CaWRKY30 Positively Regulates Pepper Immunity By Targeting CaWRKY40 Against Ralstonia Solanacearum Inoculation Through Modulating Defense-Related Genes Part 1

Abstract:

The WRKY transcription factors (TFs) network is composed of the WRKY TFs’ subset, which performs a critical role in the immunity regulation of plants. However, the functions of WRKY TFs’ network remain unclear, particularly in non-model plants such as pepper (Capsicum annum L.). This study functionally characterized CaWRKY30—a member of group III Pepper WRKY protein—for the immunity of pepper against Ralstonia solanacearum infection. The CaWRKY30 was detected in the nucleus, and its transcriptional expression levels were significantly upregulated by R. solanacearum inoculation (RSI), foliar application ethylene (ET), abscisic acid (ABA), and salicylic acid (SA). Virus-induced gene silencing (VIGS) of CaWRKY30 amplified the pepper’s vulnerability to RSI. 

Additionally, the silencing of CaWRKY30 by VIGS compromised HR-like cell death triggered by RSI and downregulated defense-associated marker genes, like CaPR1, CaNPR1, CaDEF1, CaABR1, CaHIR1, and CaWRKY40. Conversely, transient over-expression of CaWRKY30 in pepper leaves instigated HR-like cell death and upregulated defense-related marker genes. Furthermore, transient over-expression of CaWRKY30 upregulated transcriptional levels of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. 

On the other hand, transient over-expression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 upregulated transcriptional expression levels of CaWRKY30. The results recommend that newly characterized CaWRKY30 positively regulates pepper’s immunity against Ralstonia attack, which is governed by synergistically mediated signaling by phytohormones like ET, ABA, and SA, and transcriptionally assimilating into WRKY TFs networks, consisting of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. Collectively, our data will facilitate to explicate of the underlying mechanism of crosstalk between pepper’s immunity and response to RSI.

Transcription factors play a key role in the immune system, controlling the regulation of gene expression and the formation of specific cell types. The following is the relationship between transcription factors and immunity:

1. Transcription factor - NF-κB: NF-κB is a key transcription factor involved in the regulation of adaptive immune responses of T cells and B cells. NF-κB activation can promote inflammatory response, immune response, and cell apoptosis.

2. Transcription factors—STATs: STATs are transcription factors that mediate carrier signaling and cellular responses. STATs can be activated, play a key role in activated T cells and B cells, and participate in processes such as immune cell proliferation, differentiation, and activation.

3. Transcription factor - AP-1: AP-1 is a family of transcription factors involved in the regulation of various cellular functions and immune responses. AP-1 activation can promote processes such as cell proliferation, differentiation, and transformation, as well as maintain antiviral and immune responses.

4. Transcription factors—CEBPs: CEBPs are a large family of transcription factors, including CEBPα, β, γ, etc. in various tissue cells. In the immune system, CEBP is involved in the activation of macrophages and the release of cytokines, as well as the differentiation and activation of B cells and T cells.

Taken together, transcription factors play an important role in immunity, participating in the regulation and maintenance of immune system activity in different cell types and functions. Therefore, we also need to pay attention to the improvement of immunity in our daily life. Cistanche can enhance immunity, and the polysaccharides in meat can regulate the immune response of the human immune system, improve the stress ability of immune cells, and enhance the sterilization of immune cells effect.

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Keywords:

chili pepper (Capsicum annuum); Ralstonia solanacearum; CaWRKY30; WRKY TFs.

1. Introduction

Plants frequently get exposed to various biotic and abiotic stresses during their life span due to their immobile nature [1,2]. Plants have developed several defense mechanisms in response to repeated selection pressure from major ecological and environmental constraints. Different transcriptional factors (TFs) interconnect to develop sophisticated transcriptional networks for regulating these classic defense mechanisms at a transcriptional level [3,4]. Defense reactions to various stresses are properly synchronized and controlled as they are critical for plants in the form of energy expenditure and development [5]. The defense mechanisms might vary in different plant species because of varied environmental circumstances affecting their acclimation [6–8]. 

