Transcriptome analysis reveals the cotton defense mechanism in response to Verticillium dahliae in the presence of the biocontrol fungus Chaetomium globosum CEF-082

Background: Verticillium wilt of cotton is a serious soil-borne disease that causes a substantial reduction in cotton yields. A previous study showed that the endophytic fungus Chaetomium globosum CEF-082 could control Verticillium wilt of cotton, and induce a defense response in cotton plants. However, the comprehensive molecular mechanism governing this response is not clear. Results: To study the signalling mechanism induced by CEF-082, the transcriptome of cotton seedlings pretreated with CEF-082 was sequenced. The results revealed 5638 DEGs at 24 h post inoculation with CEF-082, and 2921 and 2153 DEGs at 12 and 48 h post inoculation with Verticillium dahliae, respectively. At 24 h post inoculation with CEF-082, KEGG enrichment analysis indicated that the DEGs were enriched mainly in the plant-pathogen interaction, MAPK signalling pathway-plant, flavonoid biosynthesis, and phenylpropanoid biosynthesis pathways. There were 1209 DEGs specifically induced only in cotton plants inoculated with V. dahliae in the presence of the biocontrol fungus CEF-082, and not when cotton plants were only inoculated with V. dahliae. GO enrichment analysis revealed that these DEGs were enriched mainly in the following terms: ROS metabolic process, H2O2 metabolic process, defense response, superoxide dismutase activity, and antioxidant activity. Moreover, many genes, such as ERF, CNGC, FLS2, MYB, GST and CML genes, were identified that regulate crucial points in defence-related pathways and that may contribute to V. dahliae resistance in cotton. These results provide a basis for the understanding of the molecular mechanism by which biocontrol fungus CEF-082 increased the resistance of cotton to Verticillium wilt. Conclusions: The results of this study showed that CEF-082 could regulate multiple

levels in plants in response to biocontrol agents, but these studies have mainly involved A. thaliana. An analysis of gene expression changes in A. thaliana in the presence of Trichoderma harzianum T34 revealed that salicylic acid (SA) -and jasmonic acid (JA)-related genes were down-regulated [17]. PDF1.1, a putative defensin, was upregulated after A. thaliana was treated with Bradyrhizobium sp. strain ORS278 independently of pathogen attack [18].
In previous studies, we found that the endophytic fungus Chaetomium globosum CEF-082 isolated from upland cotton plants suppressed the growth of V. dahliae and increased cotton resistance to Verticillium wilt [19] . However, the signalling mechanism induced by CEF-082 is unknown. Therefore, the purpose of this study was to reveal the molecular mechanism by which CEF-082 increased cotton resistance to Verticillium wilt via RNA sequence analysis.
by Professor Heqin Zhu from State Key Laboratory of Cotton Biology, Institute of Cotton Research of Chinese Academy of Agricultural Sciences. It is a hybrid [(Jihan 4× Ke 4104) F_2 × 74Yu102]. The seeds were sterilized with 70% alcohol for 1 minute and then with 1.05% sodium hypochlorite for 10 minutes, after which the seeds were washed with sterile water 5 times. The cotton seeds were planted in vermiculite and transferred to plastic pots (25 cm × 15 cm) that contained 2000 mL of liquid culture solution after emergence. The cultivation solution was prepared according to the methods of Zhang et al. [21], with some modifications. In this study, 2 mM NaCl was used instead of 2.5 mM KCl, while the other 9 mineral nutrients were the same. A black cystosepiment with 20 holes was placed on the plastic pot, and cotton plants were placed into the holes and supported by a sponge. Twenty plants were cultivated per pot per treatment, and each treatment was repeated three times. Twenty cotton plants in each treatment were removed from the plastic pots, and inoculated with CEF-082 by soaking the cotton roots in 300 mL of a 1×10 5 spore/mL spore suspension for 40 minutes prior to the first true leaf flattening . Instead of the CEF-082 spore suspension, water was used as the control group. The cotton plants were then returned to the pots. At 0 h, 6 h and 24 h later, 5 leaves were randomly taken at each time point for each biological replicate in each treatment, and 24 h was considered 0 h before inoculation with V. dahliae (24 h (0 h)). Twenty four hours post inoculation with CEF-082, the same method was used to inoculate V. dahliae VD1070-2 (1×10 7 spore/mL) in the treatment group and the control group. Leaf samples were then collected at 12 (Table   S1).

Control effect of CEF-082 on Verticillium wilt of cotton and the H 2 O 2 content
The disease index was 18.61 in the control group (water+ V. dahliae) and 7.62 in the treatment group (CEF-082+ V. dahliae) 14 d after V. dahliae inoculation (Fig.   1A). The results showed that CEF-082 enhanced the resistance of cotton to Verticillium wilt, and the biocontrol effect was 59.1% (Fig. 1C).
The H 2 O 2 content in the treatment group was higher than that in the control group throughout the majority of the duration of the experiment and lower than that in the control group at 5 dpi with V. dahliae. The H 2 O 2 content in the treatment group was highest at 2 dpi (12.80 μmol/g), while the H 2 O 2 content in the control group was highest at 1 dpi (10.38 μmol/g). The changes in the two groups were similar and were stable 5 d later (Fig. 1B).
Verification of RNA-Seq Analysis by qRT-PCR Twelve DEGs were randomly selected. The gene expression levels in the control and upregulated and 1 was downregulated. The GO classification showed that there were 18, 14 and 12 terms in biological process, cellular component and molecular function, respectively, and the KEGG classification indicated that the DEGs mainly belonged to the metabolism pathway (2856 DEGs).

