Ralstonia Solanacearum Inoculation Research Articles

Silicon Application for the Modulation of Rhizosphere Soil Bacterial Community Structures and Metabolite Profiles in Peanut under Ralstonia solanacearum Inoculation.

Silicon (Si) has been shown to promote peanut growth and yield, but whether Si can enhance the resistance against peanut bacterial wilt (PBW) caused by Ralstonia solanacearum, identified as a soil-borne pathogen, is still unclear. A question regarding whether Si enhances the resistance of PBW is still unclear. Here, an in vitro R. solanacearum inoculation experiment was conducted to study the effects of Si application on the disease severity and phenotype of peanuts, as well as the microbial ecology of the rhizosphere. Results revealed that Si treatment significantly reduced the disease rate, with a decrement PBW severity of 37.50% as compared to non-Si treatment. The soil available Si (ASi) significantly increased by 13.62-44.87%, and catalase activity improved by 3.01-3.10%, which displayed obvious discrimination between non-Si and Si treatments. Furthermore, the rhizosphere soil bacterial community structures and metabolite profiles dramatically changed under Si treatment. Three significantly changed bacterial taxa were observed, which showed significant abundance under Si treatment, whereas the genus Ralstonia genus was significantly suppressed by Si. Similarly, nine differential metabolites were identified to involve into unsaturated fatty acids via a biosynthesis pathway. Significant correlations were also displayed between soil physiochemical properties and enzymes, the bacterial community, and the differential metabolites by pairwise comparisons. Overall, this study reports that Si application mediated the evolution of soil physicochemical properties, the bacterial community, and metabolite profiles in the soil rhizosphere, which significantly affects the colonization of the Ralstonia genus and provides a new theoretical basis for Si application in PBW prevention.

CabZIP23 Integrates in CabZIP63-CaWRKY40 Cascade and Turns CabZIP63 on Mounting Pepper Immunity against Ralstonia solanacearum via Physical Interaction.

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.

CaWRKY30 Positively Regulates Pepper Immunity by Targeting CaWRKY40 against Ralstonia solanacearum Inoculation through Modulating Defense-Related Genes.

The WRKY transcription factors (TFs) network is composed of WRKY TFs’ subset, which performs a critical role in immunity regulation of plants. However, functions of WRKY TFs’ network remain unclear, particularly in non-model plants such as pepper (Capsicum annuum L.). This study functionally characterized CaWRKY30—a member of group III Pepper WRKY protein—for immunity of pepper against Ralstonia solanacearum infection. The CaWRKY30 was detected in nucleus, and its transcriptional expression levels were significantly upregulated by R. solanacearum inoculation (RSI), and foliar application ethylene (ET), abscisic acid (ABA), and salicylic acid (SA). Virus induced gene silencing (VIGS) of CaWRKY30 amplified pepper’s vulnerability to RSI. Additionally, the silencing of CaWRKY30 by VIGS compromised HR-like cell death triggered by RSI and downregulated defense-associated marker genes, like CaPR1, CaNPR1, CaDEF1, CaABR1, CaHIR1, and CaWRKY40. Conversely, transient over-expression of CaWRKY30 in pepper leaves instigated HR-like cell death and upregulated defense-related maker genes. Furthermore, transient over-expression of CaWRKY30 upregulated transcriptional levels of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. On the other hand, transient over-expression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 upregulated transcriptional expression levels of CaWRKY30. The results recommend that newly characterized CaWRKY30 positively regulates pepper’s immunity against Ralstonia attack, which is governed by synergistically mediated signaling by phytohormones like ET, ABA, and SA, and transcriptionally assimilating into WRKY TFs networks, consisting of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. Collectively, our data will facilitate to explicate the underlying mechanism of crosstalk between pepper’s immunity and response to RSI.

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

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.

A conserved double-W box in the promoter of CaWRKY40 mediates autoregulation during response to pathogen attack and heat stress in pepper.

