Transcription factor CaHDZ15 promotes pepper basal thermotolerance by activating HEAT SHOCK FACTORA6a.

High temperature stress (HTS) is a serious threat to plant growth and development and to crop production in the context of global warming, and plant response to HTS is largely regulated at the transcriptional level by the actions of various transcription factors (TFs). However, whether and how homeodomain-leucine zipper (HD-Zip) TFs are involved in thermotolerance are unclear. Herein, we functionally characterized a pepper (Capsicum annuum) HD-Zip I TF CaHDZ15. CaHDZ15 expression was upregulated by HTS and abscisic acid in basal thermotolerance via loss- and gain-of-function assays by virus-induced gene silencing in pepper and overexpression in Nicotiana benthamiana plants. CaHDZ15 acted positively in pepper basal thermotolerance by directly targeting and activating HEAT SHOCK FACTORA6a (HSFA6a), which further activated CaHSFA2. In addition, CaHDZ15 interacted with HEAT SHOCK PROTEIN 70-2 (CaHsp70-2) and glyceraldehyde-3-phosphate dehydrogenase1 (CaGAPC1), both of which positively affected pepper thermotolerance. CaHsp70-2 and CaGAPC1 promoted CaHDZ15 binding to the promoter of CaHSFA6a, thus enhancing its transcription. Furthermore, CaHDZ15 and CaGAPC1 were protected from 26S proteasome-mediated degradation by CaHsp70-2 via physical interaction. These results collectively indicate that CaHDZ15, modulated by the interacting partners CaGAPC1 and CaHsp70-2, promotes basal thermotolerance by directly activating the transcript of CaHSFA6a. Thus, a molecular linkage is established among CaHsp70-2, CaGAPC1, and CaHDZ15 to transcriptionally modulate CaHSFA6a in pepper thermotolerance.

Temperature-dependent action of pepper mildew resistance locus O 1 in inducing pathogen immunity and thermotolerance.

Plant diseases tend to be more serious under conditions of high-temperature/high-humidity (HTHH) than under moderate conditions, and hence disease resistance under HTHH is an important determinant for plant survival. However, how plants cope with diseases under HTHH remains poorly understood. In this study, we used the pathosystem consisting of pepper (Capsicum annuum) and Ralstonia solanacearum (bacterial wilt) as a model to examine the functions of the protein mildew resistance locus O 1 (CaMLO1) and U-box domain-containing protein 21 (CaPUB21) under conditions of 80% humidity and either 28 °C or 37 °C. Expression profiling, loss- and gain-of-function assays involving virus-induced gene-silencing and overexpression in pepper plants, and protein-protein interaction assays were conducted, and the results showed that CaMLO1 acted negatively in pepper immunity against R. solanacearum at 28 °C but positively at 37 °C. In contrast, CaPUB21 acted positively in immunity at 28 °C but negatively at 37 °C. Importantly, CaPUB21 interacted with CaMLO1 under all of the tested conditions, but only the interaction in response to R. solanacearum at 37 °C or to exposure to 37 °C alone led to CaMLO1 degradation, thereby turning off defence responses against R. solanacearum at 37 °C and under high-temperature stress to conserve resources. Thus, we show that CaMLO1 and CaPUB21 interact with each other and function distinctly in pepper immunity against R. solanacearum in an environment-dependent manner.

Capsicum annuum HsfB2a Positively Regulates the Response to Ralstonia solanacearum Infection or High Temperature and High Humidity Forming Transcriptional Cascade with CaWRKY6 and CaWRKY40.

