyzed transcriptional changes of TaSERK genes using RNA-sequencing (RNA-seq) online data derived from wheat seedlings infected with F. pseudograminearum (http://www.wheat-expression.com/; Ma et al., 2014). Upon F. pseudograminearum infection, transcript levels of TaSERK1 copies on chromosomes 2A, 2B, and 2D were higher than those of other TaSERK genes (Figure S15). Real-time qPCR results showed that the expression of TaSERK1 in the resistant wheat cultivar CI12633 was significantly induced by F. pseudograminearum infection (Figure S16A). After inoculation with F. pseudograminearum, the transcript levels of TaSERK1 were significantly higher in FCR-resistant cultivars (CI12633 and Shanhongmai) than in susceptible cultivars Yangmai 18 and Yangmai 9 (Figure S16B). Importantly, at ~30 dpi with F. pseudograminearum, TaSERK1-silenced wheat CI12633 plants exhibited more serious disease symptoms than the control plants (Figures S17–19). In two independent VIGS batches of plants, the average DIs of TaSERK1-silenced plants were significantly higher than those of control wheat plants, as were the corresponding ITs (Figure S19). Similar to TaSERK1-silenced plants, the premature-stop mutants Taserk1-2A, Taserk1-2B, and Taserk1-2D displayed higher disease severity than the Cadenza plants (Figure 1I). The average DIs of Taserk1-2A, Taserk1-2B, and Taserk1-2D mutant plants were significantly higher than those of Cadenza (Figure 1J), as were the corresponding ITs (Figure S20). These results suggested that TaSERK1 genes (especially TaSERK1-2D) are required for wheat resistance to F. pseudograminearum. Interestingly, analyses of the different haplotypes of TaSERK1-2A, TaSERK1-2B, and TaSERK1-2D indicated that four haplotypes of TaSERK1-2D were present among 364 wheat accessions, and that the cultivars with Hap4 had the lowest FCR DI in two environments (Figure S21). We then used RT-qPCR to examine whether the expression of defense-related genes was modulated by TaRLK-6A in wheat at 4 dpi with F. pseudograminearum. The transcript levels of TaSERK1, TaRLCK1B, TaMPK3, TaERF3, TaDefensin, and TaChitinase2 were significantly upregulated in three independent lines of TaRLK-6A overexpression plants compared with the WT plants (Figure S22A). In contrast, transcript abundances of these defense-related genes were significantly lower in TaRLK-6A-silenced plants than in the control plants (Figure S22B). These data suggested that TaRLK-6A positively modulates the expression of defense-related genes in wheat. Additionally, the transcript levels of these six genes were also significantly lower in TaSERK1-silenced plants than in the control plants (Figure S22C), suggesting that TaSERK1 also positively modulated the same defense-related genes that were regulated by TaRLK-6A in wheat. This study provides evidence that TaRLK-6A interacts with TaSERK1 and regulates the expression of defense-related genes in wheat, resulting in enhanced FCR resistance (Figure S23). Overexpression of TaRLK-6A increased the resistance of wheat plants to FCR by heightening the expression of defense-related genes, and loss-of-function mutants of TaRLK-6A had the opposite effects. Hap2 of TaRLK-6A and Hap4 of TaSERK1-2D are excellent haplotypes for providing FCR resistance in wheat, and appropriate Kompetitive allele-specific PCR (KASP) markers can be developed based on these haplotypes for use in marker-assisted wheat breeding for FCR resistance. This study not only broadens our current knowledge of PSKR proteins and FCR resistance in a key crop plant but also defines TaRLK-6A as a promising gene for wheat breeding efforts to improve FCR resistance. The authors are very grateful to Professor Jorge Dubcovsky (University of California, Davis, USA) for providing Cadenza mutants, to Professor Jinfeng Yu and Dr. Li Zhang (Shandong Agricultural University, China) for providing Fusarium pseudograminearum strain WHF220, to Professor Youzhi Ma (Institute of Crop Sciences, Chinese Academy of Agricultural Sciences) for supporting experimental platform, and to Professor Jizhong Wu (Jiangsu Academy of Agricultural Sciences), Professor Derong Gao and Dr. Xujiang