CaSK23, a Putative GSK3/SHAGGY-Like Kinase of Capsicum annuum, Acts as a Negative Regulator of Pepper’s Response to Ralstonia solanacearum Attack

GSK3-like kinases have been mainly implicated in the brassinosteroids (BR) pathway and, therefore, in plant growth, development, and responses to abiotic stresses; however, their roles in plant immunity remain poorly understood. Herein, we present evidence that CaSK23, a putative GSK3/SHAGGY-like kinase in pepper, acts as a negative regulator in pepper’s response to Ralstonia solanacearum (R. solanacearum) inoculation (RSI). Data from quantitative RT-PCR (qRT-PCR) showed that the constitutively-expressed CaSK23 in pepper leaves was down-regulated by RSI, as well as by exogenously-applied salicylic acid (SA) or methyl jasomonate (MeJA). Silencing of CaSK23 by virus-induced gene silencing (VIGS) decreased the susceptibility of pepper plants to RSI, coupled with up-regulation of the tested genes encoding SA-, JA-, and ethylene (ET)-dependent pathogenesis-related (PR) proteins. In contrast, ectopic overexpression (OE) of CaSK23 conferred a compromised resistance of tobacco plants to RSI, accompanied by down-regulation of the tested immunity-associated SA-, JA-, and ET-dependent PR genes. In addition, transient overexpression of CaSK23 in pepper plants consistently led to down-regulation of the tested SA-, JA-, and ET-dependent PR genes. We speculate that CaSK23 acts as a negative regulator in pepper immunity and its constitutive expression represses pepper immunity in the absence of pathogens. On the other hand, its decreased expression derepresses immunity when pepper plants are attacked by pathogens.

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CaAIL1 Acts Positively in Pepper Immunity against Ralstonia solanacearum by Repressing Negative Regulators

Plant and Cell Physiology ◽

10.1093/pcp/pcab125 ◽

2021 ◽

Author(s):

Yutong Zheng ◽

Shicong He ◽

Weiwei Cai ◽

Lei Shen ◽

Xueying Huang ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Ralstonia Solanacearum ◽

Plant Immunity ◽

Cis Elements ◽

Virus Induced Gene Silencing ◽

Mobility Shift ◽

Positive Role ◽

Genes Encoding ◽

Chromatin Immunoprecipitation Sequencing

Abstract APETALA2 (AP2) subfamily transcription factors participate in plant growth and development, but their roles in plant immunity remain unclear. Here, we discovered that the AP2 transcription factor CaAIL1 functions in immunity against Ralstonia solanacearum infection (RSI) in pepper (Capsicum annuum). CaAIL1 expression was upregulated by RSI, and loss- and gain-of-function assays using virus-induced gene silencing and transient overexpression, respectively, revealed that CaAIL1 plays a positive role in immunity to RSI in pepper. Chromatin immunoprecipitation sequencing (ChIP-seq) uncovered a subset of transcription-factor-encoding genes, including CaRAP2-7, CaGATA17, CaGtf3a and CaTCF25, that were directly targeted by CaAIL1 via their cis-elements, such as GT or AGGCA motifs. ChIP-qPCR and electrophoretic mobility shift assays confirmed these findings. These genes, encoding transcription factors with negative roles in immunity, were repressed by CaAIL1 during pepper response to RSI, whereas genes encoding positive immune regulators such as CaEAS were derepressed by CaAIL1. Importantly, we showed that the atypical EAR motif (LXXLXXLXX) in CaAIL1 is indispensable for its function in immunity. These findings indicate that CaAIL1 enhances the immunity of pepper against RSI by repressing a subset of negative immune regulators during the RSI response through its binding to several cis-elements in their promoters.

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Pepper CaMLO6 Negatively Regulates Ralstonia solanacearum Resistance and Positively Regulates High Temperature and High Humidity Responses

Plant and Cell Physiology ◽

10.1093/pcp/pcaa052 ◽

2020 ◽

Vol 61 (7) ◽

pp. 1223-1238

Author(s):

Sheng Yang ◽

Yuanyuan Shi ◽

Longyun Zou ◽

Jinfeng Huang ◽

Lei Shen ◽

...