The responses of pepper (Capsicum annuum) plants to inoculation with the pathogenic bacterium Ralstonia solanacearum and to high-temperature-high-humidity (HTHH) conditions were previously found to be coordinated by the transcription factors CaWRKY6 and CaWRKY40; however, the underlying molecular mechanism was unclear. Herein, we identified and functionally characterized CaHsfB2a, a nuclear-localized heat shock factor involved in pepper immunity to R. solanacearum inoculation (RSI) and tolerance to HTHH. CaHsfB2a is transcriptionally induced in pepper plants by RSI or HTHH and by exogenous application of salicylic acid (SA), methyl jasmonate (MeJA), ethylene (ETH), or abscisic acid (ABA). Virus-induced gene silencing (VIGS) of CaHsfB2a significantly impaired pepper immunity to RSI, hampered HTHH tolerance, and curtailed expression of immunity- and thermotolerance-associated marker genes such as CaHIR1, CaNPR1, CaABR1, and CaHSP24. Likewise, transient overexpression of CaHsfB2a in pepper leaves induced hypersensitive response (HR)-like cell death and H2O2 accumulation and upregulated the above-mentioned marker genes as well as CaWRKY6 and CaWRKY40. Chromatin immunoprecipitation (ChIP) and microscale thermophoresis (MST) analysis revealed that CaHsfB2a bound the promoters of both CaWRKY6 and CaWRKY40. In a parallel experiment, we determined by ChIP-PCR and MST that CaHsfB2a was regulated directly by CaWRKY40 but indirectly by CaWRKY6. Cumulatively, our results suggest that CaHsfB2a positively regulates plant immunity against RSI and tolerance to HTHH, via transcriptional cascades and positive feedback loops involving CaWRKY6 and CaWRKY40.

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.

CaC3H14 encoding a tandem CCCH zinc finger protein is directly targeted by CaWRKY40 and positively regulates the response of pepper to inoculation by Ralstonia solanacearum.

Tandem CCCH zinc finger (TZnF) proteins have been implicated in plant defence, but their role in pepper (Capsicum annuum) is unclear. In the present study, the role of CaC3H14, a pepper TZnF protein, in the immune response of pepper plants to Ralstonia solanacearum infection was characterized. When fused to the green fluorescent protein, CaC3H14 was localized exclusively to the nuclei in leaf cells of Nicotiana benthamiana plants transiently overexpressing CaC3H14. Transcript abundance of CaC3H14 was up-regulated by inoculation with R. solanacearum. Virus-induced silencing of CaC3H14 increased the susceptibility of the plants to R. solanacearum and down-regulated the genes associated with the hypersensitive response (HR), specifically HIR1 and salicylic acid (SA)-dependent PR1a. By contrast, silencing resulted in the up-regulation of jasmonic acid (JA)-dependent DEF1 and ethylene (ET) biosynthesis-associated ACO1. Transient overexpression of CaC3H14 in pepper triggered an intensive HR, indicated by cell death and hydrogen peroxide (H2 O2 ) accumulation, up-regulated PR1a and down-regulated DEF1 and ACO1. Ectopic overexpression of CaC3H14 in tobacco plants significantly decreased the susceptibility of tobacco plants to R. solanacearum. It also up-regulated HR-associated HSR515, immunity-associated GST1 and the SA-dependent marker genes NPR1 and PR2, but down-regulated JA-dependent PR1b and ET-dependent EFE26. The CaC3H14 promoter and was bound and its transcription was up-regulated by CaWRKY40. Collectively, these results indicate that CaC3H14 is transcriptionally targeted by CaWRKY40, is a modulator of the antagonistic interaction between SA and JA/ET signalling, and enhances the defence response of pepper plants to infection by R. solanacearum.

A novel leucine-rich repeat protein, CaLRR51, acts as a positive regulator in the response of pepper to Ralstonia solanacearum infection.

