A Verticillium dahliae exoglucanase as potential HIGS target interacts with a cotton cysteine protease to confer resistance to cotton Verticillium wilt

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.

Figure 1.

VdEXG silencing and ectopic overexpression of its interacting protein GhRD21A confers V. dahliae resistance. (a) VdEXG expression in cotton following Vd inoculation. (b–c) Penetration assay (b) and carbon use (c) of ΔVdEXG, ΔVdEXG‐C and Vd strain. (d) Pathogenicity assay in VdEXG‐RNAi lines. (e–f) Disease index (e) and fungal biomass (f) of Vd at 14 dpi in cotton plants. (g) VdEXG expression in Vd after infected cotton plants at 96 hpi. (h) Detection of VdEXG‐targeting siRNAs in the VdEXG‐RNAi lines. (i) Validation of siRNA production in VdEXG‐RNAi cotton plants using small RNA hybridization. (j) VdEXG secretion analysis. (k) Cytotoxicity analysis of VdEXG in Nicotiana benthamiana leaves. (l–m) The interaction between VdEXG and GhRD21A analysed using Y2H assay (l) and bimolecular fluorescence complementation (m). (n) GhRD21A expression detected using RT‐qPCR in cotton. (o) GhRD21A expression in Col‐0 and GhRD21A‐overexpressing lines. (p) Arabidopsis phenotypes following Vd inoculation. (q) Fungal biomass determined using RT‐qPCR in Col‐0 and transgenic Arabidopsis lines. Significant differences tested using Dunnett's test are represented with different letters (P < 0.05).

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.

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval statement

This article does not contain any studies with human or animal subjects.

Supporting information

Figure S1 (a) VdEXG coding region and VdEXG knockout via replacement with a HPT box. (b) Diagram of the VdEXG‐RNAi vector in cotton plants. (c) Protein sequence characteristics of VdEXG, including the signal peptide (SP) and the glycosyl hydrolase family (GH7) domain. (d) Diagram of the vector for ectopic GhRD21A overexpression in Col‐0.

Table S1 Primers used in the current study.

Data availability statement

All the data used for this study are presented in the paper or the Supplementary materials. The data of sRNA‐sequencing can be found here: NCBI, PRJNA1013902.