Keyword(s):

High Temperature ◽

Ralstonia Solanacearum ◽

Negative Regulator ◽

Marker Genes ◽

Plant Responses ◽

Resistance Locus ◽

High Humidity ◽

Virus Induced Gene Silencing ◽

Transcriptional Responses ◽

Pepper Plants

Abstract Plant mildew-resistance locus O (MLO) proteins influence susceptibility to powdery mildew. However, their roles in plant responses to other pathogens and heat stress remain unclear. Here, we showed that CaMLO6, a pepper (Capsicum annuum) member of MLO clade V, is a protein targeted to plasma membrane and probably endoplasmic reticulum. The transcript expression level of CaMLO6 was upregulated in the roots and leaves of pepper plants challenged with high temperature and high humidity (HTHH) and was upregulated in leaves but downregulated in roots of plants infected with the bacterial pathogen Ralstonia solanacearum. CaMLO6 was also directly upregulated by CaWRKY40 upon HTHH but downregulated by CaWRKY40 upon R. solanacearum infection. Virus-induced gene silencing of CaMLO6 significantly decreased pepper HTHH tolerance and R. solanacearum susceptibility. Moreover, CaMLO6 overexpression enhanced the susceptibility of Nicotiana benthamiana and pepper plants to R. solanacearum and their tolerance to HTHH, effects that were associated with the expression of immunity- and thermotolerance-associated marker genes, respectively. These results suggest that CaMLO6 acts as a positive regulator in response to HTHH but a negative regulator in response to R. solanacearum. Moreover, CaMLO6 is transcriptionally affected by R. solanacearum and HTHH; these transcriptional responses are at least partially regulated by CaWRKY40.

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The Hypersensitive Induced Reaction and Leucine-Rich Repeat Proteins Regulate Plant Cell Death Associated with Disease and Plant Immunity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-10-0030 ◽

2011 ◽

Vol 24 (1) ◽

pp. 68-78 ◽

Cited By ~ 45

Author(s):

Hyong Woo Choi ◽

Young Jin Kim ◽

Byung Kook Hwang

Keyword(s):

Cell Death ◽

Xanthomonas Campestris ◽

Plant Immunity ◽

Yeast Cells ◽

Leucine Rich Repeat ◽

Virus Induced Gene Silencing ◽

Susceptible Cell ◽

Gene Transcripts ◽

Induced Reaction ◽

Pepper Plants

Pathogen-induced programmed cell death (PCD) is intimately linked with disease resistance and susceptibility. However, the molecular components regulating PCD, including hypersensitive and susceptible cell death, are largely unknown in plants. In this study, we show that pathogen-induced Capsicum annuum hypersensitive induced reaction 1 (CaHIR1) and leucine-rich repeat 1 (CaLRR1) function as distinct plant PCD regulators in pepper plants during Xanthomonas campestris pv. vesicatoria infection. Confocal microscopy and protein gel blot analyses revealed that CaLRR1 and CaHIR1 localize to the extracellular matrix and plasma membrane (PM), respectively. Bimolecular fluorescent complementation and coimmunoprecipitation assays showed that the extracellular CaLRR1 specifically binds to the PM-located CaHIR1 in pepper leaves. Overexpression of CaHIR1 triggered pathogen-independent cell death in pepper and Nicotiana benthamiana plants but not in yeast cells. Virus-induced gene silencing (VIGS) of CaLRR1 and CaHIR1 distinctly strengthened and compromised hypersensitive and susceptible cell death in pepper plants, respectively. Endogenous salicylic acid levels and pathogenesis-related gene transcripts were elevated in CaHIR1-silenced plants. VIGS of NbLRR1 and NbHIR1, the N. benthamiana orthologs of CaLRR1 and CaHIR1, regulated Bax- and avrPto-/Pto-induced PCD. Taken together, these results suggest that leucine-rich repeat and hypersensitive induced reaction proteins may act as cell-death regulators associated with plant immunity and disease.