CaWRKY40 was previously found to be transcriptionally up‐regulated by Ralstonia solanacearum inoculation (RSI) or heat stress (HS), but the underlying mechanism remains unknown. Herein, we report that a double‐W box‐element (DWE) in the promoter of CaWRKY40 is critical for these responses. The upstream W box unit WI of this composite element is crucial for preferential binding by CaWRKY40 and responsiveness to RSI or HS. DWE‐driven CaWRKY40 can be transcriptionally and nonspecifically regulated by itself and by CaWRKY58 and CaWRKY27. The DWE was also found in the promoters of CaWRKY40 orthologs, including AtWRKY40, VvWRKY40, GmWRKY40, CplWRKY40, SaWRKY40, SpWRKY40, NtWRKY40, and NaWRKY40. DWEAtWRKY40 was analogous to DWECaWRKY40 by responding to RSI or HS and AtWRKY40 expression. These data suggest that a conserved response of plants to pathogen infection or HS is probably mediated by binding of the DWE by WRKY40.

CaCML13 Acts Positively in Pepper Immunity Against Ralstonia solanacearum Infection Forming Feedback Loop with CabZIP63

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.

Culturing Simpler and Bacterial Wilt Suppressive Microbial Communities from Tomato Rhizosphere.

Plant phenotype is affected by a community of associated microorganisms which requires dissection of the functional fraction. In this study, we aimed to culture the functionally active fraction of an upland soil microbiome, which can suppress tomato bacterial wilt. The microbiome fraction (MF) from the rhizosphere of Hawaii 7996 treated with an upland soil or forest soil MF was successively cultured in a designed modified M9 (MM9) medium partially mimicking the nutrient composition of tomato root exudates. Bacterial cells were harvested to amplify V3 and V4 regions of 16S rRNA gene for QIIME based sequence analysis and were also treated to Hawaii 7996 prior to Ralstonia solanacearum inoculation. The disease progress indicated that the upland MM9 1st transfer suppressed the bacterial wilt. Community analysis revealed that species richness was declined by successive cultivation of the MF. The upland MM9 1st transfer harbored population of phylum Proteobacteria (98.12%), Bacteriodetes (0.69%), Firmicutes (0.51%), Actinobacteria (0.08%), unidentified (0.54%), Cyanobacteria (0.01%), FBP (0.001%), OD1 (0.001%), Acidobacteria (0.005%). The family Enterobacteriaceae of Proteobacteria was the dominant member (86.76%) of the total population of which genus Enterobacter composed 86.76% making it a potential candidate to suppress bacterial wilt. The results suggest that this mixed culture approach is feasible to harvest microorganisms which may function as biocontrol agents.

Cloning and expression analysis of the StCUL1 gene in potato

The Cullin-RING E3 ubiquitin ligase (CRL) complex is the most common E3 ligase, and the SCF complex (CRL1) has the most diverse functions. Cullin1(CUL1) is a scaffolding protein for assembly of the complex. SCF has been shown to participate in the non-biological stress response pathways. In this study, a classic CUL1 protein was identified in Solanum tuberosum, StCUL1. A full-length cDNA of the StCUL1 gene was obtained from ED13 (a potato variety) by Ralstonia solanacearum inoculation using the RACE method. Sequence analysis indicated that the gene comprised 2662 bp, with an open reading frame of 2229 bp encoding 743 amino acids. The expression levels of the StCUL1 gene in potato treated with R. solanacearum and exogenous hormones (such as salicylic acid, jasmonic acid methyl ester and abscisic acid) at different time points were determined by real-time PCR. The results indicated that StCUL1 was induced not only by pathogenic bacteria, but also by exogenous hormones, with sustained high expression. However, there were some differences in the modes of expression. Tissue localization analysis indicated that its expression was tissue specific, and it was mainly in the phloem of the vascular system of stems and leaves.

CabZIP53 is targeted by CaWRKY40 and act as positive regulator in pepper defense against Ralstonia solanacearum and thermotolerance