The leucine-rich repeat (LRR) proteins play important roles in the recognition of corresponding ligands and signal transduction networks in plant defence responses. Herein, a novel LRR protein from Capsicum annuum, CaLRR51, was identified and characterized. It was localized to the plasma membrane and transcriptionally up-regulated by Ralstonia solanacearum infection (RSI), as well as the exogenous application of salicylic acid (SA), jasmonic acid (JA) and ethephon (ETH). Virus-induced gene silencing of CaLRR51 significantly increased the susceptibility of pepper to RSI. By contrast, transient overexpression of CaLRR51 in pepper plants activated hypersensitive response (HR)-like cell death, and up-regulated the defence-related marker genes, including PO2, HIR1, PR1, DEF1 and ACO1. Moreover, ectopic overexpression of CaLRR51 in transgenic tobacco plants significantly enhanced the resistance to RSI. Transcriptional expression of the corresponding defence-related marker genes in transgenic tobacco plants was also found to be enhanced by the overexpression of CaLRR51, which was potentiated by RSI. These loss- and gain-of-function assays suggest that CaLRR51 acts as a positive regulator in the response of pepper to RSI. In addition, the putative signal peptide and transmembrane region were found to be required for plasma membrane targeting of CaLRR51, which is indispensable for the role of CaLRR51 in plant immunity.

The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids.

In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes's response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery.

Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum.

As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs) play important roles in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a genome wide comprehensive analysis of genes encoding CDPKs and CDPK-related protein kinases (CRKs) was performed in pepper, a total of 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CDPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the C. annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families.

SRC2-1 is required in PcINF1-induced pepper immunity by acting as an interacting partner of PcINF1.

Elicitins are elicitors that can trigger hypersensitive cell death in most Nicotiana spp., but their underlying molecular mechanism is not well understood. The gene Phytophthora capsici INF1 (PcINF1) coding for an elicitin from P. capsici was characterized in this study. Transient overexpression of PcINF1 triggered cell death in pepper (Capsicum annuum L.) and was accompanied by upregulation of the hypersensitive response marker, Hypersensitive Induced Reaction gene 1 (HIR1), and the pathogenesis-related genes SAR82, DEF1, BPR1, and PO2. A putative PcINF1-interacting protein, SRC2-1, was isolated from a pepper cDNA library by yeast two-hybrid screening and was observed to target the plasma membrane. The interaction between PcINF1 and SRC2-1 was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Simultaneous transient overexpression of SRC2-1 and PcINF1 in pepper plants triggered intensive cell death, whereas silencing of SRC2-1 by virus-induced gene silencing blocked the cell death induction of PcINF1 and increased the susceptibility of pepper plants to P. capsici infection. Additionally, membrane targeting of the PcINF1-SRC2-1 complex was required for cell death induction. The C2 domain of SRC2-1 was crucial for SRC2-1 plasma membrane targeting and the PcINF1-SRC2-1 interaction. These results suggest that SRC2-1 interacts with PcINF1 and is required in PcINF1-induced pepper immunity.

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.

Overexpression of a pepper CaERF5 gene in tobacco plants enhances resistance to Ralstonia solanacearum infection.

ETHYLENE RESPONSE FACTORs (ERF) transcription factors (TFs) constitute a large transcriptional regulator family belonging to the AP2/ERF superfamily and are implicated in a range of biological processes. However, the specific roles of individual ERF family members in biotic or abiotic stress responses and the underlying molecular mechanism still need to be elucidated. In the present study, a cDNA encoding a member of ethylene response factor (ERF) transcription factor, CaERF5, was isolated from pepper. Sequence analysis showed that CaERF5 contains a typical 59 amino acid AP2/ERF DNA-binding domain, two highly conserved amino acid residues (14th alanine (A) and 19th aspartic acid (D)), a putative nuclear localisation signal (NLS), a CMIX-2 motif in the N-terminal region and two putative MAP kinase phosphorylation site CMIX-5 and CMIX-6 motifs. It belongs to group IXb of the ERF subfamily. A CaERF5-green fluorescence protein (GFP) fusion transiently expressed in onion epidermal cells localised to the nucleus. CaERF5 transcripts were induced by Ralstonia solanacearum infection, salicylic acid (SA), methyl jasmonate (MeJA) and ethephon (ETH) treatments. Constitutive expression of the CaERF5 gene in tobacco plants upregulated transcript levels of a set of defence- related genes and enhanced resistance to R. solanacearum infection. Our results suggest that CaERF5 acts as a positive regulator in plant resistance to R. solanacearum infection and show that overexpression of this transcription factor can be used as a tool to enhance disease resistance in crop species.