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HvWRKY10, HvWRKY19, and HvWRKY28 Regulate Mla-Triggered Immunity and Basal Defense to Barley Powdery Mildew

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-04-12-0082-r ◽

2012 ◽

Vol 25 (11) ◽

pp. 1492-1505 ◽

Cited By ~ 22

Author(s):

Yan Meng ◽

Roger P. Wise

Keyword(s):

Powdery Mildew ◽

Time Course ◽

Expression Profiles ◽

Single Cells ◽

Direct Interaction ◽

Large Family ◽

Negative Regulator ◽

Virus Induced Gene Silencing ◽

Effector Triggered Immunity ◽

Transient Overexpression

WRKY proteins represent a large family of transcription factors (TF), involved in plant development and defense. In all, 60 unique barley TF have been annotated that contain the WRKY domain; 26 of these are represented on the Barley1 GeneChip. Time-course expression profiles of these 26 HvWRKY TF were analyzed to investigate their role in mildew locus a (Mla)-mediated immunity to Blumeria graminis f. sp. hordei, causal agent of powdery mildew disease. Inoculation-responsive, Mla-specified interactions with B. graminis f. sp. hordei revealed that 12 HvWRKY were differentially expressed: 10 highly upregulated and two significantly downregulated. Barley stripe mosaic virus-induced gene silencing of HvWRKY10, HvWRKY19, and HvWRKY28 compromised resistance-gene-mediated defense to powdery mildew in genotypes harboring both Rar1-dependent and Rar1-independent Mla alleles, indicating that these WRKY TF play key roles in effector-triggered immunity. Comprehensive yeast two-hybrid analyses, however, did not reveal a direct interaction between these three nuclear-localized WRKY TF and MLA. Transient overexpression of all three WRKY TF in single cells expressing Mlo, which encodes a negative regulator of penetration resistance, significantly decreased susceptibility. Taken together, these loss- and gain-of-function studies demonstrate that HvWRKY10, HvWRKY19, and HvWRKY28 positively regulate the barley transcriptome in response to invasion by B. graminis f. sp. hordei.

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Ca14-3-3 Interacts With CaWRKY58 to Positively Modulate Pepper Response to Low-Phosphorus Starvation

Frontiers in Plant Science ◽

10.3389/fpls.2020.607878 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jinsen Cai ◽

Weiwei Cai ◽

Xueying Huang ◽

Sheng Yang ◽

Jiayu Wen ◽

...

Keyword(s):

Ralstonia Solanacearum ◽

Phosphorus Deficiency ◽

Negative Regulator ◽

Bimolecular Fluorescence Complementation ◽

Virus Induced Gene Silencing ◽

Mobility Shift ◽

Natural Habitats ◽

Low Phosphorus ◽

Low Phosphorus Stress ◽

Electrophoretic Mobility Shift Assays

Low-phosphorus stress (LPS) and pathogen attack are two important stresses frequently experienced by plants in their natural habitats, but how plant respond to them coordinately remains under-investigated. Here, we demonstrate that CaWRKY58, a known negative regulator of the pepper (Capsicum annuum) response to attack by Ralstonia solanacearum, is upregulated by LPS. Virus-induced gene silencing (VIGS) and overexpression of CaWRKY58 in Nicotiana benthamiana plants in combination with chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) demonstrated that CaWRKY58 positively regulates the response of pepper to LPS by directly targeting and regulating genes related to phosphorus-deficiency tolerance, including PHOSPHATE STARVATION RESPONSE1 (PHR1). Yeast two-hybrid assays revealed that CaWRKY58 interacts with a 14-3-3 protein (Ca14-3-3); this interaction was confirmed by pull-down, bimolecular fluorescence complementation (BiFC), and microscale thermophoresis (MST) assays. The interaction between Ca14-3-3 and CaWRKY58 enhanced the activation of PHR1 expression by CaWRKY58, but did not affect the expression of the immunity-related genes CaNPR1 and CaDEF1, which are negatively regulated by CaWRKY58 in pepper upon Ralstonia solanacearum inoculation. Collectively, our data indicate that CaWRKY58 negatively regulates immunity against Ralstonia solanacearum, but positively regulates tolerance to LPS and that Ca14-3-3 transcriptionally activates CaWRKY58 in response to LPS.

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Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion

Journal of Bacteriology ◽

10.1128/jb.188.6.2027-2037.2006 ◽

2006 ◽

Vol 188 (6) ◽

pp. 2027-2037 ◽

Cited By ~ 83

Author(s):

Eva Brombacher ◽

Andrea Baratto ◽

Corinne Dorel ◽

Paolo Landini

Keyword(s):

Biofilm Formation ◽

Gene Expression Regulation ◽

Constitutive Expression ◽

Cell Aggregation ◽

Negative Regulator ◽

Cellulose Biosynthesis ◽

Transcription Activator ◽

E Coli ◽

Genes Encoding ◽

And Control

ABSTRACT Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene.

