CaWRKY30 Positively Regulates Pepper Immunity by Targeting CaWRKY40 against Ralstonia solanacearum Inoculation through Modulating Defense-Related Genes

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


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][7][8]. Hence, defense system employed by model plants cannot be fully implied to non-model plants. Therefore, 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, 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 of 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., group I-III) based on number of WRKY domains and structure of zinc-finger motif. The group II of WRKY TFs is again subdivided into five sub-groups (IIa, IIb, IIc, IId, and IIe) [11][12][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][15][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][18][19][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][22][23][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, 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, has 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 capsica and Ralstonia solanacearum, respectively [28,29]. The coincidence of these pathogens imparts devastating impacts on pepper production under high temperature and high humidity (HTHH). The HTHH impairs pepper's immunity mediated by 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][31][32]. Conversely, simultaneous occurrence of pathogen infection and HTHH might not affect 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 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 is 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 coexist 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][40][41]. It recommends 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 subset of HSE elements and W-boxes in their promoter region and in the promoters of other WRKY TFs, inferring the role of WRKY networks in 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 its response to pathogen attack.
In the current study, a full-length cDNA for WRKY TFs family of pepper was isolated and named CaWRKY30. We characterized it expressionally and functionally and found that CaWRKY30was 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 H 2 O 2 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.

Cloning and Sequence Analysis of CaWRKY30
A new WRKY gene CaWRKY30 (CA01g34480) was identified by genome wide analysis (http://passport.pepper.snu.ac.kr (accessed on 25 October 2021). 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 deduced amino acid sequence of CaWRKY30 was 307 amino acids 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).

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). igure 1. Structural analysis of CaWRKY30. Comparison of CaWRKY30 deduced amino acid sequence with representative elated proteins from Solanum tuberosum StWRKY53 (XP_015166339.1), Capsicum chinese CcWRKY53 (PHU08488.1), Nicotina attenuate NaWRKY53 (XP_019233122.1), and Nicotiana sylvestris NsWRKY53 (XP_009768977.1). Green shading indicates 0-75% identity, pink shading depicts 75-100% identity, and blue shading indicates 100% identity. Analysis was assayed y using DNAMAN5.

Transcriptional Expression of CaWRKY30 under RSI and Treatment with Va Phytohormones
The presence of immunity-associated cis-elements in the promoter regio CaWRKY30 indicated its potential role in pepper's immunity RSI. To check the no qRT-PCR analysis was carried out to assess the transcriptional levels of CaWRY30 u RSI. The transcriptional levels of CaWRKY30 were increased in Ralstonia-treated le compared to mock-treated leaves (Figure 2A). The enhanced CaWRKY30 transcript expression levels were consistent between 0 h to 48 h post treatment (hpt). The hig transcriptional expression levels were recorded at 48 hpt ( Figure 2A).
Phytohormones, including ET, ABA, SA, and JA mediated signaling pathways key regulators of plant reactions to biotic and abiotic stresses. The regulatio CaWRKY30 by phytohormones-mediated signaling pathways was studied by foliar a cation of ET, ABA, SA, and JA using qRT-PCR analysis. The results indicated that rel transcriptional expression levels of CaWRKY30 were enhanced from 0 h to 48 hpt with µM ET as compared to mock. These transcriptional levels were highest at 48 hpt wit 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 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 mock. Transcriptional levels reached to the highest level at 12 hpt with ABA ( Figure 2C). Results indicated that transcriptional accumulation of CaWRKY30 was significantly increased after application of 1 mM SA, as compared to mock. These enhanced transcriptional expression levels were highest at 24 hpt ( Figure 2D). 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 phytohormone-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 the Fisher's protected LSD test.