Hence, the defense system employed by model plants cannot be fully implied to non-model plants. Therefore, the defense systems of plants have been a prime focus during past decades; however, these studies were mostly focused on model plants, i.e., Arabidopsis thaliana and rice (Oryza sativa L.). Nevertheless, the functional synchronization of these transcriptional complexes to regulate plants’ response to various stresses has been poorly investigated, mainly in non-model plants.

The WRKY proteins comprise the largest TFs family in plants. The WRKY TFs contain one or two WRKY domains and commonly WRKYGQK sequence at the N-terminus followed by C2HC or C2H2 zinc finger motif [4,9,10]. The WRKY proteins are phylogenetically divided into three main groups (i.e., groups I–III) based on several WRKY domains and the structure of the zinc-finger motif. 

Group II of WRKY TFs is again subdivided into five sub-groups (IIa, IIb, IIc, IId, and IIe) [11–13]. The WRKY members mainly bind with W-boxes [TTGAC(C/T)] found in promoter regions of various target genes by inserting an exclusive wedge vertically into DNA’s major groove [14] by WRKY GQK motif on the second b strand, which stimulates transcriptional modulation of the expression of target genes [14–16]. The WRKYs have been involved in several biological systems in plants, including seed dormancy, seed germination, senescence, and seed development. Similarly, these are also involved in response to biotic and abiotic stresses and switching transcription of their target genes [17–20].

It is reported that a group of WRKY TFs or one WRKY TF transcriptionally induced by only one stress modulates several stresses. The W-boxes are enriched inside promoter regions [21–24]. These studies depict the presence of WRKY networks implicated in response to a particular stress, or a combination of two or more. The role of WRKY proteins and underlying mechanisms in response to biotic and abiotic stresses have been frequently investigated in the past decade; however, the emphasis of these studies was primarily on one gene in response to one stress in model plants, e.g., A. thaliana and O. sativa. Recently, an important functional variance among close homologs of WRKY TFs from various plant species has been suggested [24,25]. The functions of WRKYs and WRKY web in non-model plants such as Capsicum annuum L., in response to single or combined stresses, have been poorly investigated until now.

Pepper is globally known for its economic importance. It is sown in uplands during temperate seasons [26,27]. Pepper is exposed to various soil-borne diseases such as pepper blight and bacterial wilt caused by bacterial pathogens, i.e., Phytophthora capsicum and Ralstonia solanacearum, respectively [28,29]. The coincidence of these pathogens imparts devastating impacts on pepper production under high temperatures and high humidity (HTHH). The HTHH impairs pepper’s immunity mediated by the R protein and speeds up pathogen growth and development. Furthermore, pathogen attack and HTHH simultaneously exert natural selection pressure on pepper, which has affected pepper’s evolution in the past [30–32]. Conversely, the simultaneous occurrence of pathogen infection and HTHH might not affect the evolution of model plants, like A. thaliana and O. sativa. Hence, pepper seems more appropriate for research regarding immunity against biotic or abiotic stresses, i.e., RSI or HTHH.

Ralstonia solanacearum causes bacterial wilt in numerous crops, i.e., pepper, tomato, potato, etc. It is a soilborne gram-negative β-proteobacterium [33]. It forms rapid colonies in the xylem tissues, which leads to bacterial wilt disease in the infested plants [34].

The responses of plant species to biotic and abiotic stress are known to be regulated by phytohormones [35]. Phytohormones are tiny molecules involved in plant growth regulation, plant reproduction, and survival. Salicylic acid (SA) and jasmonic acid (JA) are critical for the immune responses of plant species. These two are regarded as critical foundations of the immune responses of plants against pathogens. Biosynthesis and signaling of SA are vital for defense against biotrophic pathogens, while JA provides defense against necrotrophic pathogens [36,37]. Ethylene (ET) assists in defense responses against several plant–pathogen interactions [37].

