Requirement of the cytosolic interaction between PATHOGENESIS-RELATED PROTEIN10 and LEUCINE-RICH REPEAT PROTEIN1 for cell death and defense signaling in pepper

Plants recruit innate immune receptors such as leucine-rich repeat (LRR) proteins to recognize pathogen attack and activate defense genes. Here, we identified the pepper (Capsicum annuum) pathogenesis-related protein10 (PR10) as a leucine-rich repeat protein1 (LRR1)-interacting partner. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the specific interaction between LRR1 and PR10 in planta. Avirulent Xanthomonas campestris pv vesicatoria infection induces PR10 expression associated with the hypersensitive cell death response. Transient expression of PR10 triggers hypersensitive cell death in pepper and Nicotiana benthamiana leaves, which is amplified by LRR1 coexpression as a positive regulator. LRR1 promotes the ribonuclease activity and phosphorylation of PR10, leading to enhanced cell death signaling. The LRR1-PR10 complex is formed in the cytoplasm, resulting in its secretion into the apoplastic space. Engineered nuclear confinement of both proteins revealed that the cytoplasmic localization of the PR10-LRR1 complex is essential for cell death-mediated defense signaling. PR10/LRR1 silencing in pepper compromises resistance to avirulent X. campestris pv vesicatoria infection. By contrast, PR10/LRR1 overexpression in Arabidopsis thaliana confers enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that the cytosolic LRR-PR10 complex is responsible for cell death-mediated defense signaling.     

The pepper mannose-binding lectin gene CaMBL1 is required to regulate cell death and defense responses to microbial pathogens

Plant mannose-binding lectins (MBLs) are crucial for plant defense signaling during pathogen attack by recognizing specific carbohydrates on pathogen surfaces. In this study, we isolated and functionally characterized a novel pepper (Capsicum annuum) MBL gene, CaMBL1, from pepper leaves infected with Xanthomonas campestris pv vesicatoria (Xcv). The CaMBL1 gene contains a predicted Galanthus nivalis agglutinin-related lectin domain responsible for the recognition of high-mannose N-glycans but lacks a middle S-locus glycoprotein domain and a carboxyl-terminal PAN-Apple domain. The CaMBL1 protein exhibits binding specificity for mannose and is mainly localized to the plasma membrane. Immunoblotting using a CaMBL1-specific antibody revealed that CaMBL1 is strongly expressed and accumulates in pepper leaves during avirulent Xcv infection. The transient expression of CaMBL1 induces the accumulation of salicylic acid (SA), the activation of defense-related genes, and the cell death phenotype in pepper. The G. nivalis agglutinin-related lectin domain of CaMBL1 is responsible for cell death induction. CaMBL1-silenced pepper plants are more susceptible to virulent or avirulent Xcv infection compared with unsilenced control plants, a phenotype that is accompanied by lowered reactive oxygen species accumulation, reduced expression of downstream SA target genes, and a concomitant decrease in SA accumulation. In contrast, CaMBL1 overexpression in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to Pseudomonas syringae pv tomato and Alternaria brassicicola infection. Together, these data suggest that CaMBL1 plays a key role in the regulation of plant cell death and defense responses through the induction of downstream defense-related genes and SA accumulation after the recognition of microbial pathogens.     

Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens

Reactive oxygen species (ROS) are responsible for mediating cellular defense responses in plants. Controversy has existed over the origin of ROS in plant defense. We have isolated a novel extracellular peroxidase gene, CaPO2, from pepper (Capsicum annuum). Local or systemic expression of CaPO2 is induced in pepper by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection. We examined the function of the CaPO2 gene in plant defense using the virus-induced gene silencing technique and gain-of-function transgenic plants. CaPO2-silenced pepper plants were highly susceptible to Xcv infection. Virus-induced gene silencing of the CaPO2 gene also compromised hydrogen peroxide (H(2)O(2)) accumulation and hypersensitive cell death in leaves, both locally and systemically, during avirulent Xcv infection. In contrast, overexpression of CaPO2 in Arabidopsis (Arabidopsis thaliana) conferred enhanced disease resistance accompanied by cell death, H(2)O(2) accumulation, and PR gene induction. In CaPO2-overexpression Arabidopsis leaves infected by Pseudomonas syringae pv tomato, H(2)O(2) generation was sensitive to potassium cyanide (a peroxidase inhibitor) but insensitive to diphenylene iodonium (an NADPH oxidase inhibitor), suggesting that H(2)O(2) generation depends on peroxidase in Arabidopsis. Together, these results indicate that the CaPO2 peroxidase is involved in ROS generation, both locally and systemically, to activate cell death and PR gene induction during the defense response to pathogen invasion.     

Proteomics and functional analyses of pepper abscisic acid-responsive 1 (ABR1), which is involved in cell death and defense signaling

Abscisic acid (ABA) is a key regulator of plant growth and development, as well as plant defense responses. A high-throughput in planta proteome screen identified the pepper (Capsicum annuum) GRAM (for glucosyltransferases, Rab-like GTPase activators, and myotubularins) domain-containing ABA-RESPONSIVE1 (ABR1), which is highly induced by infection with avirulent Xanthomonas campestris pv vesicatoria and also by treatment with ABA. The GRAM domain is essential for the cell death response and for the nuclear localization of ABR1. ABR1 is required for priming cell death and reactive oxygen species production, as well as ABA-salicylic acid (SA) antagonism. Silencing of ABR1 significantly compromised the hypersensitive response but enhanced bacterial pathogen growth and ABA levels in pepper. High levels of ABA in ABR1-silenced plants antagonized the SA levels induced by pathogen infection. Heterologous transgenic expression of ABR1 in Arabidopsis thaliana conferred enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis infection. The susceptibility of the Arabidopsis ABR1 putative ortholog mutant, abr1, to these pathogens also supports the involvement of ABR1 in disease resistance. Together, these results reveal ABR1 as a novel negative regulator of ABA signaling and suggest that the nuclear ABR1 pool is essential for the cell death induction associated with ABA-SA antagonism.     