CaWRKY30 Was Found to Be Localized in Nucleus
Sequence analysis by using WoLFPSORT ("http://www.genscript.com/psort/wolf_psort.html\T1\textquotedblrightolf_psort.htm (accessed on 29 October 2021)) depicted that predicted CaWRKY30 protein sequence possesses a putative nuclear localization signal (Figure 1), showing its potential targeting in nucleus. To validate this perception, a CaWRKY30 GFP fusion construct was generated that was driven by constitutive promoter of CaMV35S. Afterwards, this constituted vector was transferred into Agrobacterium strain GV3101. We transiently over-expressed CaWRKY30 GFP construct in leaves of Nicotiana benthamiana by A. tumefaciens injection, and GFP signals were examined by confocal fluorescence microscope. Results depicted that GFP signals of CaWRKY30-GFP was present in the nuclei, while GFP of control was detected in various subcellular portions, like cytoplasm and nuclei ( Figure 3). The transcriptional abundance in RSI-treated leaves was compared with MgCl 2 -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 ddH 2 O-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 the Fisher's protected LSD test.

CaWRKY30 Was Found to Be Localized in Nucleus
Sequence analysis by using WoLFPSORT ("http://www.genscript.com/psort/wolf_ psort.html\T1\textquotedblrightolf_psort.htm (accessed on 29 October 2021)) depicted that predicted CaWRKY30 protein sequence possesses a putative nuclear localization signal (Figure 1), showing its potential targeting in nucleus. To validate this perception, a CaWRKY30 GFP fusion construct was generated that was driven by constitutive promoter of CaMV35S. Afterwards, this constituted vector was transferred into Agrobacterium strain GV3101. We transiently over-expressed CaWRKY30 GFP construct in leaves of Nicotiana benthamiana by A. tumefaciens injection, and GFP signals were examined by confocal fluorescence microscope. Results depicted that GFP signals of CaWRKY30-GFP was present in the nuclei, while GFP of control was detected in various subcellular portions, like cytoplasm and nuclei ( Figure 3).

Silencing of CaWRKY30by 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's immunity. A total of 50 CaWRKY30-unsilenced (TRV:00) and 50 CaWRKY30silenced (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 (sign of H2O2 production) and HR-like cell death indicated by dark trypan blue staining was 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 hpi ( 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 hpi ( Figure 4G).

Silencing of CaWRKY30by 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's immunity. A total of 50 CaWRKY30-unsilenced (TRV:00) and 50 CaWRKY30silenced (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 H 2 O 2 production and cell necrosis in Ralstonia inoculated CaWRKY30 silenced (TRV:CaWRKY30), and unsilenced (TRV:00) pepper leaves. A dark DAB staining (sign of H 2 O 2 production) and HR-like cell death indicated by dark trypan blue staining was 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 hpi ( 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 hpi ( Figure 4G).

