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Xanthomonas effector AvrBsT interacts with plant defense proteins and triggers cell death and defense response. This review highlights our current understanding of the molecular functions of AvrBsT and its host interactor proteins. The AvrBsT protein is a member of a growing family of effector proteins in both plant and animal pathogens. Xanthomonas type III effector AvrBsT, a member of the YopJ/AvrRxv family, suppresses plant defense responses in susceptible hosts, but triggers cell death signaling leading to hypersensitive response (HR) and defense responses in resistant plants. AvrBsT interacts with host defense-related proteins to trigger the HR cell death and defense responses in plants. Here, we review and discuss recent progress in understanding the molecular functions of AvrBsT and its host interactor proteins in pepper (Capsicum annuum). Pepper arginine decarboxylase1 (CaADC1), pepper aldehyde dehydrogenase1 (CaALDH1), pepper heat shock protein 70a (CaHSP70a), pepper suppressor of the G2 allele of skp1 (CaSGT1), pepper SNF1-related kinase1 (SnRK1), and Arabidopsis acetylated interacting protein1 (ACIP1) have been identified as AvrBsT interactors in pepper and Arabidopsis. Gene expression profiling, virus-induced gene silencing, and transient transgenic overexpression approaches have advanced the functional characterization of AvrBsT-interacting proteins in plants. AvrBsT is localized in the cytoplasm and forms protein–protein complexes with host interactors. All identified AvrBsT interactors regulate HR cell death and defense responses in plants. Notably, CaSGT1 physically binds to both AvrBsT and pepper receptor-like cytoplasmic kinase1 (CaPIK1) in the cytoplasm. During infection with Xanthomonas campestris pv. vesicatoria strain Ds1 (avrBsT), AvrBsT is phosphorylated by CaPIK1 and forms the active AvrBsT–CaSGT1–CaPIK1 complex, which ultimately triggers HR cell death and defense responses. Collectively, the AvrBsT interactor proteins are involved in plant cell death and immunity signaling.

Phosphoenolpyruvate carboxykinase, a member of the lyase family, is involved in the metabolic pathway of gluconeogenesis in organisms. Although the major function of PEPCK in gluconeogenesis is well established, it is unclear whether this enzyme is involved in plant immunity. Here, we isolated and identified the pepper (Capsicum annuum) PEPCK (CaPEPCK1) gene from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaPEPCK1 was strongly expressed in pepper leaves during the incompatible interaction with avirulent Xcv and in response to environmental stresses, especially salicylic acid (SA) treatment. PEPCK activity was low in healthy leaves but dramatically increased in avirulent Xcv-infected leaves. Knock-down expression of CaPEPCK1 by virus-induced gene silencing resulted in high levels of susceptibility to both virulent and avirulent Xcv infection. CaPEPCK1 silencing in pepper compromised induction of the basal defense-marker genes CaPR1 (pathogenesis-related 1 protein), CaPR10 (pathogenesis-related 10 protein) and CaDEF1 (defensin) during Xcv infection. SA accumulation was also significantly suppressed in the CaPEPCK1-silenced pepper leaves infected with Xcv. CaPEPCK1 in an Arabidopsis overexpression (OX) line inhibited the proliferation of Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis (Hpa). CaPEPCK1-OX plants developed more rapidly, with enlarged leaves, compared to wild-type plants. The T-DNA insertion Arabidopsis orthologous mutants pck1-3 and pck1-4 were more susceptible to the bacterial Pst and oomycete Hpa pathogens than the wild type. Taken together, these results suggest that CaPEPCK positively contributes to plant innate immunity against hemibiotrophic bacterial and obligate biotrophic oomycete pathogens.

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.

Pepper leucine-rich repeat protein (CaLRR1) interacts with defense response proteins to regulate plant cell death and immunity. This review highlights the current understanding of the molecular functions of CaLRR1 and its interactor proteins. Plant cell death and immune responses to microbial pathogens are controlled by complex and tightly regulated molecular signaling networks. Xanthomonas campestris pv. vesicatoria (Xcv)-inducible pepper (Capsicum annuum) leucine-rich repeat protein 1 (CaLRR1) serves as a molecular marker for plant cell death and immunity signaling. In this review, we discuss recent advances in elucidating the functional roles of CaLRR1 and its interacting plant proteins, and understanding how they are involved in the cell death and defense responses. CaLRR1 physically interacts with pepper pathogenesis-related proteins (CaPR10 and CaPR4b) and hypersensitive-induced reaction protein (CaHIR1) to regulate plant cell death and defense responses. CaLRR1 is produced in the cytoplasm and trafficked to the extracellular matrix. CaLRR1 binds to CaPR10 in the cytoplasm and CaPR4b and CaHIR1 at the plasma membrane. CaLRR1 synergistically accelerates CaPR10-triggered hypersensitive cell death, but negatively regulates CaPR4b- and CaHIR1-triggered cell death. CaHIR1 interacts with Xcv filamentous hemagglutinin (Fha1) to trigger disease-associated cell death. The subcellular localization and cellular function of these CaLRR1 interactors during plant cell death and defense responses were elucidated by Agrobacterium-mediated transient expression, virus-induced gene silencing, and transgenic overexpression studies. CaPR10, CaPR4b, and CaHIR1 positively regulate defense signaling mediated by salicylic acid and reactive oxygen species, thereby activating hypersensitive cell death and disease resistance. A comprehensive understanding of the molecular functions of CaLRR1 and its interacting protein partners in cell death and defense responses will provide valuable information for the molecular genetics of plant disease resistance, which could be exploited as a sustainable disease management strategy.

Summary Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.

Formate dehydrogenase (FDH; EC 1.2.1.2) is an NAD-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide. Here, we report the identification and characterization of pepper (Capsicum annuum) mitochondrial FDH1 as a positive regulator of cell death and defense responses. Transient expression of FDH1 caused hypersensitive response (HR)-like cell death in pepper and Nicotiana benthamiana leaves. The D-isomer-specific 2-hydroxyacid dehydrogenase signatures of FDH1 were required for the induction of HR-like cell death and FDH activity. FDH1 contained a mitochondrial targeting sequence at the N-terminal region; however, mitochondrial localization of FDH1 was not essential for the induction of HR-like cell death and FDH activity. FDH1 silencing in pepper significantly attenuated the cell death response and salicylic acid levels but stimulated growth of Xanthomonas campestris pv vesicatoria. By contrast, transgenic Arabidopsis (Arabidopsis thaliana) overexpressing FDH1 exhibited greater resistance to Pseudomonas syringae pv tomato in a salicylic acid-dependent manner. Arabidopsis transfer DNA insertion mutant analysis indicated that AtFDH1 expression is required for basal defense and resistance gene-mediated resistance to P. syringae pv tomato infection. Taken together, these data suggest that FDH1 has an important role in HR-like cell death and defense responses to bacterial pathogens.

Xanthomonas campestris pv vesicatoria type III effector protein, AvrBsT, triggers hypersensitive cell death in pepper (Capsicum annuum). Here, we have identified the pepper SGT1 (for suppressor of the G2 allele of skp1) as a host interactor of AvrBsT and also the pepper PIK1 (for receptor-like cytoplasmic kinase1). PIK1 specifically phosphorylates SGT1 and AvrBsT in vitro. AvrBsT specifically binds to the CHORD-containing protein and SGT1 domain of SGT1, resulting in the inhibition of PIK1-mediated SGT1 phosphorylation and subsequent nuclear transport of the SGT1-PIK1 complex. Liquid chromatography-tandem mass spectrometry of the proteolytic peptides of SGT1 identified the residues serine-98 and serine-279 of SGT1 as the major PIK1-mediated phosphorylation sites. Site-directed mutagenesis of SGT1 revealed that the identified SGT1 phosphorylation sites are responsible for the activation of AvrBsT-triggered cell death in planta. SGT1 forms a heterotrimeric complex with both AvrBsT and PIK1 exclusively in the cytoplasm. Agrobacterium tumefaciens-mediated coexpression of SGT1 and PIK1 with avrBsT promotes avrBsT-triggered cell death in Nicotiana benthamiana, dependent on PIK1. Virus-induced silencing of SGT1 and/or PIK1 compromises avrBsT-triggered cell death, hydrogen peroxide production, defense gene induction, and salicylic acid accumulation, leading to the enhanced bacterial pathogen growth in pepper. Together, these results suggest that SGT1 interacts with PIK1 and the bacterial effector protein AvrBsT and promotes the hypersensitive cell death associated with PIK1-mediated phosphorylation in plants.

