CaWRKY28 Cys249 is Required for Interaction with CaWRKY40 in the Regulation of Pepper Immunity to Ralstonia solanacearum.

WRKY transcription factors have been implicated in plant response to pathogens but how WRKY-mediated networks are organized and operate to produce appropriate transcriptional outputs remains largely unclear. Here, we identify a member of the WRKY family from pepper (Capsicum annuum), CaWRKY28, that physically interacts with CaWRKY40, a positive regulator of pepper immunity and thermotolerance. We confirmed CaWRKY28-CaWRKY40 interaction by coimmunoprecipitation, bimolecular fluorescence complementation, and microscale thermophoresis. Our findings supported the idea that CaWRKY28 is a nuclear protein that acts as positive regulator in pepper responses to infection by the pathogenic bacterium Ralstonia solanacearum. It performs its function not by directly modulating the W-box containing immunity-related genes but by promoting CaWRKY40 via physical interaction to bind and activate its immunity-related target genes, including CaPR1, CaNPR1, CaDEF1, and CaABR1, but not its thermotolerance-related target gene, CaHSP24. All of these data indicate that CaWRKY28 interacts with and potentiates CaWRKY40 in regulating immunity against R. solanacearum infection but not thermotolerance. Importantly, we discovered that CaWRKY28 Cys249, shared by CaWRKY28 and its orthologs probably only in the family Solanaceae, is crucial for the CaWRKY28-CaWRKY40 interaction. These results highlight how CaWRKY28 associates with CaWRKY40 during the establishment of WRKY networks, and how CaWRKY40 achieves its functional specificity during pepper responses to R. solanacearum infection.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cloning and overexpression of the ascorbate peroxidase gene from the yam (Dioscorea alata) enhances chilling and flood tolerance in transgenic Arabidopsis.

Minghuai 1 (MH1) is a yam (Dioscorea alata) cultivar with high tolerance to flooding but sensitivity to chilling. MH1 responded differently to chilling and flooding according to various physiological parameters and antioxidant enzymes. Flooding led to an increase in ascorbate peroxidase (APX) activity in both roots and leaves, while chilling did not affect APX activity. The full length DaAPX ORF sequence from MH1 (750 bp) was then cloned. Phylogenetic analysis showed that plant cytosolic APXs into four major clusters and DaAPX was closely related to Oncidium. The DaAPX gene driven by a 35S promoter was transferred into Arabidopsis. The gene expression and enzyme activity of APX in the DaAPX transgenic lines 1-3 were significantly higher than in wild type (WT) plants. Compared to WT plants, seedling growth characteristics were significantly better in all transgenic lines under chilling, flooding, and oxidative stresses, indicating that the overexpression of DaAPX in Arabidopsis enhanced tolerance to several abiotic stresses. MH1 plants supplied with H2O2 presented an increase in the activity of APX leading to enhanced tolerance to chilling. Functional characterization of the APX gene should improve our understanding of the chilling- and flood-response mechanism in the yam.

CaWRKY40b in Pepper Acts as a Negative Regulator in Response to Ralstonia solanacearum by Directly Modulating Defense Genes Including CaWRKY40.

WRKY transcription factors (TFs) have been implicated in plant growth, development, and in response to environmental cues; however, the function of the majority of pepper WRKY TFs remains unclear. In the present study, we functionally characterized CaWRKY40b, a homolog of AtWRKY40, in pepper immunity. Ralstonia solanacearum inoculation (RSI) in pepper plants resulted in downregulation of CaWRKY40b transcript, and green fluorescent protein (GFP)-tagged CaWRKY40b was localized to the nuclei when transiently overexpressed in the leaves of Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of CaWRKY40b significantly decreased pepper’ susceptibility to RSI. Consistently, the transient over-expression of CaWRKY40b-SRDX (chimeric repressor version of CaWRKY40b) triggered cell death, as indicated by darker trypan blue and DAB staining. CaWRKY40b targets a number of immunity-associated genes, including CaWRKY40 JAR, RLK1, EIN3, FLS2, CNGIC8, CDPK13, and heat shock cognate protein 70 (HSC70), which were identified by ChIP-seq and confirmed using ChIP-real time PCR. Among these target genes, the negative regulator HSC70 was upregulated by transient overexpression of CaWRKY40b and downregulated by silencing of CaWRKY40b, whereas other positive regulators as well as two non-target genes, CaNPR1 and CaDEF1, were downregulated by the transient overexpression of CaWRKY40b and upregulated by CaWRKY40b silencing or transient overexpression of CaWRKY40b-SRDX. In addition, CaWRKY40b exhibited a positive feedback regulation at transcriptional level by directly targeting the promoter of itself. In conclusion, the findings of the present study suggest that CaWRKY40b acts as a negative regulator in pepper immunity against R. solanacearum by transcriptional modulation of a subset of immunity-associated genes; it also represses immunity in the absence of a pathogen, and derepresses immunity upon pathogen challenge.