Broad range transcriptional changes after pathogen invasion decide immune responses to these invaders. The responses of Capsicum annuum plants to bacterial pathogen Ralstonia solanacearum and high-temperature-high-humidity (HTHH) conditions are coordinated by different transcription factors. However, the basic molecular mechanism is not clear. The functions of different Capsicum annuum bZIP transcription factors (TFs) in plant defense are obscured in spite of their great importance in responses to biotic stress. In present study, we functionally characterized a nucleus localized bZIP TF, CabZIP53, for its involvement in pepper defense to Ralstonia solanacearum inoculation (RSI) and HTHH. CabZIP53 is transcriptionally induced in pepper plants by RSI or HTHH. Loss of function studies by Virus-induced gene silencing (VIGS) of CabZIP53 showed significantly impaired pepper basal defense against RSI, increased pathogen growth, decreased HTHH tolerance and hampered expression of immunity- and thermotolerance-associated marker genes such as CaHIR1, CaPR1, CaNPR1, CaABR1, CabZIP63 and CaHSP24 respectively. Similarly, transient overexpression of CabZIP53 mediated by Agrobacterium cells harbouring 35S::CabZIP53-HA in pepper leaves induced hypersensitive response (HR)-like mimic cell death, H2O2 accretion and up-regulated the immunity/thermotoelrance associated marker genes as well as CaWRKY6 CaWRKY40, CabZIP63 and CaCDPK15. qRT-PCR analysis revealed that CabZIP53 makes a positive feed back loop with CaWRKY40. In parallel, we determined that CabZIP53 was regulated directly by CaWRKY40 but indirectly by CaWRKY6. On the whole, our results suggest that CabZIP53 positively regulates plant immunity to RSI and tolerance against HTHH by means of transcriptional networks and positive feedback loops involving CaWRKY40.

CaWRKY22 Acts as a Positive Regulator in Pepper Response to RalstoniaSolanacearum by Constituting Networks with CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58.

The WRKY web, which is comprised of a subset of WRKY transcription factors (TFs), plays a crucial role in the regulation of plant immunity, however, the mode of organization and operation of this network remains obscure, especially in non-model plants such as pepper (Capsicum annuum). Herein, CaWRKY22, a member of a subgroup of IIe WRKY proteins from pepper, was functionally characterized in pepper immunity against Ralstonia Solanacearum. CaWRKY22 was found to target the nuclei, and its transcript level was significantly upregulated by Ralstonia Solanacearum inoculation (RSI) and exogenously applied salicylic acid (SA), Methyl jasmonate (MeJA), or ethephon (ETH). Loss-of-function CaWRKY22, caused by virus-induced gene silencing (VIGS), enhanced pepper’s susceptibility to RSI. In addition, the silencing of CaWRKY22 perturbed the hypersensitive response (HR)-like cell death elicited by RSI and downregulated defense-related genes including CaPO2, CaPR4, CaACC, CaBPR1, CaDEF1, CaHIR1, and CaWRKY40. CaWRKY22 was found to directly bind to the promoters of CaPR1, CaDEF1, and CaWRKY40 by chromatin immuno-precipitation (ChIP) analysis. Contrastingly, transient overexpression of CaWRKY22 in pepper leaves triggered significant HR-like cell death and upregulated the tested immunity associated maker genes. Moreover, the transient overexpression of CaWRKY22 upregulated the expression of CaWRKY6 and CaWRKY27 while it downregulated of the expression of CaWRKY58. Conversely, the transient overexpression of CaWRKY6, CaWRKY27, and CaWRKY40 upregulated the expression of CaWRKY22, while transient overexpression of CaWRKY58 downregulated the transcript levels of CaWRKY22. These data collectively recommend the role of CaWRKY22 as a positive regulator of pepper immunity against R. Solanacearum, which is regulated by signaling synergistically mediated by SA, jasmonic acid (JA), and ethylene (ET), integrating into WRKY networks with WRKY TFs including CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58.

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.

Expression and functional evaluation of CaZNF830 during pepper response to Ralstonia solanacearum or high temperature and humidity

