Transcription factor GhWRKY70 from Gossypium hirsutum enhances resistance to Verticillium wilt via the jasmonic acid pathway

doi:10.21203/rs.3.rs-1965649/v1

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Research Article

Transcription factor GhWRKY70 from Gossypium hirsutum enhances resistance to Verticillium wilt via the jasmonic acid pathway

https://doi.org/10.21203/rs.3.rs-1965649/v1

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published 14 Mar, 2023

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Background

The transcription factor WRKYs play significant roles in plant growth, development, and defense responses. However, in cotton, the molecular mechanism of most WRKY proteins and their involvement in Verticillium wilt tolerance are not well understood.

Results

GhWRKY70 was greatly up-regulated in cotton by Verticillium dahliae. Subcellar localization suggested that the GhWRKY70 was only located in the nucleus. Transcriptional activation of GhWRKY70 was further demonstrated that the GhWRKY70 functioned as a transcriptional activator. The transgenic Arabidopsis with GhWRKY70 plants exhibited better growth performance, higher lignin content, antioxidant enzyme activities and Jasmonic acid (JA) levels compared to wild type after infection with V. dahliae. In addition, the transgenic Arabidopsis resulted in enhanced expression level of AtAOS1, related JA synthesis gene, further leading to an increase in higher JA accumulation compared to wild type. However, the proportion of disease index (DI) values of the VIGS-treated cotton plants with TRV:WRKY70 plants were also significantly higher than VIGS-treated with TRV:00. Chlorophyll, lignin content and JA level of TRV:WRKY70 plants were significantly lower than that of TRV:00 plants. The GhAOS1 expression and JA abundance in TRV:WRKY70 plants was decreased. The GhWRKY70 protein was confirmed to bind to the W-box element in the promoter region of GhAOS by yeast one-hybrid assay and transient expression.

Conclusions

These results indicate that the GhWRKY70 transcription factor was a positive regulator in Verticillium wilt tolerance of cotton, and may promote production of JA via regulation of GhAOS1 expression.

Cotton

Transcription factor

GhWRKY70

Verticillium wilt

jasmonic acid pathway

Cotton (Gossypium hirsutum L.) is an excellent source of oil and fiber, widely considered one of the most economically important crops worldwide. In the growth and development process, cotton is highly vulnerable to attack by V. dahliae, which is a typical soil borne pathogenic fungus that results in Verticillium wilt disease in more than 400 dicotyledon plant species, including annual herbs, perennials, and woody plants [1]. The yield of cotton was declined by 10–35% because of Verticillium wilt. The production practice has demonstrated that genetic engineering by expressing important disease-resistant genes is an effective approach for developing transgenic plants with enhanced disease resistance [2, 3]. In this connection, it is an urgent task for understanding the underlying physiology and molecular mechanism in the cotton defense against V. dahliae infection.

A large spectrum of resistance related genes were regulated to participate in the synthesis of various metabolites that resist pathogen infection in plants. JA plays an important role in diverse physiological and developmental processes and biological and abiotic stresses [4–7]. In the JA signal transduction pathway, Jasmonate ZIM domain (JAZ) protein is a negative regulator and JA induces its degradation to activate JA responsive gene expression [8–10]. MYC2, one of the master transcription factors in the JA signaling pathway, is the target gene of JAZs protein which operates in diverse JA dependent functions and various plant environmental responses [11–13]. As a stress signaling compound, rapid accumulation of JA cannot be fulfilled without Lipoxygenase (LOX) and Allene Oxides Synthase (AOS), two key enzymes in the synthesis of JA [14]. RNAi-mediated silence of GhLOX2 decreased cotton resistance to V. dahliae and was coupled with suppression of JA-related genes both after inoculation with V. dahliae or MeJA treatment [15]. The AOS mutant of Arabidopsis thaliana was susceptible to Sclerotinia sclerotiorum accompanied by deficiency of JA biosynthesis [16]. Although it has been reported the LOX2 and AOS may contribute to responses to biological stresses by regulating JA levels, the gene expression mechanism remains unclear.

Plants challenged by pathogen infection can produce ROS, which can induce plants to activate some stress-related genes to resist environmental changes [17, 18]. Hydrogen peroxide (H₂O₂), as a more physiologically important ROS, is a relatively stable ROS that can diffuse into subcellular compartments. H₂O₂ can be generated and cleared by many enzymes, such as peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), which are involved in the antioxidant system. The role of H₂O₂ in extracellular matrix is not only limited to participating in a defense response, but also involved in regulating the synthesis of cell wall components, such as lignin synthesis [19]. Over-accumulation of H₂O₂ can damage plant’s biological macromolecules, disturbing its normal physiological and metabolic processes in plant cells [20–23]. Therefore, plants adjust the concentration of H₂O₂ in vivo by regulating the activities of antioxidant enzymes to resist pathogen invasion.