An efficient, flexible perovskite solar module exceeding 8% prepared with an ultrafast PbI2 deposition rate.

Large-area, pinhole-free CH3NH3PbI3 perovskite thin films were successfully fabricated on 5 cm × 5 cm flexible indium tin oxide coated polyethylene naphthalate (ITO-PEN) substrates through a sequential evaporation/spin-coating deposition method in this research. The influence of the rate-controlled evaporation of PbI2 films on the quality of the perovskite layer and the final performance of the planar-structured perovskite solar cells were investigated. An ultrafast evaporation rate of 20 Å s-1 was found to be most beneficial for the conversion of PbI2 to CH3NH3PbI3 perovskite. Based on this high-quality CH3NH3PbI3 film, a resultant flexible perovskite solar sub-module (active area of 16 cm2) with a power conversion efficiency of more than 8% and a 1.2 cm2 flexible perovskite solar cell with a power conversion efficiency of 12.7% were obtained.

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.

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

CaWRKY40 is known to act as a positive regulator in the response of pepper (Capsicum annuum) to Ralstonia solanacearum inoculation (RSI) or high temperature-high humidity (HTHH), but the underlying mechanism remains elusive. Herein, we report that CabZIP63, a pepper bZIP family member, participates in this process by regulating the expression of CaWRKY40. CabZIP63 was found to localize in the nuclei, be up-regulated by RSI or HTHH, bind to promoters of both CabZIP63(pCabZIP63) and CaWRKY40(pCaWRKY40), and activate pCabZIP63- and pCaWRKY40-driven ß-glucuronidase expression in a C- or G-box-dependent manner. Silencing of CabZIP63 by virus-induced gene silencing (VIGS) in pepper plants significantly attenuated their resistance to RSI and tolerance to HTHH, accompanied by down-regulation of immunity- or thermotolerance-associated CaPR1, CaNPR1, CaDEF1, and CaHSP24. Hypersensitive response-mediated cell death and expression of the tested immunity- and thermotolerance-associated marker genes were induced by transient overexpression (TOE) of CabZIP63, but decreased by that of CabZIP63-SRDX. Additionally, binding of CabZIP63 to pCaWRKY40 was up-regulated by RSI or HTHH, and the transcript level of CaWRKY40 and binding of CaWRKY40 to the promoters of CaPR1, CaNPR1, CaDEF1 and CaHSP24 were up-regulated by TOE of CabZIP63. On the other hand, CabZIP63 was also up-regulated transcriptionally by TOE of CaWRKY40. The data suggest collectively that CabZIP63 directly or indirectly regulates the expression of CaWRKY40 at both the transcriptional and post-transcriptional level, forming a positive feedback loop with CaWRKY40 during pepper's response to RSI or HTHH. Altogether, our data will help to elucidate the underlying mechanism of crosstalk between pepper's response to RSI and HTHH.

SGT1 is required in PcINF1/SRC2-1 induced pepper defense response by interacting with SRC2-1.

PcINF1 was previously found to induce pepper defense response by interacting with SRC2-1, but the underlying mechanism remains uninvestigated. Herein, we describe the involvement of SGT1 in the PcINF1/SRC2-1-induced immunity. SGT1 was observed to be up-regulated by Phytophthora capsici inoculation and synergistically transient overexpression of PcINF1/SRC2-1 in pepper plants. SGT1-silencing compromised HR cell death, blocked H2O2 accumulation, and downregulated HR-associated and hormones-dependent marker genes' expression triggered by PcINF1/SRC2-1 co-overexpression. The interaction between SRC2-1 and SGT1 was found by the yeast two hybrid system and was further confirmed by bimolecular fluorescence complementation and co-immunoprecipitation analyses. The SGT1/SRC2-1 interaction was enhanced by transient overexpression of PcINF1 and Phytophthora capsici inoculation, and SGT1-silencing attenuated PcINF1/SRC2-1 interaction. Additionally, by modulating subcellular localizations of SRC2-1, SGT1, and the interacting complex of SGT1/SRC2-1, it was revealed that exclusive nuclear targeting of the SGT1/SRC2-1 complex blocks immunity triggered by formation of SGT1/SRC2-1, and a translocation of the SGT1/SRC2-1 complex from the plasma membrane and cytoplasm to the nuclei upon the inoculation of P. capsici. Our data demonstrate that the SGT1/SRC2-1 interaction, and its nucleocytoplasmic partitioning, is involved in pepper's immunity against P. capsici, thus providing a molecular link between Ca(2+) signaling associated SRC2-1 and SGT1-mediated defense signaling.

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.