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

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.

scholarly journals A cysteine-rich receptor-like protein kinase CaCKR5 modulates immune response against Ralstonia solanacearum infection in pepper

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shaoliang Mou ◽  
Qianqian Meng ◽  
Feng Gao ◽  
Tingting Zhang ◽  
Weihong He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ralstonia Solanacearum ◽  
Green Fluorescence Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Pcr Analysis ◽  
Virus Induced Gene Silencing ◽  
Mobility Shift ◽  
Positive Role ◽  
Physiological Processes ◽  
Mobility Shift Assay

Abstract Background Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and play vital roles in diverse physiological processes in regulating plant growth and development. Results CaCRK5 transcripts were induced in pepper upon the infection of Ralstonia solanacearum and treatment with salicylic acid. The fusions between CaCRK5 and green fluorescence protein were targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing (VIGS) made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied with decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 increased resistance against R. solanacearum in Nicotiana benthamiana. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation coupled with quantitative real-time PCR analysis revealed that a homeodomain zipper I protein CaHDZ27 can active the expression of CaCRK5 through directly binding to its promoter. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses suggested that CaCRK5 heterodimerized with the homologous member CaCRK6 on the plasma membrane. Conclusions Our data revealed that CaCRK5 played a positive role in regulating immune responses against R. solanacearum infection in pepper.

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

2018 ◽  
Vol 19 (9) ◽  
pp. 2698 ◽  
Author(s):  
Ailian Qiu ◽  
Ji Wu ◽  
Yufen Lei ◽  
Yiting Cai ◽  
Song Wang ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Plant Immunity ◽  
Constitutive Expression ◽  
Negative Regulator ◽  
Virus Induced Gene Silencing ◽  
Pr Genes ◽  
Down Regulation ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Pepper Plants

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.

scholarly journals The LysR-Type Transcriptional Regulator CrgA Negatively Regulates the Flagellar Master Regulator flhDC in Ralstonia solanacearum GMI1000

2020 ◽  
Vol 203 (1) ◽  
Author(s):  
Xiaojing Fan ◽  
Zhiwen Zhao ◽  
Tingyan Sun ◽  
Wei Rou ◽  
Caiying Gui ◽  
...  
Keyword(s):  
Cell Motility ◽  
Ralstonia Solanacearum ◽  
Host Plants ◽  
Transcriptional Regulator ◽  
Growth Defect ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Gus Reporter ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC. In addition, the ΔcrgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC. Despite these alterations, the ΔcrgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the β-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato (Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants. IMPORTANCE Ralstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum because it allows this pathogen to efficiently invade and colonize host plants. In R. solanacearum, motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum.

scholarly journals A Cysteine-Rich Receptor-Like Protein Kinase CaCKR5 Contributes to Immune Defense Against Ralstonia Solanacearum in Pepper

2020 ◽  
Author(s):  
Shaoliang Mou ◽  
Feng Gao ◽  
Tingting Zhang ◽  
Weihong He ◽  
Qianqian Meng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescence Protein ◽  
Immune Defense ◽  
Bimolecular Fluorescence Complementation ◽  
Pcr Analysis ◽  
Virus Induced Gene Silencing ◽  
Mobility Shift ◽  
Positive Role ◽  
Physiological Processes ◽  
Mobility Shift Assay

Abstract Background: Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and have important roles in numerous different physiological processes in plants. Results: CaCRK5 transcripts were observed to accumulate after the inoculation of R. solanacearum and treatment with salicylic acid. The fusion between CaCRK5 and the green fluorescence protein was targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied by decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 in tobacco conferred increased resistance against R. solanacearum. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation with quantitative real-time PCR analysis verified that a homeodomain zipper I protein CaHDZ27 can bind to the CaCRK5 promoter, and directly active its expression. Yeast two-hybrid and bimolecular fluorescence complementation analyses suggested that CaCRK5 could heterodimerize with the homologous member CaCRK6 in the plasma membrane. Conclusions: Our data indicated that CaCRK5 played a positive role in pepper resistance against R. solanacearum infection.

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

2020 ◽  
Vol 33 (7) ◽  
pp. 945-957 ◽  
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.

scholarly journals A NAC transcription factor and its interaction protein hinder abscisic acid biosynthesis by synergistically repressing NCED5 in Citrus reticulata

2020 ◽  
Vol 71 (12) ◽  
pp. 3613-3625
Author(s):  
Feng Zhu ◽  
Tao Luo ◽  
Chaoyang Liu ◽  
Yang Wang ◽  
Li Zheng ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Abscisic Acid ◽  
Citrus Fruit ◽  
Bimolecular Fluorescence Complementation ◽  
Citrus Reticulata ◽  
Mobility Shift ◽  
Abscisic Acid Biosynthesis ◽  
Multiple Transcription Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Aba Biosynthesis

Abstract Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5.

scholarly journals CaHDZ27, a Homeodomain-Leucine Zipper I Protein, Positively Regulates the Resistance to Ralstonia solanacearum Infection in Pepper

2017 ◽  
Vol 30 (12) ◽  
pp. 960-973 ◽  
Author(s):  
Shaoliang Mou ◽  
Zhiqin Liu ◽  
Feng Gao ◽  
Sheng Yang ◽  
Meixia Su ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Plant Immunity ◽  
Bimolecular Fluorescence Complementation ◽  
Marker Genes ◽  
Plant Responses ◽  
Dependent Manner ◽  
Virus Induced Gene Silencing ◽  
Green Fluorescent

Homeodomain-leucine zipper class I (HD-Zip I) transcription factors have been functionally characterized in plant responses to abiotic stresses, but their roles in plant immunity are poorly understood. Here, a HD-Zip I gene, CaHZ27, was isolated from pepper (Capsicum annum) and characterized for its role in pepper immunity. Quantitative real-time polymerase chain reaction showed that CaHDZ27 was transcriptionally induced by Ralstonia solanacearum inoculation and exogenous application of methyl jasmonate, salicylic acid, or ethephon. The CaHDZ27-green fluorescent protein fused protein was targeted exclusively to the nucleus. Chromatin immunoprecipitation demonstrated that CaHDZ27 bound to the 9-bp pseudopalindromic element (CAATAATTG) and triggered β-glucuronidase expression in a CAATAATTG-dependent manner. Virus-induced gene silencing of CaHDZ27 significantly attenuated the resistance of pepper plants against R. solanacearum and downregulated defense-related marker genes, including CaHIR1, CaACO1, CaPR1, CaPR4, CaPO2, and CaBPR1. By contrast, transient overexpression of CaHDZ27 triggered strong cell death mediated by the hypersensitive response and upregulated the tested immunity-associated marker genes. Ectopic CaHDZ27 expression in tobacco enhances its resistance against R. solanacearum. These results collectively suggest that CaHDZ27 functions as a positive regulator in pepper resistance against R. solanacearum. Bimolecular fluorescence complementation and coimmunoprecipitation assays indicate that CaHDZ27 monomers bind with each other, and this binding is enhanced significantly by R. solanacearum inoculation. We speculate that homodimerization of CaHZ27 might play a role in pepper response to R. solanacearum, further direct evidence is required to confirm it.

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

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.

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

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.

Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

1995 ◽  
Vol 73 (01) ◽  
pp. 039-048 ◽  
Author(s):  
A Bierhaus ◽  
Ch J Hemmer ◽  
N Mackman ◽  
R Kutob ◽  
R Ziegler ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Falciparum Malaria ◽  
De Novo ◽  
Neutralizing Antibody ◽  
Mobility Shift ◽  
Before And After ◽  
Tf Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Antiparasitic Treatment ◽  
Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

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