CaWRKY30 Transient Over-Expression Stimulated HR-Like Cell Death, Production of H2O2, and Transcriptional Upregulation of Immunity Related Marker Genes
The VIGS experiments indicated that CaWRKY30 positively regulates pepper's immunity to RSI. Transient over-expression experiments were performed by infiltrating GV3101 cells carrying 35S:00 (EV) or 35S:CaWRKY30 into pepper leaves for further validation of this speculation. We studied the role of CaWRKY30 transient over-expression on induction of HR-like cell death and H2O2 accumulation in pepper plants. The expression of CaWRKY30 in pepper plants was established by western blotting experiments ( Figure  5A). The HR like cell death and H2O2 accumulation was confirmed by trypan blue and DAB staining, respectively. Results revealed that transient over-expression of CaWRKY30 caused HR-like cell death and H2O2 accumulation in pepper leaves manifested by darker trypan blue staining and dark brown DAB staining ( Figure 5B). Conductivity was performed to study the intensity of cell necrosis caused by transient over-expression of CaWRKY30, and results indicated that pepper leaves transiently over-expressing CaWRKY30 showed more ion leakage at 24 and 48 h after Agro-infiltration as compared   Figure 5B). Conductivity was performed to study the intensity of cell necrosis caused by transient over-expression of CaWRKY30, and results indicated that pepper leaves transiently over-expressing CaWRKY30 showed more ion leakage at 24 and 48 h after Agro-infiltration as compared with pepper leaves over-expressing empty vector ( Figure 5C). Relative transcriptional expression levels of defense-associated marker genes such as ET-biosynthesis related CaPR1, SA-related CaNPR1, ABA-responsive CaABR1, and HR-associated marker gene CaHIR1 were detected by qRT-PCR analysis.
Our results depicted that transient over-expression of CaWRKY30 in pepper leaves notably amplified transcriptional accumulation of CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1 in pepper as compared to transiently over-expressing empty vector pepper plants at 48 hpi ( Figure 5D). These results portray that CaWRKY30 acts as a positive regulator of plant HR-like cell death, H 2 O 2 production, and transcriptional upregulation of immunity related marker genes. with pepper leaves over-expressing empty vector ( Figure 5C). Relative transcriptional expression levels of defense-associated marker genes such as ET-biosynthesis related CaPR1, SA-related CaNPR1, ABA-responsive CaABR1, and HR-associated marker gene CaHIR1 were detected by qRT-PCR analysis.
Our results depicted that transient over-expression of CaWRKY30 in pepper leaves notably amplified transcriptional accumulation of CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1 in pepper as compared to transiently over-expressing empty vector pepper plants at 48 hpi ( Figure 5D). These results portray that CaWRKY30 acts as a positive regulator of plant HR-like cell death, H2O2 production, and transcriptional upregulation of immunity related marker genes. Figure 5. Transient over-expression of CaWRKY30 induced HR-like cell death, H2O2 accumulation, and expression of immunity-associated marker genes in pepper leaves. Successful over-expression of CaWRKY30-Flag was confirmed by western blotting experiment (A), HR-like cell-death triggered by transient over-expression of 35S:CaWRKY30, confirmed by phenotype detection, exposure under UV light, and DAB and Trypan Blue staining at 48 hpi, respectively; Scale bar = 50 µm (B), Measurement of electrolyte leakage (ion conductivity) to estimate the cell death response in pepper leaf discs at 24 and 48 h after agro-infiltration, respectively (C), qRT-PCR assay to check the transcriptional expression levels of immunity-associated marker genes, including CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1 in 35S: CaWRKY30 expressed pepper leaves at 48 hpi, respectively (D). The relative expression level of known defense-related marker-genes in pepper leaves transiently overexpressing the empty vector was set to "1". Data representing the means ± SE from four biological replicates. Error bars are standard errors of the means. Different letters above the bars indicates significant differences among the means.

Inter-Relationship between CaWRKY30 and CaWRKY40
It is reported that different WRKY TFs form networks together and we previously reported that various members of WRKY TFs, i.e., CaWRKY6, CaWRKY22, and CaWRKY40, are expressionally and functionally linked to each other. Therefore, we postulate that CaWRKY30 might relate to other members of the WRKY family, including CaWRKY40, which is a positive regulator of pepper's immunity against RSI. The qRT-PCR analysis was performed for further investigation of possible feedback regulation of CaWRKY40 by CaWRKY30, and to study the potential modulation of CaWRKY30 by CaWRKY40 transient over-expression assay or by silencing of CaWRKY40 by VIGS.
Our results expressed transcriptional levels of CaWRKY30 in pepper leaves transiently over-expressing CaWRKY40 increased at 24 and 48 hpi in comparison with plants leakage (ion conductivity) to estimate the cell death response in pepper leaf discs at 24 and 48 h after agro-infiltration, respectively (C), qRT-PCR assay to check the transcriptional expression levels of immunity-associated marker genes, including CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1 in 35S: CaWRKY30 expressed pepper leaves at 48 hpi, respectively (D). The relative expression level of known defense-related marker-genes in pepper leaves transiently over-expressing the empty vector was set to "1". Data representing the means ± SE from four biological replicates. Error bars are standard errors of the means. Different letters above the bars indicates significant differences among the means.