WRKYs, as transcription factors (TFs), are important constituents of plant signaling pathways and play crucial roles in controlling many important biological processes [24, 25]. Based on the number of WRKY domains and the structure of their zinc-finger motifs, WRKY proteins can be classified into three main groups: I, II, and III. Group III is composed of C₂HC (C-X₇-C-X₂₃-H-X₁-C) zinc-finger motif. These compounds are notably different from C₂H₂ of Group I and II, and are expressed only in higher plants which most expressed proteins are related to biological stress. Group I is expressed in ferns and some eukaryotic cells as well as higher plants [26, 27]. Therefore, Group III TFs, may have evolved as a result to acquire adaptations to different environmental pressure [28, 29]. AtWRKY70 (Group III) is a node of convergence for salicylic acid (SA) and JA mediated defense signaling pathways. Together with WRKY54 (Group III), AtWRKY70 enhances resistance to the hemibiotroph Pseudomonas syringae pv tomato (Pst) DC3000, but increased susceptibility to the necrotrophic pathogens Pectobacterium carotovorum and Botrytis cinerea in Arabidopsis [30]. Although identifications and characteristics of the WRKY genes family have been performed in Gossypium arboreum, Gossypium raimondii, and G. hirsutum [31, 32], only a few WRKY family genes resistant to V. dahliae have been identified in cotton, such as GbWRKY1 [33], GhWRKY70 [34, 35]. There is unclear about the molecular mechanisms of cotton resistance to V. dahliae invasion.

There are many studies related to biotic and abiotic stress of WRKY transcription factor family. Cotton is considered one of the most economically important crops worldwide. It’s of great significance to analyze the molecular mechanism of Verticillium wilt tolerance for cotton. However, the molecular mechanism of WRKY genes responding to Verticillium wilt tolerance has been still unclear in cotton. Here, we identified a new WRKY transcription factor, GhWRKY70, which can regulate GhAOS1 expression by directly binding to the w-box existed in GhAOS1 promoter. Furthermore, our study indicated that GhWRKY70 play a positive regulator role in response to invasion of V. dahliae, which was at least in part through increasing content of JA via promoting GhAOS1 expression.

Isolation and bioinformatics analysis of GhWRKY70

A significantly induced WRKY TF named GhWRKY70 was obtained from a transcriptome of G. hirsutum following V. dahliae infection. Bioinformatics analysis show that the length of GhWRKY70 was 2141 base pairs (bp). GhWRKY70 is comprised from a section of spliced DNA containing three exons and two introns. The sizes of the introns were 505 bp and 715 bp, respectively (Fig. 1A). To determine the cis-acting elements, promoter regions 2.0 kb of DNA sequences upstream from the codons of GhWRKY70, were identified and analyzed by plant CARE (Fig. 1B). Some basic elements, including TATA elements and CAAT-box, were found. In addition, Cis elements related to abiotic stress and hormone regulation were also identified in the promoter region. Examples include LTR (cis-acting element involved in low-temperature responsiveness), MBS (MYB binding site involved in drought-inducibility), CGTCA-motif and TGACG element (cis-acting element involved in the MeJA response). GhWRKY70 contains a 921 bp open reading frame (ORF) and encodes a predicted polypeptide of 306 amino acid residues with a calculated molecular mass of 34.3 kDa and an isoelectric point of 6.06. A phylogenetic tree was constructed based on the GhWRKY70 used a total of 72 WRKYs from Arabidopsis demonstrated that they could be classified into four major groups, and GhWRKY70 had the closest relationship with AtWRKY70 due to its high identity, belongs to the Group III (Fig. 1C). The gene was designated as GhWRKY70. Multiple sequence alignment showed that GhWRKY70 protein possesses a highly conserved WRKY domain comprised of 60 amino acids (133–192) (Fig. 1D).

Expression patterns of GhWRKY70 in response to abiotic and biotic stresses

According to cis elements of GhWRKY70 in the promoter region, the expression of GhWRKY70 under different abiotic and biotic stresses, including V. dahliae, MeJA, PEG and low temperature, was investigated by qPCR. The results showed that GhWRKY70 transcript level quickly accumulated at 2 h after infection with V. dahliae. (higher than tenfold induction), and decreased to the low value at the last time point until 24 h, then increased at 48 h (Fig. 2A). After MeJA treatment, the transcript abundance of GhWRKY70 first decreased then increased to the maximum value at 24 h (Fig. 2B). In the case of PEG treatment, the expression level of GhWRKY70 was slightly up-regulated (Fig. 2C). When subjected to cold treatment, the expression of GhWRKY70 eventually declined at 48 h, probably because the mRNA degraded gradually after long-term cold treatment of plants (Fig. 2D).

Subcellular localization of GhWRKY70

Subcellular localization of GhWRKY70 was predicted by examining GhWRKY70::GFP. The nuclear signal peptide fused to a RFP protein mKate was used as a positive control. The GhWRKY70::GFP vector and positive control with red fluorescent protein (RFP)-mKATE were co-transformed into Nicotiana tabacum leaves. The green fluorescence from GhWRKY70::GFP perfectly overlapped with red fluorescence from (RFP)-mKATE was exclusively detected under confocal microscope (Fig. 3A–D). However, green fluorescence was observed in the entire cell region when the only GFP plasmid and red fluorescent protein (RFP)-mKATE vector co-transformed into tobacco leaves. The results indicated that GhWRKY70 was localized in the nucleus. (Fig. 3E–H).

