Knockout of GRAIN WIDTH2 has a dual effect on enhancing leaf rust resistance and increasing grain weight in wheat

Wheat leaf rust, caused by the fungal pathogen Puccinia triticina Eriksson (Pt), poses a major threat to global wheat production. The widespread presence and rapid evolution of Pt races lead to frequent epidemics, particularly in favourable climates, causing yield losses of 30%–50% (Prasad et al., 2020). Employing resistant genes and cultivars remains the most effective strategy against leaf rust. However, the limited number of cloned resistance genes often correlates with undesirable agronomic traits, and single resistance gene usage may lead to rapid resistance breakdown due to continuous Pt evolution (Dracatos et al., 2023). Therefore, discovering new resistance genes and balancing yield with disease resistance is essential for sustainable breeding strategies. In our research, we inoculated wheat variety Fielder with a highly virulent Pt race THT and collected samples at 0, 12 and 24 h post-inoculation (hpi) for RNA-Seq analysis (Appendix S1). The analysis highlighted significant up-regulation of differentially expressed genes (DEGs) involved in cellular protein modification, including E3 ubiquitin ligases crucial for protein degradation (Figure 1a,b). Among 255 Pt-induced E3 genes, we identified wheat GRAIN WIDTH2 (TaGW2) homoeologs that negatively regulate wheat grain width and weight (Qin et al., 2014). Their expression increased over 3.8-fold at 12 hpi and 3-fold at 24 hpi (Figure 1c; Table S1). RT-qPCR assay confirmed significant up-regulation of TaGW2 at 12 and 24 hpi (Figure S1), indicating its involvement in wheat's response to leaf rust. To investigate the role of TaGW2 in wheat leaf rust resistance, we generated TaGW2-6A overexpression (OE) transgenic plants and TaGW2 knockout (KO) plants via CRISPR/Cas9 technology in Fielder cultivar (Figure S2; Table S2). After THT inoculation, OE plants had more uredia than wild type (WT), while KO plants had fewer at 10 days post-inoculation (dpi) during the seedling stage (Figure 1d,e). Microscopic analysis showed increased H2O2 accumulation in KO at 48 hpi and reduced hyphal length and number at 120 hpi, unlike the OE plants (Figure 1f). Furthermore, pathogenesis-related gene transcription levels were higher in KO and lower in OE (Figure S3). The adult-plant resistance phenotypes were further examined, revealing that KO plants had fewer and smaller sporulations than OE plants after THT inoculation, aligning with the seedling-stage resistance phenotypes (Figure 1g,h). Thus, the loss of TaGW2 function enhances wheat resistance to Pt, indicating TaGW2 as a negative regulator of leaf rust resistance. In exploring the molecular mechanisms of TaGW2-mediated plant immunity, we used the N-terminus of TaGW2 in a yeast two-hybrid (Y2H) assay, avoiding its C-terminus due to self-activation. The Y2H screening identified nine potential interacting proteins, including the Suppressor of the G2 allele of SKP1 (SGT1) (Table S3; Figure 1i). Recognizing the vital role of SGT1 in plant immunity (Wang et al., 2022), we validated its interaction with TaGW2 using firefly luciferase complementation imaging (LCI) and bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves (Figure 1j,k). A pull-down assay further confirmed their direct interaction in vitro (Figure 1l). These results suggest a physical interaction between TaGW2 and TaSGT1. Previous studies identified TaSGT1 as involved in Lr21-mediated resistance against wheat leaf rust (Scofield et al., 2005). We found its transcription significantly induced by the Pt race THT, similar to TaGW2 (Figure S4). Using barley stripe mosaic virus (BSMV)-mediated gene silencing, we knocked down TaSGT1 expression in Fielder (Figure S5a,b). TaSGT1 silencing led to increased uredia, decreased H2O2 accumulation and more hypha (Figure 1m,n; Figure S5c). These findings imply that TaSGT1 limits Pt proliferation in wheat leaf tissue, acting as a positive regulator in resistance against leaf rust. Given TaGW2's interaction with TaSGT1 and its role as a RING-type E3 ubiquitin ligase (Liu et al., 2020), we hypothesized that TaGW2 could ubiquitinate TaSGT1. We tested this through an in vitro ubiquitination assay using recombinant His-TaGW2 and MBP-TaSGT1 proteins. The assay revealed that MBP-TaSGT1 could be ubiquitinated by TaGW2 in the presence of Ub, E1 and E2, while the MBP control could not (Figure 1o). This suggests that TaGW2 may regulate wheat resistance to leaf rust by mediating the ubiquitination and degradation of TaSGT1. As a well-established regulator of grain weight and width, we assessed the effect of TaGW2 on grain traits, such as thousand-grain weight (TGW), grain length (GL) and grain width (GW) under both non-inoculated and inoculated conditions. All lines showed a reduction in GW and TGW after inoculation. However, KO plants experienced only a 5.6%–7.4% decrease in TGW, compared to a 15.7%–17.2% decrease in OE plants, mainly due to different declines of GW in KO and OE (Figure 1p–r). As a result, KO plants showed a 5.1%–6.6% increase in GW and a 4.5–5.5 g rise in TGW compared to WT, while OE plants experienced an 8.0%–11.0% reduction in GW and a 5.5–6.3 g decrease in TGW (Figure 1q,r; Table S4). No significant changes in GL were observed in any line with non-inoculation and inoculation (Figure S6). These results suggest that the increased resistance of TaGW2-KO plants to Pt aids in preserving wheat yield from leaf rust damage. In conclusion, our findings reveal that TaGW2, beyond its known role in negatively regulating grain weight, also negatively regulates wheat leaf rust resistance by mediating the ubiquitination of TaSGT1. By employing CRISPR/Cas9 to edit TaGW2, we achieved simultaneous increases in grain weight and leaf rust resistance, offering a novel approach to enhancing wheat's resistance to leaf rust without sacrificing yield. This research was financially supported by grants from the National Key Research and Development Program of China (2023YFF1000400) and the Hebei Province Key Research and Development Program (22326306D). The authors declare that they have no conflict of interest. T.L., X.Z. and X.C. designed the research. S.L., H.L., M.G. and Y.P. performed the experiments. T.L., S.L., C.H., J.H. and L.Y. analysed the data. T.L. and S.L. wrote the manuscript. The data that supports the findings of this study are available in the supplementary material of this article Table S1 List of genes encoding E3 ubiquitin ligases among the upregulated DEGs. Table S2 A summary of primer information. Table S3 List of potentially interacting proteins of TaGW2 identified through Y2H screening. Table S4 Comparison of disease severity, TGW, GW, and GL among WT, OE, and KO lines with and without inoculation. Figure S1 Relative expression level of TaGW2 at different stages of Pt infection. Figure S2 Characterization of TaGW2-6A overexpression (OE) lines and CRISPR/Cas9-based TaGW2 knockout (KO) plants. Figure S3 Relative expression levels of pathogenesis-related genes TaPR1, TaPR2 and TaPR5 in WT, OE and KO lines at 0, 12, 24 and 48 hpi with Pt infection. Figure S4 Relative expression level of TaSGT1 at different stages of Pt infection. Figure S5 Silencing TaSGT1 promotes Pt proliferation. Figure S6 Grain length phenotype of WT, OE and KO lines with non-inoculation and inoculation. Appendix S1 Materials and methods. 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.