Fusarium head blight (FHB) is one of the devastating diseases for wheat production worldwide, which causes significant yield losses and reduces grain quality because of mycotoxins contamination in wheat grains. As wheat relatives, Thinopyrum elongatum and Th. ponticum are important genetic resources that can be used to improve wheat FHB resistance. Using recombinant inbred lines derived from a cross between two Thatcher-Th. ponticum substitution lines, K11463 (7E1/7D) and K2620 (7E2/7D), the major FHB resistance locus Fhb7 was mapped to the very distal region of the long arm of chromosome 7E2 (Guo et al., 2015). Wang et al. (2020) sequenced the genome of Th. elongatum and cloned the glutathione S-transferase-encoding Fhb7 by genetic mapping. Relying on the recombination between Th. elongatum chromosome 7E and Th. ponticum chromosome 7E1, a resistant gene Fhb-7EL for FHB resistance was located to the long arm of 7E (Ceoloni et al., 2017).

To transfer the resistant gene Fhb-7EL to common wheat, hundreds of wheat-Th. elongatum translocation lines were developed by irradiating the pollen of the wheat-Th. elongatum addition line Chinese Spring (CS)-7EL at anthesis, among which Zhongke 1878 proved to carry an approximately 100 Mb 7EL chromatin on chromosome 6DL (Figure 1a, Figure S1, Appendix S1). After backcrossing Zhongke 1878 with the highly susceptible variety Jimai 22 for six generations, FHB resistance evaluation showed that the translocated chromosome could significantly increase the FHB resistance of Jimai 22 to the level of Sumai 3 by decreasing the number of diseased spikelets from 13.43 to 1.43 (Figure 1b,c).

To explore the nature of the FHB resistance gene, we inoculated the spikes of the line Zhongke 1878 with F. graminearum and performed single-molecule real-time isoform sequencing after 96 h. Removing the transcripts derived from wheat and Fusarium species by blasting wheat reference genome and nucleotide database on NCBI, 25 transcripts were identified derived from alien chromatin by PCR in Zhongke 1878 (Figure S2, Tables S1 and S2). To study the mechanisms of FHB resistance in the line Zhongke 1878, next-generation sequencing-based transcriptomic analysis was performed on these 25 transcripts. Annotated as a GST protein, the expression of the transcript T26102 was significantly increased 48 h after inoculation with F. graminearum (Figure 1d,e, Table S1).

To illustrate the association between T26102 and FHB resistance, the distribution of T26102 was checked in a series of wheat-Thinopyrum derivatives. The homologue of T26102 was not only detected in wheat-Th. ponticum amphiploid SNTE20 but also in the wheat-Th. ponticum translocation lines 4460 and 4462 (Figure 1f, Figures S3, S4). After sequencing the amplified product, two different T26102 homologues were discovered in lines CS-7EL, Zhongke 1878 and SNTE20 respectively (Figure 1f, Figure S4). Although SNTE20, 4460 and 4462 were proven to carry the GST-encoding Fhb7 homologues, all three lines were identified as susceptible to FHB as well as the susceptible control Jimai 22 (Figure 1g,h). Expression analysis revealed that Fhb7 homologues were induced in lines 4460, 4462 and SNTE20 after inoculating with F. graminearum (Figure 1i). Similar results were also reported in the wheat-Th. ponticum partial amphiploid SNTE122 and translocation line TNT-B (Guo et al., 2022). More puzzling was that the Fhb7 homologue and its promoter shared by 4460 and 4462 were identical to the one in the wheat-Th. ponticum substitution line 7E2/7D used as the resistant parent to map Fhb7 (Figure 1f, Figures S4, S5). All these results casted our doubt on the FHB-resistant function of the GSTs.

To verify the FHB resistance function of T26102, we transformed the overexpression vector pUbi:T26102 into three common wheat accessions 19AS161, Jimai 22 and Zhongmai 175. The transgenic positive wheat plants overexpressing T26102 were used for FHB resistance evaluation (Figure S6). A few bleached spikelets were observed on all spikes of both wild types and T₀ transgenic plants 7 days after inoculation with F. graminearum (Figure 1j). Statistical analysis was performed between the wild type and the transgenic plants; no difference was discovered between them (Figure 1k). To rule out the effect of amino acid variation on the function of T26102, we also expressed the GST-encoding Fhb7 under the ubiquitin promoter and the same native promoter as reported by Wang et al. (2020) in common wheat varieties Zhengmai 7698 and Kenong 199. Regardless of the vector driven by the ubiquitin promoter or the native promoter, nearly half the inoculated spikes bleached in the Zhengmai 7698 transgenic plants expressing Fhb7 (Figure 1l). Except for the FHB evaluation on the T₀ generation, the T₁ transgenic plants on Kenong 199 background were chosen to verify the function of Fhb7. With obvious bleached spikelets on the inoculated spikes, no statistical difference in FHB resistance was discovered between the T₁ transgenic plants and the control Kenong 199 (Figure 1m,n). These results suggested the GST-encoding Fhb7 also failed to confer wheat FHB resistance. All these results suggested that GSTs from Thinopyrum, including the GST-encoding Fhb7 and its homologues, were not decisive for FHB resistance.