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Arabidopsis CALMODULIN-BINDING PROTEIN 60b plays dual roles in plant immunity

10.1101/2021.04.15.440066 ◽

2021 ◽

Author(s):

Weijie Huang ◽

Zhongshou Wu ◽

Hainan Tian ◽

Xin Li ◽

Yuelin Zhang

Keyword(s):

Systemic Acquired Resistance ◽

Binding Protein ◽

Plant Immunity ◽

Interleukin 1 ◽

Constitutive Expression ◽

Defense Responses ◽

Negative Regulator ◽

Loss Of Function ◽

Constitutive Defense ◽

Calmodulin Binding

AbstractArabidopsis SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g) are two master transcription factors that regulate many defense-related genes in plant immunity. They are required for immunity downstream of the receptor-like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2). Constitutive defense responses in the gain-of-function autoimmune snc2-1D mutant are modestly affected by either sard1 or cbp60g single mutants, but completely suppressed by the sard1 cbp60g double mutant. Here we report that CBP60b, another member of the CBP60 family, also functions as a positive regulator of SNC2-mediated immunity. Loss-of-function mutations of CBP60b suppress the constitutive expression of SARD1 and enhanced disease resistance in cbp60g-1 snc2-1D, whereas over-expression of CBP60b leads to elevated SARD1 expression and constitutive defense responses. In addition, transient expression of CBP60b in Nicotiana benthamiana activates the expression of the pSARD1::luciferase reporter gene. Chromatin immunoprecipitation assay further showed that CBP60b is recruited to the promoter region of SARD1, suggesting that it directly regulates SARD1 expression. Interestingly, knocking out CBP60b in the wild type background leads to ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-dependent autoimmunity, suggesting that CBP60b is required for the expression of a guardee/decoy or a negative regulator in immunity mediated by receptors carrying an N-terminal TIR (Toll-interleukin-1 receptor-like) domain.Significance statementArabidopsis SARD1 serves as a master transcription factor in plant immunity. In this study, we showed that CBP60b positively regulates SARD1 expression, and TIR signaling is activated when CBP60b is inactivated.

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Ectopic Expression of Tsi1 in Transgenic Hot Pepper Plants Enhances Host Resistance to Viral, Bacterial, and Oomycete Pathogens

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.10.983 ◽

2002 ◽

Vol 15 (10) ◽

pp. 983-989 ◽

Cited By ~ 64

Author(s):

Ryoung Shin ◽

Jeong Mee Park ◽

Jong-Min An ◽

Kyung-Hee Paek

Keyword(s):

Xanthomonas Campestris ◽

Phytophthora Capsici ◽

Regulatory Protein ◽

Ectopic Expression ◽

Constitutive Expression ◽

Bacterial Pathogen ◽

Hot Pepper ◽

Pr Genes ◽

Transcriptional Regulatory ◽

Pepper Plants

In many plants, including hot pepper plants, productivity is greatly affected by pathogen attack. We reported previously that tobacco stress-induced gene 1 (Tsi1) may play an important role in regulating stress responsive genes and pathogenesis-related (PR) genes. In this study, we demonstrated that overexpression of Tsi1 gene in transgenic hot pepper plants induced constitutive expression of several PR genes in the absence of stress or pathogen treatment. The transgenic hot pepper plants expressing Tsi1 exhibited resistance to Pepper mild mottle virus (PMMV) and Cucumber mosaic virus (CMV). Furthermore, these transgenic plants showed increased resistance to a bacterial pathogen, Xanthomonas campestris pv. vesicatoria and also an oomycete pathogen, Phytophthora capsici. These results suggested that ectopic expression of Tsi1 in transgenic hot pepper plants enhanced the resistance of the plants to various pathogens, including viruses, bacteria, and oomycete. These results suggest that using transcriptional regulatory protein genes may contribute to developing broad-spectrum resistance in crop plants.

