Shaoliang Mou

CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection.

WRKY proteins form a large family of plant transcription factors implicated in the modulation of numerous biological processes, such as growth, development and responses to various environmental stresses. However, the roles of the majority WRKY family members, especially in non-model plants, remain poorly understood. We identified CaWRKY40 from pepper. Transient expression in onion epidermal cells showed that CaWRKY40 can be targeted to nuclei and activates expression of a W-box-containing reporter gene. CaWRKY40 transcripts are induced in pepper by Ralstonia solanacearum and heat shock. To assess roles of CaWRKY40 in plant stress responses we performed gain- and loss-of-function experiments. Overexpression of CaWRKY40 enhanced resistance to R. solanacearum and tolerance to heat shock in tobacco. In contrast, silencing of CaWRKY40 enhanced susceptibility to R. solanacearum and impaired thermotolerance in pepper. Consistent with its role in multiple stress responses, we found CaWRKY40 transcripts to be induced by signalling mechanisms mediated by the stress hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Overexpression of CaWRKY40 in tobacco modified the expression of hypersensitive response (HR)-associated and pathogenesis-related genes. Collectively, our results suggest that CaWRKY40 orthologs are regulated by SA, JA and ET signalling and coordinate responses to R. solanacearum attacks and heat stress in pepper and tobacco.

CaWRKY6 transcriptionally activates CaWRKY40, regulates Ralstonia solanacearum resistance, and confers high-temperature and high-humidity tolerance in pepper

High temperature (HT), high humidity (HH), and pathogen infection often co-occur and negatively affect plant growth. However, these stress factors and plant responses are generally studied in isolation. The mechanisms of synergistic responses to combined stresses are poorly understood. We isolated the subgroup IIb WRKY family member CaWRKY6 from Capsicum annuum and performed quantitative real-time PCR analysis. CaWRKY6 expression was upregulated by individual or simultaneous treatment with HT, HH, combined HT and HH (HTHH), and Ralstonia solanacearum inoculation, and responded to exogenous application of jasmonic acid (JA), ethephon, and abscisic acid (ABA). Virus-induced gene silencing of CaWRKY6 enhanced pepper plant susceptibility to R. solanacearum and HTHH, and downregulated the hypersensitive response (HR), JA-, ethylene (ET)-, and ABA-induced marker gene expression, and thermotolerance-associated expression of CaHSP24, ER-small CaSHP, and Chl-small CaHSP. CaWRKY6 overexpression in pepper attenuated the HTHH-induced suppression of resistance to R. solanacearum infection. CaWRKY6 bound to and activated the CaWRKY40 promoter in planta, which is a pepper WRKY that regulates heat-stress tolerance and R. solanacearum resistance. CaWRKY40 silencing significantly blocked HR-induced cell death and reduced transcriptional expression of CaWRKY40. These data suggest that CaWRKY6 is a positive regulator of R. solanacearum resistance and heat-stress tolerance, which occurs in part by activating CaWRKY40.

Overexpression of a Chinese cabbage BrERF11 transcription factor enhances disease resistance to Ralstonia solanacearum in tobacco

Ethylene-responsive factors (ERFs) play diverse roles in plant growth, developmental processes and stress responses. However, the roles and underlying mechanism of ERFs remain poorly understood, especially in non-model plants. In this study, a full length cDNA of ERF gene was isolated from the cDNA library of Chinese cabbage. According to sequence alignment, we found a highly conservative AP2/ERF domain, two nuclear localization signals, and an ERF-associated Amphiphilic Repression (EAR) motif in its C-terminal region. It belonged to VIIIa group ERFs sharing the highest sequence identity with AtERF11 in all of the ERFs in Arabidopsis and designated BrERF11. BrERF11-green fluorescence protein (GFP) transient expressed in onion epidermis cells localized to the nucleus. The transcript levels of BrERF11 were induced by exogenous salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), and hydrogen peroxide (H(2)O(2)). Constitutive expression of BrERF11 enhanced tolerance to Ralstonia solanacearum infection in transgenic tobacco plants, which was coupled with hypersensitive response (HR), burst of H(2)O(2) and upregulation of defense-related genes including HR marker genes, SA-, JA-dependent pathogen-related genes and ET biosynthesis associated genes and downregulation of CAT1, suggesting BrERF11 may participate in pathogen-associated molecular pattern (PAMP)- and effector-triggered immunity (PTI and ETI) mediated by SA-, JA- and ET-dependent signaling mechanisms.