Phospholipases hydrolyze phospholipids into fatty acids and other lipophilic substances. Phospholipid signaling is crucial for diverse cellular processes in plants. However, the precise role of phospholipases in plant cell death and defense signaling is not fully understood. Here, we identified a pepper (Capsicum annuum) patatin-like phospholipase (CaPLP1) gene that is transcriptionally induced in pepper leaves by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaPLP1 containing an N-terminal signal peptide localized to the cytoplasm and plasma membrane, leading to the secretion into the apoplastic regions. Silencing of CaPLP1 in pepper conferred enhanced susceptibility to Xcv infection. Defense responses to Xcv, including the generation of reactive oxygen species (ROS), hypersensitive cell death and the expression of the salicylic acid (SA)-dependent marker gene CaPR1, were compromised in the CaPLP1-silenced pepper plants. Transient expression of CaPLP1 in pepper leaves induced the accumulation of fluorescent phenolics, expression of the defense marker genes CaPR1 and CaSAR82A, and generation of ROS, ultimately leading to the hypersensitive cell death response. Overexpression (OX) of CaPLP1 in Arabidopsis also conferred enhanced resistance to Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis infection. CaPLP1-OX leaves showed reduced Pst growth, enhanced ROS burst and electrolyte leakage, induction of the defense response genes AtPR1, AtRbohD and AtGST, as well as constitutive activation of both the SA-dependent gene AtPR1 and the JA-dependent gene AtPDF1.2. Together, these results suggest that CaPLP1 is involved in plant defense and cell death signaling in response to microbial pathogens.

Osmotin or osmotin-like protein, a PR-5 family member, is differentially induced in plants by abiotic and biotic stresses. Here, we demonstrate that the pepper (Capsicum annuum) osmotin-like protein 1 gene, CaOSM1, was required for the defense and hypersensitive cell death response and oxidative burst signaling during Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaOSM1 protein was localized to the plasma membrane in leaf cells of Nicotiana benthamiana. Agrobacterium-mediated transient expression of CaOSM1 in pepper distinctly induced the hypersensitive cell death response and H2O2 accumulation. Knock-down of CaOSM1 in pepper by virus-induced gene silencing increased the susceptibility to Xcv infection, which was accompanied by attenuation of the cell death response and decreased accumulation of H2O2. CaOSM1 overexpression in transgenic Arabidopsis conferred reduced susceptibility and accelerated cell death response and H2O2 accumulation to infection by Pseudomonas syringe pv. tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that CaOSM1 is involved in cell death and oxidative burst responses during plant defense against microbial pathogens.

The Xanthomonas campestris pv vesicatoria (Xcv) effector AvrBsT induces a hypersensitive cell death in pepper (Capsicum annuum). However, the molecular mechanisms underlying AvrBsT-triggered cell death are not fully understood. Here, we identified pepper arginine decarboxylase (CaADC1) as an AvrBsT-interacting protein, which is early and strongly induced in incompatible pepper-Xcv interactions. Bimolecular fluorescence complementation and coimmunoprecipitation assays showed that the CaADC1-AvrBsT complex was localized to the cytoplasm. Transient coexpression of CaADC1 with avrBsT in Nicotiana benthamiana leaves specifically enhanced AvrBsT-triggered cell death, accompanied by an accumulation of polyamines, nitric oxide (NO), and hydrogen peroxide (H2O2) bursts. Among the polyamines, spermine application strongly induced NO and H2O2 bursts, ultimately leading to cell death. CaADC1 silencing in pepper leaves significantly compromised NO and H2O2 accumulation and cell death induction, leading to the enhanced avirulent Xcv growth during infection. The levels of salicylic acid, polyamines, and γ-aminobutyric acid (GABA), and the expression of defense response genes during avirulent Xcv infection, were distinctly lower in CaADC1-silenced plants than those in the empty vector control plants. GABA application significantly inhibited avirulent Xcv growth in CaADC1-silenced leaves and the empty vector control plants. Together, these results suggest that CaADC1 may act as a key defense and cell death regulator via mediation of polyamine and GABA metabolism.

Proteomics and functional analyses of the Arabidopsis - Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection. Notably, avirulent Pst DC3000 (avrRpt2) infection led to significant induction of AtNIT2 and AtNIT4 in leaves. Pst DC3000 and Pst DC3000 (avrRpt2) significantly grew well in leaves of nitrilase transgenic (nit2i-2) and mutant (nit1-1 and nit3-1) lines compared to the wild-type leaves. In contrast, NIT2 overexpression in nit2 mutants led to significantly high growth of the two Pst strains in leaves. The nitrilase transgenic and mutant lines exhibited enhanced susceptibility to Hyaloperonospora arabidopsidis infection. The nit2 mutation enhanced Pst DC3000 (avrRpt2) growth in salicylic acid (SA)-deficient NahG transgenic and sid2 and npr1 mutant lines. Infection with Pst DC3000 or Pst DC3000 (avrRpt2) induced lower levels of indole-3-acetic acid (IAA) in nit2i and nit2i NahG plants than in wild-type plants, but did not alter the IAA level in NahG transgenic plants. This suggests that Arabidopsis nitrilase 2 is involved in IAA signaling of defense and R gene-mediated resistance responses to Pst infection. Quantification of SA in these transgenic and mutant plants demonstrates that Arabidopsis nitrilase 2 is not required for SA-mediated defense response to the virulent Pst DC3000 but regulates SA-mediated resistance to the avirulent Pst DC3000 (avrRpt2). These results collectively suggest that Arabidopsis nitrilase genes are involved in plant defense and R gene-mediated resistant responses to microbial pathogens.

Plants produce various proteinaceous inhibitors to protect themselves against microbial pathogen attack. A xyloglucan-specific endo-β-1,4-glucanase inhibitor1 gene, CaXEGIP1, was isolated and functionally characterized in pepper (Capsicum annuum) plants. CaXEGIP1 was rapidly and strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria, and purified CaXEGIP1 protein significantly inhibited the hydrolytic activity of the glycoside hydrolase74 family xyloglucan-specific endo-β-1,4-glucanase from Clostridium thermocellum. Soluble-modified green fluorescent protein-tagged CaXEGIP1 proteins were mainly localized to the apoplast of onion (Allium cepa) epidermal cells. Agrobacterium tumefaciens-mediated overexpression of CaXEGIP1 triggered pathogen-independent, spontaneous cell death in pepper and Nicotiana benthamiana leaves. CaXEGIP1 silencing in pepper conferred enhanced susceptibility to virulent and avirulent X. campestris pv vesicatoria, accompanied by a compromised hypersensitive response and lowered expression of defense-related genes. Overexpression of dexamethasone:CaXEGIP1 in Arabidopsis (Arabidopsis thaliana) enhanced resistance to Hyaloperonospora arabidopsidis infection. Comparative histochemical and proteomic analyses revealed that CaXEGIP1 overexpression induced a spontaneous cell death response and also increased the expression of some defense-related proteins in transgenic Arabidopsis leaves. This response was also accompanied by cell wall thickening and darkening. Together, these results suggest that pathogen-inducible CaXEGIP1 positively regulates cell death-mediated defense responses in plants.