赤霉病 (FHB) 是世界范围内小麦生产的破坏性病害之一，由于小麦籽粒中的霉菌毒素污染，导致产量显着下降并降低谷物质量。作为小麦的近亲，Thinopyrum elongatum和Th. ponticum是重要的遗传资源，可用于提高小麦 FHB 抗性。使用源自两个 Thatcher- Th 之间杂交的重组近交系。桥体替代品系 K11463 (7E1/7D) 和 K2620 (7E2/7D)，主要的 FHB 抗性位点Fhb7被定位到染色体 7E2 长臂的最远端区域（Guo等人，  2015 年）。王等人。( 2020) 对 Th 的基因组进行了测序。elongatum并通过遗传作图克隆了编码谷胱甘肽 S-转移酶的Fhb7 。依靠Th之间的重组。elongatum染色体 7E 和Th。桥脑染色体 7E1，一个抗 FHB 的抗性基因Fhb-7EL位于 7E 的长臂上 (Ceoloni et al .,  2017 )。

为了将抗性基因Fhb-7EL转移到普通小麦中，数百个小麦- Th。elongatum易位系是通过辐照小麦的花粉开发的。elongatum添加系 Chinese Spring (CS)-7EL 在花期，其中中科 1878 被证明在染色体 6DL 上携带约 100 Mb 的 7EL 染色质（图 1a，图 S1，附录 S1）。中科1878与高感品种济麦22回交6代后，FHB抗性评价表明，易位染色体可显着提高济麦22的FHB抗性，达到苏麦3号的水平，病小穗数从13.43个减少到1.43个（图 1b,c)。

为了探索 FHB 抗性基因的性质，我们将禾谷镰刀菌接种到中科 1878 品系的穗状花序上，并在 96 小时后进行单分子实时亚型测序。通过在 NCBI 上爆破小麦参考基因组和核苷酸数据库去除来自小麦和镰刀菌属物种的转录本，通过 PCR 在中科 1878 中鉴定出 25 个来自外来染色质的转录本（图 S2，表 S1 和 S2 ）。为了研究品系中科 1878 的 FHB 抗性机制，对这 25 个转录本进行了基于下一代测序的转录组学分析。注释为 GST 蛋白，转录物 T26102 的表达在接种禾谷镰刀菌后 48 小时显着增加（图 1d，e，表 S1）。

为了说明 T26102 和 FHB 抗性之间的关联，检查了 T26102 在一系列小麦-噻吩啉衍生物中的分布。T26102 的同系物不仅在小麦-Th 中检测到。ponticum二倍体 SNTE20 但也存在于小麦- Th。桥脑易位线 4460 和 4462（图 1f，图 S3、S4）。对扩增产物进行测序后，分别在 CS-7EL、Zhongke 1878 和 SNTE20 品系中发现了两个不同的 T26102 同系物（图 1f，图 S4）。尽管 SNTE20、4460 和 4462 被证明携带 GST 编码的Fhb7同源物，但所有三个品系都被确定为对 FHB 以及易感对照 Jimai 22 敏感（图 1g，h）。表达分析表明在用禾谷镰刀菌接种后，在品系 4460、4462 和 SNTE20 中诱导了Fhb7同系物（图 1i）。在小麦- Th 中也报道了类似的结果。桥体部分二倍体 SNTE122 和易位系 TNT-B (Guo et al .,  2022 )。更令人费解的是， 4460 和 4462 共享的Fhb7同系物及其启动子与小麦-Th 中的相同。ponticum替代品系 7E2/7D 用作绘制Fhb7 的抗性亲本（图 1f，图 S4，S5）。所有这些结果使我们对 GST 的抗 FHB 功能产生了怀疑。

为了验证 T26102 的 FHB 抗性功能，我们将过表达载体 pUbi:T26102 转化到三个普通小麦种质 19AS161、济麦 22 和中麦 175 中。过表达 T26102 的转基因阳性小麦植株用于 FHB 抗性评价（图 S6）。在接种禾谷镰刀菌 7 天后，在野生型和 T _{0转基因植物的所有穗状花序上}都观察到了一些漂白的小穗（图 1j）。在野生型和转基因植物之间进行统计分析；它们之间没有发现差异（图 1k）。为了排除氨基酸变异对 T26102 功能的影响，我们还在泛素下表达了编码 GST 的Fhb7启动子和 Wang等人报道的同一本地启动子。( 2020 ) 在普通小麦品种郑麦 7698 和克农 199 中。无论由泛素启动子还是天然启动子驱动的载体，表达Fhb7的郑麦 7698 转基因植物中近一半的接种穗发生漂白（图 1l）。_{除对T 0}代进行FHB评价外，选用科农199背景的T _{1转基因植株验证}Fhb7的功能。接种的穗上有明显的漂白小穗，T _{1之间没有发现 FHB 抗性的统计差异}转基因植物和对照 Kenong 199（图 1m，n）。这些结果表明编码 GST 的Fhb7也未能赋予小麦 FHB 抗性。所有这些结果表明，来自Thinopyrum的 GST ，包括编码 GST 的Fhb7及其同系物，对 FHB 抗性不是决定性的。