Red light induces salicylic acid accumulation by activating CaHY5 to enhance pepper resistance against Phytophthora capsici.

Pepper (Capsicum annuum L.) is frequently challenged by various pathogens, among which Phytophthora capsici is the most devastating to pepper production. Red light signal acts as a positive induction of plant resistance against multiple pathogens. However, little is known about how the red light signal affects pepper resistance to P. capsici infection (PCI). Here, we report that red light regulates salicylic acid (SA) accumulation by activating elongated hypocotyl5 (CaHY5), a basic leucine zipper (bZIP) transcription factor, thereby decreasing pepper susceptibility to PCI. Exogenous SA treatment reduced pepper susceptibility to PCI, while silencing of CaPHYB (a red light photoreceptor) increased its susceptibility. PCI significantly induced CaHY5 expression, and silencing of CaHY5 reduced SA accumulation, accompanied by decreases in the expression levels of phenylalanine ammonia-lyase 3 (CaPAL3), CaPAL7, pathogenesis-related 1 (CaPR1), and CaPR1L, which finally resulted in higher susceptibility of pepper to PCI. Moreover, CaHY5 was found to activate the expression of CaPAL3 and CaPAL7, which are essential for SA biosynthesis, by directly binding to their promoters. Further analysis revealed that exogenous SA treatment could restore the resistance of CaHY5-silenced pepper plants to PCI. Collectively, this study reveals a critical mechanism through which red light induces SA accumulation by regulating CaHY5-mediated CaPAL3 and CaPAL7 expression, leading to enhanced resistance to PCI. Moreover, red light-induced CaHY5 regulates pepper resistance to PCI, which may have implications for PCI control in protected vegetable production.

SlWRKY30 and SlWRKY81 synergistically modulate tomato immunity to Ralstonia solanacearum by directly regulating SlPR-STH2.

Bacterial wilt is a devastating disease of tomato (Solanum lycopersicum) caused by Ralstonia solanacearum that severely threatens tomato production. Group III WRKY transcription factors (TFs) are implicated in the plant response to pathogen infection; however, their roles in the response of tomato to R. solanacearum infection (RSI) remain largely unexplored. Here, we report the crucial role of SlWRKY30, a group III SlWRKY TF, in the regulation of tomato response to RSI. SlWRKY30 was strongly induced by RSI. SlWRKY30 overexpression reduced tomato susceptibility to RSI, and also increased H2O2 accumulation and cell necrosis, suggesting that SlWRKY30 positively regulates tomato resistance to RSI. RNA sequencing and reverse transcription-quantitative PCR revealed that SlWRKY30 overexpression significantly upregulated pathogenesis-related protein (SlPR-STH2) genes SlPR-STH2a, SlPR-STH2b, SlPR-STH2c, and SlPR-STH2d (hereafter SlPR-STH2a/b/c/d) in tomato, and these SlPR-STH2 genes were directly targeted by SlWRKY30. Moreover, four group III WRKY proteins (SlWRKY52, SlWRKY59, SlWRKY80, and SlWRKY81) interacted with SlWRKY30, and SlWRKY81 silencing increased tomato susceptibility to RSI. Both SlWRKY30 and SlWRKY81 activated SlPR-STH2a/b/c/d expression by directly binding to their promoters. Taking these results together, SlWRKY30 and SlWRKY81 synergistically regulate resistance to RSI by activating SlPR-STH2a/b/c/d expression in tomato. Our results also highlight the potential of SlWRKY30 to improve tomato resistance to RSI via genetic manipulations.

Characterization of the B-BOX gene family in pepper and the role of CaBBX14 in defense response against Phytophthora capsici infection.