Transcriptional activation assay of GhWRKY70 in Yeast

The GAL4 yeast expression system was used to detect the transcriptional activation of GhWRKY70. Yeast strain AH109 was transformed with the construction pGBKT7-GhWRKY70, PGBKT7-GAL4 as positive controls and pGBKT7 as negative controls, respectively. The yeast cells transformed with pGBKT7-GhWRKY70 and PGBKT7-GAL4 grew well and turned blue on the SD-THA/X medium with X-α-gal. Meanwhile, the yeast cells transformed with pGBKT7 could only exist on the SD/-TH/X medium (Fig. 4). The results demonstrated that the GhWRKY70 functioned as a transcriptional activator.

Over-expression of GhWRKY70 improved Arabidopsis resistance to V. dahliae

As GhWRKY70 transcript level was induced by V. dahliae and MeJA in strong a manner, whereas transgenic Arabidopsis plants overexpressing GhWRKY70 were generated to characterize the role of GhWRKY70 in response to V. dahliae. Two transgenic lines with higher transcriptional levels of GhWRKY70 (named as L1 and L2) were selected for the resistance experiment test. On 9 days after germination, the roots of the transgenic lines were longer and stronger than that of the wild type (WT) (Fig. 5A). To assess the effect of damage caused by V. dahliae, the four-week-old WT and transgenic plants were inoculated with spore suspensions of V. dahliae, and the control was treated with water instead of V. dahliae. After withholding and culturing for 15 days, transgenic plants displayed better resistance than the WT plants (Fig. 5B). Disease index (DI), is a comprehensive index for measuring incidence rate and severity of plant disease. In our study, the DI was approximate 46.1% and 48.3% in two transgenic lines lower than 61.0% of wild type, respectively (Fig. 5C), suggesting that Verticillium wilt damage of transgenic GhWRKY70 lines was more milder than WT Arabidopsis. The height and fresh weights of WT inoculated plants were 46.6% and 41.8% lower than the mock control, respectively. The height and fresh weights from the two inoculated transgenic L1 and L2, by contrast, were 34.4%, 37.6% and 36.5%, 38.5% lower than the control, respectively (Fig. 5D, E). Similarly, chlorophyll content of transgenic lines was remarkable higher than that of WT (Fig. 5F). The lignin staining showed that lignin content of transgenic lines increased (Fig. 5G). Take together; our results showed that transgenic Arabidopsis had obviously increased V. dahliae resistance. Moreover, there is a JA-inducing element in the promoter sequence of GhWRKY70, and the expression level of GhWRKY70 was induced by MeJA. Therefore, JA levels of the WT and transgenic lines were quantitatively examined in this study. As shown in Fig. 5H, JA levels of the transgenic L1 and L2 increased by 26.8% and 16.2% compared with the WT, respectively. JA levels of the WT and transgenic lines all increased, while the increase of JA content in transgenic lines was significantly higher than those of WT plants under V. dahliae induction.

Silencing of GhWRKY70 in cotton confers sensitivity to V. dahliae

To further elucidate the role of GhWRKY70 in resistance to V. dahliae, the GhWRKY70 silenced plants were obtained by a virus-induced gene silencing (VIGS) method. When the TRV:GhCLA1 plants showed a leaf bleaching phenotype (Fig. 6A), the transcripts of GhWRKY70 was analyzed in the in the silenced plants and TRV:00 plants. The result showed that the expression of GhWRKY70 were reduced in the pTRV2-GhWRKY70 plants compared to TRV:00 plants (Fig. 6B). When subjected to V. dahliae treatment for 15 days, the proportion of DI values was investigated and showed the silenced plants were significantly higher than those of the TRV:00 plants (Fig. 6C). Meanwhile, lignin content and chlorophyll of silenced plants were significantly lower than those of TRV:00 plants (Fig. 6D and E). Moreover, JA levels of TRV:00 plants increased by 50.1% after inoculation with V. dahliae, while TRV:WRKY70 plants increased by only 29.6% (Fig. 6F).

Silencing GhWRKY70 affected antioxidant and defense enzymes of transformed plants