SRC2-1 is required in PcINF1-induced pepper immunity by acting as an interacting partner of PcINF1

Elicitins are elicitors that can trigger hypersensitive cell death in most Nicotiana spp., but their underlying molecular mechanism is not well understood. The gene Phytophthora capsici INF1 (PcINF1) coding for an elicitin from P. capsici was characterized in this study. Transient overexpression of PcINF1 triggered cell death in pepper (Capsicum annuum L.) and was accompanied by upregulation of the hypersensitive response marker, Hypersensitive Induced Reaction gene 1 (HIR1), and the pathogenesis-related genes SAR82, DEF1, BPR1, and PO2. A putative PcINF1-interacting protein, SRC2-1, was isolated from a pepper cDNA library by yeast two-hybrid screening and was observed to target the plasma membrane. The interaction between PcINF1 and SRC2-1 was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Simultaneous transient overexpression of SRC2-1 and PcINF1 in pepper plants triggered intensive cell death, whereas silencing of SRC2-1 by virus-induced gene silencing blocked the cell death induction of PcINF1 and increased the susceptibility of pepper plants to P. capsici infection. Additionally, membrane targeting of the PcINF1-SRC2-1 complex was required for cell death induction. The C2 domain of SRC2-1 was crucial for SRC2-1 plasma membrane targeting and the PcINF1-SRC2-1 interaction. These results suggest that SRC2-1 interacts with PcINF1 and is required in PcINF1-induced pepper immunity.

Functional Analysis and Expressional Characterization of Rice Ankyrin Repeat-Containing Protein, OsPIANK1, in Basal Defense against Magnaporthe oryzae Attack

The ankyrin repeat-containing protein gene OsPIANK1 (AK068021) in rice (Oryza sativa L.) was previously shown to be upregulated following infection with the rice leaf blight pathogen Xanthomonas oryzae pv oryzae (Xoo). In this study, we further characterized the role of OsPIANK1 in basal defense against Magnaporthe oryzae (M.oryzae) by 5′ deletion analysis of its promoter and overexpression of the gene. The promoter of OsPIANK1 with 1,985 bps in length was sufficient to induce the OsPIANK1 response to inoculation with M.oryzae and to exogenous application of methyl jasmonate (MeJA) or salicylic acid (SA), but not to exogenous application of abscisic acid (ABA). A TCA-element present in the region between −563 bp and −249 bp may be responsible for the OsPIANK1 response to both M.oryzae infection and exogenous SA application. The JERE box, CGTCA-box, and two MYB binding sites locating in the region between −1985 bp and −907 bp may be responsible for the response of OsPIANK1 to exogenous MeJA. OsPIANK1 expression was upregulated after inoculation with M.oryzae and after treatment with exogenous SA and MeJA. Overexpression of OsPIANK1 enhanced resistance of rice to M.oryzae, although it did not confer complete resistance. The enhanced resistance to M.oryzae was accompanied by enhanced transcriptional expression of SA- and JA-dependent genes such as NH1, WKRY13, PAL, AOS2, PR1b, and PR5. This evidence suggests that OsPIANK1 acted as a positive regulator in rice basal defense mediated by SA- and JA-signaling pathways.

Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum

The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum

As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs) play important roles in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a genome wide comprehensive analysis of genes encoding CDPKs and CDPK-related protein kinases (CRKs) was performed in pepper, a total of 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CDPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the C. annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families.

Pepper CabZIP63 acts as a positive regulator during Ralstonia solanacearum or high temperature–high humidity challenge in a positive feedback loop with CaWRKY40

Highlight CabZIP63, indirectly activated by CaWRKY40, positively modulates transcription of CabZIP63 and CaWRKY40, enhances the binding of CaWRKY40 to its target promoters, and, therefore, increases resistance to Ralstonia solanacearum and thermotolerance.

CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling.

CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity.

A novel leucine-rich repeat protein, CaLRR51, acts as a positive regulator in the response of pepper to Ralstonia solanacearum infection.

The leucine-rich repeat (LRR) proteins play important roles in the recognition of corresponding ligands and signal transduction networks in plant defence responses. Herein, a novel LRR protein from Capsicum annuum, CaLRR51, was identified and characterized. It was localized to the plasma membrane and transcriptionally up-regulated by Ralstonia solanacearum infection (RSI), as well as the exogenous application of salicylic acid (SA), jasmonic acid (JA) and ethephon (ETH). Virus-induced gene silencing of CaLRR51 significantly increased the susceptibility of pepper to RSI. By contrast, transient overexpression of CaLRR51 in pepper plants activated hypersensitive response (HR)-like cell death, and up-regulated the defence-related marker genes, including PO2, HIR1, PR1, DEF1 and ACO1. Moreover, ectopic overexpression of CaLRR51 in transgenic tobacco plants significantly enhanced the resistance to RSI. Transcriptional expression of the corresponding defence-related marker genes in transgenic tobacco plants was also found to be enhanced by the overexpression of CaLRR51, which was potentiated by RSI. These loss- and gain-of-function assays suggest that CaLRR51 acts as a positive regulator in the response of pepper to RSI. In addition, the putative signal peptide and transmembrane region were found to be required for plasma membrane targeting of CaLRR51, which is indispensable for the role of CaLRR51 in plant immunity.