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.

小麦叶锈病由真菌病原体小麦叶锈病（Pt ）引起，对全球小麦生产构成重大威胁。Pt种群的广泛存在和快速进化导致流行病频繁发生，特别是在有利的气候下，造成 30%–50% 的产量损失（Prasad等，  2020）。采用抗性基因和品种仍然是对抗叶锈病的最有效策略。然而，克隆的抗性基因数量有限，通常与不良农艺性状相关，并且单一抗性基因的使用可能会因持续的Pt进化而导致抗性快速崩溃（Dracatos等，  2023）。因此，发现新的抗性基因并平衡产量与抗病性对于可持续育种策略至关重要。

在我们的研究中，我们用高毒力的Pt种族 THT 接种了小麦品种 Fielder，并在接种后 0、12 和 24 小时 (hpi) 收集样本进行 RNA 测序分析（附录 S1）。该分析强调了参与细胞蛋白质修饰的差异表达基因 (DEG) 的显着上调，包括对蛋白质降解至关重要的 E3 泛素连接酶（图 1a、b）。在 255 个Pt诱导的E3基因中，我们鉴定了小麦GRAIN WIDTH2 ( TaGW2 ) 同源基因，对小麦籽粒宽度和重量具有负调节作用 (Qin et al .,  2014 )。它们的表达在 12 hpi 时增加了 3.8 倍以上，在 24 hpi 时增加了 3 倍（图 1c；表 S1）。RT-qPCR 测定证实TaGW2在 12 和 24 hpi 时显着上调（图 S1），表明其参与小麦对叶锈病的反应。

为了研究TaGW2在小麦叶锈病抗性中的作用，我们通过Fielder品种中的CRISPR/Cas9技术生成了TaGW2-6A过表达（OE）转基因植物和TaGW2敲除（KO）植物（图S2；表S2）。THT 接种后，OE 植物比野生型 (WT) 具有更多的尿素，而 KO 植物在幼苗阶段接种后 10 天 (dpi) 的尿素较少（图 1d、e）。显微镜分析显示，与 OE 植物不同，48 hpi 时 KO 中的H ₂ O _{2积累增加，120 hpi 时菌丝长度和数量减少（图 1f）。}此外，发病机制相关基因转录水平在 KO 中较高，在 OE 中较低（图 S3）。进一步检查了成年植物的抗性表型，发现接种 THT 后，KO 植物比 OE 植物具有更少且更小的孢子形成，与苗期抗性表型一致（图 1g，h）。因此， TaGW2功能的丧失增强了小麦对Pt的抗性，表明TaGW2是叶锈病抗性的负调节因子。

在探索 TaGW2 介导的植物免疫的分子机制中，我们在酵母双杂交 (Y2H) 测定中使用了 TaGW2 的 N 末端，避免了由于自我激活而使用的 C 末端。Y2H 筛选鉴定出九种潜在的相互作用蛋白，包括 SKP1 (SGT1) 的 G2 等位基因的抑制子（表 S3；图 1i）。认识到SGT1在植物免疫中的重要作用(Wang et al .,  2022 )，我们使用萤火虫荧光素酶互补成像 (LCI) 和双分子荧光互补 (BiFC) 检测在本塞姆氏烟草叶子中验证了其与 TaGW2 的相互作用（图 1j,k） 。Pull-down 测定进一步证实了它们在体外的直接相互作用（图 1l）。这些结果表明 TaGW2 和 TaSGT1 之间存在物理相互作用。

先前的研究确定TaSGT1参与Lr21介导的小麦叶锈病抗性（Scofield等，  2005）。我们发现它的转录受到Pt种族 THT 的显着诱导，类似于TaGW2（图 S4）。使用大麦条纹花叶病毒 (BSMV) 介导的基因沉默，我们敲低了Fielder 中TaSGT1 的表达（图 S5a、b）。TaSGT1沉默导致尿素增加、H ₂ O ₂积累减少和菌丝增多（图 1m,n；图 S5c）。这些发现表明，TaSGT1限制了小麦叶片组织中的Pt增殖，充当抗叶锈病的正调节因子。

鉴于 TaGW2 与 TaSGT1 的相互作用及其作为 RING 型 E3 泛素连接酶的作用 (Liu et al .,  2020 )，我们假设 TaGW2 可以泛素化 TaSGT1。我们通过使用重组 His-TaGW2 和 MBP-TaSGT1 蛋白的体外泛素化测定对此进行了测试。该测定表明，在 Ub、E1 和 E2 存在的情况下，MBP-TaSGT1 可以被 TaGW2 泛素化，而 MBP 对照则不能（图 1o）。这表明TaGW2可能通过介导TaSGT1的泛素化和降解来调节小麦对叶锈病的抗性。

作为成熟的粒重和粒宽调节剂，我们评估了TaGW2在未接种和接种条件下对籽粒性状的影响，例如千粒重 (TGW)、粒长 (GL) 和粒宽 (GW)状况。所有品系在接种后均表现出 GW 和 TGW 的减少。然而，KO 工厂的 TGW 仅下降了 5.6%–7.4%，而 OE 工厂下降了 15.7%–17.2%，这主要是由于 KO 和 OE 的 GW 下降不同（图 1p–r）。结果，与 WT 相比，KO 植物的 GW 增加了 5.1%–6.6%，TGW 增加了 4.5–5.5 g，而 OE 植物的 GW 减少了 8.0%–11.0%，TGW 减少了 5.5–6.3 g。 TGW（图 1q、r；表 S4）。在任何未接种和接种的品系中均未观察到 GL 的显着变化（图 S6）。这些结果表明，TaGW2-KO 植物对Pt的抗性增强有助于保护小麦产量免受叶锈病损害。

总之，我们的研究结果表明，TaGW2 除了负向调节粒重的已知作用外，还通过介导 TaSGT1 的泛素化来负向调节小麦叶锈病抗性。通过使用CRISPR/Cas9编辑TaGW2，我们实现了粒重和叶锈病抗性的同步增加，为在不牺牲产量的情况下增强小麦对叶锈病的抗性提供了一种新方法。