To assess the changes of antioxidant defense system created by over-expression and silence of GhWRKY70, the activities of antioxidant and defense enzymes in transgenic Arabidopsis lines (L1 and L2) and silenced cotton plants were measured and the results are shown in Fig. 7. The activities of antioxidant and defense enzymes in the two transgenic lines exhibited the same levels in comparison to the WT plants except for PPO. And the SOD and POD activities were also increased with 28.7%, 19.7% in L1 and 25.7%, 15.7% in L2 after 15 days inoculation in comparison to the WT plants, respectively. (Fig. 7A and B). While CAT activities in L1 and 2 were 15.4% and 17.1% lower than that in WT plants, respectively (Fig. 7C). Similarly, PAL and PPO in transgenic L1 and L2 plants were 28.9%, 19.7% and 22.9%, 30.6% higher than those in the WT plants, respectively (Fig. 7D and E). Meanwhile, DAB staining assay showed that the accumulation of H₂O₂ in both transgenic lines increased after 15 days inoculation (Fig. 7F). The activities of PPO in GhWRKY70 silenced plants is lower than that of the TRV:00 plants, while the activities of other antioxidant and defense enzymes showed no obvious differences between TRV:GhWRKY70 and TRV:00 plants. Fifteen days after inoculation, the SOD and POD activities in pTRV2-GhWRKY70 were 13.4% and 14.8% lower than those in control plants, respectively (Fig. 7G and H), whereas the CAT activity was 6.57% higher than that in TRV:00 plants (Fig. 7I). The PAL and PPO activities decreased 23.8% and 34.6% in pTRV2-GhWRKY70 compare with control plants (Fig. 7J and K). In addition, the accumulation of H₂O₂ in TRV:GhWRKY70 plants decreased more than that in TRV:00 plants after 15 days of inoculation (Fig. 7L).

Expression analysis of stress-responsive genes of transformed plants

Our above data showed that the JA content in overexpression lines and silenced cotton plants had significantly changed after V. dahliae treatment. Therefore, the genes related the JA signaling pathway in the transformed plants were analyzed by qRT-PCR assay. The expression of all genes in transgenic lines and silencing plants were similar to those in the control plants under normal conditions. However, the AtLOX1, AtAOS (for JA biosynthesis) in both transgenic lines had more abundant expression level after V. dahliae treatment (Fig. 8A and B). For JA signal response gene, the level of AtJAZ3 gene expression reduced, whereas the AtMYC2 expression increased (Fig. 8C and D). Interestingly, GhLOX1, GhAOS1 and GhMYC2 were significantly inhibited in TRV:GhWRKY70 plants after V. dahliae treatment (Fig. 8E, F and H), whereas the expression of GhJAZ3 increased significantly (Fig. 8G). The results perfectly matched with their JA level in transgenic lines and silenced plants (Fig. 5H, and Fig. 6F). Meanwhile, the V. dahliae induction caused dramatical up-regulation of GhLOX1 and GhAOS1 in the TRV:00 plants, but there were no significant changes in the TRV:GhWRKY70 plants. All results suggested that the GhWRKY70 functions in response to V. dahliae by enhancement of the expression of the JA biosynthesis genes, especially for GhLOX1 or GhAOS1.

GhWRKY70 directly interacts with the promoter of GhAOS1

Expression levels of GhAOS1 were strongly induced in GhWRKY70-overexpressing lines and decreased in GhWRKY70-silencing lines. We propose that GhAOS1 might be the potential target gene regulated by GhWRKY70. The results of GhAOS1 promoter sequence bioinformatics analysis showed that one potential W-box (TTGACT) element existed at the upstream region. To investigate the interaction between GhWRKY70 and GhAOS1 promoter, yeast one-hybrid (Y1H) assay was conducted. The result showed that the yeast cells of positive controls and those co-transformed pGADT7-GhWRKY70 with bait 150bp fragment containing the W-box prey or W-box mutant (negative controls) grew normally in SD/-Ura/-Leu medium. However, when 200 ng/mL AbA was added in SD/-Ura/-Leu medium, growth of the positive control and bait-prey transformants survived, while the negative control was completely inhibited (Fig. 9A). Transient expression assay showed that the LUC/REN ratio in the tobacco leaf transformed with the P2 containing reporter and effector was significantly higher than that transformed with mP2 and effector (Fig. 9B).

WRKY TFs are one of the largest gene families, forming a vital component of plant signal transduction network for modulating multiple stress response processes in plants [28]. In recent years, many WRKY proteins have been identified from a variety of plants such as rice [36], soybean [37], cotton [31], maize [38], and wheat [39]. Relative to the number of WRKYs identified of cotton, knowledge about their function and molecular mechanism is still unclear, although part of the genome has been comprehensively analyzed according to their functions in the model plants [40]. Therefore, characterization and function of WRKYs in cotton will obtain novel insights into the regulatory mechanism mediated by WRKYs under stress conditions. Here, except for the identification of a WRKY TF GhWRKY70 from transcriptome and verification of its function in resistance to Verticillium Wilt, we further demonstrated that GhWRKY70 is a positive regulator of GhAOS1 expression, a key enzyme in the JA biosynthetic pathway. Thus, our study reveals a new mechanism of GhWRKY70 and links the function of WRKY to JA biosynthesis.