Inter-Relationship between CaWRKY30 and CaWRKY40
It is reported that different WRKY TFs form networks together and we previously reported that various members of WRKY TFs, i.e., CaWRKY6, CaWRKY22, and CaWRKY40, are expressionally and functionally linked to each other. Therefore, we postulate that CaWRKY30 might relate to other members of the WRKY family, including CaWRKY40, which is a positive regulator of pepper's immunity against RSI. The qRT-PCR analysis was performed for further investigation of possible feedback regulation of CaWRKY40 by CaWRKY30, and to study the potential modulation of CaWRKY30 by CaWRKY40 transient over-expression assay or by silencing of CaWRKY40 by VIGS.
Our results expressed transcriptional levels of CaWRKY30 in pepper leaves transiently over-expressing CaWRKY40 increased at 24 and 48 hpi in comparison with plants transiently over-expressing empty vector (EV) (Figure 6A), whereas the transcriptional levels of CaWRKY40 increased in those plants transiently over-expressing CaWRKY30 as compared to plants transiently over-expressing EV ( Figure 6B). Contrastingly, our results depicted that transcriptional accumulation of CaWRKY40 was notably downregulated in Ralstonia-infected CaWRKY30-silenced plants as compared to un-silenced plants ( Figure 6C). Additionally, transcriptional accumulation of known immunity linked marker genes, e.g., CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1 was fully or partially suppressed in CaWRKY30 silenced plants that was triggered by CaWRK40 ( Figure 6D).
This data indicates transcriptional regulation of CaWRKY40 by CaWRKY30 and the presence of positive regulatory loop involving CaWRKY30 and CaWRKY40. transiently over-expressing empty vector (EV) (Figure 6A), whereas the transcriptional levels of CaWRKY40 increased in those plants transiently over-expressing CaWRKY30 as compared to plants transiently over-expressing EV ( Figure 6B). Contrastingly, our results depicted that transcriptional accumulation of CaWRKY40 was notably downregulated in Ralstonia-infected CaWRKY30-silenced plants as compared to un-silenced plants ( Figure  6C). Additionally, transcriptional accumulation of known immunity linked marker genes, e.g., CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1 was fully or partially suppressed in CaWRKY30 silenced plants that was triggered by CaWRK40 ( Figure 6D). This data indicates transcriptional regulation of CaWRKY40 by CaWRKY30 and the presence of positive regulatory loop involving CaWRKY30 and CaWRKY40.

Inter-Relationship among CaWRY30 and CaWRKY6, CaWRKY22, and CaWRKY27
In our previous studies, CaWRKY40 was detected to be associated expressionally and functionally to different WRKY TFs, such as CaWRKY6 and CaWRKY22. The relationship among CaWRKY30 and CaWRKY40 indicates that CaWRKY30 is possibly linked with different WRKY TFs involved in pepper's immunity against RSI. To confirm this assumption, qRT-PCR analysis was performed to study relationship among CaWRKY30 and different WRKY TFs which have been previously reported to be involved in pepper's immunity against RSI, including CaWRKY6, CaWRKY22, and CaWRKY27. Our results indicated that transcriptional accumulation of CaWRKY30 was amplified in CaWRKY6, CaWRKY22, and CaWRKY27 transiently over-expressing pepper plants at 48 hpi ( Figure 7A), while transcriptional accumulation of CaWRKY6, CaWRKY22, and CaWRKY27 were also increased in pepper leaves transiently over-expressing CaWRKY30 at 48 hpi ( Figure 7B). Our results of qRT-PCR assay imply that CaWRKY30 and CaWRKY6, CaWRKY22, and CaWRKY27 are expressionally and functionally inter-related.

