Molecular basis for the interference of the Arabidopsis WRKY54-mediated immune response by two sequence-unrelated bacterial effectors.

Arabidopsis thaliana WRKY proteins are potential targets of pathogen-secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well-studied Arabidopsis nucleotide-binding and leucine-rich repeat (NLR) immune receptor carrying a C-terminal WRKY domain that functions as an integrated decoy. RRS1-R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death-inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54-induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54-induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA-binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4-mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses.

Phylogenetic analysis of ABCG subfamily proteins in plants: functional clustering and coevolution with ABCGs of pathogens.

ABCG subfamily proteins are highly enriched in terrestrial plants. Many of these proteins secrete secondary metabolites that repel or inhibit pathogens. To establish why the ABCG subfamily proteins proliferated extensively during evolution, we constructed phylogenetic trees from a broad range of eukaryotic organisms. ABCG proteins were massively duplicated in land plants and in oomycetes, a group of agronomically important plant pathogens, which prompted us to hypothesize that plant and pathogen ABCGs coevolved. Supporting this hypothesis, full-size ABCGs in host plants (Arabidopsis thaliana and Glycine max) and their pathogens (Hyaloperonospora arabidopsidis and Phytophthora sojae, respectively) had similar divergence times and patterns. Furthermore, generalist pathogens with broad ranges of host plants have diversified more ABCGs than their specialist counterparts. The hypothesis was further tested using an example pair of ABCGs that first diverged during multiplication in a host plant and its pathogen: AtABCG31 of A. thaliana and HpaP802307 of H. arabidopsidis. AtABCG31 expression was activated following infection with H. arabidopsidis, and disrupting AtABCG31 led to increased susceptibility to H. arabidopsidis. Together, our results suggest that ABCG genes in plants and their oomycete pathogens coevolved in an arms race, to extrude secondary metabolites involved in the plant's defense response against pathogens.

Structural analysis of Phytophthora suppressor of RNA silencing 2 (PSR2) reveals a conserved modular fold contributing to virulence.

Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae (PsPSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of PsPSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the PsPSR2-like arrangement, that is, containing a W-Y motif as the "start" unit, various numbers of L-W-Y motifs as the "middle" units, and a degenerate L-W-Y as the "end" unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of PsPSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a "building block" to accelerate effector evolution in Phytophthora.

A Phytophthora Effector Suppresses Trans-Kingdom RNAi to Promote Disease Susceptibility.

RNA silencing (RNAi) has a well-established role in anti-viral immunity in plants. The destructive eukaryotic pathogen Phytophthora encodes suppressors of RNAi (PSRs), which enhance plant susceptibility. However, the role of small RNAs in defense against eukaryotic pathogens is unclear. Here, we show that Phytophthora infection of Arabidopsis leads to increased production of a diverse pool of secondary small interfering RNAs (siRNAs). Instead of regulating endogenous plant genes, these siRNAs are found in extracellular vesicles and likely silence target genes in Phytophthora during natural infection. Introduction of a plant siRNA in Phytophthora leads to developmental deficiency and abolishes virulence, while Arabidopsis mutants defective in secondary siRNA biogenesis are hypersusceptible. Notably, Phytophthora effector PSR2 specifically inhibits secondary siRNA biogenesis in Arabidopsis and promotes infection. These findings uncover the role of siRNAs as antimicrobial agents against eukaryotic pathogens and highlight a defense/counter-defense arms race centered on trans-kingdom gene silencing between hosts and pathogens.

A Conserved EAR Motif Is Required for Avirulence and Stability of the Ralstonia solanacearum Effector PopP2 In Planta.

