The bacterial effector DspA/E is toxic in Arabidopsis thaliana and is required for multiplication and survival of fire blight pathogen

The type III effector DspA/E is an essential pathogenicity factor of the phytopathogenic bacterium Erwinia amylovora. We showed that DspA/E was required for transient bacterial growth in nonhost Arabidopsis thaliana leaves, as an E. amylovora dspA/E mutant was unable to grow. We expressed DspA/E in A. thaliana transgenic plants under the control of an oestradiol‐inducible promoter, and found that DspA/E expressed in planta restored the growth of a dspA/E mutant. DspA/E expression in these transgenic plants led to the modulation by at least two‐fold of the expression of 384 genes, mostly induced (324 genes). Both induced and repressed genes contained high proportions of defence genes. DspA/E expression ultimately resulted in plant cell death without requiring a functional salicylic acid signalling pathway. Analysis of A. thaliana transgenic seedlings expressing a green fluorescent protein (GFP):DspA/E fusion indicated that the fusion protein could only be detected in a few cells per seedling, suggesting the degradation or absence of accumulation of DspA/E in plant cells. Consistently, we found that DspA/E repressed plant protein synthesis when injected by E. amylovora or when expressed in transgenic plants. Thus, we conclude that DspA/E is toxic to A. thaliana: it promotes modifications, among which the repression of protein synthesis could be determinant in the facilitation of necrosis and bacterial growth.     

WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants

Pantoea stewartii subsp. stewartii ( Pnss ) causes Stewart's bacterial wilt of sweet corn and leaf blight of maize. The pathogenicity of Pnss depends on synthesis of extracellular polysaccharide and an Hrp type III secretion system. WtsE, a type III secreted effector protein, is essential for the virulence of Pnss on corn. It belongs to the AvrE family of effectors, which includes DspA/E from Erwinia amylovora and AvrE1 from Pseudomonas syringae . Previously, WtsE was shown to cause disease-associated cell death in its host plant, sweet corn. Here, we examine the biological activity of WtsE in several non-host plants. WtsE induced cell death in Nicotiana benthamiana , tobacco, beet and Arabidopsis thaliana when it was transiently produced in plant cells following agroinfiltration or translocated into plant cells from Pnss , Escherichia coli or Pseudomonas syringae pv. phaseolicola ( Pph ). WtsE-induced cell death in N. benthamiana , tobacco and beet resembled a hypersensitive response and in N. benthamiana it was delayed by cycloheximide. Interestingly, WtsE strongly promoted the growth of Pnss in N. benthamiana prior to the onset of cell death. Deletion derivatives of WtsE that failed to induce cell death in N. benthamiana and tobacco also did not complement wtsE mutants of Pnss for virulence in sweet corn, indicating a correlation between the two activities. WtsE also induced cell death in A. thaliana , where it suppressed basal defences induced by Pph . Thus, WtsE has growth-promoting, defence-suppressing and cell death-inducing activities in non-host plants. Expression of WtsE also prevented the growth of yeast, possibly due to an innate toxicity to eukaryotic cells.     

DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants

Erwinia amylovora is responsible for fire blight, a necrotic disease of apples and pears. E. amylovora relies on a type III secretion system (TTSS) to induce disease on hosts and hypersensitive response (HR) on nonhost plants. The DspA/E protein is essential for E. amylovora pathogenicity and is secreted via the TTSS in vitro. DspA/E belongs to a type III effector family that is conserved in several phytopathogenic bacteria. In E. amylovora, DspA/E has been implicated in the generation of an oxidative stress during disease and the suppression of callose deposition. We investigated the fate of DspA/E in planta. DspA/E delivered artificially to apple or tobacco cells by agroinfection induced necrotic symptoms, indicating that DspA/E was probably injected via the TTSS. We confirmed that DspA/E acts as a major cell-death inducer during disease and HR, because the dspA/E mutant is severely impaired in its ability to induce electrolyte leakage in apple and tobacco leaves. Expression of the defense marker gene PR1 was delayed when dspA/E was transiently expressed in tobacco, suggesting that DspA/E-mediated necrosis may be associated with an alteration of defense responses.     

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.     

Erwinia amylovora type three-secreted proteins trigger cell death and defense responses in Arabidopsis thaliana

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting plants of the rosaceous family. E. amylovora pathogenicity requires a functional type three secretion system (T3SS). We show here that E. amylovora triggers a T3SS-dependent cell death on Arabidopsis thaliana. The plants respond by inducing T3SS-dependent defense responses, including salicylic acid (SA)-independent callose deposition, activation of the SA defense pathway, reactive oxygen species (ROS) accumulation, and part of the jasmonic acid/ethylene defense pathway. Several of these reactions are similar to what is observed in host plants. We show that the cell death triggered by E. amylovora on A. thaliana could not be simply explained by the recognition of AvrRpt2 ea by the resistance gene product RPS2. We then analyzed the role of type three-secreted proteins (T3SPs) DspA/E, HrpN, and HrpW in the induction of cell death and defense reactions in A. thaliana following infection with the corresponding E. amylovora mutant strains. HrpN and DspA/E were found to play an important role in the induction of cell death, activation of defense pathways, and ROS accumulation. None of the T3SPs tested played a major role in the induction of SA-independent callose deposition. The relative importance of T3SPs in A. thaliana is correlated with their relative importance in the disease process on host plants, indicating that A. thaliana can be used as a model to study their role.     