Inter-Relationship among CaWRY30 and CaWRKY6, CaWRKY22, and CaWRKY27
In our previous studies, CaWRKY40 was detected to be associated expressionally and functionally to different WRKY TFs, such as CaWRKY6 and CaWRKY22. The relationship among CaWRKY30 and CaWRKY40 indicates that CaWRKY30 is possibly linked with different WRKY TFs involved in pepper's immunity against RSI. To confirm this assumption, qRT-PCR analysis was performed to study relationship among CaWRKY30 and different WRKY TFs which have been previously reported to be involved in pepper's immunity against RSI, including CaWRKY6, CaWRKY22, and CaWRKY27. Our results indicated that transcriptional accumulation of CaWRKY30 was amplified in CaWRKY6, CaWRKY22, and CaWRKY27 transiently over-expressing pepper plants at 48 hpi ( Figure 7A), while transcriptional accumulation of CaWRKY6, CaWRKY22, and CaWRKY27 were also increased in pepper leaves transiently over-expressing CaWRKY30 at 48 hpi ( Figure 7B). Our results of qRT-PCR assay imply that CaWRKY30 and CaWRKY6, CaWRKY22, and CaWRKY27 are expressionally and functionally inter-related.

Discussion
The WRKY protein is one of the largest families of plant transcription factors (TFs). A group of WRKY TFs family members, reported from Arabidopsis thaliana and Oryza sativa, is known to take part and play a crucial part in plants' immunity regulation. Considerable functional variations among closely structural homologs of WRKYs have been reported [17,48], and functions of WRKY TFs in immunity of non-model plants, such as pepper, is inadequately investigated.
The CaWRKY30, a group III WRKY gene, has been functionally characterized in the current study. Results suggested that CaWRKY30 positively regulates pepper's immunity against RSI, and it is a vital constituent of WRKY web comprising CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. The entanglement of CaWRKY30 in pepper's immunity is endorsed by pathogen responsive cis elements, including TGA element, TGACG-motif, TATC-box, TATA-box, and W-box present in the promoter region of CaWRKY30. This assumption was consistent with upregulation of transcriptional levels of CaWRKY30 after RSI, and upon exogenously supplied ETH, ABA, and SA [49,50].
Since genes triggered by exposure to a certain stress factor have been often known to be involved in response to that stress [51], we speculated that CaWRKY30 might positively regulate pepper's immunity to RSI. This speculation was supported by the data of silencing of CaWRKY30 by VIGS, and by CaWRKY30 transient over-expression assay. This curtailed immunity due to loss of function of CaWRKY30 was accompanied by amplified growth of RSI and downregulation of immunity related marker genes like SA dependent CaPR1 [52], CaNPR1 [53], JA related CaDEF1 [54], ABA associated CaABR1 [55], and HRassociated CaHIR1 [56]. On the other hand, HR-like cell death and H2O2 secretion was triggered by the transient over-expression of CaWRKY30 accompanied with transcriptional up regulation of immunity related marker genes like CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1. This suggests the role of CaWRKY30 as positive regulator of HRlike cell death and pepper's immunity against RSI. Likewise, it has been observed in previous studies that AtWRKY30, a homolog of CaWRKY30 in Arabidopsis, positively regulates plants' immunity against biotic and abiotic stresses [57,58]. This can be suggested that CaWRKY30 is upregulated upon Ralstonia infection, leading to decreased vulnerability of pepper to bacterial pathogen infection.
Phytohormones such as ET, ABA, and SA are essential signaling molecules implicated in response of plants to pathogen attack and high-temperature stress. These are also

