Verticillium wilt, caused by the soil-borne pathogenic fungus Verticillium dahliae (Vd), represents a devastating disease impacting cotton (Gossypium spp.). However, the limited efficacy of measures to control Verticillium wilt arises because Vd colonizes the host vascular system, as well as the inherent resilience of Vd resting structures (microsclerotia), to various environmental influences (Fradin and Thomma, 2010). Breeding-resistant cotton cultivars are the most economical and efficient approach to increasing host resistance to pathogens (Koch et al., 2019). One such strategy involves the utilization of host-induced gene silencing (HIGS) to target Vd effector genes.

We previously employed HIGS to transiently silence the Vd gene encoding an exoglucanase (VdEXG, VDAG_02898) with the typical glycosyl hydrolase family (GH7) domain, which improved host resistance to Vd. However, the underlying molecular mechanisms require further elucidation (Su et al., 2020; Zhao et al., 2015). In this study, we investigated the VdEXG expression pattern in Vd-infected cotton seedlings using reverse transcription quantitative PCR (RT-qPCR) (Figure 1a). The VdEXG transcript levels increased continuously upon Vd infection and peaked at 12 h post-inoculation (hpi). To elucidate the role of VdEXG in fungal pathogenicity, we knocked out VdEXG in Vd (designated as ΔVdEXG mutant) using a hygromycin resistance cassette by homologous recombination (Figure S1a). The penetration capability of ΔVdEXG through a cellophane membrane was notably lower than that of the Vd and ΔVdEXG-complemented (ΔVdEXG-C) strains (Figure 1b). Furthermore, ΔVdEXG exhibited substantially reduced growth compared with that of Vd and ΔVdEXG-C strains when cultured in media containing various carbon sources (Figure 1c), signifying the indispensable role of VdEXG in vegetative Vd growth.

Subsequently, we constructed a recombinant HIGS plasmid targeting the 486-bp VdEXG coding sequences (Figure S1b), which was integrated into the cotton genome. This led to the generation of two independent transgenic cotton lines (VdEXG-RNAi-1/2) that displayed heightened resistance to Vd, resulting in decreased fungal biomass compared with that observed in WT (Figure 1d–f). Meanwhile, VdEXG expression at 96 hpi was substantially lower in Vd-infected VdEXG-RNAi transgenic cotton compared with that in WT (Figure 1g). Furthermore, siRNA sequencing corroborated the generation of VdEXG-targeting siRNAs in Vd-infected VdEXG-RNAi transgenic cotton (Figure 1h). Using RNA hybridization, we observed prominent siVdEXG signals (21–24 nt) in the VdEXG-RNAi lines but not in WT (Figure 1i). These data reveal that the small interfering RNAs (siRNAs) targeting VdEXG reduce the ability of Vd to infect its host and VdEXG as a potential HIGS target to control Vd.

Concurrently, fungal glycoside hydrolases are effectors that activate and inhibit host resistance (Cui et al., 2015). SignalP (version 5.0) predicted that VdEXG possesses an N-terminal signal peptide, which was subsequently validated using the yeast signal trap and 2,3,5-triphenyl tetrazolium chloride (TTC) assays (Figure S1c). Yeast harbouring the Avr1b effector from Phytophthora sojae and the full-length VdEXG (VdEXG^FL) displayed normal growth and caused TTC to turn red, whereas VdEXG lacking the signal peptide sequence (VdEXG^NS) and negative controls exhibited no growth and remained colorless (Figure 1j). Transient VdEXG^NS expression in Nicotiana benthamiana leaves resulted in cell death at 48 hpi (Figure 1k), which is consistent with Bcl-2-associated protein X (BAX) rather than eGFP (Cheng et al., 2017). Therefore, we hypothesized that VdEXG functions as an effector to modulate the host immune system.

To validate this hypothesis, we identified the cotton cysteine proteinase RD21A (GhRD21A, XM_016851915.2) as a candidate protein interacting with VdEXG from a Vd-inoculated cotton cDNA library. We confirmed the interaction in the yeast two-hybrid (Y2H) assay (Figure 1l). Subsequently, we used a bimolecular fluorescence complementation assay in N. benthamiana leaves to verify that VdEXG interacts with RD21A in vivo (Figure 1m). Co-expression of VdEXG-nYFP and RD21A-cYFP in plant cells generated a yellow fluorescent signal in the nucleus, indicating the interaction between VdEXG and RD21A. Given the significant reduction in VdEXG expression observed in Vd-infected VdEXG-RNAi cotton lines (Figure 1g), we hypothesized that GhRD21A was also inhibited. Concordantly, GhRD21A expression was inhibited in the VdEXG-RNAi lines compared with that in WT at 96 hpi (Figure 1n). Furthermore, we ectopically overexpressed GhRD21A in an Arabidopsis ecotype (Col-0) to evaluate its function (Figures S1d and 1o). The transgenic lines had significantly increased resistance to Vd infection, with reduced necrosis and fungal biomass compared with that in Col-0 (Figure 1p,q), which was consistent with the results of a previous study (Zhang et al., 2019). These findings suggest that GhRD21A interacts with VdEXG during Vd infection to promote cotton resistance.

In conclusion, our findings suggest that GhRD21A recognized VdEXG to enhance cotton resistance to Vd, while HIGS targeting VdEXG limited the Vd pathogenicity and conferred disease resistance in cotton. These results provide a new strategy for using secretory proteins involved in pathogenicity to breed wilt-resistant cultivars.