[Function of nitric oxide in initiating production of lignin degrading peroxidases by Phanerochaete chrysosporium].

OBJECTIVE: By analyzing the function and mechanism of nitric oxide in initiating producing lignin peroxidases by phanerochaete chrysosporium, we studied the regulation mechanism triggering the secondary metabolism of white-rot fungi. METHODS: Mutant (pcR5305) and wild-type (pc530) strains of phanerochaete chrysosporium were respectively cultured under both the conditions of nitrogen limitation and nitrogen sufficiency. To compare their lignin peroxidases (LiP)-production and nitric oxide(NO)-production kinetics and their different influences on producing LiP after the NO donor Sodium Nitroprusside (SNP) and scavenger cPTIO were respectively added to the nitrogen limitation or sufficiency culture medium to show the function and mechanism of nitric oxide in initiating production of lignin peroxidases by white-rot fungi. RESULTS: Both strains produced nitric oxide (NO) under the two opposite nutritional conditions, but the levels of NO produced were related with the type of strain and the nutritional conditions. Strain pc530 produced NO requiring nutrition depletion and producing of NO was strongly delayed and reduced when it was cultured under nitrogen sufficiency condition. On the contrary, pcR5305 did not require nitrogen depletion to trigger and the levels of NO were higher than that of pc530. The results indicate that LiP content had positive correlation with NO value except the occurrence time of LiP peak value was later than that of NO. The ability of producing LiP was promoted after the NO donor SNP added, but SNP affected more on pc530 than pcR5305 in promoting producing LiP. 15mM cPTIO would greatly repress producing LiP, but could not completely restrain the synthesis of LiP for both strains. CONCLUSION: By producing NO, Phanerochaete chrysosporium triggers LiP synthesis. However, the evidences do not indicate that NO participates or effect directly in LiP synthesis. It is more likely that NO is reacting as an upstream signal molecule. Besides NO, there are other signal molecules that have a positive effect on NO levels also involving in the regulation producing LiP. The mechanism of the resistance to nutritional repression of pcR5305 in synthesizing lignin degrading peroxidases may be the answer to the different NO production mechanism of pcR5305 from pc530.

Functional analysis and expressional characterization of rice ankyrin repeat-containing protein, OsPIANK1, in basal defense against Magnaporthe oryzae attack.

The ankyrin repeat-containing protein gene OsPIANK1 (AK068021) in rice (Oryza sativa L.) was previously shown to be upregulated following infection with the rice leaf blight pathogen Xanthomonas oryzae pv oryzae (Xoo). In this study, we further characterized the role of OsPIANK1 in basal defense against Magnaporthe oryzae (M.oryzae) by 5' deletion analysis of its promoter and overexpression of the gene. The promoter of OsPIANK1 with 1,985 bps in length was sufficient to induce the OsPIANK1 response to inoculation with M.oryzae and to exogenous application of methyl jasmonate (MeJA) or salicylic acid (SA), but not to exogenous application of abscisic acid (ABA). A TCA-element present in the region between -563 bp and -249 bp may be responsible for the OsPIANK1 response to both M.oryzae infection and exogenous SA application. The JERE box, CGTCA-box, and two MYB binding sites locating in the region between -1985 bp and -907 bp may be responsible for the response of OsPIANK1 to exogenous MeJA. OsPIANK1 expression was upregulated after inoculation with M.oryzae and after treatment with exogenous SA and MeJA. Overexpression of OsPIANK1 enhanced resistance of rice to M.oryzae, although it did not confer complete resistance. The enhanced resistance to M.oryzae was accompanied by enhanced transcriptional expression of SA- and JA-dependent genes such as NH1, WKRY13, PAL, AOS2, PR1b, and PR5. This evidence suggests that OsPIANK1 acted as a positive regulator in rice basal defense mediated by SA- and JA-signaling pathways.

CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection.