Identification and expression profiling of tomato genes differentially regulated during a resistance response to Xanthomonas campestris pv. vesicatoria

The gram-negative bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of spot disease in tomato and pepper. Plants of the tomato line Hawaii 7981 are resistant to race T3 of X. campestris pv. vesicatoria expressing the type III effector protein AvrXv3 and develop a typical hypersensitive response upon bacterial challenge. A combination of suppression subtractive hybridization and microarray analysis identified a large set of cDNAs that are induced or repressed during the resistance response of Hawaii 7981 plants to X. campestris pv. vesicatoria T3 bacteria. Sequence analysis of the isolated cDNAs revealed that they correspond to 426 nonredundant genes, which were designated as XRE (Xanthomonas-regulated) genes and were classified into more than 20 functional classes. The largest functional groups contain genes involved in defense, stress responses, protein synthesis, signaling, and photosynthesis. Analysis of XRE expression kinetics during the tomato resistance response to X. campestris pv. vesicatoria T3 revealed six clusters of genes with coordinate expression. In addition, by using isogenic X. campestris pv. vesicatoria T2 strains differing only by the avrXv3 avirulence gene, we found that 77% of the identified XRE genes were directly modulated by expression of the AvrXv3 effector protein. Interestingly, 64% of the XRE genes were also induced in tomato during an incompatible interaction with an avirulent strain of Pseudomonas syringae pv. tomato. The identification and expression analysis of X. campestris pv. vesicatoria T3-modulated genes, which may be involved in the control or in the execution of plant defense responses, set the stage for the dissection of signaling and cellular responses activated in tomato plants during the onset of spot disease resistance.     

Differential Induction and Accumulation of β-1,3-Glucanase and Chitinase Isoforms in the Intercellular Space and Leaf Tissues of Pepper by Xanthomonas campestris pv. vesicatoria Infection

The virulent strain Ds 1 of Xanthomonas campestris pv. vesicatoria multiplied in pepper (cv. Hanbyul) leaves better than did the avirulent strain 81–23, which formed localized necrosis at the onset of pathogenesis. Infection of pepper leaves by X. campestris pv. vesicatoria induced the synthesis and accumulation of β-1,3-glucanase and chitinase in the intercellular space and leaf tissue of pepper plants. In the uninoculated controls, the two hydrolases remained at a very low level. High levels of the two enzymes were found in an incompatible interaction of pepper leaves with X. campestris pv. vesicatoria . In particular, chitinase activity in the intercellular washing fluids (IWF) was higher in the incompatible than in the compatible interactions. The direct detection of acidic β-1,3-glucanases on 10% native PAGE gels revealed only two isoform bands (Ga 1 and Ga 2). Isoelectric focusing identified two acidic β-1,3-glucanase isoforms with pl 5.0 and 5.2, and four basic isoforms with pl 7.1, 7.4, 7.9, and 8.8 in the IWF and extracts of infected leaf tissues. Some of the isoforms disappeared during pathogenesis and the others appeared during symptom expression. The acidic chitinase isoforms (Ca 1, Ca 2, and Ca 3) were located primarily in the intercellular spaces. Synthesis of high levels of the acidic isoform Ca 3 in infected pepper leaves was seen. Several basis chitinase isoforms accumulated only in diseased leaf tissue, and especially more in the incompatible than the compatible interaction. By using isoelectric focusing, the three acidic and seven basic chitinase isoforms in the IWF and leaf extracts were detected on chitin overlay gels.     

A gene from Xanthomonas campestris pv. vesicatoria that determines avirulence in tomato is related to avrBs3.

Strains of Xanthomonas campestris pv. vesicatoria that were avirulent in tomato leaves but virulent in pepper leaves were identified. A cloned gene, avrBsP, from one of the strains, Xv 87-7, converted a virulent strain in tomato to avirulent in tomato. A 1.7-kb subclone containing the avirulence gene cross-hybridized with the avirulence gene, which determines race 1 within the pepper group of strains (avrBs3). However, the two avirulence genes differ in their biological activity. The base sequences of the two avirulence genes were almost identical through the 1.7-kb segment of avrBsP, with significant differences only in some bases in the repeat region.     

Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance

XA21 is a receptor-like kinase protein in rice (Oryza sativa) that confers gene-for-gene resistance to specific races of the causal agent of bacterial blight disease, Xanthomonas oryzae pv oryzae. We identified XA21 binding protein 3 (XB3), an E3 ubiquitin ligase, as a substrate for the XA21 Ser and Thr kinase. The interaction between XB3 and the kinase domain of XA21 has been shown in yeast and in vitro, and the physical association between XB3 and XA21 in vivo has also been confirmed by coimmunoprecipitation assays. XB3 contains an ankyrin repeat domain and a RING finger motif that is sufficient for its interaction with the kinase domain of XA21 and for its E3 ubiquitin ligase activity, respectively. Transgenic plants with reduced expression of the Xb3 gene are compromised in resistance to the avirulent race of X. oryzae pv oryzae. Furthermore, reduced levels of Xb3 lead to decreased levels of the XA21 protein. These results indicate that Xb3 is necessary for full accumulation of the XA21 protein and for Xa21-mediated resistance.