The B-box (BBX) transcription factors are widely implicated in plant growth, development, and response to various biotic and abiotic stresses. However, their roles in the response of pepper to Phytophthora capsici infection (PCI) remain largely unexplored. Here, we report a total of 25 CaBBX genes with an uneven distribution were identified in pepper genome, and their characteristics, phylogenetic relationships, gene structures, conserved domains, and expression profiles were validated. CaBBXs were classified into five major clades (I to V) based on their phylogenetic relationships and conserved domains (presence of one or two B-box domains and a CCT domain). Gene duplication analysis demonstrated that there are two segmental duplication events but no tandem duplication event within pepper genome. Conserved motif and gene structure analysis revealed that the CaBBXs in the same clade have relatively similar motif arrangements and exon-intron patterns. Expression analysis revealed that the CaBBX genes have different expression levels in various tissues, and some of which were significantly induced during PCI and exogenous salicylic acid (SA) treatment. Among them, CaBBX14 displayed remarkable changed expression during PCI and SA treatment. The silencing of CaBBX14 increases pepper susceptibility to PCI, and also decreases in SA content and expression of pathogenesis-related (PR) and SA-related genes compared with control plants. Together, these findings advance our knowledge base on biological functions of CaBBXs in pepper during PCI through the SA signaling pathway, and we provide an example demonstrating that the potential of CaBBX14 to improve pepper resistance to PCI.

ZAT10 plays dual roles in cadmium uptake and detoxification in Arabidopsis.

Cadmium (Cd) is a harmful heavy metal that is risky for plant growth and human health. The zinc-finger transcription factor ZAT10 is highly conserved with ZAT6 and ZAT12, which are involved in Cd tolerance in plants. However, the definite function of ZAT10 in Cd tolerance remains uncertain. Here, we demonstrated that ZAT10 negatively regulated Cd uptake and enhanced Cd detoxification in Arabidopsis. The expression of ZAT10 in plants is induced by Cd treatment. The zat10 mutant plants exhibited a greater sensitivity to Cd stress and accumulated more Cd in both shoot and root. Further investigations revealed that ZAT10 repressed the transcriptional activity of IRT1, which encodes a key metal transporter involved in Cd uptake. Meanwhile, ZAT10 positively regulated four heavy metal detoxification-related genes: NAS1, NAS2, IRT2, and MTP3. We further found that ZAT10 interacts with FIT, but their regulatory relationship is still unclear. In addition, ZAT10 directly bound to its own promoter and repressed its transcription as a negative feedback regulation. Collectively, our findings provided new insights into the dual functions of ZAT10 on Cd uptake and detoxification in plants and pointed to ZAT10 as a potential gene resource for Cd tolerance improvement in plants.

CaWRKY27 negatively regulates salt and osmotic stress responses in pepper.

WRKY transcription factors are key regulatory components of plant responses to both biotic and abiotic stresses. In pepper (Capsicum annuum), CaWRKY27 positively regulates resistance to the pathogenic bacterium Ralstonia solanacearum and negatively regulates thermotolerance. Here, we report that CaWRKY27 functions in the response to salinity and osmotic stress. CaWRKY27 transcription was induced by salinity, osmotic, and abscisic acid (ABA) treatments, as determined using qPCR and GUS assays. Transgenic Arabidopsis thaliana and tobacco (Nicotiana tabacum) plants heterologously expressing CaWRKY27 had an increased sensitivity to salinity and osmotic stress, with a higher inhibition of both root elongation and whole plant growth, more severe chlorosis and wilting, lower germination rates, and an enhanced germination sensitivity to ABA than the corresponding wild-type plants. Furthermore, most marker genes associated with reactive oxygen species (ROS) detoxification and polyamine and ABA biosynthesis, as well as stress-responsive genes NtDREB3, were downregulated in plants transgenically expressing CaWRKY27 upon exposure to salinity or osmotic stress. Consistently, silencing of CaWRKY27 using virus-induced gene silencing conferred tolerance to salinity and osmotic stress in pepper plants. These findings suggest that CaWRKY27 acts as a molecular link in the antagonistic crosstalk regulating the expression of defense-related genes in the responses to both abiotic and biotic stresses by acting either as a transcriptional activator or repressor in pepper.

A feedback loop between CaWRKY41 and H2O2 coordinates the response to Ralstonia solanacearum and excess cadmium in pepper.