WRKY proteins form a large family of plant transcription factors implicated in the modulation of numerous biological processes, such as growth, development and responses to various environmental stresses. However, the roles of the majority WRKY family members, especially in non-model plants, remain poorly understood. We identified CaWRKY40 from pepper. Transient expression in onion epidermal cells showed that CaWRKY40 can be targeted to nuclei and activates expression of a W-box-containing reporter gene. CaWRKY40 transcripts are induced in pepper by Ralstonia solanacearum and heat shock. To assess roles of CaWRKY40 in plant stress responses we performed gain- and loss-of-function experiments. Overexpression of CaWRKY40 enhanced resistance to R. solanacearum and tolerance to heat shock in tobacco. In contrast, silencing of CaWRKY40 enhanced susceptibility to R. solanacearum and impaired thermotolerance in pepper. Consistent with its role in multiple stress responses, we found CaWRKY40 transcripts to be induced by signalling mechanisms mediated by the stress hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Overexpression of CaWRKY40 in tobacco modified the expression of hypersensitive response (HR)-associated and pathogenesis-related genes. Collectively, our results suggest that CaWRKY40 orthologs are regulated by SA, JA and ET signalling and coordinate responses to R. solanacearum attacks and heat stress in pepper and tobacco.

CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection.

WRKY transcription factors are encoded by large gene families across the plant kingdom. So far, their biological and molecular functions in nonmodel plants, including pepper (Capsicum annuum) and other Solanaceae, remain poorly understood. Here, we report on the functional characterization of a new group I WRKY protein from pepper, termed CaWRKY58. Our data indicate that CaWRKY58 can be localized to the nucleus and can activate the transcription of the reporter ß-glucuronidase (GUS) gene driven by the 35S core promoter with two copies of the W-box in its proximal upstream region. In pepper plants infected with the bacterial pathogen Ralstonia solanacearum, CaWRKY58 transcript levels showed a biphasic response, manifested in an early/transient down-regulation and late up-regulation. CaWRKY58 transcripts were suppressed by treatment with methyl jasmonate and abscisic acid. Tobacco plants overexpressing CaWRKY58 did not show any obvious morphological phenotypes, but exhibited disease symptoms of greater severity than did wild-type plants. The enhanced susceptibility of CaWRKY58-overexpressing tobacco plants correlated with the decreased expression of hypersensitive response marker genes, as well as various defence-associated genes. Consistently, CaWRKY58 pepper plants silenced by virus-induced gene silencing (VIGS) displayed enhanced resistance to the highly virulent R. solanacearum strain FJC100301, and this was correlated with enhanced transcripts of defence-related pepper genes. Our results suggest that CaWRKY58 acts as a transcriptional activator of negative regulators in the resistance of pepper to R. solanacearum infection.

Overexpression of a Chinese cabbage BrERF11 transcription factor enhances disease resistance to Ralstonia solanacearum in tobacco.

Ethylene-responsive factors (ERFs) play diverse roles in plant growth, developmental processes and stress responses. However, the roles and underlying mechanism of ERFs remain poorly understood, especially in non-model plants. In this study, a full length cDNA of ERF gene was isolated from the cDNA library of Chinese cabbage. According to sequence alignment, we found a highly conservative AP2/ERF domain, two nuclear localization signals, and an ERF-associated Amphiphilic Repression (EAR) motif in its C-terminal region. It belonged to VIIIa group ERFs sharing the highest sequence identity with AtERF11 in all of the ERFs in Arabidopsis and designated BrERF11. BrERF11-green fluorescence protein (GFP) transient expressed in onion epidermis cells localized to the nucleus. The transcript levels of BrERF11 were induced by exogenous salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), and hydrogen peroxide (H(2)O(2)). Constitutive expression of BrERF11 enhanced tolerance to Ralstonia solanacearum infection in transgenic tobacco plants, which was coupled with hypersensitive response (HR), burst of H(2)O(2) and upregulation of defense-related genes including HR marker genes, SA-, JA-dependent pathogen-related genes and ET biosynthesis associated genes and downregulation of CAT1, suggesting BrERF11 may participate in pathogen-associated molecular pattern (PAMP)- and effector-triggered immunity (PTI and ETI) mediated by SA-, JA- and ET-dependent signaling mechanisms.