Various indigenous pepper cultivars of subtropical areas demonstrate accelerated disease resistance, even under HTHH [38]. Heat-related cis-element HSE generally coexists with phytohormones, including SA, JA, ET, ABA, or pathogen-related cis-elements in promoter regions of most MAPKs and CDPKs involved in plant immunity [39–41]. It recommends the presence of crosstalk between immunity and HTHH. Pepper genome is 27 and 7.5 times bigger as compared to A. thaliana and O. sativa, respectively. However, it has only 73 WRKY genes, whereas smaller genomes of A. thaliana and O. sativa have 72 and 122 WRKY genes, respectively [9,42,43]. We previously detected that CaWRKY6 [44], CaWRKY22 [9], CaWRKY40 [45], and CaWRKY58 [46] have been involved in pepper’s response against RSI. 

Among these, CaWRKY6, -22, -27, and -40 act as positive regulators of plant immunity, whereas CaWRKY58 is a negative regulator. These genes possess a subset of HSE elements and W-boxes in their promoter region and the promoters of other WRKY TFs, inferring the role of WRKY networks in the regulation of pepper’s response against RSI. Additionally, CaWRKY40 was directly regulated by CaWRKY6 [44] and CabZIP63 [16], while CaWRKY40 was indirectly regulated by CaCDPK15 [47]. Conversely, most of the pepper’s WRKY TFs have not been characterized yet for their response to pathogen attacks.

In the current study, a full-length cDNA for the WRKY TFs family of pepper was isolated and named CaWRKY30. We characterized it expressional and functionally and found that CaWRKY30 was induced by RSI and foliar application of SA, JA, ET, and ABA. Transient over-expression of CaWRKY30 in pepper leaves triggered HR-like cell death and H2O2 production. Silencing of CaWRKY30 in pepper compromised the immunity of pepper to RSI. These results suggest that CaWRKY30 positively regulates pepper’s immunity to RSI.

2. Results

2.1. Cloning and Sequence Analysis of CaWRKY30

A new WRKY gene CaWRKY30 (CA01g34480) was identified by genome-wide analysis. The CaWRKY30 gene was chosen for functional characterization since the existence of immunity-related cis-elements such as TGA (TGACG motif-binding factor), TGACG-motif, TATC-box, TATAbox, and W-box in the promoter region designate its possible role in pepper’s immunity (Figure S1). A cDNA fragment of CaWRKY30 (CA01g34480) of 924 bp open-reading-frame (ORF) was cloned by using gene-specific primers (Table S1). The length of the deduced amino acid sequence of CaWRKY30 was 307 amino acid residues, possessing conserved WRKY domain, and it was categorized into group III (Figure 1). The predicted protein has a size and theoretical pI of 34.55 kDa and 7.32, respectively. The CaWRKY30 shares 88, 60, 68, and 57% amino acid identity with NsWRKY53, CcWRKY53, NaWRKY53, and StWRKY53, respectively (Figure S2).

2.2. Transcriptional Expression of CaWRKY30 under RSI and Treatment with Various Phytohormones

The presence of immunity-associated cis-elements in the promoter region of CaWRKY30 indicated its potential role in pepper’s immunity RSI. To check the notion, qRT-PCR analysis was carried out to assess the transcriptional levels of CaWRY30 upon RSI. The transcriptional levels of CaWRKY30 were increased in Ralstonia-treated leaves compared to mock-treated leaves (Figure 2A). The enhanced CaWRKY30 transcriptional expression levels were consistent between 0 h to 48 h post-treatment (HPT). The highest transcriptional expression levels were recorded at 48 hpt (Figure 2A).

