Pepper pathogenesis‐related protein 4c is a plasma membrane‐localized cysteine protease inhibitor that is required for plant cell death and defense signaling

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.     

Cell wall-bound invertase limits sucrose export and is involved in symptom development and inhibition of photosynthesis during compatible interaction between tomato and Xanthomonas campestris pv vesicatoria

Cell wall-bound invertase (cw-Inv) plays an important role in carbohydrate partitioning and regulation of sink-source interaction. There is increasing evidence that pathogens interfere with sink-source interaction, and induction of cw-Inv activity has frequently been shown in response to pathogen infection. To investigate the role of cw-Inv, transgenic tomato (Solanum lycopersicum) plants silenced for the major leaf cw-Inv isoforms were generated and analyzed during normal growth and during the compatible interaction with Xanthomonas campestris pv vesicatoria. Under normal growth conditions, activities of sucrolytic enzymes as well as photosynthesis and respiration were unaltered in the transgenic plants compared with wild-type plants. However, starch levels of source leaves were strongly reduced, which was most likely caused by an enhanced sucrose exudation rate. Following X. campestris pv vesicatoria infection, cw-Inv-silenced plants showed an increased sucrose to hexose ratio in the apoplast of leaves. Symptom development, inhibition of photosynthesis, and expression of photosynthetic genes were clearly delayed in transgenic plants compared with wild-type plants. In addition, induction of senescence-associated and pathogenesis-related genes observed in infected wild-type plants was abolished in cw-Inv-silenced tomato lines. These changes were not associated with decreased bacterial growth. In conclusion, cw-Inv restricts carbon export from source leaves and regulates the sucrose to hexose ratio in the apoplast. Furthermore, an increased apoplastic hexose to sucrose ratio can be linked to inhibition of photosynthesis and induction of pathogenesis-related gene expression but does not significantly influence bacterial growth. Indirectly, bacteria may benefit from low invertase activity, since the longevity of host cells is raised and basal defense might be dampened.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity

? The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen. ? In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression. ? In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). ? XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI-related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.     

Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). The PGIPs directly reduce the aggressive potential of PGs. Here, we isolated and functionally characterized three members of the pepper (Capsicum annuum) PGIP gene family. Each was up-regulated at a different time following stimulation of the pepper leaves by Phytophthora capcisi and abiotic stresses including salicylic acid, methyl jasmonate, abscisic acid, wounding and cold treatment. Purified recombinant proteins individually inhibited activity of PGs produced by Alternaria alternata and Colletotrichum nicotianae, respectively, and virus-induced gene silencing in pepper conferred enhanced susceptibility to P. capsici. Because three PGIP genes acted similarily in conferring resistance to infection by P. capsici, and because individually purified proteins showed consistent inhibition against PG activity of both pathogens, CaPGIP1 was selected for manipulating transgenic tobacco. The crude proteins from transgenic tobacco exhibited distinct enhanced resistance to PG activity of both fungi. Moreover, the transgenic tobacco showed effective resistance to infection and a significant reduction in the number of infection sites, number of lesions and average size of lesions in the leaves. All results suggest that CaPGIPs may be involved in plant defense response and play an important role in a plant’s resistance to disease.     

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.     

The Pepper 9-Lipoxygenase Gene CaLOX1 Functions in Defense and Cell Death Responses to Microbial Pathogens