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CaCBL1 Acts as a Positive Regulator in Pepper Response to Ralstonia solanacearum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-19-0241-r ◽

2020 ◽

Vol 33 (7) ◽

pp. 945-957 ◽

Cited By ~ 1

Author(s):

Lei Shen ◽

Sheng Yang ◽

Feng Yang ◽

Deyi Guan ◽

Shuilin He

Keyword(s):

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Marker Genes ◽

Virus Induced Gene Silencing ◽

Mobility Shift ◽

Positive Regulator ◽

Positive Regulators ◽

Mobility Shift Assay ◽

Transient Overexpression ◽

Important Disease

Bacterial wilt caused by Ralstonia solanacearum is an important disease of pepper (Capsicum annuum), an economically important solanaceous vegetable worldwide, in particular, under high temperature (HT) conditions. However, the molecular mechanism underlying pepper immunity against bacterial wilt remains poorly understood. Herein, CaCBL1, a putative calcineurin B-like protein, was functionally characterized in the pepper response to R. solanacearum inoculation (RSI) under HT (RSI/HT). CaCBL1 was significantly upregulated by RSI at room temperature (RSI/RT), HT, or RSI/HT. CaCBL1-GFP fused protein targeted to whole epidermal cells of Nicotiana benthamiana when transiently overexpressed. CaCBL1 silencing by virus-induced gene silencing significantly enhanced pepper susceptibility to RSI under RT or HT, while its transient overexpression triggered hypersensitive response mimic cell death and upregulation of immunity-associated marker genes, including CabZIP63, CaWRKY40, and CaCDPK15, the positive regulators in the pepper response to RSI or HT found in our previous studies. In addition, by chromatin immunoprecipitation PCR and electrophoretic mobility shift assay, CaCBL1 was found to be directly targeted by CaWRKY40, although not by CaWRKY27 or CaWRKY58, via the W-box-2 within its promoter, and its transcription was found to be downregulated by silencing of CaWRKY40 while it was enhanced by its transient overexpression. These results suggest that CaCBL1 acts as a positive regulator in pepper immunity against R. solanacearum infection, constituting a positive feedback loop with CaWRKY40.

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The Pepper Mitogen-Activated Protein Kinase CaMAPK7 Acts as a Positive Regulator in Response to Ralstonia solanacearum Infection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.664926 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lanping Shi ◽

Kan Zhang ◽

Linjing Xie ◽

Mingxing Yang ◽

Baixue Xie ◽

...

Keyword(s):

Protein Kinase ◽

Ralstonia Solanacearum ◽

Target Genes ◽

Vital Role ◽

Mitogen Activated Protein Kinase ◽

Marker Genes ◽

Virus Induced Gene Silencing ◽

Mitogen Activated Protein ◽

Pepper Genome ◽

Pepper Plants

Mitogen-activated protein kinase (MAPK) pathways play a vital role in multiple plant processes, including growth, development, and stress signaling, but their involvement in response to Ralstonia solanacearum is poorly understood, particularly in pepper plants. Herein, CaMAPK7 was identified from the pepper genome and functionally analyzed. The accumulations of CaMAPK7 transcripts and promoter activities were both significantly induced in response to R. solanacearum strain FJC100301 infection, and exogenously applied phytohormones, including methyl jasmonate (MeJA), brassinolide (BR), salicylic acid (SA), and ethephon (ETN), were decreased by abscisic acid (ABA) treatment. Virus-induced gene silencing (VIGS) of CaMAPK7 significantly enhanced the susceptibility of pepper plants to infection by R. solanacearum and downregulated the defense-related marker genes, including CaDEF1, CaPO2, CaSAR82A, and CaWRKY40. In contrast, the ectopic overexpression of CaMAPK7 in transgenic tobacco enhanced resistance to R. solanacearum and upregulated the defense-associated marker genes, including NtHSR201, NtHSR203, NtPR4, PR1a/c, NtPR1b, NtCAT1, and NtACC. Furthermore, transient overexpression of CaMAPK7 in pepper leaves triggered intensive hypersensitive response (HR)-like cell death, H2O2 accumulation, and enriched CaWRKY40 at the promoters of its target genes and drove their transcript accumulations, including CaDEF1, CaPO2, and CaSAR82A. Taken together, these data indicate that R. solanacearum infection induced the expression of CaMAPK7, which indirectly modifies the binding of CaWRKY40 to its downstream targets, including CaDEF1, CaPO2, and CaSAR82A, ultimately leading to the activation of pepper immunity against R. solanacearum. The protein that responds to CaMAPK7 in pepper plants should be isolated in the future to build a signaling bridge between CaMAPK7 and CaWRKY40.

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