黄萎病由土传病原真菌大丽黄萎病( Vd ) 引起，是一种影响棉花 ( Gossypium spp.) 的毁灭性疾病。然而，控制黄萎病的措施效果有限，因为Vd定植于宿主血管系统，以及Vd静止结构（微菌核）对各种环境影响的固有弹性（Fradin 和 Thomma，  2010）。抗育种棉花品种是提高宿主对病原体抵抗力的最经济、最有效的方法（Koch等，  2019）。其中一种策略涉及利用宿主诱导的基因沉默（HIGS）来靶向Vd效应基因。

我们之前使用 HIGS 暂时沉默编码具有典型糖基水解酶家族 (GH7) 结构域的外切葡聚糖酶 ( VdEXG 、VDAG_02898) 的Vd基因，从而提高了宿主对Vd的抵抗力。然而，潜在的分子机制需要进一步阐明（Su等，  2020；Zhao等，  2015）。在本研究中，我们使用逆转录定量 PCR (RT-qPCR)研究了Vd感染的棉花幼苗中的VdEXG表达模式（图 1a）。 VdEXG转录水平在Vd感染后持续增加，并在接种后 12 小时 (hpi) 达到峰值。为了阐明VdEXG在真菌致病性中的作用，我们使用潮霉素抗性盒通过同源重组敲除Vd中的VdEXG（称为ΔVdEXG突变体）（图 S1a）。 ΔVdEXG通过玻璃纸膜的渗透能力明显低于Vd和ΔVdEXG互补 ( ΔVdEXG- C) 菌株（图 1b）。此外，当在含有各种碳源的培养基中培养时，与Vd和ΔVdEXG- C菌株相比，ΔVdEXG表现出显着减少的生长（图1c），这表明VdEXG在Vd营养生长中不可或缺的作用。

随后，我们构建了一个针对 486 bp VdEXG编码序列的重组 HIGS 质粒（图 S1b），并将其整合到棉花基因组中。这导致产生了两个独立的转基因棉花品系（VdEXG -RNAi-1/2），它们对Vd表现出更高的抗性，导致真菌生物量与WT中观察到的相比减少（图1d-f）。同时，与WT相比，Vd感染的VdEXG -RNAi转基因棉花在96 hpi时的VdEXG表达显着降低（图1g）。此外，siRNA测序证实了Vd感染的VdEXG -RNAi转基因棉花中VdEXG-靶向siRNA的产生（图1h）。使用 RNA 杂交，我们在VdEXG -RNAi 系中观察到显着的 si VdEXG信号（21-24 nt），但在 WT 中未观察到（图 1i）。这些数据表明，针对VdEXG 的小干扰 RNA (siRNA)降低了Vd感染宿主的能力，并且VdEXG作为控制Vd的潜在 HIGS 靶点。

同时，真菌糖苷水解酶是激活和抑制宿主抵抗力的效应器（Cui et al .,  2015）。 SignalP（版本 5.0）预测 VdEXG 具有 N 末端信号肽，随后使用酵母信号捕获和 2,3,5-三苯基氯化四唑 (TTC) 测定法对其进行了验证（图 S1c）。含有大豆疫霉Avr1b 效应子和全长 VdEXG (VdEXG ^FL ) 的酵母显示正常生长并导致 TTC 变红，而缺乏信号肽序列 (VdEXG ^NS ) 的 VdEXG 和阴性对照则没有生长并保持无色（图1j）。本塞姆氏烟草叶片中瞬时VdEXG ^NS表达导致 48 hpi 时的细胞死亡（图 1k），这与 Bcl-2 相关蛋白 X (BAX) 而不是 eGFP 一致（Cheng等人，  2017）。因此，我们假设 VdEXG 作为调节宿主免疫系统的效应子发挥作用。

为了验证这一假设，我们从接种Vd的棉花 cDNA 文库中鉴定出棉花半胱氨酸蛋白酶 RD21A (GhRD21A，XM_016851915.2) 作为与 VdEXG 相互作用的候选蛋白。我们在酵母双杂交 (Y2H) 测定中证实了相互作用（图 1l）。随后，我们在本塞姆氏烟草叶子中使用双分子荧光互补测定来验证 VdEXG在体内与 RD21A 相互作用（图 1m）。VdEXG-nYFP和RD21A-cYFP在植物细胞中的共表达在细胞核中产生黄色荧光信号，表明 VdEXG 和 RD21A 之间的相互作用。鉴于在Vd感染的VdEXG -RNAi 棉花品系中观察到的VdEXG表达显着降低（图 1g），我们假设GhRD21A也受到抑制。一致地，与 WT 中的表达相比，在 96 hpi 时，VdEXG -RNAi 系中的GhRD21A表达受到抑制（图 1n）。此外，我们在拟南芥生态型（Col-0）中异位过度表达GhRD21A以评估其功能（图 S1d 和 1o）。与Col-0相比，转基因株系对Vd感染的抵抗力显着增强，坏死和真菌生物量减少（图1p，q），这与之前的研究结果一致（Zhang et al .,  2019）。这些发现表明，GhRD21A 在Vd感染期间与 VdEXG 相互作用，以促进棉花抗性。

总之，我们的研究结果表明，GhRD21A 识别 VdEXG 以增强棉花对Vd 的抗性，而针对VdEXG的 HIGS限制了Vd致病性并赋予棉花抗病性。这些结果为利用参与致病性的分泌蛋白培育抗枯萎品种提供了新策略。