Lipoxygenases (LOXs) are crucial for lipid peroxidation processes during plant defense responses to pathogen infection. A pepper (Capsicum annuum) 9-LOX gene, CaLOX1, which encodes a 9-specific lipoxygenase, was isolated from pepper leaves. Recombinant CaLOX1 protein expressed in Escherichia coli catalyzed the hydroperoxidation of linoleic acid, with a K_m value of 113. 9 μm. Expression of CaLOX1 was differentially induced in pepper leaves not only during Xanthomonas campestris pv vesicatoria (Xcv) infection but also after exposure to abiotic elicitors. Transient expression of CaLOX1 in pepper leaves induced the cell death phenotype and defense responses. CaLOX1-silenced pepper plants were more susceptible to Xcv and Colletotrichum coccodes infection, which was accompanied by reduced expression of defense-related genes, lowered lipid peroxidation, as well as decreased reactive oxygen species and lowered salicylic acid accumulation. Infection with Xcv, especially in an incompatible interaction, rapidly stimulated LOX activity in unsilenced, but not CaLOX1-silenced, pepper leaves. Furthermore, overexpression of CaLOX1 in Arabidopsis (Arabidopsis thaliana) conferred enhanced resistance to Pseudomonas syringae pv tomato, Hyaloperonospora arabidopsidis, and Alternaria brassicicola. In contrast, mutation of the Arabidopsis CaLOX1 ortholog AtLOX1 significantly increased susceptibility to these three pathogens. Together, these results suggest that CaLOX1 and AtLOX1 positively regulate defense and cell death responses to microbial pathogens.To effectively combat invasion by microbial pathogens, plants activate distinct defense responses that are specifically effective. Despite the presence of plant immune systems, many pathogens can evade or suppress host defense mechanisms. Lipoxygenase (LOX) pathways are crucial for lipid peroxidation processes during plant defense responses to pathogen infection (Casey and Hughes, 2004). Plant LOXs are key enzymes involved in the generation of fatty acid derivatives in oxylipin metabolism.LOXs comprise a family of non-heme-iron-containing fatty acid dioxygenases, which are ubiquitous in plants and animals (Brash, 1999). LOXs catalyze the conversion of polyunsaturated fatty acids such as linoleic acid into hydroperoxides that are in turn converted to oxylipins. These primary products, which may cause oxidative damage to plant membranes during the hypersensitive response (HR; Slusarenko, 1996), are enzymatically metabolized into traumatin, jasmonic acid (JA), and methyl jasmonate (MeJA). These latter compounds are involved in diverse physiological functions in plant growth and development, senescence, and stress responses. Plant LOXs can be classified as 9-LOXs or 13-LOXs according to the position at which oxygen is incorporated into linoleic acid or linolenic acid, the most important substrates for LOX catalysis in plants (Feussner and Wasternack, 2002). LOX enzymatic activity initiates the different biosynthetic pathways that result in the accumulation of distinct oxylipins. The most understood functional aspects of oxylipin pathways have come mainly from studies of JA produced through the action of 13-LOXs but not 9-LOXs. The metabolism of 13-LOX has been described in tobacco (Nicotiana tabacum) leaves infected by an avirulent strain of Pseudomonas syringae pv phaseolicola (Kenton et al., 1999). During bacterial infection, JA accumulates in tobacco leaves prior to cell death (Kenton et al., 1999). The level of LOX activity and gene expression also increases in tobacco plants during infection with Phytophthora parasitica var nicotianae (Christophe et al., 1996; Rancé et al., 1998). However, the defense-related functions of 9-LOXs are not fully understood. Both 9-LOXs and oxidative processes are proposed to be involved in the HR of tobacco induced by the avirulent pathogen Pseudomonas syringae pv syringae (Montillet et al., 2005). The production of free fatty acid hydroperoxides via the 9-LOX pathway in tobacco is crucial for hypersensitive cell death induced by cryptogein, a purified protein from Phytophthora cryptogea (Rusterucci et al., 1999). The function of LOXs in defense against pathogens is likely to be related to the synthesis of fatty acid hydroperoxides and of volatile products with signaling functions (Rusterucci et al., 1999) and antimicrobial activity (Croft et al., 1993; Weber et al., 1999). Gao et al. (2007) recently suggested that oxylipin metabolism mediated by a specific 9-LOX, ZmLOX3, may be involved in fungal pathogenesis in maize (Zea mays). ZmLOX3 loss-of-function mutants are susceptible to Aspergillus flavus and Aspergillus nidulans infection (Gao et al., 2009).LOX activity may initiate the synthesis of signal molecules or induce structural and metabolic changes in the cell, ultimately leading to cell death that has been termed the HR (Maccarrone et al., 2001). Plant cell death occurs during various phases of development, senescence, and responses to abiotic and biotic stresses, and in particular, in response to pathogen invasion (Morel and Dangl, 1997). Activation of LOXs in plants may be involved in cell death induced by pathogens (Buonaurio and Servili, 1999; Rusterucci et al., 1999). The induction of HR-like cell death by the activation of the 9-LOX-encoding gene GhLOX1 was shown in cotton (Gossypium hirsutum) plants during Xanthomonas campestris pv malvacearum infection (Marmey et al., 2007). LOX activity increases in parallel with the induction of HR symptoms in tobacco; however, in compatible interactions, LOX activity is delayed and reaches much lower levels (Montillet et al., 2002). In cotton, high LOX activity supports cell death during X. campestris pv malvacearum infection (Sayegh-Alhamdia et al., 2008). The HR, an important defense reaction of plants to pathogen infection, is accompanied by lipid peroxidation processes. In particular, 9-LOX-dependent lipid peroxidation operates during cryptogein-induced HR in tobacco leaves (Rusterucci et al., 1999). In potato (Solanum tuberosum), lipid peroxidation occurs as a controlled and directed process that is facilitated by the action of a specific 9-LOX during the HR (Göbel et al., 2003; Montillet et al., 2005). GhLOX1 is associated with salicylic acid (SA) accumulation during the HR of cotton to X. campestris pv malvacearum (Marmey et al., 2007).The bacterial plant pathogen Xanthomonas campestris pv vesicatoria (Xcv) is the causative agent of bacterial spot disease on pepper (Capsicum annuum) and tomato (Solanum lycopersicum) plants. To identify genes involved in the HR-based innate immune response in pepper, we have isolated and functionally characterized defense-related genes encoding PR1 (for pathogenesis-related protein 1; Kim and Hwang, 2000; Hong and Hwang, 2005), chitinase (Hong et al., 2000), chitin-binding protein (Lee et al., 2001), thionin (Lee et al., 2000), SAR 8.2 (Lee and Hwang, 2003), peroxidase (Choi et al., 2007), and menthone reductase (Choi et al., 2008) from pepper leaves infected with the Xcv avirulent strain Bv5-4a. In this study, we used a cDNA macroarray method (Jung and Hwang, 2000) to isolate a novel pepper gene, CaLOX1, which encodes a 9-LOX and is specifically induced by avirulent Xcv infection of pepper leaves. The purified CaLOX1 protein was expressed in Escherichia coli and investigated for LOX activity. Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are the most popular for VIGS. Recently, a VIGS method was established for the functional characterization of defense-related genes in pepper (Baulcombe, 1999; Burch-Smith et al., 2006; Choi et al., 2007; Chung et al., 2007). Here, we analyzed the effect of CaLOX1 loss of function during pathogen infection using TRV-based VIGS of the CaLOX1 gene. Arabidopsis (Arabidopsis thaliana) plants that constitutively overexpressed CaLOX1 were also examined to determine the gain-of-function phenotype of CaLOX1 in plant defense. We further functionally characterized the Arabidopsis mutants lox1-1 and lox1-2, which have T-DNA insertions in AtLOX1, a putative CaLOX1 ortholog. Analysis of the function of CaLOX1 in pepper and Arabidopsis plants provided insight into the role of CaLOX1 expression in defense responses and the hypersensitive cell death of plants following pathogen invasion.     

Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O₂-resistant photosynthesis. The apparent K_m for H₂O₂ was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.