Each WRKY TF possesses two or one domains composed of 60 amino acids with a highly conserved WRKYGQK motif, and there is Cys2His2 or Cys2HisCys zinc-finger motif behind the WRKYGQK motif [28]. GhWRKY70 was classified into the Group III category according to its conserved WRKYGQK domain and Zinc finger structure (Fig. 1D). It has been previously demonstrated that most Group III TFs of plants were involved in different plant defense signaling pathways [29, 41–43]. In this study, the results from gene relationship analysis, subcellular localization and transcriptional activation analysis (Figs. 1, 3 and 4) were in accordance with its putative role as a transcription factor. Overexpression of GhWRKY70 conferred enhanced Arabidopsis resistance to V. dahliae, suggesting that GhWRKY70 may play positive regulator in response to V. dahliae. Taken together, previous studies and our findings indicated that WRKY group III genes play significant and complex role in defense against biotic stresses.

Since expression of GhWRKY70 was obvious induced by V. dahliae and MeJA treatment, we attempted to confirm its role in response to V. dahliae by obtaining transgenic Arabidopsis with GhWRKY70. Here, the transgenic lines with over-expression of GhWRKY70 exhibited better root, height, fresh weight, cand hlorophyll and JA content than WT under treatment with V. dahliae, indicating that over-expression of GhWRKY70 significantly enhanced V. dahliae resistance mediate JA signaling pathway. Consistent with previous studies, GhWRKY70A05a acted as cotton's resistance against V. dahliae via inhibiting JA signaling pathway, meanwhile promoting SA signaling pathway [34]. GhWRKY70D13 negatively regulates cotton's response to V.dahliae infection by down-regulating the ET and JA signaling pathways, a mechanism different from that of GhWRKY70A05a [35]. However, our study showed that down-regulation of GhWRKY70 in cotton significantly reduced accumulation of JA, suggesting that each WRKY plays a different role and might hold great potential for stress tolerance.

Previous study showed that H₂O₂ accumulation is closely related to biotic or abiotic stress. Plant cells depend greatly on antioxidant defense system for maintaining H₂O₂ relative balance, such as SOD, CAT and POD [44]. Research has illustrated that H₂O₂ can diffuse across membranes and act as a signal during cell wall synthesis and fortification associated with disease resistance [15, 45]. The present study indicated that the content of H₂O₂ in TRV:GhWRKY70 cotton exhibited a relatively lower than that in TRV:00 under the V. dahliae infection. Further work showed that SOD activity decreased in TRV:00 plants, TRV:GhWRKY70 plants, WT and over-expression Arabidopsis plants after V. dahliae infection. The results indicated that V. dahliae can destroy the homeostasis of active oxygen metabolism system in plants. However, the SOD activity of WT Arabidopsis and TRV: GhWRKY70 decreased rapidly, while the SOD activity of transgenic Arabidopsis and TRV:00 plants decreased relatively gently, indicating that the latter plants have resistance to V. dahliae to a certain extent. The CAT activity of overexpression plants decreased significantly than that of WT, while the CAT activity of TRV:GhWRKY70 slightly increased more than that of TRV:00 after treatment with V. dahliae. This may be one of the reasons why the content of H₂O₂ in transgenic Arabidopsis is higher than that in WT, while the opposite was true for gene silenced plants. Studies have shown that CAT expression also relate to JA [46]. Over-expressing CATALASE2 increase plant JA content and resistance to infection of Botrytis cinerea B05.10 [47]. In addition, MYC2 could directly bind to the promoter of CAT2 and inhibit its expression [48], which may be one of the reasons for increased H₂O₂ in over-expressed plants. Therefore, we speculated that H₂O₂ may be one of the key downstream factors of JA in the immune reaction against V. dahliae.

PAL and PPO have been confirmed to be involved in plant resistance to fungal infection [49–51] and can be used as innate immunity markers in plant [68]. PPO can not only promote the synthesis of quinine by catalyzing the oxidation of phenolic compounds, but also produce pre-benzoic acid, which is the precursor of lignin synthesis [50, 51]. The expression of PPO gene in resistant varieties of olives was significantly higher than that in sensitive varieties after V. dahliae inoculation, and the contents of phenolic compounds and lignin were also higher than that in susceptible varieties [53]. In our study, the increase of defensive enzyme activities were significantly induced and inhibited in GhWRKY70 overexpressed and silenced plants, respectively following V. dahliae inoculation (Fig. 7), which further confirmed that GhWRKY70 might play a positive regulator in resistance to V. dahliae.

To further explore GhWRKY70 function in defense against V. dahliae, relative expression levels of JA biosynthesis genes LOX and AOS, JA signal response gene JAZ3 and MYC2 were monitored before and after V. dahliae treatment in overexpressed and gene silenced plants. Contrary to gene silenced plants, the expression of LOX and AOS enhanced in transgenic Arabidopsis after V. dahliae infection. The results were consistent with the changes of JA content in the over expressed and gene silenced plants. We speculated that GhWRKY70 may be related to JA synthesis. In this study, a W-Box element exists in GhAOS1 promoter, and interaction between GhWRKY70 and the GhAOS1 promoter was further verified by transient expression assays. These data suggested that GhAOS1 is a target gene of GhWRKY70. JAZ3, as a negative regulator and early response gene of JA signal pathway, is ubiquitinated and degraded when JA content is high [6, 54]. MYC2, as the target gene of JAZs protein, is released and activates the expression of JA signaling pathway downstream related genes accompanying JAZs protein degradation. After infection with V. dahliae, a high level of JA will be rapidly formed in the overexpressed Arabidopsis, and JAZs protein was degraded and MYC2 was released, which maintain the continuous opening of JA signal pathway and enhances the disease resistance of the plant. However, the expression of JAZ3 increased and MYC2 declined in silenced plants, and then the JA signaling pathway was closed, which reduced the resistance to V. dahliae.

