The number of DEGs identified at 12 h and 48 h was lower than that identified at 0 h. It has been suggested that the number of DEGs decreased because both plants were infected with V. dahliae and began to respond defensively. For CEF–082 treatment and CEF–082+ V. dahliae treatment, DEGs were mainly enriched in 5 signalling pathways, plant-pathogen interaction, MAPK signalling pathway-plant, flavonoid biosynthesis, phenylpropanoid biosynthesis, and glutathione metabolism. The pathways of plant-pathogen interaction and flavonoid biosynthesis were also induced in sunflower infected with V. dahliae [13], and the results were also consistent with those of Tan [18], who reported that most DEGs in tomato were associated with phenylpropanoid metabolism and plant-pathogen interaction pathways. However, the glutathione metabolism pathway has rarely been reported in the transcriptome of cotton plants treated with V. dahliae.
DEGs related to ET, SA, JA, brassinosteroid (BR) and cytokinin were upregulated or downregulated upon V. dahliae infection in cotton [3]. In this study, we also found that DEGs in ABA, auxin and gibberellin were significantly induced not only after treatment with CEF–082 but also after inoculation with V. dahliae. The 8 plant hormones were also induced after infection with V. dahliae in sunflower [13]. The responses of the A. thaliana auxin receptors TIR1, AFB1 and AFB3 and auxin transporter AXR4 were impaired upon infection with V. dahliae [19]. Therefore, both CEF–082 and V. dahliae can induce changes in hormones.
Previously, it was shown that after plants were infected with pathogens, the FLS2 pattern recognition receptors recognized pathogens, and the hypersensitive response (HR) was activated through ROS, JA, WRKYs and the NO signalling pathways [20–21] and mediated by CNGC, RBOH, CaM/CML and FLS2 [22–24]. These results are consistent with the results from this study. In this study, 24 h after treatment with CEF–082, the DEGs of FLS2, Rboh, CDPK, CNGCs and GST in the plants were also upregulated or downregulated to varying degrees (Fig. 5). In addition, most of the genes coding peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were also upregulated. These genes were related to the accumulation of ROS. Forty-eight hours after treatment with V. dahliae, the genes encoding CNGC, CaM/CML and FLS2 were upregulated. However, in this study, the NO signalling pathway was not induced.
Phenylpropane synthesis is related to cotton defence mechanisms [25], while flavonoids are known were to buffer substantial stress-induced alterations in ROS homeostasis and to modulate the ROS-signalling cascade [26]. Plant CNGC subunits and CaM constitute a molecular switch that either opens or closes calcium channels [27]. Previous reports have shown that calcium-dependent CDPK4 and CDPK5 regulate ROS production by phosphorylating NADPH oxidase in potatoes [28]. ROS are important not only for signalling mechanisms for defence [29] but also for regulating programmed cell death via the establishment of the HR [30]. MAPK family members can improve resistance to Verticillium wilt in cotton [31]. In this study, 24 h after CEF–082 inoculation, certain signal transduction pathways may have been involved in the plant response to CEF–082 (Fig. 11). After inoculation with CEF–082, FLS2 recognized CEF–082, MAPK signal transduction was induced, and calcium channels were opened. Then, H2O2 was produced, leading to ROS burst. Plant hormones were also induced, including ET, SA, JA, ABA, BR, auxin, gibberellin and cytokinin. The signalling pathways of flavonoids and phenylpropane synthesis were also involved in this process. In addition, lignin synthesis was also induced after treatment with CEF–082 (Fig. 12). C4H and C3H were not induced in T0h-vs-C0h, T12h-vs-C12h, or T48h-vs-C48h but were induced in C12h-vs-C0h, which was similar to the results of Xu et al. [32], who indicated that C4H–1 and C4H–3 were upregulated after treatment with V. dahliae. Three days after inoculation with V. dahliae, lignin was detected, and the pith diameter of CEF–082 + V. dahliae-treated plants was slightly larger than that of water + V. dahliae-treated plants (Fig. S3). The defence response at T12h and T48h was similar to that at T0h, and only some key points induced were different in the pathways shown in Fig. 11 and Fig. 12. Thus, it is speculated that CEF–082 can control cotton Verticillium wilt because inoculation with CEF–082 can prime signalling pathways to defend against V. dahliae upon its infection.
When pathogens infect plants, they induce a series of defence responses. GST participates in plant defences and can remove ROS [34]. Plant GSTs can be subdivided into eight categories, phi, zeta, tau, theta, lambda, dehydroascorbate reductase (DHAR), elongation factor 1 gamma (EF1G) and tetrachlorohydroquinone dehalogenase (TCHQD) [35]. GSTF8 was used as a marker in early stress and defence responses [36], and salicylic acid, methyl jasmonate, ABA and H2O2 can induce GST expression [37–39]. LrGSTU5 was obviously upregulated after treatment with Fusarium oxysporum [40], and the GST genes were also upregulated in G. barbadense treated with V. dahliae [41]. In this study, the GST genes were also significantly induced 24 h after treatment with CEF–082 (Fig. 5), and GST genes were upregulated in cotton treated with Water + V. dahliae. These results are consistent with those of Han et al. and Zhang et al. [40–41]. Certain GST genes were also significantly induced in the treatment group but were not significantly induced in the control group after treatment with V. dahliae. The GST gene Gh_A09G1509 enhanced resistance against Verticillium wilt in tobacco [42]. Hence, we suggest that CEF–082 can induce specific GST genes to protect cotton from V. dahliae.
V. dahliae can induce a defence response after it infects cotton [3]. In this study, susceptible cotton varieties were inoculated with the biocontrol fungus CEF–082 and V. dahliae, which also induced a series of defence responses. Compared with plants inoculated with water +V. dahliae, the plants inoculated with CEF–082 + V. dahliae had significantly upregulated or downregulated expression levels of resistance-related genes. Therefore, it is speculated that the defence response was strengthened after inoculation with the biocontrol fungus CEF–082. In addition, we obtained 1209 specific DEGs, which could not be induced in plants inoculated with water +V. dahliae, and GO enrichment showed that these genes were involved in the metabolic process of ROS. The disease resistance of cotton was enhanced after CEF–082 treatment, and thus, we inferred that these specific DEGs might be genes related to plant disease resistance.