Discussion
The WRKY protein is one of the largest families of plant transcription factors (TFs). A group of WRKY TFs family members, reported from Arabidopsis thaliana and Oryza sativa, is known to take part and play a crucial part in plants' immunity regulation. Considerable functional variations among closely structural homologs of WRKYs have been reported [17,48], and functions of WRKY TFs in immunity of non-model plants, such as pepper, is inadequately investigated.
The CaWRKY30, a group III WRKY gene, has been functionally characterized in the current study. Results suggested that CaWRKY30 positively regulates pepper's immunity against RSI, and it is a vital constituent of WRKY web comprising CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. The entanglement of CaWRKY30 in pepper's immunity is endorsed by pathogen responsive cis elements, including TGA element, TGACG-motif, TATC-box, TATA-box, and W-box present in the promoter region of CaWRKY30. This assumption was consistent with upregulation of transcriptional levels of CaWRKY30 after RSI, and upon exogenously supplied ETH, ABA, and SA [49,50].
Since genes triggered by exposure to a certain stress factor have been often known to be involved in response to that stress [51], we speculated that CaWRKY30 might positively regulate pepper's immunity to RSI. This speculation was supported by the data of silencing of CaWRKY30 by VIGS, and by CaWRKY30 transient over-expression assay. This curtailed immunity due to loss of function of CaWRKY30 was accompanied by amplified growth of RSI and downregulation of immunity related marker genes like SA dependent CaPR1 [52], CaNPR1 [53], JA related CaDEF1 [54], ABA associated CaABR1 [55], and HR-associated CaHIR1 [56]. On the other hand, HR-like cell death and H 2 O 2 secretion was triggered by the transient over-expression of CaWRKY30 accompanied with transcriptional up regulation of immunity related marker genes like CaPR1, CaNPR1, CaDEF1, CaABR1, and CaHIR1. This suggests the role of CaWRKY30 as positive regulator of HR-like cell death and pepper's immunity against RSI. Likewise, it has been observed in previous studies that AtWRKY30, a homolog of CaWRKY30 in Arabidopsis, positively regulates plants' immunity against biotic and abiotic stresses [57,58]. This can be suggested that CaWRKY30 is upregulated upon Ralstonia infection, leading to decreased vulnerability of pepper to bacterial pathogen infection.
Phytohormones such as ET, ABA, and SA are essential signaling molecules implicated in response of plants to pathogen attack and high-temperature stress. These are also involved in crosstalk between plants' responses to biotic and abiotic stresses [59]. The SA instigates resistance against biotrophic pathogen, while ET plays a vital role in immunity of plants against necrotrophic pathogens [60]. Generally, synthesis of SA, ET, and JA is coupled with PAMP-triggered immunity (PTI) or effector-triggered-immunity (ETI). These phytohormones can play their role both synergistically and antagonistically based on their concentrations during defense signaling [61]. Synergistic association between these three signaling elements has been detected in PTI, whereas compensatory association has been detected in ETI [62]. The CaWRKY30 was constantly induced by exogenously supplied phytohormones. The known SA, ET, and ABA-dependent immunity-related marker genes such as CaPR1, CaNPR1, CaDEF1, and CaABR1 were downregulated by silencing of CaWRK30; however, they were upregulated by transient over-expression of CaWRKY30 in pepper plants, showing that CaWRKY30 takes part in synergistically mediated defense signaling by SA, ET, and ABA, leading to PTI.

Generation of Vectors
The gateway-cloning technique was employed to generate vectors. To construct satellite vectors, CaWRKY30 full length ORF (with terminal codon or without terminal codon) was cloned into entry vector pDONR207 by using a BP reaction. Afterwards, this construct was transferred into destination vectors pMDC83, CD3687 (HA-tag), CD3688 (Flag-tag), and Pk7WG2 by performing LR reaction to generate vectors for sub-cellular localization and over-expression, respectively.
For the construction of vectors for VIGS, 364 bps fragment in 3 -untranslated region (UTR) of CaWRKY30 was selected and the specificity was confirmed by BLAST against genome sequence in database of CM334 (http://peppergenome.snu.ac.kr/ (accessed on 29 October 2021)) and Zunla-1 (http://peppersequence.genomics.cn/page/species/blast. jsp (accessed on 29 October 2021)). Next, the BP reaction was performed to clone this fragment into pDONR207/201, after this construct was transferred into vector PYL279 by performing LR reaction.