Our work suggested that GhWRKY70 plays the role of a positive regulator in defenses against V. dahliae, which may be partly attributed to its role in influencing JA biosynthesis via regulating AOS1 expression. Invasion of V. dahliae promote GhWRKY70 expression, which further regulates GhAOS1 expression by interaction between GhWRKY70 and W-box of GhAOS1 promoter. Up-regulation of the GhAOS1 enhances JA content and further activates the response gene of the JA signaling pathway, accompanied by H₂O₂ generation and scavenging, improving its ability to resist V. dahliae. These results indicate that GhWRKY70 in involved in a complex signal regulatory network in response to V. dahliae. This work provides a novel insight into the molecular mechanisms of JA synthesis and H₂O₂ relative homeostasis in resistance to infection with V. dahliae. In the future, more work is needed to detect other components in connection with GhWRKY70 in order to obtain a clearer picture of the molecular mechanisms about GhWRKY70 function in resistance to V. dahliae.

Plant materials and stress treatments

G. hirsutum cv. Zhongzhimian 12 (V. dahliae-resistant cultivar) and Arabidopsis thaliana (ecotype Columbia-0) were obtained from State Key Laboratory of North China Crop Improvement and Regulation, Hebei Agricultural University, Baoding, China. Plants were cultured in a greenhouse with a 16:8 light:dark cycle at 25°C. All plants were watered weekly with Hoagland's nutrient solution.

Two-leaf stage cotton seedlings from greenhouse were treated with either 100 µmol L^− 1 MeJA or 15% (m/v) polyethylene glycol (PEG–6000). Moreover, for cold stress, the seedlings were moved to a growth chamber set at 4°C. Cotton leaves were harvested at 0, 2, 6, 12, 24, and 48 h after treatment. For each treatment, at least three randomly collected seedlings at each designated time point and flash frozen in liquid nitrogen and stored at − 80°C until use [34].

A highly aggressive V. dahliae strain linxi2-1, from state key laboratory of north china crop improvement and regulation, was cultured on potato dextrose agar (PDA) medium at 25°C for 7–10 days. Colonies were then moved to Czapek medium by shake culture (150 r min^− 1) for 10 days at 25°C. The culture solution was diluted to 10⁷ conidia mL^− 1 spore suspensions and then watered (10 mL per treatment) into the pots with soil containing cotton seedlings and Arabidopsis plants [55]. The roots of cotton and Arabidopsis were cut at 0, 6, 12, 24, and 48 h after infection with V. dahliae, flash frozen in liquid nitrogen, and stored at − 80°C for transcription analysis.

Isolation and bioinformatics analysis of GhWRKY70

In previous work, WRKY TF (CotAD_14615.1) with high sequence homology to WRKY70 was found to be up-regulated in the transcriptome of cotton treated with V. dahliae. A pair of gene-specific primers (GSP1, Table S1) was designed according to the sequence for amplification GhWRKY70 in Zhongzhimian 12. The total RNA was isolated and cDNA template was prepared. PCR products were purified, linked into the pMDT-19 vector and sequenced by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Multiple sequence alignments were performed using Clustal W [56], and a phylogenetic tree was constructed with MEGA 7.0 using the neighbor-joining method [57]. Approximately 2.0 kb of DNA sequence upstream from the codons of GhWRKY70 was downloaded from the cotton genome project (https://cottonfgd.org/analyze/). PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to analyze the promoter region of GhWRKY70.

RNA isolation and expression pattern analysis

Total RNA was extracted from Arabidopsis and cotton roots or leaves using a RN09-EASYspin RNA Plant Mini Kit (Aidlab Biotechnologies Co. Ltd., Beijing, China), and digested with DNase I (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) to eliminate DNA contamination. Approximately 2µg of total RNA was reversely transcribed into first-strand cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology (Dalian) Co., Ltd.). The control GhUBQ7 or AtACTIN2, genes involved in the JA signaling pathway, were analyzed by qRT-PCR with SYBR Green Mix (Takara) and the Roche LightCycler 2.0 (Roche, Germany). The primer pairs used for RT-qPCR analysis were designed with Primer5 (Table S1). Expression data obtained from three replicate experiments were analyzed by the 2 ^−△△CT method [58] and presented as means ± SD.