Loss-of-function alleles of the mildew resistance locus O (MLO) gene provide broad-spectrum powdery mildew disease resistance. Here, we identified a pepper (Capsicum annuum) MLO gene (CaMLO2) that is transcriptionally induced by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Topology and subcellular localization analyses reveal that CaMLO2 is a plasma membrane-anchored and amphiphilic Ca²⁺-dependent calmodulin-binding protein. CaMLO2 expression is up-regulated by Xcv and salicylic acid, as well as abiotic stresses. Silencing of CaMLO2 in pepper plants confers enhanced resistance against virulent Xcv, but not against avirulent Xcv. This resistance is accompanied by a compromised susceptibility cell-death response and reduced bacterial growth, as well as an accelerated reactive oxygen species burst. Virulent Xcv infection drastically induces expression of the salicylic acid-dependent defense marker gene CaPR1 in CaMLO2-silenced leaves. CaMLO2 over-expression in Arabidopsis enhances susceptibility to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. Leaves of plants over-expressing CaMLO2 exhibit a susceptibility cell-death response and high bacterial growth during virulent Pst DC3000 infection. These are accompanied by enhanced electrolyte leakage but compromised induction of some defense response genes and the reactive oxygen species. Together, our results suggest that CaMLO2 is involved in the susceptibility cell-death response and bacterial and oomycete proliferation in pepper and Arabidopsis.

The regulation of gene expression via post-transcriptional modification by RNA-binding proteins is crucial for plant disease and innate immunity. Here, we report the identification of the pepper (Capsicum annuum) RNA-binding protein1 gene (CaRBP1) as essential for hypersensitive cell death and defense signaling in the cytoplasm. CaRBP1 contains an RNA recognition motif and is rapidly and strongly induced in pepper by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaRBP1 displays in vitro RNA- and DNA-binding activity and in planta nucleocytoplasmic localization. Transient expression of CaRBP1 in pepper leaves triggers cell-death and defense responses. Notably, cytoplasmic localization of CaRBP1, mediated by the N-terminal region of CaRBP1, is essential for the hypersensitive cell-death response. Silencing of CaRBP1 in pepper plants significantly enhances susceptibility to avirulent Xcv infection. This is accompanied by compromised hypersensitive cell death, production of reactive oxygen species in oxidative bursts, expression of defense marker genes and accumulation of endogenous salicylic acid and jasmonic acid. Over-expression of CaRBP1 in Arabidopsis confers reduced susceptibility to infection by the biotrophic oomycete Hyaloperonospora arabidopsidis. Together, these results suggest that cytoplasmic localization of CaRBP1 is required for plant signaling of hypersensitive cell-death and defense responses.

Recognition of bacterial effector proteins by plant cells is crucial for plant disease and defense response signaling. The Xanthomonas campestris pv. vesicatoria (Xcv) type III effector protein, AvrBsT, is secreted into plant cells from Xcv strain Bv5-4a. Here, we demonstrate that dexamethasone (DEX): avrBsT overexpression triggers cell death signaling in healthy transgenic Arabidopsis plants. AvrBsT overexpression in Arabidopsis also reduced susceptibility to infection with the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Overexpression of avrBsT significantly induced some defense-related genes in Arabidopsis leaves. A high-throughput in planta proteomics screen identified TCP-1 chaperonin, SEC7-like guanine nucleotide exchange protein and calmodulin-like protein, which were differentially expressed in DEX:avrBsT-overexpression (OX) Arabidopsis plants during Hp. arabidopsidis infection. Treatment with purified GST-tagged AvrBsT proteins distinctly inhibited the growth and sporulation of Hp. arabidopsidis on Arabdiopsis cotyledons. In contrast, DEX:avrBsT-OX plants exhibited enhanced susceptibility to Pseudomonas syringae pv. tomato (Pst) DC3000 infection. Notably, susceptible cell death and enhanced electrolyte leakage were significantly induced in the Pst-infected leaves of DEX:avrBsT-OX plants. Together, these results suggest that Xcv effector AvrBsT overexpression triggers plant cell death, disease and defense signaling leading to both disease and defense responses to microbial pathogens of different lifestyles.

In plants, biotic and abiotic stresses regulate the expression and activity of various peroxidase isoforms. Capsicum annuum EXTRACELLULAR PEROXIDASE 2 (CaPO2) was previously shown to play a role in local and systemic reactive oxygen species bursts and disease resistance during bacterial pathogen infection. Here, we report CaPO2 expression patterns and functions during conditions of biotic and abiotic stress. In pepper plants, CaPO2 expression was strongly induced by abscisic acid, but not by defense-related plant hormones such as salicylic acid, ethylene and jasmonic acid. CaPO2 was also strongly induced by abiotic and biotic stress treatments, including drought, cold, high salinity and infection by the hemibiotrophic fungal pathogen Colletotrichum coccodes. Loss-of-function of CaPO2 in virus-induced gene silenced pepper plants led to increased susceptibility to salt- and osmotic-induced stress. In contrast, CaPO2 overexpression in transgenic Arabidopsis thaliana plants conferred enhanced tolerance to high salt, drought, and oxidative stress, while also enhancing resistance to infection by the necrotrophic fungal pathogen Alternaria brassicicola. Taken together, these results provide evidence for the involvement of pepper extracellular peroxidase CaPO2 in plant defense responses to various abiotic stresses and plant fungal pathogens.

Proteomics facilitates our understanding of cellular processes and network functions in the plant defense response during abiotic and biotic stresses. Here, we demonstrate that the ectopic expression of the Capsicum annuum antimicrobial protein CaAMP1 gene in Arabidopsis thaliana confers enhanced tolerance to methyl viologen (MV)-induced oxidative stress, which is accompanied by lower levels of lipid peroxidation. Quantitative comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified some of the oxidative stress- and disease-related proteins that are differentially regulated by CaAMP1 overexpression in Arabidopsis leaves. Antioxidant- and defense-related proteins, such as 2-cys peroxiredoxin, L-ascorbate peroxidase, peroxiredoxin, glutathione S-transferase and copper homeostasis factor, were up-regulated in the CaAMP1 transgenic leaf tissues. In contrast, GSH-dependent dehydroascorbate reductase and WD-40 repeat family protein were down-regulated by CaAMP1 overexpression. In addition, CaAMP1 overexpression enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and also H(2)O(2) accumulation in Arabidopsis. The identified antioxidant- and defense-related genes were differentially expressed during MV-induced oxidative stress and Pst DC3000 infection. Taken together, we conclude that CaAMP1 overexpression can regulate the differential expression of defense-related proteins in response to environmental stresses to maintain reactive oxygen species (ROS) homeostasis.

Summary Asparagine synthetase is a key enzyme in the production of the nitrogen-rich amino acid asparagine, which is crucial to primary nitrogen metabolism. Despite its importance physiologically, the roles that asparagine synthetase plays during plant defense responses remain unknown. Here, we determined that pepper (Capsicum annuum) asparagine synthetase 1 (CaAS1) is essential for plant defense to microbial pathogens. Infection with Xanthomonas campestris pv. vesicatoria (Xcv) induced early and strong CaAS1 expression in pepper leaves and silencing of this gene resulted in enhanced susceptibility to Xcv infection. Transgenic Arabidopsis (Arabidopsis thaliana) plants that overexpressed CaAS1 exhibited enhanced resistance to Pseudomonas syringae pv. tomato DC3000 and Hyaloperonospora arabidopsidis. Increased CaAS1 expression influenced early defense responses in diseased leaves, including increased electrolyte leakage, reactive oxygen species and nitric oxide bursts. In plants, increased conversion of aspartate to asparagine appears to be associated with enhanced resistance to bacterial and oomycete pathogens. In CaAS1-silenced pepper and/or CaAS1-overexpressing Arabidopsis, CaAS1-dependent changes in asparagine levels correlated with increased susceptibility or defense responses to microbial pathogens, respectively. Linking transcriptional and targeted metabolite studies, our results suggest that CaAS1 is required for asparagine synthesis and disease resistance in plants.