[Carbon-nitrogen regulation of a laccase-producing mutant of Phanerochaete chrysosporium resisting carbon and nitrogen nutritional repression].

OBJECTIVE: Comparing the effects of different carbon-nitrogen nutrition and their consumption on laccase production, we studied the ecophysiological characteristics of Phanerochaete chrysosporium resisting nutritional repression, and the carbon-nitrogen physiological regulation mechanism of the white-rot fungi. METHODS: The mutant and the wild-type strains were respectively cultured under the conditions of: carbon and nitrogen limitation, carbon limitation and nitrogen sufficiency, carbon sufficiency and nitrogen limitation, carbon and nitrogen sufficiency, to compare their laccase-production kinetics, cell growth and glucose and ammonia nitrogen consumption to show the characteristics and the regulation pathway of carbon-nitrogen nutrition on laccase production. RESULTS: The wild-type strain produced 0.107 U/L, 0.029 U/L,12.84 U/L and 18.05 U/L of laccase respectively on 11th,14th, 19th and 19th day when glucose or ammonia nitrogen was consumed to the lowest value; the mutant produced laccase throughout the whole process with two peaks respectively on 8th, 7th, 12th and 12th day with laccase of 298.83 U/L, 343.14U/L, 271.22 U/L and 251.49 U/L and on 12th, 13th, 19th and 19th day with laccase of 257.69 U/L, 298.78 U/L, 213.81 U/L and 216.93 U/L. The enzyme-production kinetics trends were similar between the two strains on the condition of the same initial carbon concentration but were different on the same initial nitrogen concentration, which showed that carbon source had more effect on laccase production. CONCLUSION: The laccase production of the wild-type strain was regulated by carbon or nitrogen starvation. Under different conditions, it was regulated by different nutrient. For example, under carbon limitation condition it was started by the glucose starvation, however under carbon sufficient condition the ammonia nitrogen starvation aroused it. The laccase production of the mutant didn't repress by carbon and nitrogen nutrition. Maybe it referred to a global regulation change which relieved nutritional repression on the laccase production.

[Breeding and characterization of laccase-producing Phanerochaete chrysosporium mutant resistant to nutritional repression].

OBJECTIVE: To screen Phanerochaete chrysosporium mutants resisting nutritional repression and to characterize laccase produced by the mutants. METHODS: We used repeated UV mutagenesis and screened the mutant strains by using the guaiacol nitrogen sufficient differential medium. We characterized enzymes production mechanism of the nutritional regulation through comparing the differences of cell growth and enzyme-production kinetics under different nutritional conditions; We validated production of laccase by Phanerochaete chrysosporium through measurements of the heat treatment, removal of manganese ion and addition of the catalase. RESULTS: Three different methods were validated that both strains of pcR5305 and pcR5324 can produce laccase under the nitrogen limitation (N-L) and nitrogen sufficient (N-S) conditions. Under the N-L conditions, pcR5305 can produce 203.5 U/L laccase and pcR5324 can produce 187.6 U/L laccase; Under the N-S conditions, pcR5305 can produce 220.6 U/L laccase and pcR5324 can produce 183.9 U/L laccase. The original strain pc530 only can produce very little laccase under either conditions. The laccase-production regulation mechanisms of the two strains are different: Production of laccase and the cell growth by pcR5305 are in synchronism. However production of the laccase by pcR5324 is repressed by nutrition. Both strains have the capacity of resisting nutritional repression and produce lignin peroxidase and manganese peroxidase with high yield. (LiP 1343.2, MnP 252.2 U/L and LiP 1169.5, MnP 172.4 U/L respectively). CONCLUSION: The mutants of Phanerochaete chrysosporium can produce laccase. At same time they showed the capacity of resisting nutritional repression and production of laccase, lignin peroxidase and manganese peroxidase. Our results possess high value for production, application and fundamental research. We provided new strains and established a very good foundation for the further research of metabolic regulation of ligninolytic enzymes production.