Phytohormones, including ET, ABA, SA, and JA-mediated signaling pathways, are key regulators of plant reactions to biotic and abiotic stresses. The regulation of CaWRKY30 by phytohormones-mediated signaling pathways was studied by foliar application of ET, ABA, SA, and JA using qRT-PCR analysis. The results indicated that relative transcriptional expression levels of CaWRKY30 were enhanced from 0 h to 48 hpt with 100 µM ET as compared to the mock. These transcriptional levels were highest at 48 hpt with ET (Figure 2B). Foliar application of 100 µM ABA significantly increased transcriptional levels of CaWRKY30 as compared to the mock. Transcriptional levels reached the highest level at 12 hpt with ABA (Figure 2C). Results indicated that transcriptional accumulation of CaWRKY30 was significantly increased after the application of 1 mM SA, as compared to the mock. These enhanced transcriptional expression levels were highest at 24 hpt (Figure 2D).

Figure 2. The qRT-PCR assay of CaWRKY30 relative transcriptional expression levels in pepper leaves subjected to Ralstonia solanacearum inoculation (RSI), and foliar application of various phytohormones. The qRT-PCR analysis was used to check the transcriptional levels of CaWRKY30 in pepper leaves subjected to various treatments including RSI (A), application of 100 µm ET (B), application of 100 µm ABA (C), and application of 1 mM SA (D) at different time intervals. The transcriptional abundance in RSI-treated leaves was compared with MgCl2 -treated control leaves (mock), whose relative expression level was set to “1”. (B–D) The transcriptional expression levels in phyto-hormone-treated leaves were compared with ddH2O-treated leaves (mock), whose expression level was set to “1”. The height of the bar indicates means and error bars indicate the standard error of means. Different letters above the bars show a significant difference between the means based on Fisher’s protected LSD test.

2.3. CaWRKY30 Was Found to Be Localized in the Nucleus

Sequence analysis by using WoLFPSORT depicted that the predicted CaWRKY30 protein sequence possesses a putative nuclear localization signal (Figure 1), showing its potential targeting in the nucleus. To validate this perception, an aCaWRKY30 GFP fusion construct was generated that was driven by a constitutive promoter of CaMV35S. Afterward, this constituted vector was transferred into Agrobacterium strainGV3101. We transiently over-expressed the CaWRKY30 GFP construct in leaves of Nicofiamabenthamiana by A. tumefacient injection, and GFP signals were examined by confocal fluores.science microscope. Results depicted that GFP signals of CaWRKY30-GFP were present in the nuclei, while GFP of control was detected in various subcellular portions, like cytoplasm and nuclei (Figure 3).

Figure 3. Subcellular localization of CaWRKY30. The CaWRKY30 has fully located in the nucleus of nicotiana benthaminna leaves. The green color demonstrates GFP Blue color demonstrates DAPI staining of the nucleus. The cyan color demonstrates the fusion of green CFP and blue DAPI-stained nuclei. CPP signaGreen) for the control, N. benthamiana leaves were detected all over the cell. images were captured by confocal microscopy at 48 h post-inoculation. Bars = 25 um.

2.4. Silencing of CaWRKY30 by VIGS Curtailed Pepper’s Immunity to RSI and Downregulated Immunity Associated Marker Genes.

The CaWRKY30 was silenced by virus-induced gene silencing (VIGS) to study its role in pepper immunity. A total of 50 CaWRKY30-unsilenced (TRV:00) and 50 CaWRKY30- silenced (TRV: CaWRKY30) plants were acquired. Six CaWRKY30 silenced plants were randomly selected to check their efficiency of gene silencing by root inoculation with cells of compatible virulent R. solanacearum strain. Our findings indicated that transcriptional levels of CaWRKY30 were decreased by ~30% in Ralstonia-treated CaWRKY30 silenced plants as compared to unsilenced plants, validating the silencing of CaWRKY30 (Figure 4A). 