Subcellular localization of GhWRKY70

The complete ORF sequence of GhWRKY70 without a stop codon was amplified by RT-PCR using specific primers (GSP2, Table S1), and then linked into the pSuper1300-GFP vector using HindⅢand SalⅠto obtain a fusion protein GhWRKY70::GFP. The nuclear signal peptide (MDPKKKRKV) was fused to the far-RFP mKate of pBWA(V)HS vector to get pBWA(V)HS-nls-mKate-RFP vector [59]. Both pSuper1300-GhWRKY70-GFP and pBWA(V)HS-nls-mKate-RFP were co-transformed into tobacco leaves. Empty vector pSuper1300-GFP was used as a control. After transformation for 48–72h, GFP emission signal was collected at an excitation wavelength of 488 nm under a confocal microscope (Leica), and the RFP signal was observed between 587 and 610 nm.

Analysis of transcriptional activation in Yeast

The full-length coding sequence of GhWRKY70 was amplified using primers (GSP3, Table S1) with EcoRI and SalI restriction sites, fused to the pGBKT7 vector to construct pGBKT7-GhWRKY70. Empty vector pGBKT7 was taken as a control. The construction and empty vector pGBKT7 were introduced into the yeast strain AH109 separately by the lithium acetate method. Yeast cells were cultured on selective medium without tryptophan (SD-T). The positive clones identified by PCR were cultured on the SD-T, SD without tryptophan, histidine, and adenine (SD-T/H/A) and SD-T/H/A with X-D-Galactosidase (X-gal) medium at 30℃. The results were observed after 3 d to detect the transcriptional activation.

Generation of GhWRKY70-Overexpressing Arabidopsis plants

The ORF of GhWRKY70 was cloned into expression vector pCamE carrying the hygromycin B resistance gene after PCR amplification using a primer pair (GSP4, Table S1) with BamHI and KpnI restriction sites. Binary vector pCamE-GhWRKY70 was introduced into Agrobacterium tumefaciens strain GV3101 and then transformed WT Arabidopsis plants using the floral-dip method [60].

VIGS and pathogen inoculation

Tobacco rattle virus (TRV)-based vectors and A. tumefaciens were used for VIGS [61]. pTRV2:GhCLAI (Cloroplastos Alterados 1) were selected for positive control [62]. A fragment of GhWRKY70 cDNA was digested with BamHI and KpnI (GSP4, Table S1) and then cloned into the pTRV2 plasmid to construct the VIGS vector of pTRV2:GhWRKY70. The constructions were introduced into A. tumefaciens GV3101 as described previously [3, 63]. pTRV1 and pTRV2:GhWRKY70 Agrobacterium (OD600 = 1.0) mixture (1:1 ratio, v/v) was agro-infiltrated into the cotyledons of 7-day-old cotton seedlings. These treated seedlings were cultured for 12 h in darkness and then grown in a controlled environment greenhouse. Leaves of the TRV:GhWRKY70 and the TRV:00 plants were picked to RNA isolation after the TRV:GbCLA1 plants were observed to have a leaf bleaching phenotype. Some of TRV:GhWRKY70 and TRV:00 plants were infected by V. dahliae to observe phenotypic traits and to score disease index (DI). The plant DI was calculated according to the method described by Xu et al. [64].

Analysis of antioxidant enzyme and defense enzyme activities

For SOD, POD and CAT activities detection, fresh tissue (0.4 g) was homogenized with 0.1 M phosphate buffer saline (5 mL, pH 7.5). Then the homogenate was centrifuged at 12000×g at 4 ◦C for 15 min, and the supernatant was collected for enzymatic detection [65]. SOD and POD activities were analyzed according to Giannopolitis and Ries [66] and Doerge et al. [67], respectively. CAT activity was measured by monitoring the consumption of hydrogen peroxide (H₂O₂) at 240 nm [68]. The enzymes’ activities were expressed as Ug^− 1 fresh weight (FW). To determine Phenylalanine ammonialyase (PAL) and polyphenol oxidase (PPO) enzymes activities, fresh tissue (0.4 g) was grinded using 4 mL 0.2 mM boric acid buffer (pH 8.8) containing 10% (w/v) PVP, 5 mM β-mercaptoethanol, 1mM EDTA and 0.1 M of sodium phosphate buffer (pH 7.8) containing 1% (w/v) PVP. The homogenates were centrifuged at 12000×g for 15 min at 4 ◦C, and the supernatants were used for enzyme assays [69].

DAB staining and lignin histochemical staining

For H₂O₂ determination, leaves were soaked in 1 mg/mL pH 3.8 DAB-HCl (Sigma-Aldrich, USA) for 8 h in the dark, and then cleared by boiling in 95% ethanol for 10 min. The reddish color of the leaves served as visual marker of H₂O₂ production. After V. dahliae inoculation, the stem base of plants was taken for transverse section. The free-hand sections (approximately 0.5 mm thick) were soaked in lignin acidification solution for 5 min. Then, the same amount of phloroglucinol staining solution was added to the transverse section and immersed for 10 min. The lignin staining results were observed under the optical microscope.

