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CabZIP63 and CaWRKY40 were previously found to be shared in the pepper defense response to high temperature stress (HTS) and to Ralstonia solanacearum inoculation (RSI), forming a transcriptional cascade. However, how they activate the two distinct defense responses is not fully understood. Herein, using a revised genetic approach, we functionally characterized CabZIP23 in the CabZIP63–CaWRKY40 cascade and its context specific pepper immunity activation against RSI by interaction with CabZIP63. CabZIP23 was originally found by immunoprecipitation-mass spectrometry to be an interacting protein of CabZIP63-GFP; it was upregulated by RSI and acted positively in pepper immunity against RSI by virus induced gene silencing in pepper plants, and transient overexpression in Nicotiana benthamiana plants. By chromatin immunoprecipitation (ChIP)-qPCR and electrophoresis mobility shift assay (EMSA), CabZIP23 was found to be directly regulated by CaWRKY40, and CabZIP63 was directly regulated by CabZIP23, forming a positive feedback loop. CabZIP23–CabZIP63 interaction was confirmed by co-immunoprecipitation (CoIP) and bimolecular fluorescent complimentary (BiFC) assays, which promoted CabZIP63 binding immunity related target genes, including CaPR1, CaNPR1 and CaWRKY40, thereby enhancing pepper immunity against RSI, but not affecting the expression of thermotolerance related CaHSP24. All these data appear to show that CabZIP23 integrates in the CabZIP63–CaWRKY40 cascade and the context specifically turns it on mounting pepper immunity against RSI.

CabZIP63 acts positively in the resistance of pepper (Capsicum annuum) to bacterial wilt caused by Ralstonia solanacearum or tolerance to high-temperature/high-humidity stress, but it is unclear how CabZIP63 achieves its functional specificity against R. solanacearum. Here, CaASR1, an abscisic acid-, stress-, and ripening-inducible protein of C. annuum , was functionally characterized in modulating the functional specificity of CabZIP63 during the defense response of pepper to R. solanacearum. In pepper plants inoculated with R. solanacearum , CaASR1 was up-regulated before 24 h post-inoculation but down-regulated thereafter, and was down-regulated by high-temperature/high-humidity stress. Data from gene silencing and transient overexpression experiments indicated that CaASR1 acts as a positive regulator in the immunity of pepper against R. solanacearum and a negative regulator of thermotolerance. Pull-down combined with mass spectrometry revealed that CaASR1 interacted with CabZIP63 upon R. solanacearum infection; the interaction was confirmed by microscale thermophoresis and bimolecular fluorescence complementation assays. CaASR1 silencing upon R. solanacearum inoculation repressed CabZIP63-mediated transcription from the promoters of the salicylic acid (SA)-dependent CaPR1 and CaNPR1 , but derepressed transcription of CaHSP24 and the jasmonic acid (JA)-dependent CaDEF1. Our findings suggest that CaASR1 acts as a positive regulator of the defense response of pepper to R. solanacearum by interacting with CabZIP63, enabling it to promote SA-dependent but repress JA-dependent immunity and thermotolerance during the early stages of infection. [ABSTRACT FROM AUTHOR]

Ca2+-signaling—which requires the presence of calcium sensors such as calmodulin (CaM) and calmodulin-like (CML) proteins—is crucial for the regulation of plant immunity against pathogen attack. However, the underlying mechanisms remain elusive, especially the roles of CMLs involved in plant immunity remains largely uninvestigated. In the present study, CaCML13, a calmodulin-like protein of pepper that was originally found to be upregulated by Ralstonia solanacearum inoculation (RSI) in RNA-seq, was functionally characterized in immunity against RSI. CaCML13 was found to target the whole epidermal cell including plasma membrane, cytoplasm and nucleus. We also confirmed that CaCML13 was upregulated by RSI in pepper roots by quantitative real-time PCR (qRT-PCR). The silencing of CaCML13 significantly enhanced pepper plants’ susceptibility to RSI accompanied with downregulation of immunity-related CaPR1, CaNPR1, CaDEF1 and CabZIP63. In contrast, CaCML13 transient overexpression induced clear hypersensitivity-reaction (HR)-mimicked cell death and upregulation of the tested immunity-related genes. In addition, we also revealed that the G-box-containing CaCML13 promoter was bound by CabZIP63 and CaCML13 was positively regulated by CabZIP63 at transcriptional level. Our data collectively indicate that CaCML13 act as a positive regulator in pepper immunity against RSI forming a positive feedback loop with CabZIP63.

CaWRKY40 is known to act as a positive regulator in the response of pepper (Capsicum annuum) to Ralstonia solan-acearum 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 β-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. [ABSTRACT FROM AUTHOR]

CabZIP63 and CaWRKY40 were previously found to be shared in the pepper defense response to high temperature stress (HTS) and to Ralstonia solanacearum inoculation (RSI), forming a transcriptional cascade. However, how they activate the two distinct defense responses is not fully understood. Herein, using a revised genetic approach, we functionally characterized CabZIP23 in the CabZIP63–CaWRKY40 cascade and its context specific pepper immunity activation against RSI by interaction with CabZIP63. CabZIP23 was originally found by immunoprecipitation-mass spectrometry to be an interacting protein of CabZIP63-GFP; it was upregulated by RSI and acted positively in pepper immunity against RSI by virus induced gene silencing in pepper plants, and transient overexpression in Nicotiana benthamiana plants. By chromatin immunoprecipitation (ChIP)-qPCR and electrophoresis mobility shift assay (EMSA), CabZIP23 was found to be directly regulated by CaWRKY40, and CabZIP63 was directly regulated by CabZIP23, forming a positive feedback loop. CabZIP23–CabZIP63 interaction was confirmed by co-immunoprecipitation (CoIP) and bimolecular fluorescent complimentary (BiFC) assays, which promoted CabZIP63 binding immunity related target genes, including CaPR1, CaNPR1 and CaWRKY40, thereby enhancing pepper immunity against RSI, but not affecting the expression of thermotolerance related CaHSP24. All these data appear to show that CabZIP23 integrates in the CabZIP63–CaWRKY40 cascade and the context specifically turns it on mounting pepper immunity against RSI. [ABSTRACT FROM AUTHOR]

Ca2+-signaling—which requires the presence of calcium sensors such as calmodulin (CaM) and calmodulin-like (CML) proteins—is crucial for the regulation of plant immunity against pathogen attack. However, the underlying mechanisms remain elusive, especially the roles of CMLs involved in plant immunity remains largely uninvestigated. In the present study, CaCML13, a calmodulin-like protein of pepper that was originally found to be upregulated by Ralstonia solanacearum inoculation (RSI) in RNA-seq, was functionally characterized in immunity against RSI. CaCML13 was found to target the whole epidermal cell including plasma membrane, cytoplasm and nucleus. We also confirmed that CaCML13 was upregulated by RSI in pepper roots by quantitative real-time PCR (qRT-PCR). The silencing of CaCML13 significantly enhanced pepper plants' susceptibility to RSI accompanied with downregulation of immunity-related CaPR1, CaNPR1, CaDEF1 and CabZIP63. In contrast, CaCML13 transient overexpression induced clear hypersensitivity-reaction (HR)-mimicked cell death and upregulation of the tested immunity-related genes. In addition, we also revealed that the G-box-containing CaCML13 promoter was bound by CabZIP63 and CaCML13 was positively regulated by CabZIP63 at transcriptional level. Our data collectively indicate that CaCML13 act as a positive regulator in pepper immunity against RSI forming a positive feedback loop with CabZIP63. [ABSTRACT FROM AUTHOR]

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 β-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., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)