The CaWRKY30 silenced plants showed significantly higher susceptibility to RSI as compared to unsilenced plants. Pepper’s susceptibility to pathogen was increased by showing a rise in Ralstonia population growth, marked by higher cfu values in CaWRKY30 silenced pepper plants as compared with unsilenced plants at 3 d and 5 days post inoculation (dpi) (Figure 4B). Histochemical staining assay was done to detect the H2O2 production and cell necrosis in Ralstonia inoculated CaWRKY30 silenced (TRV: CaWRKY30), and unsilenced (TRV:00) pepper leaves. A dark DAB staining (a sign of H2O2 production) and HR-like cell death indicated by dark trypan blue staining were noticed in unsilenced pepper leaves at 48 hpi (hours post inoculation). Conversely, the concentrations of trypan blue and DAB staining were significantly decreased in CaWRKY30 silenced pepper leaves (Figure 4C). Electrical conductivity—an indicator of ion leakage—was calculated to study plasma membrane damage and cell death after RSI. 

The results exhibited that unsilenced plants treated with RSI exhibited higher ion leakage than Ralstonia-treated CaWRKY30 silenced plants at 24 and 48 dpi (Figure 4D). Relative disease index was estimated up to 10 dpi for inferring the level of disease in CaWRKY30-silenced and un-silenced plants after RSI (Supplementary Table S2). Prominent disease symptoms were noticed in CaWRKY30 silenced plants at 10 dpi, while unsilenced plants expressed little disease symptoms (Figure 4E). Six CaWRKY30-silenced and un-silenced plants were randomly chosen and infiltrated with R. solanacearum in the roots for phenotype assay. Obvious wilting disease symptoms were noticed in CaWRKY30 silenced pepper at 10 dpi, while unsilenced plants expressed very feeble wilting symptoms (Figure 4F). The qRT-PCR analysis was carried out to study the transcriptional accumulation of immunity-related marker genes and results expressed that transcriptional expression levels of immunity-associated marker genes such as CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1 were reduced in CaWRKY30-silenced plants as compared to un-silenced plants at 48 dpi (Figure 4G).

Figure 4. Silencing of CaWRKY30 by VIGS curtailed pepper’s resistance to RSI and upregulated immunity-associated marker genes. The qRT-PCR assay of CaWRKY30 expressional levels in R. solanacearum-inoculated, mock (inoculated with MgCl2 solution) CaWRKY30-silenced pepper plants (TRV: CaWRKY30), and control plants (TRV:00) (A). The difference in the growth of R. solanacearum between CaWRKY30-silenced and un-silenced (control) pepper plants inoculated with R. solanacearum at 3 and 5 dpi (B). Histochemical staining (DAB and trypan blue staining) in R. solanacearum-inoculated CaWRKY30-silenced (TRV: CaWRKY30) and un-silenced (TRV:00) pepper leaves at 48 dpi. Scale bar = 50 µm (C). Electrolyte leakage measurement as ion conductivity to evaluate the cell-death responses in the leaf discs of CaWRKY30-silenced (TRV: CaWRKY22) and un-silenced (TRV:00) pepper plants at 24 and 48 hpi with and without R. solanacearum (D). CaWRKY30-silencing by VIGS enhanced pepper plants' susceptibility to R. solanacearum infection. 

The disease index significantly escalated with time. CaWRKY30-silenced (TRV: CaWRKY30) plants expressed high susceptibility to R. solanacearum infection as compared to un-silenced (TRV:00) plants with time. Averages are based on four biological replicates with five plants per replication (E). The phenotypic effect of R. solanacearum treatment on CaWRKY30-silenced (TRV: CaWRKY30) and un-silenced (TRV:00) pepper plants at 10 dpi (F). qRT-PCR assay of transcriptional levels of defense-associated marker genes in CaWRKY30-silenced (TRV: CaWRKY30) and un-silenced (TRV:00) pepper plants at 48 h post-inoculation with R. solanacearum (G). The relative transcriptional expression level of mock-treated un-silenced plants was set to “1”. The height of the bar indicates means and error bars indicate the standard error of means. Data represent the means ± SE from four biological replicates. Different letters above the bars show significant differences among means.

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