Isolation of GhAOS1 promoter and Y1H assay

The promoter sequence of GhAOS1 was acquired by RT-PCR using special primer (GSP5, Table S1). Potential cis-acting elements related to stress resistance were predicted on PLACE. Based on the characteristic of the promoter sequence, the fragment (683bp to 854bp) containing potential cis-acting elements W-box with HindIII and XhoI restriction sites was chemically synthesized and integrated into the pAbAi to generate pAbAi-GhAOS1 reporter vector. Meanwhile, GhWRKY70 full-length ORF of stop codon removed was amplified using GSP6 (Table S1) and ligated into the EcoⅠand XholⅠsites of pGADT7 to obtain the pGADT7-GhWRKY70 effector vector. Following the instruction of Matchmaker Gold Y1H Library Screening System (Clontech, Dalian, China), the interaction of GhWRKY70 and the GhAOS1 promoter was examined by yeast one-hybrid. The pAbAi-GhAOS1 and the pGADT7-GhWRKY70 were co-transformed into the yeast cells. The transformed yeast cells were cultured on SD/-Ura/-Leu medium either with or without 200 ng/mL Aureobasidin A (AbA) for 3 days.

Transient expression assay

The coding region of GhWRKY70 was amplified using primers (GSP7, Table S1) containing the BamHⅠand EcoRⅠrestriction sites and inserted into pGreenII 62-SK digested by the same enzymes. A 150-bp promoter fragment of GhAOS1 with W-box element (P2) and its mutated sequence (mP2) were chemical synthesized containing either Pst I or BamH I restriction sites and ligated into the reporter vector, pGreen II 0800-LUC [70]. The reporter and effector constructs were transformed into A. tumefaciens GV3101 cells. Assays for transient expression in tobacco leaves were performed as described previously [71]. The transformed tobacco were placed in dark at 25°C for 18 h, and then analyzed the activity of Renilla Luciferase (REN) and firefly luciferase (LUC) by the Dual- Luciferase-Reporter Assay System (Promega, Madison, WI, USA) with an Infinite200 Pro reader (Tecan, M€annedorf, Switzerland). The promoter activity was described as the ratio of LUC/REN.

Statistical analysis

All data were expressed as the means ± standard deviation (SD) and compared by two-group t-test comparisons at P < 0.05 or P < 0.01 using Origin software 8.5.

Acknowledgements

None.

Authors’contributions

Xingfen Wang conceived and designed the experiments. Shuling Zhang, Lijun Dong, Lizhu Wu and Xue Zhang performed most of experiments and analyzed the data. Xiaohong Fu and Lin Zhao assisted in experiments. Jianfeng Liu wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the State Key Laboratory of North China Crop Improvement and Regulation, the Natural Science Foundation of Hebei Province, China (No.C2020201001 and C2021201043).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files; Sequence data from this article can be found in the TAIR/GenBank data libraries under the following accession numbers: AtWRKY30, At5g24110; AtWRKY38, AT5G22570; AtWRKY41, AT4G11070; AtWRKY46, AT2G46400; AtWRKY53, AT4G23810; AtWRKY54, AT2G40750; AtWRKY55, AT2G40740; AtWRKY62, AT5G01900; AtWRKY63, AT1G66600; AtWRKY64, AT1G66560; AtWRKY66, AT1G80590; AtWRKY67, AT1G66550; AtWRKY70, AT3G56400; AtLOX1, AT1G55020; AtAOS, AT5G42650; AtJAZ3, AT3G17860; AtMYC2, AT1G32640; AtACTIN2, AT3G18780; GhLOX1, XM_041108768.1; GhAOS1, XM_016842008.2; GhJAZ3, XM_041107142; GhMYC2, 1.XM_016865820.2; GhUBQ7, XM_016855110.2.

Ethics approval and consent to participate

The upland cotton cultivar (Zhongzhimian 12) and Arabidopsis thaliana (ecotype Columbia-0) used is from our laboratory and we have permission to use it. Field studies were conducted in accordance with local legislation.

Consent to publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹School of Life Sciences, Institute of Life Science and Green Development, Hebei University, No.180, Wusi East Road, 071000 Baoding, China. ²State Key Laboratory of North China Crop Improvement and Regulation, Hebei Agricultural University, No. 289, Lingyusi Street, 071001 Baoding, China

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Transcription factor GhWRKY70 from Gossypium hirsutum enhances resistance to Verticillium wilt via the jasmonic acid pathway

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Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Isolation and bioinformatics analysis of GhWRKY70

Subcellular localization of GhWRKY70

Transcriptional activation assay of GhWRKY70 in Yeast

Silencing GhWRKY70 affected antioxidant and defense enzymes of transformed plants

Expression analysis of stress-responsive genes of transformed plants

GhWRKY70 directly interacts with the promoter of GhAOS1

Discussion

Conclusion

Methods

Plant materials and stress treatments

Isolation and bioinformatics analysis of GhWRKY70

RNA isolation and expression pattern analysis

Subcellular localization of GhWRKY70

Analysis of transcriptional activation in Yeast

VIGS and pathogen inoculation

Analysis of antioxidant enzyme and defense enzyme activities

DAB staining and lignin histochemical staining

Isolation of GhAOS1 promoter and Y1H assay

Transient expression assay

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

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