WRKY transcription factors have been implicated in both plant immunity and plant responses to cadmium (Cd); however, the mechanism underlying the crosstalk between these processes is unclear. Here, we characterized the roles of CaWRKY41, a group III WRKY transcription factor, in immunity against the pathogenic bacterium Ralstonia solanacearum and Cd stress responses in pepper (Capsicum annuum). CaWRKY41 was transcriptionally up-regulated in response to Cd exposure, R. solanacearum inoculation, and H2O2 treatment. Virus-induced silencing of CaWRKY41 increased Cd tolerance and R. solanacearum susceptibility, while heterologous overexpression of CaWRKY41 in Arabidopsis impaired Cd tolerance, and enhanced Cd and zinc (Zn) uptake and H2O2 accumulation. Genes encoding reactive oxygen species-scavenging enzymes were down-regulated in CaWRKY41-overexpressing Arabidopsis plants, whereas genes encoding Zn transporters and enzymes involved in H2O2 production were up-regulated. Consistent with these findings, the ocp3 (overexpressor of cationic peroxidase 3) mutant, which has elevated H2O2 levels, displayed enhanced sensitivity to Cd stress. These results suggest that a positive feedback loop between H2O2 accumulation and CaWRKY41 up-regulation coordinates the responses of pepper to R. solanacearum inoculation and Cd exposure. This mechanism might reduce Cd tolerance by increasing Cd uptake via Zn transporters, while enhancing resistance to R. solanacearum.

CaWRKY27 Negatively Regulates H2O2-Mediated Thermotolerance in Pepper (Capsicum annuum).

Heat stress, an important and damaging abiotic stress, regulates numerous WRKY transcription factors, but their roles in heat stress responses remain largely unexplored. Here, we show that pepper (Capsicum annuum) CaWRKY27 negatively regulates basal thermotolerance mediated by H2O2 signaling. CaWRKY27 expression increased during heat stress and persisted during recovery. CaWRKY27 overexpression impaired basal thermotolerance in tobacco (Nicotiana tabacum) and Arabidopsis thaliana, CaWRKY27-overexpressing plants had a lower survival rate under heat stress, accompanied by decreased expression of multiple thermotolerance-associated genes. Accordingly, silencing of CaWRKY27 increased basal thermotolerance in pepper plants. Exogenously applied H2O2 induced CaWRKY27 expression, and CaWRKY27 overexpression repressed the scavenging of H2O2 in Arabidopsis, indicating a positive feedback loop between H2O2 accumulation and CaWRKY27 expression. Consistent with this, CaWRKY27 expression was repressed under heat stress in the presence H2O2 scavengers and CaWRKY27 silencing decreased H2O2 accumulation in pepper leaves. These changes may result from changes in levels of reactive oxygen species (ROS)-scavenging enzymes, since the heat stress-challenged CaWRKY27-silenced pepper plants had significantly higher expression of multiple genes encoding ROS-scavenging enzymes, such as CaCAT1, CaAPX1, CaAPX2, CaCSD2, and CaSOD1. Therefore, CaWRKY27 acts as a downstream negative regulator of H2O2-mediated heat stress responses, preventing inappropriate responses during heat stress and recovery.

Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection.

WRKY proteins are encoded by a large gene family and are linked to many biological processes across a range of plant species. The functions and underlying mechanisms of WRKY proteins have been investigated primarily in model plants such as Arabidopsis and rice. The roles of these transcription factors in non-model plants, including pepper and other Solanaceae, are poorly understood. Here, we characterize the expression and function of a subgroup IIe WRKY protein from pepper (Capsicum annuum), denoted as CaWRKY27. The protein localized to nuclei and activated the transcription of a reporter GUS gene construct driven by the 35S promoter that contained two copies of the W-box in its proximal upstream region. Inoculation of pepper cultivars with Ralstonia solanacearum induced the expression of CaWRKY27 transcript in 76a, a bacterial wilt-resistant pepper cultivar, whereas it downregulated the expression of CaWRKY27 transcript in Gui-1-3, a bacterial wilt-susceptible pepper cultivar. CaWRKY27 transcript levels were also increased by treatments with salicylic acid (SA), methyl jasmonate (MeJA) and ethephon (ETH). Transgenic tobacco plants overexpressing CaWRKY27 exhibited resistance to R. solanacearum infection compared to that of wild-type plants. This resistance was coupled with increased transcript levels in a number of marker genes, including hypersensitive response genes, and SA-, JA- and ET-associated genes. By contrast, virus-induced gene silencing (VIGS) of CaWRKY27 increased the susceptibility of pepper plants to R. solanacearum infection. These results suggest that CaWRKY27 acts as a positive regulator in tobacco resistance responses to R. solanacearum infection through modulation of SA-, JA- and ET-mediated signaling pathways.

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.

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.

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.

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.