Certain protein kinases have been shown to be crucial for plant cell signaling pathways associated with plant immune responses. Here we identified a pepper (Capsicum annuum) receptor-like cytoplasmic protein kinase (RLCK) gene (CaPIK1) that is transcriptionally activated by infection with Xanthomonas campestris pv. vesicatoria (Xcv). Silencing of CaPIK1 in pepper plants confers enhanced susceptibility to Xcv infection. Salicylic acid-dependent defense responses are attenuated in the CaPIK1-silenced plants, including expression of salicylic acid-dependent genes, but not of a jasmonic acid-regulated gene. Induction of salicylic acid accumulation by Xcv infection is compromised in CaPIK1-silenced plants. The functional CaPIK1 protein not only autophosphorylates, but also phosphorylates myelin basic protein. CaPIK1 exists in the cytoplasm and also localizes to the plasma membrane of plant cells via its N-terminus. Transient expression of CaPIK1 in pepper leaves leads to generation of reactive oxygen species (ROS), ultimately leading to hypersensitive cell death. Over-expression (OX) of CaPIK1 in Arabidopsis enhances the basal resistance to infection with Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis, associated with elevated ROS bursts. Salicylic acid levels in CaPIK1-OX plants are higher than those in wild-type plants. Together, these results suggest that CaPIK1 modulates the signaling required for the salicylic acid-dependent defense response to pathogen infection.

Pathogen-induced programmed cell death (PCD) is intimately linked with disease resistance and susceptibility. However, the molecular components regulating PCD, including hypersensitive and susceptible cell death, are largely unknown in plants. In this study, we show that pathogen-induced Capsicum annuum hypersensitive induced reaction 1 (CaHIR1) and leucine-rich repeat 1 (CaLRR1) function as distinct plant PCD regulators in pepper plants during Xanthomonas campestris pv. vesicatoria infection. Confocal microscopy and protein gel blot analyses revealed that CaLRR1 and CaHIR1 localize to the extracellular matrix and plasma membrane (PM), respectively. Bimolecular fluorescent complementation and coimmunoprecipitation assays showed that the extracellular CaLRR1 specifically binds to the PM-located CaHIR1 in pepper leaves. Overexpression of CaHIR1 triggered pathogen-independent cell death in pepper and Nicotiana benthamiana plants but not in yeast cells. Virus-induced gene silencing (VIGS) of CaLRR1 and CaHIR1 distinctly strengthened and compromised hypersensitive and susceptible cell death in pepper plants, respectively. Endogenous salicylic acid levels and pathogenesis-related gene transcripts were elevated in CaHIR1-silenced plants. VIGS of NbLRR1 and NbHIR1, the N. benthamiana orthologs of CaLRR1 and CaHIR1, regulated Bax- and avrPto-/Pto-induced PCD. Taken together, these results suggest that leucine-rich repeat and hypersensitive induced reaction proteins may act as cell-death regulators associated with plant immunity and disease.

Plant cytochrome P450 enzymes are involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates, plant hormones, or defensive compounds. Herein, we have identified the pepper cytochrome P450 gene CaCYP450A, which is differentially induced during Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaCYP450A contains a heme-binding motif, PXFXXGXRXCXG, located in the C-terminal region and a hydrophobic membrane anchor region at the N terminal. Knock-down of CaCYP450A by virus-induced gene silencing (VIGS) led to increased susceptibility to Xcv infection in pepper. CaCYP450A-overexpressing Arabidopsis plants exhibited lower pathogen growth and reduced disease symptoms, and they were more resistant to Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis than wild-type plants. Overexpression of CaCYP450A also enhanced H(2)O(2) accumulation and cell death. However, CaCYP450A Arabidopsis ortholog CYP94B3 mutants showed enhanced susceptibility to virulent Pst DC3000, but not to avirulent Pst DC3000 avrRpm1 or virulent H. arabidopsidis infection. Taken together, these results suggest that CaCYP450A is required for defense responses to microbial pathogens in plants. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number HM581974.

Analysis of the promoters of defense-related genes is valuable for determining stress signaling and transcriptional activation during pathogen infection. Here, we have isolated and functionally characterized the promoter region of the pepper (Capsicum annuum) pectin methylesterase inhibitor 1 (CaPMEI1) gene in transiently transformed tobacco plants and stably transformed Arabidopsis plants. Among four 5' deletion constructs analyzed, the -958-bp CaPMEI1 promoter induced a high level of GUS reporter activity in tobacco leaf tissue, driven by pathogen infection as well as by ethylene and methyl jasmonate (MeJA) treatment. The 204-bp region from -958 bp to -754 bp of the CaPMEI1 promoter is responsible for the stress-responsive expression. In addition, the pepper transcription factor CARAV1 activated the CaPMEI1 promoter in tobacco leaves, whereas the transcription factor CAbZIP1 did not. In the transgenic Arabidopsis plants, the -958 bp CaPMEI1 promoter was functionally regulated by developmental cues, bacterial and oomycete pathogen infections, and treatment with ethylene and MeJA. Histochemical GUS staining analyses of Arabidopsis tissues revealed that the CaPMEI1 promoter was mainly activated in leaf veins in response to various biotic and abiotic stimuli. Together, these results suggest that CaPMEI1 promoter activation may be a critical molecular event for host defense response and ethylene- and MeJA-mediated CaPMEI1 gene expression.

In healthy, untreated pepper plants, transcripts of thionin (CATHION1) gene were induced in an organ-specific manner, as revealed by RNA blot analysis and in situ hybridization. The CATHION1 mRNAs were expressed in stems and flowers as well as roots, but not in leaves and fruits. In the stem and root tissues, the CATHION1 mRNA was present in endodermis and the phloem. In contrast, the transcripts were uniformly located in all the cells of flower tissues. Northern blot analysis and in situ hybridization were also performed to examine the temporal and spatial expression patterns of thionin (CATHION1) mRNA in leaf and fruit tissues of pepper (Capsicum annuum L.) infected by anthracnose pathogens Colletotrichum coccodes and C. gloeosporioides, respectively. Transcripts of the CATHION1 gene were more rapidly induced in pepper leaves at the 4- than at the 8-leaf stage by C. coccodes infection. Typical anthracnose symptoms were observed on green pepper fruits inoculated with C. gloeosporioides, but not on red pepper fruits. The transcript accumulation was more pronounced in green fruits than in red fruits upon C. gloeosporioides infection. The CATHION1 transcripts were detected in the phloem cells infected by anthracnose pathogens.

Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1 (for Capsicum annuum ANTIMICROBIAL PROTEIN1), was isolated from pepper (C. annuum) leaves infected with Xanthomonas campestris pv vesicatoria. Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad-spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was localized mainly in the external and intercellular regions of onion (Allium cepa) epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to X. campestris pv vesicatoria and Colletotrichum coccodes infection, accompanied by reduced PATHOGENESIS-RELATED (PR) gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis (Arabidopsis thaliana) conferred broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiolae and Alternaria brassicicola. CaAMP1 overexpression induced the salicylic acid pathway-dependent genes PR1 and PR5 but not the jasmonic acid-dependent defense gene PDF1.2 during P. syringae pv tomato infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad-spectrum resistance to bacterial and fungal pathogen infection.

Plant integral membrane proteins have essential roles in diverse internal and external physiological processes as signal receptors or ion transporters. The pepper CaPIMP1 gene encoding a putative integral membrane protein with four transmembrane domains was isolated and functionally characterized from pepper leaves infected with the avirulent strain Xanthomonas campestris pv. vesicatoria (Xcv). CaPIMP1-green fluorescence protein (GFP) fusions localized to the plasma membrane in onion cells, as observed by confocal microscopy. CaPIMP1 was expressed in an organ-specific manner in healthy pepper plants. Infection with Xcv induced differential accumulation of CaPIMP1 transcripts in pepper leaf tissues during compatible and incompatible interactions. The function of CaPIMP1 was examined by using the virus-induced gene silencing technique in pepper plants and by overexpression in Arabidopsis. CaPIMP1-silenced pepper plants were highly susceptible to Xcv infection and expressed lower levels of the defense-related gene CaSAR82A. CaPIMP1 overexpression (CaPIMP1-OX) in transgenic Arabidopsis conferred enhanced resistance to P. syringae pv. tomato infection, accompanied by enhanced AtPDF1.2 gene expression. In contrast, CaPIMP1-OX plants were highly susceptible to the biotrophic oomycete Hyaloperonospora parasitica. Taken together, we propose that CaPIMP1 plays distinct roles in both bacterial disease resistance and oomycete disease susceptibility.