Pathogen's Growth and Inoculation Procedures
We isolated a compatible virulent strain of R. solanacearum from pepper plants infected by Ralstonia from Dera Ghazi Khan, Pakistan. The tetrazolium chloride method was used to purify above-ground vascular tissue portion exudates of the pathogen-infected plants [9,45]. The isolated R. solanacearum was cultivated in SPA medium in thermo control shaker (200 g potato, 20 g sucrose, 3 g beef extract, 5 g tryptone, and 1 L of double-distilled H 2 O) at 200 rpm and 28 • C for overnight. Next, cultivated R. solanacearum strain was centrifuged at 6500 rpm and 28 • C for 10 min. Liquid supernatant after centrifugation was poured out, and pellets in the bottom of centrifuge tube were diluted in sterilized 10 mM MgCl 2 solution. Bacterial cell density was adjusted to 10 8 cfu ml −1 (OD600 = 0.8). To study the effects of RSI on transcriptional levels of CaWRKY30 and on the resistance of pepper plants to R. solanacearum attack, the top third leaf of pepper plants was infiltrated with 10 µL of R. solanacearum solution by a syringe without a needle. Pathogen-treated leaves samples were harvested at specific time points for histochemical staining experiments (e.g., DAB staining or trypan blue staining), electrical conductivity, cfu, and RNA extraction for more experiments. For the phenotype experiment of CaWRKY30-silenced and unsilenced plants, roots were injured by using a glass rod and infiltrated with compatible virulent R. solanacearum. Plants were kept in a growth chamber in control conditions at 28 ± 2 • C temperature, 60-70 µmol photons m −2 s −1 light intensity, 70% relative humidity, and under a 16-hlight/8-h dark photoperiod after R. solanacearum inoculation. Pictures of phenotype were captured after RSI at specific time intervals.

Foliar Application of Phytohormones
To study the effects of phytohormones, healthy pepper plants were sprayed with 100 µM ETH, 100 µM ABA, and 1 mM SA at four leaf stages. Mock (control) plants were treated with sterile ddH 2 O. Samples treated with phytohormones were collected at desired time points for RNA extraction and for further study.

Examination of CaWRKY30 Sub-Cellular Localization
Agrobacterium cells having 35S:CaWRKY30-GFP or 35S:GFP (control) were cultivated overnight in LB medium possessing corresponding antibiotics. Cultured media was centrifuged at 6500 rpm, liquid supernatant was poured out, and the pellet in the bottom was diluted in induction medium (10 mM MES, 10 mM MgCl2, pH 5.7, and 150 µM acetosyringone), and set to OD600 = 0.8. Agrobacterium containing 35S:CaWRKY30-GFP and 35S:GFP were injected into leaves of N. benthamiana by using a syringe without needle. The previously described method was used to do 4,6-diamidino-2-phenylindole staining (DAPI) [9]. At 48 hpt, GFP and DAPI fluorescence signals were observed, and pictures were captured by a laser scanning confocal microscope (TCS SP8, Leica, Solms, Germany), with an excitation wavelength of 488 nm and a 505-530 nm band-pass emission filter.

Histochemical Staining
Histochemical staining (Trypan blue and 3, 3 -diaminobenzidine) were carried out as described previously [9,11,79]. To perform trypan blue staining assay, leaves of pepper were boiled in trypan blue solution (10 mL lactic acid, 10 mL glycerol, 10 mL phenol, 40 mL ethanol, 10 mL ddH 2 O, and 1 mL trypan blue) for 30 min. Afterwards, leaves were kept at room temperature for 8 h, immersed into solution of chloral hydrate (2.5 g of chloral hydrate dissolved in 1 mL of distilled water), and de-stained by boiling for 30 min. Chloral hydrate solution was changed multiple times to reduce the background, and then samples were mounted in 70% glycerol. Leaves were placed in 1 mg/mL of DAB solution for overnight for DAB staining assay. The DAB stained leaves were boiled in lactic acid:glycerol:absolute ethanol [1:1:3 (v/v/v)] and then de-stained in absolute ethanol for overnight [80]. The pictures of trypan blue and DAB-stained leaves were taken by a camera and by a light microscope (Leica, Wetzlar, Germany).