Wu (Institute of Agricultural Science of Lixiahe District in Jiangsu Province) for providing the wheat cultivars. This study was funded by the National Key Project for Research on Transgenic Biology, China (Grant No. 2016ZX08002001 to Z.Z.) and the NSFC Program (Grant No. 31771789 to Z.Z.). The authors declare no conflict of interest. Z.Z. conceived the study, designed the experiments, and wrote and revised the manuscript. H.Q. performed the majority of the experiments, analyzed the data, and wrote the draft manuscript. X.Z. and W.S. analyzed some data. X.Y. and F.C. analyzed the different haplotypes of TaRLK-6A and TaSERK1-2D among 364 wheat cultivars. C.Z. tested the mutants. G.L. performed wheat transformation. X.W. assessed these wheat materials. All authors approved the submitted version. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13596/suppinfo Figure S1. Transcript levels of TaRLK-6A, TaRLK-6B, and TaRLK-6D in susceptible and resistant wheat plants, as determined using RNA-sequencing (RNA-seq) online data Figure S2. Transcript levels of TaRLK-6A in plants of the Fusarium crown rot (FCR)-resistant wheat cultivar CI12633 and the highly susceptible wheat cultivar Yangmai 9 following inoculation with F. pseudograminearum Figure S3. Transcript levels of TaRLK-6A in four wheat cultivars after F. pseudograminearum infection or without such infection Figure S4. Tissue-specific expression patterns of TaRLK-6A, TaRLK-6B, and TaRLK-6D in the wheat cultivar Chinese Spring as determined from RNA-sequencing (RNA-seq) online data Figure S5. TaRLK-6A haplotypes and association of haplotype 2 with Fusarium crown rot (FCR) resistance Figure S6. LUC reporter-based analysis of TaRLK-6A promoter activity and relative transcript levels for the two haplotypes Figure S7. Identification of TaRLK-6A-overexpressing wheat plants Figure S8. Characteristics of recombinants BSMV:TaRLK-6A vector Figure S9. Silencing of TaRLK-6A increases wheat susceptibility to F. pseudograminearum Figure S10. The transcript levels of TaRLK-6A and TaRLK-6B were significantly lower in BSMV:TaRLK-6A-infected wheat CI12633 plants at 4 d post inoculation (dpi) with F. pseudograminearum Figure S11. Premature-stop mutants of TaRLK-6A and TaRLK-6B have reduced resistance to F. pseudograminearum infection Figure S12. Gene and protein structure and phylogenetic analysis of TaRLK-6A Figure S13. Subcellular localization of TaRLK-6A-GFP in wheat protoplasts Figure S14. Subcellular localization of TaSERK1-2D-GFP in wheat protoplasts Figure S15. The transcript levels of TaSERK genes in the wheat Fusarium crown rot (FCR) RNA-sequencing online data Figure S16. The expression of TaSERK1 is upregulated in response to Fusarium crown rot (FCR) Figure S17. Characteristics of recombinants BSMV:TaSERK1 vector Figure S18. Transcript levels of TaSERK1-2A, TaSERK1-2B, and TaSERK1-2D were significantly lower in TaSERK1-infected CI12633 plants Figure S19. Silencing of TaSERK1 increases wheat susceptibility to F. pseudograminearum Figure S20. Average infection types of premature-stop mutants Taserk1-2A, Taserk1-2B, Taserk1-2D, and wild-type (WT) Cadenza plants at 30 d post inoculation (dpi) with F. pseudograminearum Figure S21. TaSERK1-2D haplotypes and the association of Hap4 with wheat resistance to Fusarium crown rot (FCR) Figure S22. Expression levels of defense-related genes regulated by TaRLK-6A and TaSERK1 Figure S23. A working model of TaRLK-6A positive regulating wheat resistance to Fusarium crown rot (FCR) Figure S24. Schematic diagram of seedling inoculation and identification for Fusarium crown rot (FCR) (Materials and methods section) Table S1. The different putative cis-acting elements in promoters between the two haplotypes of TaRLK-6A Table S2. The infection types and disease indexes of Fusarium crown rot (FCR) in three TaRLK-6A-overexpressing and wild-type (WT) (Yangmai18) wheat plants in the T1 and T2 generations Table S3. The potential TaRLK-6A-interacting proteins based on yeast-two-hybrid screening Table S4. Primers used in this study (Materials and methods section) 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.