As environmental and commercial requirements for new fungicides are increasingly demanding, antifungal compounds of microbial origin attract tremendous interest as a starting point in the development of environmentally sound agricultural fungicides. As seen in fenpiclonil, fludioxonil and synthetic derivatives of the strobilurins such as azoxystrobin and krexosim-methyl, this approach for the development of microbial fungicides has proven to be a promising and effective strategy for developing new fungicides. As a result, microbial metabolites face a revival as lead compounds. Recently, numerous antifungal compounds were discovered from diverse microbial sources using traditional activity-based screening techniques. These microbial compounds showed potent control efficacy against various plant diseases, including chronic diseases which are difficult to control with conventional synthetic fungicides. Advances in screening systems directed to specific targets of fungal metabolism have increased the opportunities to discover novel antifungal agents with selectivity over non-target organisms. Microbial metabolites have also been exploited as a source for non-fungicidal disease control agents that do not inhibit vegetative hyphal growth, but rather interfere specifically with the infection process of pathogenic fungi. The specificity of microbial fungicides is a highly preferred characteristic in terms of impacting the environment, where it is closely related to the occurrence of fungicide resistance. The most recently developed fungicides from microbial metabolites, the strobilurins, provide a cue for the high risk of resistance development of site-specific fungicides.

GDSL-type lipase is a hydrolytic enzyme whose amino acid sequence contains a pentapeptide motif (Gly-X-Ser-X-Gly) with active serine (Ser). Pepper GDSL-type lipase (CaGLIP1) gene was isolated and functionally characterized from pepper leaf tissues infected by Xanthomonas campestris pv. vesicatoria (Xcv). The CaGLIP1 protein was located in the vascular tissues of Arabidopsis root. The CaGLIP1 gene was preferentially expressed in pepper leaves during the compatible interaction with Xcv. Treatment with salicylic acid, ethylene and methyl jasmonate induced CaGLIP1 gene expression in pepper leaves. Sodium nitroprusside, methyl viologen, high salt, mannitol-mediated dehydration and wounding also induced early and transient CaGLIP1 expression in pepper leaf tissues. Virus-induced gene silencing of CaGLIP1 in pepper conferred enhanced resistance to Xcv, accompanied by the suppressed expression of basic PR1 (CaBPR1) and defensin (CaDEF1) genes. The CaGLIP1 lipase produced in Escherichia coli hydrolyzed the substrates of short and long chain nitrophenyl esters. The CaGLIP1-overexpressing Arabidopsis exhibited enhanced hydrolytic activity toward short and long chain nitrophenyl ester, as well as enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato and the biotrophic oomycete Hyaloperonospora parasitica. SA-induced expression of AtPR1 and AtGST1, also was delayed in CaGLIP1-overexpressing plants by SA application. During seed germination and plant growth, the CaGLIP1 transgenic plants showed drought tolerance and differential expression of drought- and abscisic acid (ABA)-inducible genes AtRD29A, AtADH and AtRab18. ABA treatment differentially regulated seed germination and gene expression in wild-type and CaGLIP1 transgenic Arabidopsis. Overexpression of CaGLIP1 also regulated glucose- and oxidative stress signaling. Together, these results indicate that CaGLIP1 modulates disease susceptibility and abiotic stress tolerance.

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 (H2O2) 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, H2O2 accumulation, and PR gene induction. In CaPO2-overexpression Arabidopsis leaves infected by Pseudomonas syringae pv tomato, H2O2 generation was sensitive to potassium cyanide (a peroxidase inhibitor) but insensitive to diphenylene iodonium (an NADPH oxidase inhibitor), suggesting that H2O2 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.

Promoter is a region of DNA to which transcription factor binds before initiating the transcription of DNA into RNA. The pepper pathogen-induced protein gene, CAPIP2, was locally or systemically induced in pepper plants infected by Xanthomonas campestris pv. vesicatoria. In this study, we isolated and transiently characterized the CAPIP2 promoter in tobacco leaves to identify the cis-acting regulatory sequences involved in CAPIP2 gene expression. The 991-bp DNA sequence upstream of the CAPIP2 gene was assessed for the activity of the CAPIP2 promoter fused to the β-glucuronidase (GUS) reporter gene, via an Agrobacterium-mediated transient expression assay. Several cis-acting elements, including GT1, MYB, RAV, and W-box, resided within the genomic sequence upstream of the CAPIP2 gene. The activation of the CAPIP2 promoter was induced by Pseudomonas syringae pv. tabaci, salicylic acid, methyl jasmonate and abscisic acid, NaCl and cold stress. The expression of the pepper transcription factors, CARAV1 and CAZFP1, was shown to activate the CAPIP2 promoter. Analysis of a series of 5′-deletions of the CAPIP2 promoter suggests that novel cis-acting elements necessary to induce gene expression by pathogens and environmental stresses are specifically localized in the CAPIP2 promoter region.

Carbohydrate-binding proteins, commonly referred to as lectins or agglutinins, function in defense responses to microbial pathogens. Pepper (Capsicum annuum) GNA-related lectin and PAN-domain protein gene CaGLP1 was isolated and functionally characterized from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaGLP1 contained an amine-terminus prokaryotic membrane lipoprotein lipid attachment site, a Galanthus nivalis agglutinin (GNA)-related lectin domain responsible for the recognition of high-mannose N-glycans, and a carboxyl-terminus PAN/apple domain. RNA gel blot and immunoblot analyses determined that CaGLP1 was strongly induced in pepper by compatible and incompatible Xcv infection. CaGLP1 protein localized primarily to the plasma membrane and exhibited mannose-binding specificity. CaGLP1-silenced pepper plants were more susceptible to compatible or incompatible Xcv infection compared with that of non-silenced control plants. CaGLP1 silencing in pepper leaves did not accumulate H2O2 and induce cell death during incompatible Xcv infection. Defense-related CaDEF1 (defensin) gene expression was significantly reduced in CaGLP1-silenced pepper plants. CaGLP1-overexpression in Arabidopsis thaliana enhanced resistance to Pseudomonas syringae pv. tomato. Defense-related AtPDF1.2 expression was elevated in CaGLP1-overexpression lines. Together, these results suggest that CaGLP1 is required for plant cell death and defense responses through the reactive oxygen species burst and downstream defense-related gene expression in response to bacterial pathogen challenge.

Plants are constantly exposed to a variety of biotic and abiotic stresses, which include pathogens and conditions of high salinity, low temperature, and drought. Abscisic acid (ABA) is a major plant hormone involved in signal transduction pathways that mediate the defense response of plants to abiotic stress. Previously, we isolated Ring finger protein gene (CaRING1) from pepper (Capsicum annuum), which is associated with resistance to bacterial pathogens, accompanied by hypersensitive cell death. Here, we report a new function of the CaRING1 gene product in the ABA-mediated defense responses of plants to dehydration stress. The expression of the CaRING1 gene was induced in pepper leaves treated with ABA or exposed to dehydration or NaCl. Virus-induced gene silencing of CaRING1 in pepper plants exhibited low degree of ABA-induced stomatal closure and high levels of transpirational water loss in dehydrated leaves. These led to be more vulnerable to dehydration stress in CaRING1-silenced pepper than in the control pepper, accompanied by reduction of ABA-regulated gene expression and low accumulation of ABA and H2O2. In contrast, CaRING1-overexpressing transgenic plants showed enhanced sensitivity to ABA during the seedling growth and establishment. These plants were also more tolerant to dehydration stress than the wild-type plants because of high ABA accumulation, enhanced stomatal closure and increased expression of stress-responsive genes. Together, these results suggest that the CaRING1 acts as positive factor for dehydration tolerance in Arabidopsis by modulating ABA biosynthesis and ABA-mediated stomatal closing and gene expression.