Extensive transcriptional reprogramming after pathogen attack determines immunity to these invaders and plant development. Zinc finger (ZNF) transcription factors regulate important processes in plants such as development, vegetative activities and plant immunity. Despite their immense significance, majority of ZNF transcription factors (TF) involved in pepper immunity and resistance to heat stress have not been focused much. Herein, we identified and functionally characterized CaZNF830 in pepper defense against Ralstonia solanacearum inoculation (RSI) and tolerance to high temperature and high humidity (HTHH). Transient expression analysis of CaZNF830-GFP fusion protein in tobacco leaves revealed its localization to the nucleus. Transcription of CaZNF830 is induced in pepper plants upon RSI or HTHH. Consistent with this, fluorometric GUS enzymatic assay driven by pCaZNF830 presented significantly enhanced activity under RSI and HTHH in comparison with the control plants. The silencing of CaZNF830 by virus induced gene silencing (VIGS) significantly compromised pepper immunity against RSI with enhanced growth of Ralstonia solanacearum in pepper plants. Silencing of CaZNF830 also impaired tolerance to HTHH coupled with decreased expression levels of immunity and thermo-tolerance associated marker genes including CaHIR1, CaNPR1, CaPR1, CaABR1 and CaHSP24. By contrast, the transient over-expression of CaZNF830 in pepper leaves by infiltration of GV3101 cells containing 35S::CaZNF830-HA induced HR mimic cell death, H2O2 accumulation and activated the transcriptions of the tested defense-relative or thermo-tolerance associated marker genes. RT-PCR and immune-blotting assay confirmed the stable expression of HA-tagged CaZNF830 mRNA and protein in pepper. All these results suggest that CaZNF830 acts as a positive regulator of plant immunity against RSI or tolerance to HTHH, it is induced by RSI or HTHH and consequently activate pepper immunity against RSI or tolerance to HTHH by directly or indirectly transcriptional modulation of many defense-linked genes.

Overexpression of NtPR-Q Up-Regulates Multiple Defense-Related Genes in Nicotiana tabacum and Enhances Plant Resistance to Ralstonia solanacearum.

Various classes of plant pathogenesis-related proteins have been identified in the past several decades. PR-Q, a member of the PR3 family encoding chitinases, has played an important role in regulating plant resistance and preventing pathogen infection. In this paper, we functionally characterized NtPR-Q in tobacco plants and found that the overexpression of NtPR-Q in tobacco Yunyan87 resulted in higher resistance to Ralstonia solanacearum inoculation. Surprisingly, overexpression of NtPR-Q led to the activation of many defense-related genes, such as salicylic acid (SA)-responsive genes NtPR1a/c, NtPR2 and NtCHN50, JA-responsive gene NtPR1b and ET production-associated genes NtACC Oxidase and NtEFE26. Consistent with the role of NtPR-Q in multiple stress responses, NtPR-Q transcripts were induced by the exogenous hormones SA, ethylene and methyl jasmonate, which could enhance the resistance of tobacco to R. solanacearum. Collectively, our results suggested that NtPR-Q overexpression led to the up-regulation of defense-related genes and enhanced plant resistance to R. solanacearum infection.

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

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.

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 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.

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.

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.

Avaliação da resistência do eucalipto à murcha-bacteriana causada por Ralstonia solanacearum

O plantio de clones de eucalipto resistentes constitui a principal estratégia para o controle de doenças no campo. Assim, este trabalho objetivou testar métodos de inoculação de Ralstonia solanacearum visando selecionar eucalipto resistente à murcha-bacteriana. Os métodos de inoculação foram selecionados em função da facilidade operacional e testados na avaliação de quatro clones (híbridos E. urophylla x E. grandis): i) aplicação de 5 mL de inóculo (10(8) ufc/mL) na região do coleto de mudas; ii) aplicação de 5 mL de inóculo (10(8) ufc/mL) na região do coleto de mudas após o ferimento do sistema radicular; iii) corte de 1/3 basal do sistema radicular e imersão das raízes por 1 min em suspensão de inóculo na mesma concentração; e iv) condução de mudas transplantadas em infectário, constituído de canaletões com areia infestada quinzenalmente (taxa de 0,25 L/m³) com suspensão bacteriana (10(8) ufc/mL) e submetidas a coletas de brotações, de forma similar ao que é feito em minijardins comerciais. A condução das mudas em infectário apresentou maiores incidências de doença (exsudação de pus bacteriano), variando de 81 a 100%. O segundo experimento objetivou avaliar a resistência de diferentes espécies de eucalipto, empregando-se o método de transplantio das mudas para o infectário. A avaliação da infecção pela presença de exsudação bacteriana apresentou resultados mais consistentes para comparação dos genótipos. A intensidade da murcha-bacteriana foi afetada pelas condições da planta e do ambiente. Todas as espécies exibiram sintomas da doença, porém E. tereticornis e E. grandis apresentaram, respectivamente, o menor (33,3%) e o maior (91,7%) percentual de genótipos infectados (suscetíveis). Os resultados deste trabalho são importantes ferramentas (método de inoculação) e fontes (espécies de eucalipto) de resistência para o controle da murcha-bacteriana do eucalipto.