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease in many high value Solanaceae crops. R. solanacearum secretes around 70 effectors into host cells in order to promote infection. Plants have, however, evolved specialized immune receptors that recognize corresponding effectors and confer qualitative disease resistance. In the model species Arabidopsis thaliana, the paired immune receptors RRS1 (resistance to Ralstonia solanacearum 1) and RPS4 (resistance to Pseudomonas syringae 4) cooperatively recognize the R. solanacearum effector PopP2 in the nuclei of infected cells. PopP2 is an acetyltransferase that binds to and acetylates the RRS1 WRKY DNA-binding domain resulting in reduced RRS1-DNA association thereby activating plant immunity. Here, we surveyed the naturally occurring variation in PopP2 sequence among the R. solanacearum strains isolated from diseased tomato and pepper fields across the Republic of Korea. Our analysis revealed high conservation of popP2 sequence with only three polymorphic alleles present amongst 17 strains. Only one variation (a premature stop codon) caused the loss of RPS4/RRS1-dependent recognition in Arabidopsis. We also found that PopP2 harbors a putative eukaryotic transcriptional repressor motif (ethylene-responsive element binding factor-associated amphiphilic repression or EAR), which is known to be involved in the recruitment of transcriptional co-repressors. Remarkably, mutation of the EAR motif disabled PopP2 avirulence function as measured by the development of hypersensitive response, electrolyte leakage, defense marker gene expression and bacterial growth in Arabidopsis. This lack of recognition was partially but significantly reverted by the C-terminal addition of a synthetic EAR motif. We show that the EAR motif-dependent gain of avirulence correlated with the stability of the PopP2 protein. Furthermore, we demonstrated the requirement of the PopP2 EAR motif for PTI suppression. A yeast two-hybrid screen indicated that PopP2 does not interact with any well-known Arabidopsis transcriptional co-repressors. Overall, this study reveals high conservation of the PopP2 effector in Korean R. solanacearum strains isolated from commercially cultivated tomato and pepper genotypes. Importantly, our data also indicate that the PopP2 conserved repressor motif could contribute to the effector accumulation in plant cells.

A bacterial acetyltransferase triggers immunity in Arabidopsis thaliana independent of hypersensitive response.

Type-III secreted effectors (T3Es) play critical roles during bacterial pathogenesis in plants. Plant recognition of certain T3Es can trigger defence, often accompanied by macroscopic cell death, termed the hypersensitive response (HR). Economically important species of kiwifruit are susceptible to Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker. Although Psa is non-pathogenic in Arabidopsis thaliana, we observed that a T3E, HopZ5 that is unique to a global outbreak clade of Psa, triggers HR and defence in Arabidopsis accession Ct-1. Ws-2 and Col-0 accessions are unable to produce an HR in response to Pseudomonas-delivered HopZ5. While Ws-2 is susceptible to virulent bacterial strain Pseudomonas syringae pv. tomato DC3000 carrying HopZ5, Col-0 is resistant despite the lack of an HR. We show that HopZ5, like other members of the YopJ superfamily of acetyltransferases that it belongs to, autoacetylates lysine residues. Through comparisons to other family members, we identified an acetyltransferase catalytic activity and demonstrate its requirement for triggering defence in Arabidopsis and Nicotiana species. Collectively, data herein indicate that HopZ5 is a plasma membrane-localized acetyltransferase with autoacetylation activity required for avirulence.

Proteomics and functional analyses of Arabidopsis nitrilases involved in the defense response to microbial pathogens.

MAIN CONCLUSION: 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.

The pepper GNA-related lectin and PAN domain protein gene, CaGLP1, is required for plant cell death and defense signaling during bacterial infection.

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.

The pepper phosphoenolpyruvate carboxykinase CaPEPCK1 is involved in plant immunity against bacterial and oomycete pathogens.

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.

Pepper mitochondrial FORMATE DEHYDROGENASE1 regulates cell death and defense responses against bacterial pathogens.

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.

The pepper cysteine/histidine-rich DC1 domain protein CaDC1 binds both RNA and DNA and is required for plant cell death and defense response.

Plant defense against microbial pathogens is coordinated by a complex regulatory network. Cysteine/histidine-rich DC1 domain proteins mediate a variety of cellular processes involved in plant growth, development and stress responses. We identified a pepper (Capsicum annuum) cysteine/histidine-rich DC1 domain protein gene, CaDC1, which positively regulates plant defense during microbial infection, based on gene silencing and transient expression in pepper, as well as ectopic expression in Arabidopsis. Induction of CaDC1 by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection was pronounced at both transcriptional and translational levels in pepper leaves. Purified CaDC1 protein bound to both DNA and RNA in vitro, especially in the presence of Zn(2+). CaDC1 was localized to both the nucleus and the cytoplasm, which was required for plant cell death signaling. The nuclear localization of CaDC1 was dependent on the divergent C1 (DC1) domain. CaDC1 silencing in pepper conferred increased susceptibility to Xcv infection, which was accompanied by reduced salicylic acid accumulation and defense-related gene expression. Ectopic expression of CaDC1 in Arabidopsis enhanced resistance to Hyaloperonospora arabidopsidis. CaDC1 binds both RNA and DNA and functions as a positive regulator of plant cell death and SA-dependent defense responses.

Pathogenesis-related protein 4b interacts with leucine-rich repeat protein 1 to suppress PR4b-triggered cell death and defense response in pepper.