Limited information is available about the roles of RING-finger proteins in plant defense. A pepper CaRFP1 encoding the C3-H-C4 type RING-finger protein that physically interacted with the basic PR-1 protein CABPR1 was isolated from pepper leaves infected by Xanthomonas campestris pv. vesicatoria. The CaRFP1 protein has VWFA domain, and N-terminal serine-rich and C-terminal cysteine-rich regions. The CaRFP1 transcripts accumulated earlier than did those of the basic PR-1 gene CABPR1 during the incompatible interaction of pepper leaves with X. campestris pv. vesicatoria, as well as in the systemic, uninoculated pepper leaf tissues. The CaRFP1 gene also was induced in pepper leaf tissues infected by Colletotrichum coccodes. The CaRFP1 gene was strongly induced much earlier by salicylic acid, ethylene and methyl jasmonate treatments, as well as environmental stresses including methyl viologen, mannitol and NaCl treatments. Overexpression of the CaRFP1 gene in the transgenic Arabidopsis plants conferred disease susceptibility to Pseudomonas syringae pv. tomato infection, accompanied by reduced PR-2 and PR-5 gene expression, suggesting that the CaRFP1 acts as an E3 ligase for polyubiquitination of target PR proteins. Exogenous salicylic acid treatment also abolished PR-2 and PR-5 gene expression in the transgenic plants. Differential osmotic stress tolerance was induced by high salt and drought in the CaRFP1-overexpressing plants during germination and seedling development, which was closely correlated with abscisic acid sensitivity of Arabidopsis plants. These results suggest that the CaRFP1 gene functions as an early defense regulator controlling bacterial disease susceptibility and osmotic stress tolerance.

Pepper SAR8.2 gene (CASAR82A) was previously reported to be locally or systemically induced in pepper plants by biotic and abiotic stresses. In this study, the physiological and molecular functions of the pepper SAR8.2 protein in the plant defense responses were investigated by generating Arabidopsis transgenic lines overexpressing the CASAR82A gene. The transgenic Arabidopsis plants grew faster than the wild-type plants, indicating that the CASAR82A gene was involved in plant development. The ectopic expression of CASAR82A in Arabidopsis was accompanied by the expression of the Arabidopsis pathogenesis-related (PR)-genes including AtPR-1, AtPR-4 and AtPR-5. CASAR82A overexpression enhanced the resistance against infections by Pseudomonas syringae pv. tomato, Fusarium oxysporum f.sp. matthiolae or Botrytis cinerea. The transgenic plants also exhibited increased NaCl and drought tolerance during all growth stages. Moreover, the methyl viologen test showed that the transgenic plants were tolerant to oxidative stress. The purified recombinant CASAR82A protein and crude protein extracts of the transgenic plants exhibited antifungal activity against some phytopathogenic fungi, indicating that the enhanced resistance of the transgenic plants to fungal pathogen infection may be due to the antifungal effect of SAR8.2 protein.

The promoter fragment of 1065 bp upstream from the translation initiation site of the CALTPIII gene encoding a basic lipid transfer protein was isolated from the genomic DNA of Capsicum annuum . Some putative cis -acting elements, including an ERE-box, a W-box, and MYB-core elements were found in the promoter region of the CALTPIII gene. In Agrobacterium -mediated transient expression assay, strong activation of the CALTPIII full promoter region (1065 bp long) occurred in tobacco leaves after infection with Pseudomonas syringae pv. tabaci , and treatments with ethylene, salicylic acid, methyl jasmonate and H 2 O 2 . However, the full promoter did not respond to abscisic acid while previous studies have shown that the CALTPIII mRNA is indeed induced by ABA. Pathogen and ethylene responses were mostly affected by the deletion between −830 and −422 bp. Salicylic acid response was lost in any deleted construct. A fine analysis showed that the crucial elements for pathogen response are located between −626 and −552 bp.

The pepper SAR8.2 gene, CASAR82A, was locally and systemically induced in pepper plants which had been infected by Xanthomonas campestris pv. vesicatoria or by Pseudomonas fluorescens. The DNA 1,283 bp sequence upstream of the CASAR82A gene was assessed with regard to the activity of the CASAR82A promoter fused to the beta-glucuronidase (GUS) reporter gene, via an Agrobacterium-mediated transient expression assay. In tobacco leaves which transiently expressed the -831 bp CASAR82A promoter, GUS activity was locally and systemically induced by Pseudomonas syringae pv. tabaci. GUS activity, which was driven by the -831 promoter, was also differentially activated in the leaves as the result of treatment with salicylic acid, ethylene, methyl jasmonate, abscisic acid, NaCl, and low temperatures. The -831 bp sequence upstream of the CASAR82A gene elicited full promoter activity in response to pathogen infection, abiotic elicitors, and environmental stresses. The expression of the pepper transcription factor, CARAV1, was shown to activate the CASAR82A promoter. Analyses of a series of 5'-deletions of the CASAR82A promoter revealed that novel cis-acting elements necessary for the induction of gene expression as the result of exposure to pathogen and abiotic elicitors appear to be localized in the promoter region between -831 and -759 bp.

The activation of the CAChi2 promoter as the result of bacterial infection and osmotic stresses was examined using the Agrobacterium-mediated transient expression assay. Several stress-related cis-acting elements were revealed within the upstream genomic sequence of the CAChi2 gene. In tobacco leaf tissues transiently transformed with the CAChi2 promoter-beta-glucuronidase (GUS) gene, the CAChi2 promoter was up-regulated by Pseudomonas syringae pv. tabaci infection. The CAChi2-GUS activation was closely related to osmotic stresses, including treatment with mannitol and NaCl. The -378 CAChi2 promoter was sufficient for the CAChi2 gene induction by salicylic acid treatment. CAChi2 overexpression in the transgenic Arabidopsis plants enhanced bacterial disease resistance against Pseudomonas syringae pv. tomato infection. CAChi2-overexpressing Arabidopsis plants also exhibited increased tolerance to NaCl-induced osmotic stresses during seed germination and seedling growth. CAChi2 overexpression induced the expression of the NaCl stress-responsive gene RD29A in the absence of NaCl stress. The CAChi2-overexpressing transgenic plants exhibited increased sensitivity to abscisic acid during seed germination.

In order to determine the role of ascorbate peroxidase, an antioxidant enzyme, in the cellular responses to oxidative stress and pathogens, transgenic tobacco plants were generated, using the Capsicum annuum ascorbate peroxidase-like 1 gene (CAPOA1), under the control of the CaMV 35S promoter. High levels of CAPOA1 gene expression were observed in the transgenic plants, with a 2-fold increase in total peroxidase activity. The constitutive expression of CAPOA1 in the tobacco exhibited no morphological abnormalities, while significantly increased growth was observed in transgenic plants, as compared to control plants. The CAPOA1-overexpressed plants exhibited increased tolerance to methyl viologen-mediated oxidative stress, and also enhanced resistance to the oomycete pathogen, Phytophthora nicotianae. However, the transgenic plants were not found to be resistant to the bacterial pathogen, Pseudomonas syringae pv. tabaci, but were weakly resistant to Ralstonia solanacearum. Our results suggested that the overproduction of ascorbate peroxidase increased peroxidase activity that enhances active oxygen scavenging system, leading to oxidative stress tolerance and oomycete pathogen resistance. # 2005 Elsevier Ireland Ltd. All rights reserved.