DEGs co-induced by CEF-082 and V. dahliae
There were 463 shared DEGs at 12 h and 48 h that were significantly enriched in 6 KEGG pathways (Table 3). In the plant-pathogen interaction pathway, 29 DEGs  (Table 5). GO analysis showed that the DEGs were enriched in superoxide dismutase activity, oxidoreductase activity, acting on superoxide radicals as acceptors, and antioxidant activity terms. Of the 96 DEGs, 9 encoded TFs and 20 encoded predicted PRGs (Table S4).
A protein-protein interaction network (Fig. S6) was constructed via the 96 DEGs shared between 12 h and 48 h and genes interacting with them in cotton. Six hub genes were obtained, including Gh_A05G1020, Gh_D09G0858, BGI_novel_G004376, Gh_A08G0125, Gh_D07G1197 and Gh_A05G3508. Among them, Gh_D07G1197 was enriched in the flavonoid biosynthesis pathway.

Putative R genes and TFs involved in resistance to Verticillium wilt
On the basis of the transcriptome analysis, a total of 65 candidate genes that may be related to the resistance of cotton to Verticillium wilt were identified, including 5 CNLs (whose members contain an NB-ARC domain), 3 CNs (members of the U-box domain-containing protein kinase family protein), 5 NLs (whose members contain an NBS-LRR domain), 7 RLPs (whose members contain an eLRR-TM-S/TPK domain), 7 Ns (whose members contain an NBS domain only), 9 TNLs (members of the TIR-NBS-LRR class), 6 Ts (members of NAC domain containing protein 17), 1 Mlo-like (a member of the Mlo-like resistant proteins) and 2 other types (which have resistance functions but do not fit the known classes). These genes mainly included a disease resistance protein, 2 probable calcium-binding protein (CML45), 3 ethyleneresponsive transcription factor (ERF), 2 cyclic nucleotide-gated ion channel 2 (CNGC2), 5 MYB TFs and 2 GST (  (Fig. 4) showed that certain genes were upregulated at 0,  [16], and the results were also consistent with those of Tan It is clear that plant responses to biotic or abiotic stress depend on interactions among several signalling pathways, including those mediated by JA, ET, SA or ABA [28][29]. Morán-Diez E et al. [17] found SA-and JA-related DEGs were downregulated in A. thaliana after 24 h of incubation in the presence of T. harzianum

T34. A set of DEGs influenced by JA or ET, was induced upon pathogen attack when
A. thaliana were previously colonized by a photosynthetic Bradyrhizobium sp. strain, ORS278 [18]. 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.
Besides, DEGs related to JA, ET, SA, BR and cytokinin were induced in cotton plants treated only with CEF-082. The 8 plant hormones were also induced after infection with V. dahliae in sunflower [16]. The responses of the A. thaliana auxin receptors TIR1, AFB1 and AFB3 and auxin transporter AXR4 were impaired upon infection with V. dahliae [30]. 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 [31][32]and mediated by CNGC, RBOH, CaM/CML and FLS2 [33][34][35]. 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. 3). 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 defense mechanisms [36], while flavonoids are known to buffer substantial stress-induced alterations in ROS homeostasis and to modulate the ROS-signalling cascade [37]. Plant CNGC subunits and CaM constitute a molecular switch that either opens or closes calcium channels [38]. Previous reports have shown that calcium-dependent CDPK4 and CDPK5 regulate ROS production by phosphorylating NADPH oxidase in potatoes [39]. ROS are important not only for signalling mechanisms for defense [40]but also for regulating programmed cell death via the establishment of the HR [41]. MAPK family members can improve resistance to Verticillium wilt of cotton [42]. In this study, 24 h after CEF-082 inoculation, certain signal transduction pathways might have been involved in the plant response to CEF-082 (Fig. 5). After inoculation with CEF-082, FLS2 recognized CEF-082, MAPK signal transduction was induced, and calcium channels opened. H 2 O 2 was then produced, leading to an ROS burst. Plant hormones were also induced, including ET, SA, JA, ABA, BR, auxin, gibberellin and cytokinin.
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. S7). The defense response at T12h and T48h was similar to that at T0h, and only some key points induced were different in the pathways, which are shown in Fig. 11 and Fig. 12. Thus, it is speculated that CEF-082 reduced the occurrence of cotton Verticillium wilt because inoculation with CEF-082 can prime signalling pathways in defense against V. dahliae upon infection.
barbadense treated with V. dahliae [52]. 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. [51][52]. 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 was shown to increase resistance to Verticillium wilt in tobacco [53]. Hence, we suggest that CEF-082 can induce specific GST genes to protect cotton from V. dahliae.
V. dahliae can induce a defense 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 presented significantly upregulated or downregulated expression levels of resistance-related genes. Therefore, it is speculated that the defense 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 induced only in plants inoculated with CEF-082 +V. dahliae. 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.    Expression levels of genes related to ROS and Ca2+. The red color represents upregulation, Figure 4 Clustering thermogram of putative R genes and genes encoding TFs.   Lignin biosynthesis pathway [43]. Enzymes coloured in red or black indicate the key points in