To control defense and cell-death signaling, plants contain an abundance of pathogen recognition receptors such as leucine-rich repeat (LRR) proteins. Here we show that pepper (Capsicum annuum) LRR1 interacts with the pepper pathogenesis-related (PR) protein 4b, PR4b, in yeast and in planta. PR4b is synthesized in the endoplasmic reticulum, interacts with LRR1 in the plasma membrane, and is secreted to the apoplast via the plasma membrane. Binding of PR4b to LRR1 requires the chitin-binding domain of PR4b. Purified PR4b protein inhibits spore germination and mycelial growth of plant fungal pathogens. Transient expression of PR4b triggers hypersensitive cell death. This cell death is compromised by co-expression of LRR1 as a negative regulator in Nicotiana benthamiana leaves. LRR1/PR4b silencing in pepper and PR4b over-expression in Arabidopsis thaliana demonstrated that LRR1 and PR4b are necessary for defense responses to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis (Hpa) infection. The mutant of the PR4b Arabidopsis ortholog, pr4, showed enhanced susceptibility to Hpa infection. Together, our results suggest that PR4b functions as a positive modulator of plant cell death and defense responses. However, the activity of PR4b is suppressed by interaction with LRR1.

Pepper osmotin-like protein 1 (CaOSM1) is an essential component for defense response, cell death, and oxidative burst in plants.

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.

Overexpression of Xanthomonas campestris pv. vesicatoria effector AvrBsT in Arabidopsis triggers plant cell death, disease 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.

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.

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.

The pepper oxidoreductase CaOXR1 interacts with the transcription factor CaRAV1 and is required for salt and osmotic stress tolerance.

RAV1 (Related to ABI3/VP1) proteins function as a transcription factor in signal transduction pathways in plants. The yeast-two-hybrid and in vivo coimmunoprecipitation assays identified the pepper (Capsicum annuum) oxidoreductase protein CaOXR1 that physically interacts with the pepper CaRAV1 transcription factor. The AP2 domain of CaRAV1 protein is essential for its direct interaction with CaOXR1. Both CaRAV1 and CaOXR1 proteins co-localize to the nuclei of plant cells. Virus-induced gene silencing of CaRAV1 and CaRAV1/CAOXR1 confers enhanced susceptibility to high salinity and osmotic stresses, which is accompanied by altered expression of the stress marker genes in pepper. Expression of CaAMP1 (pepper antimicrobial protein) and CaOSM1 (pepper osmotin) is suppressed by 1.2-6.6-fold in silenced leaves upon treatment with NaCl or mannitol. Overexpression of CaRAV1, CaOXR1 and CaOXR1/CaRAV1 in Arabidopsis also confers enhanced resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis infection. In addition, CaRAV1- and CaOXR1/CaRAV1-overexpression (OX) Arabidopsis plants are highly tolerant to high salinity and osmotic stress. Together, these results suggest that CaOXR1 protein positively controls CaRAV1-mediated plant defense during biotic and abiotic stresses.

Distinct roles of the pepper pathogen-induced membrane protein gene CaPIMP1 in bacterial disease resistance and oomycete disease susceptibility.

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.

Function of a novel GDSL-type pepper lipase gene, CaGLIP1, in disease susceptibility and abiotic stress tolerance.

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.

Functional roles of the pepper pathogen-induced bZIP transcription factor, CAbZIP1, in enhanced resistance to pathogen infection and environmental stresses.

Transcription factors often belong to multigene families and their individual contribution in a particular regulatory network remains difficult to assess. We identify and functionally characterize the pepper bZIP transcription factor CAbZIP1 gene isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria. Transient expression analysis of the CAbZIP1-GFP fusion protein in Arabidopsis protoplasts revealed that the CAbZIP1 protein is localized in the nucleus. The N-terminal region of CAbZIP1 fused to the GAL4 DNA-binding domain is required to activate transcription of reporter genes in yeast. The CAbZIP1 transcripts are constitutively expressed in the pepper root and flower, but not in the leaf, stem and fruit. The CAbZIP1 gene is locally or systemically induced in pepper plants infected by either X. campestris pv. vesicatoria or Pseudomonas fluorescens. The CAbZIP1 gene is also induced by abiotic elicitors and environmental stresses. The CAbZIP1 transgenic Arabidopsis exhibits a dwarf phenotype, indicating that CAbZIP1 may be involved in plant development. The CAbZIP1 overexpression in the transgenic Arabidopsis plants confers enhanced resistance to Pseudomonas syringae pv. tomato DC3000, accompanied by expression of the AtPR-4 and AtRD29A. The transgenic plants also exhibit increased drought and salt tolerance during all growth stages. Moreover, the transgenic plants are tolerant to methyl viologen-oxidative stress. Together, these data suggest that the CAbZIP1 transcription factor function as a possible regulator in enhanced disease resistance and environmental stress tolerance.