The pathogen- and ethylene-inducible pepper-basic pathogenesis-related (PR)-1 gene, CABPR1, was strongly expressed in pepper leaves by osmotic and oxidative stresses. The pepper CABPR1 was introduced into the Arabidopsis plants under the control of the cauliflower mosaic virus 35S promoter. Polymerase chain reaction-amplification with the Arabidopsis genomic DNA and Northern blot analyses confirmed that the pepper CABPR1 gene was integrated into the Arabidopsis genome, where it was overexpressed in the transgenic Arabidopsis plants under normal growth conditions. The constitutive overexpression of CABPR1 induced the expression of the Arabidopsis PR-genes including PR-4, PR-5 and PDF1.2. Enhanced resistance to phytopathogenic bacteria, Pseudomonas syringae pv. tomato DC3000, was also observed in the transgenic Arabidopsis plants. CABPR1 overexpression in the transgenic Arabidopsis caused enhanced seed germination under NaCI (ionic) and mannitol (non-ionic) osmotic stresses. Enhanced tolerances to high salinity and dehydration stresses during seed germination of the transgenic plants were not found at the early seedling stage. The transgenic Arabidopsis plants exhibited a higher tolerance to oxidative stress by methyl viologen at the seed germination, seedling and adult plant stages. These results suggest that the CABPR1 gene may function in the enhanced disease resistance and oxidative stress tolerance of transgenic Arabidopsis plants.

A pepper zinc-finger protein gene, CAZFP1 , encoding the Cys2/His2-type zinc-finger transcription factor was isolated from pepper leaves inoculated with an avirulent strain Bv5-4a of Xanthomonas campestris pv. vesicatoria . The CAZFP1 protein is a nuclear targeting protein, which functions as a transcriptional regulator. The full-length CAZFP1 had no transcriptional activation activity, whereas the C-terminal region of CAZFP1 had transactivation activity. The CAZFP1 transcripts were constitutively expressed in the pepper stem, root, flower and red fruit, but were not detectable in the leaf and green fruit. The CAZFP1 transcripts accumulated earlier than the CAZFP1 (PR-1) gene in the incompatible interaction of the pepper leaves with X. campestris pv. vesicatoria . The CAZFP1 transcripts were significantly induced in the systemic, uninoculated leaf tissues early after inoculation with bacterial pathogens, but gradually declined thereafter. The CAZFP1 transcripts were localized, and confined to the phloem cells of the vascular bundle in the pepper leaf midrib in response to Colletotrichum. coccodes infection, ethylene and abscisic acid. The CAZFP1 gene was also induced much earlier by abiotic elicitors and environmental stresses, compared with the CAZFP1 gene. Overexpression of the CAZFP1 gene in the transgenic Arabidopsis plants enhanced not only the resistance against infection by Pseudomonas syringae pv. tomato, but also the drought tolerance. These results suggest that the CAZFP1 gene functions as an early-defense gene to enhance disease resistance and drought tolerance.

Pepper defensin ( CADEF1 ) clone was isolated from cDNA library constructed from pepper leaves infected with avirulent strain Bv5-4a of Xanthomonas campestris pv. vesicatoria . Putative protein encoded by CADEF1 gene consists of 78 amino acids including eight conserved cysteine residues to form four structure-stabilizing disulfide bridges. Transcription of the CADEF1 gene was earlier and more strongly induced by X. campestris pv. vesicatoria infection in the incompatible than in the compatible interaction. CADEF1 mRNA was constitutively expressed in stem, root and green fruit of pepper. Transcripts of CADEF1 gene drastically accumulated in pepper leaf tissues treated with salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), hydrogen peroxide (H 2 O 2 ), benzothiadiazole (BTH) and d , l -β-amino- n -butyric acid (BABA). In situ hybridization results revealed that CADEF1 mRNA was localized in the phloem areas of vascular bundles in leaf tissues treated with exogenous SA, MeJA and ABA. Strong accumulation of CADEF1 mRNA occurred in pepper leaves in response to wounding, high salinity and drought stress. These results suggest that bacterial pathogen infection, abiotic elicitors and some environmental stresses may play a significant role in signal transduction pathway for CADEF1 gene expression.

Northern blot and in situ hybridization analyses revealed that a pepper basic class II chitinase gene (CAChi2) is constitutively expressed in floral organs and root endodermis, but not in leaf, stem and fruit of pepper. Resistance of pepper leaves to Colletotrichum coccodes infection at a late growth stage was correlated with induction of beta-1,3-glucanase and PR-1 mRNA, but not of chitinase (CAChi2) mRNA. Transcriptional activation of the CAChi2 gene in pepper leaves occurred during anthracnose development. The CAChi2 transcripts were mainly localized in phloem cells of vascular tissues of pepper leaves infected with C. coccodes. The CAChi2 gene was also differentially induced in leaf and stem tissue by treatment with abscisic acid (ABA), sodium chloride or drought. Strong accumulation of the CAChi2 transcripts occurred in pepper stem tissues due to high salt and drought, and also due to treatment with ABA. These results suggest involvement of the chitinase gene in protection of pepper plants against the pathogen, but also document cross talk with stress signals mediated by ABA, high salinity and drought.

Pepper (Capsicum annuum L.) provides a good experimental system for studying the molecular and functional genomics underlying the ability of plants to defend themselves against microbial pathogens. Cell death is a genetically programmed response that requires specific host cellular factors. Hypersensitive response (HR) is defined as rapid cell death in response to a pathogen attack. Pepper plants respond to pathogen attacks by activating genetically controlled HR- or disease-associated cell death. HR cell death, specifically in incompatible interactions between pepper and Xanthomonas campestris pv. vesicatoria, is mediated by the molecular genetics and biochemical machinery that underlie pathogen-induced cell death in plants. Gene expression profiles during the HR-like cell death response, virus-induced gene silencing and transient and transgenic overexpression approaches are used to isolate and identify HR- or disease-associated cell death genes in pepper plants. Reactive oxygen species, nitric oxide, cytosolic calcium ion and defense-related hormones such as salicylic acid, jasmonic acid, ethylene and abscisic acid are involved in the execution of pathogen-induced cell death in plants. In this review, we summarize recent molecular and cellular studies of the pepper cell death-mediated defense response, highlighting the signaling events of cell death in disease-resistant pepper plants. Comprehensive knowledge and understanding of the cellular functions of pepper cell death response genes will aid the development of novel practical approaches to enhance disease resistance in pepper, thereby helping to secure the future supply of safe and nutritious pepper plants worldwide.

Pepper CaMLO2 specifically interacts with CaCaM1 and translocates cytoplasmic CaCaM1 to the plasma membrane, leading to the suppression of Xanthomonas AvrBsT-triggered Ca 2+ influx, hypersensitive cell death and defense responses. Pathogen-induced cell death is closely linked with disease susceptibility and resistance in plants. Pepper (Capsicum annuum) mildew resistance locus O (CaMLO2) and calmodulin (CaCaM1) genes are required for disease-associated cell death and hypersensitive cell death, respectively. Here, we demonstrate that pathogen-responsive CaMLO2 interacts with CaCaM1 in yeast and in planta. Bimolecular fluorescence complementation and co-immunoprecipitation analyses confirm a specific interaction between CaMLO2 and CaCaM1 at the plasma membrane (PM) in plant cells. Subcellular localization analyses of CaCaM1 fused to green fluorescent protein reveals that treatment with Ca2+ and co-expression with CaMLO2 induce translocation of cytosolic CaCaM1 to the PM where CaMLO2 is localized. Transient CaMLO2 expression negatively regulates CaCaM1 accumulation in Nicotiana benthamiana. Xanthomonas avrBsT-triggered Ca2+ influx and hypersensitive cell death are disrupted by CaCaM1 and/or CaMLO2 expression. CaMLO2 silencing in pepper significantly enhances reactive oxygen species burst, cell death, and resistance responses to Xanthomonas campestris pv. vesicatoria Ds1 and Ds1 (avrBsT), which is accompanied by enhanced induction of CaCaM1, CaPR1 (PR-1), and CaPO2 (peroxidase). These results suggest that CaMLO2 interacts with CaCaM1 and suppresses AvrBsT-triggered cell death and defense responses.