Silencing of CaWRKY30 through Virus-Induced Gene Silencing (VIGS)
Previously described Tobacco Rattle Virus (TRV)-based virus induced gene silencing (VIGS) system was carried out for CaWRKY30 silencing in pepper plants [47,81,82]. Agrobacterium GV3101 containing PYL192, PYL279-CaWRKY30, PYL279, and PYL279-PDS (OD 600 = 0.8) constructs were mixed in a 1:1 ratio. This mix was infiltrated into 2-weeks old pepper plants' cotyledons by a syringe without needle. The infiltrated plants were incubated in a growth chamber for 56 h under dark at 16 • C temperature and 45% relative humidity, and then shifted into a growth room in controlled conditions at 25 ± 2 • C, 60-70 µmol photons m −2 s −1 , and relative humidity of 70% under a photoperiod of 16 h light/8 h dark.

CaWRKY30 Transient Over-Expression in Pepper Leaves
Agrobacterium cells GV3101 containing CaWRKY30-Flag and EV were cultivated in LB medium in a thermo control shaker, having corresponding antibiotics overnight to OD 600 = 1.0. Afterwards, these Agrobacterium GV3101 were centrifuged at 6000 rpm and 28 • C for 10 min. After centrifugation, liquid supernatant was removed and the solid pellet in the bottom was diluted to OD 600 = 0.8 into induction medium (10 mM MES, pH5.4, 10 mM MgCl 2 , 200 µM acetosyringone). This mixture was infiltrated into leaves by a syringe without needle. These transiently over-expressed leaves were observed for HR-like cell death, or samples were collected to perform DAB and trypan blue staining experiments, and for RNA isolation for more experiments.

RNA Extraction and Quantitative Real Time RT-PCR
TRIzol reagent method (Invitrogen) was used to extract total RNA from leaves samples of treated pepper plants and from mock seedlings. Afterwards, Prime Script RT-PCR kit (TaKaRa, Dalian, China) was used to reverse transcribe extracted RNA. Real-time qRT-PCR analysis was carried out to study relative transcriptional levels of targeted marker genes. Data processing was done according to previously described methods [45,82], with specific primers (Supplementary Table S3), using manufacturer ' s instructions for Bio-Rad Real time PCR system (Bio-Rad, Foster City, CA, USA) and SYBR premix Ex Taq II system (TaKaRa Perfect Real Time).

Estimation of ELECTRICAL Conductivity
Electrical conductivity (ion leakage) was calculated by using the method described previously with small changes [44,83]. Six leaf discs (4 mm in width) were obtained by using a hole-puncher. The discs were washed three times with sterilized ddH 2 O and incubated instantaneously into 10 mL ddH 2 O. After that, these leaf discs were kept in a gently shaking (60 rpm) shaker at room temperature for 1 h. A conductivity meter (Mettler Toledo 326 Mettler, Zurich, Switzerland) was used to record data of electrical conductivity.

Immuno Blotting
A previously described procedure of protein extraction buffer was used to extract protein from pepper leaves [3]. The extracted protein was incubated overnight with anti-HA agarose at 4 • C (Thermo Fisher Scientific, Waltham, MA, USA). Magnetic crack was used to collect beads and washed thrice with Tris buffer saline (TBS) and tween 20 (0.05%). Eluted protein was observed using immuno-blotting and by anti-HA-peroxidase (Abcam, Cambridge, UK).

Conclusions
In conclusion, we are of the view that CaWRKY30 transcriptionally activates immunity of pepper against RSI. Silencing of CaWRKY30 by VIGS curtailed pepper plants' immunity to bacterial pathogen infection, whereas transient over-expression of CaWRKY30 induced the resistance to R. solanacearum. Collectively, the formation of positive feed-back loop and its function in parallel with other TFs is ample evidence of its activation under biotic stress. Additionally, we propose generation of CaWRKY30 stable transgenic pepper plants and CaWRKY30 target protein to comprehensively explicate the role of this gene against bacterial pathogen and also in various metabolic pathways.