A pathogen induced pepper cyclophilin (CACYP1) cDNA clone was isolated from a pepper cDNA library from hypersensitive response (HR) lesions of leaves infected with Xanthomonas campestris pv.vesicatoria using a differential hybridization technique. The deduced amino acid sequences of thisCACYP1gene have high levels of identity with those of cyclophilin genes from tomatoes, potatoes, beans andArabidopsis . The CACYP1 mRNA was constitutively expressed in the leaf, stem, root and flower, but not in the green (unripe) and the red (ripe) fruits of pepper plants. The accumulation of CACYP1 mRNA was strongly induced in pepper plants by X. campestris pv. vesicatoria and Colletotrichum gloeosporioides infections. In the incompatible interaction, a drastic induction of the CACYP1 mRNA occurred in the bacteria inoculated leaves compared to that of the compatible interaction. Prior inoculation with an avirulent strain of X. campestris pv.vesicatoria in lower leaves resulted in a strong, systemic CACYP1 mRNA accumulation in the uninoculated upper leaves. In situ hybridization results revealed that CACYP1 mRNA was localized in the phloem cells of vascular tissues in pepper fruits infected by C. gloeosporioides. Treatment with salicyclic acid and methyl jasmonate strongly induced the CACYP1 transcripts in pepper leaves.

The compatible interaction of pepper stems with Phytophthora capsici showed more rapid and severe disease development than did the incompatible interaction, although pathogen penetration styles of host cells in compatible and incompatible interactions were similar to each other. Treatment with DL -β-amino- n -butyric acid (BABA) protected the pepper plants against P. capsici infection. Reduced hyphal growth and sporangial formation were found after P. capsici infection in BABA-induced resistant and incompatible reactions. One of the most noticeable ultrastructural features of the BABA-induced resistant reaction was the formation of electron-dense wall appositions. The thick and dense wall appositions that encased the haustoria restricted haustorial development, thus leading to limitation of further pathogen penetration into inner plant tissues. A main host response in the incompatible interaction was the occlusion of cortical cells with an amorphous material. Plugging of the intercellular spaces in the cortical cells with electron opaque material was frequently observed in the incompatible interaction, but not in the compatible interaction. Another common feature of the BABA-induced resistant and incompatible reactions was degeneration of mitochondrial structure within penetrating hyphal cytoplasm. The mitochondrial structure in the BABA-induced resistant or incompatible reactions had no distinct double membrane layer and well-shaped cristae.

A basic beta-1,3-glucanase cDNA clone (CABGLU) was isolated from the cDNA library constructed from hypersensitive response lesions of pepper leaves infected with avirulent strain of Xanthomonas campestris pv. vesicatoria. The deduced polypeptide of CABGLU which contains a C-terminal extension N-glycosylated at a single site characterized as typical structure of class I beta-1,3-glucanase has a high level of identity with tobacco basic beta-1,3-glucanase (77.4%), but only a moderate level of identity with tomato acidic beta-1,3-glucanase (42.6%). Genomic DNA gel blot analysis indicates that the pepper genome contains one or two beta-1,3-glucanase copy genes. Transcripts of the CABGLU gene were more induced in incompatible interactions than in compatible interactions, when inoculated with X. campestris pv. vesicatoria or Phytophthora capsici. Accumulation of CABGLU mRNA was strongly induced in pepper leaves by both ethephon and methyl jasmonate. The CABGLU mRNA was constitutively expressed only in the roots of all the plant organs. These data indicate that the basic beta-1,3-glucanase gene may be induced by pathogen attack and abiotic stresses.

A chitinase cDNA clone (designated CAChi2) was isolated from the cDNA library of pepper leaves infected with Xanthomonas campestris pv. vesicatoria . The 1004-bp full-length CAChi2 cDNA encodes a basic chitinase with an N-terminal 24 amino acid signal peptide followed by a catalytic region. An analysis of its sequence indicates that CAChi2 is a class II chitinase, because it does not have chitin-binding domain and C-terminal extension sequences. The deduced amino acid sequence of CAChi2 has a high level of identity with class II chitinases from potato, tomato, tobacco and petunia. Southern analysis demonstrated that the CAChi2 chitinase is encoded by a single or two copy genes in the pepper genome. Following X. campestris pv. vesicatoria or Phytophthora capsici infection, the CAChi2 chitinase mRNA was more highly expressed in the incompatible interaction, compared to expression in the compatible interaction. Treatment with ethylene-releasing ethephon resulted in a strong accumulation of the transcripts in the leaves. In contrast, dl -β-amino- n -butyric acid, salicylic acid and methyl jasmonate were not effective in inducing CAChi2 transcripts in pepper leaves.

The induction of chitinase ( CAChi2 ) mRNA started as early as 6 h after inoculation and gradually increased in the incompatible interaction of pepper stems with Phytophthora capsici. In the compatible interaction, the induction of the chitinase transcripts was detected later than that in the incompatible interaction. The CAChi2 mRNA was usually localized in the vascular tissues and their expression was constricted in the phloem-related cells. These results showed that the spatial pattern of CAChi2 mRNA expression was similar in both compatible and incompatible interactions but the temporal patterns were different from each other. In addition, the early induction of CAChi2 mRNA was quite distinct in the incompatible interaction. Immunogold labelling data showed specific labelling of chitinase on the cell wall of the oomycete in both compatible and incompatible interactions at 24 h after inoculation. In particular, numerous gold particles were deposited on the cell wall of P. capsici with a predominant accumulation over areas showing signs of degradation in the incompatible interaction. Chitinase labelling was also detected in the intercellular space and the host cytoplasm. However, healthy pepper stem tissue was nearly free of labelling.

Infection by pathogens or treatment with abiotic elicitors led to a strong expression of thionin gene in pepper (Capsicum annuum L.) leaves. The CATHION1 cDNA, which codes for thionin, was isolated from the cDNA library of pepper leaves infected with Xanthomonas campestris pv. vesicatoria. A comparison of theCATHION1 and other known thionin genes revealed that the eight cysteine residues are at the same location. Modes of transcriptional regulation of CATHION1 were compared between the compatible and incompatible interactions. Transcripts of the CATHION1 gene were more induced in the incompatible than compatible interactions of pepper leaves with X. campestris pv. vesicatoria, but occurred in a contrary manner in the interactions with Phytophthora capsici. Transcripts of the CATHION1 gene were constitutively expressed in healthy pepper stems, but considerably accumulated upon infection by virulent and avirulent isolates of P. capsici. CATHION1 mRNA was strongly induced by treatment with ethephon or methyl jasmonate (MeJA), but somewhat weakly by salicylic acid (SA) and benzothiadiazole. These results suggest that induction of CATHION1 mRNA by both SA and MeJA/ethylene may be involved in the signal transduction pathway of pepper defense- or pathogenesis-related plant responses for thionin synthesis in pepper plants.

A new basic pathogenesis-related (PR)-1 cDNA (CABPR1) was isolated from a cDNA library of pepper leaves infected with Xanthomonas campestris pv. vesicatoria. The full-length clone contains an open reading frame of 531 nucleotides encoding 177 amino acid residues with a predicted molecular mass of 20241 and pI of 8.1. The deduced amino acid sequence of CABPR1 has a high level of identity with PR-1 proteins from tobacco and tomato. Expression of the CABPR1 mRNA was greatly induced in an incompatible interaction of pepper leaves with X. campestris pv. vesicatoria. Accumulation of the basic PR-1 mRNA in the bacteria-infected leaves was intrinsically associated with the induction of ethylene biosynthesis. Treatment with 1-aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis, strongly suppressed the induction of PR-1 mRNA expression in the bacteria-infected leaves. These data suggest that ethylene may function as a strong signal elicitor in the activation of PR-1 genes, eventually mediating the plant defence response. Phytophthora capsici infection also distinctly stimulated ethylene biosynthesis, together with accumulation of PR-1 mRNA. Expression of the basic PR-1 mRNA was also induced in the pepper leaves by treatment with dl-β-amino-n-butyric acid or salicylic acid, but not by wounding. Treatment with methyl jasmonate, in combination with salicylic acid, strongly induced the PR-1 transcripts. The basic PR-1 gene expression occurred in untreated roots, flowers and green fruit of pepper but not in leaf, stem or red fruit.