SGT1 positively regulates the process of plant cell death during both compatible and incompatible plant–pathogen interactions

SGT1 (suppressor of G2 allele of Skp1), an interactor of SCF (Skp1‐Cullin‐F‐box) ubiquitin ligase complexes that mediate protein degradation, plays an important role at both G1–S and G2–M cell cycle transitions in yeast, and is highly conserved throughout eukaryotes. Plant SGT1 is required for both resistance (R) gene‐mediated disease resistance and nonhost resistance to certain pathogens. Using virus‐induced gene silencing (VIGS) in Nicotiana benthamiana, we demonstrate that SGT1 positively regulates the process of cell death during both host and nonhost interactions with various pathovars of Pseudomonas syringae. Silencing of NbSGT1 in N. benthamiana plants delays the induction of hypersensitive response (HR)‐mediated cell death against nonhost pathogens and the development of disease‐associated cell death caused by the host pathogen P. syringae pv. tabaci. Our results further demonstrate that NbSGT1 is required for Erwinia carotovora‐ and Sclerotinia sclerotiorum‐induced disease‐associated cell death. Overexpression of NbSGT1 in N. benthamiana accelerates the development of HR during R gene‐mediated disease resistance and nonhost resistance. Our data also indicate that SGT1 is required for pathogen‐induced cell death, but is not always necessary for the restriction of bacterial multiplication in planta. Therefore, we conclude that SGT1 is an essential component affecting the process of cell death during both compatible and incompatible plant–pathogen interactions.     

The HrpN effector of Erwinia amylovora, which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves

Erwinia amylovora is responsible for fire blight of apple and pear trees. Its pathogenicity depends on a type III secretion system (T3SS) mediating the translocation of effectors into the plant cell. The DspA/E effector suppresses callose deposition on apple leaves. We found that E. amylovora and Pseudomonas syringae DC3000 tts mutants or peptide flg22 do not trigger callose deposition as strongly as the dspA/E mutant on apple leaves. This suggests that, on apple leaves, callose deposition is poorly elicited by pathogen-associated molecular patterns (PAMPs) such as flg22 or other PAMPs harbored by tts mutants and is mainly elicited by injected effectors or by the T3SS itself. Callose elicitation partly depends on HrpW because an hrpW-dspA/E mutant elicits lower callose deposition than a dspA/E mutant. Furthermore, an hrpN-dspA/E mutant does not trigger callose deposition, indicating that HrpN is required to trigger this plant defense reaction. We showed that HrpN plays a general role in the translocation process. Thus, the HrpN requirement for callose deposition may be explained by its role in translocation: HrpN could be involved in the translocation of other effectors inducing callose deposition. Furthermore, HrpN may also directly contribute to the elicitation process because we showed that purified HrpN induces callose deposition.     

Role of SCF ubiquitin-ligase and the COP9 signalosome in the N gene-mediated resistance response to Tobacco mosaic virus

The tobacco N gene confers resistance to Tobacco mosaic virus (TMV) and encodes a toll-interleukin-1 receptor/nucleotide binding/Leu-rich repeat class protein. Recent evidence indicates that the Nicotiana benthamiana Rar1 gene (NbRar1), which encodes a protein with a zinc finger motif called CHORD (Cys- and His-rich domain), is required for the function of N. To investigate the role of NbRar1 in plant defense, we identified its interaction partners. We show that the NbRar1 protein interacts with NbSGT1, a highly conserved component of the SCF (Skp1/Cullin/F-box protein)-type E3 ubiquitin ligase complex involved in protein degradation. In addition, we show that NbSGT1 interacts with NbSKP1. Suppression of NbSGT1 and NbSKP1 shows that these genes play an important role in the N-mediated resistance response to TMV. Both NbRar1 and NbSGT1 associate with the COP9 signalosome, another multiprotein complex involved in protein degradation via the ubiquitin-proteasome pathway. Silencing of the NbCOP9 signalosome also compromises N-mediated resistance to TMV. Our results reveal new roles for SCF and the COP9 signalosome in plant defense signaling.     

Expression of the Bacterial Type III Effector DspA/E in Saccharomyces cerevisiae Down-regulates the Sphingolipid Biosynthetic Pathway Leading to Growth Arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest.     

Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.     

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.     

Ornithine-delta-aminotransferase and proline dehydrogenase genes play a role in non-host disease resistance by regulating pyrroline-5-carboxylate metabolism-induced hypersensitive response

Non-host disease resistance involves the production of hypersensitive response (HR), a programmed cell death (PCD) that occurs at the site of pathogen infection. Plant mitochondrial reactive oxygen species (ROS) production and red-ox changes play a major role in regulating such cell death. Proline catabolism reactions, especially pyrroline-5-carboxylate (P5C) accumulation, are known to produce ROS and contribute to cell death. Here we studied important genes related to proline synthesis and catabolism in the defence against host and non-host strains of Pseudomonas syringae in Nicotiana benthamiana and Arabidopsis. Our results show that ornithine delta-aminotransferase (δOAT) and proline dehydrogenases (ProDH1 and ProDH2) are involved in the defence against non-host pathogens. Silencing of these genes in N. benthamiana delayed occurrence of HR and favoured non-host pathogen growth. Arabidopsis mutants for these genes compromised non-host resistance and showed a decrease in non-host pathogen-induced ROS. Some of the genes involved in proline metabolism were also induced by a pathogen-carrying avirulence gene, indicating that proline metabolism is influenced during effector-triggered immunity (ETI). Our results demonstrate that δOAT and ProDH enzyme-mediated steps produce ROS in mitochondria and regulate non-host HR, thus contributing to non-host resistance in plants.     

Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in

ABSTRACT: BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. In plants, MAPK cascades are associated with responses to various abiotic and biotic stresses such as plant pathogens. MAPK cascades function through sequential phosphorylation: MAPK kinase kinases (MAPKKKs) phosphorylate MAPK kinases (MAPKKs), and phosphorylated MAPKKs phosphorylate MAPKs. Of these three types of kinase, the MAPKKKs exhibit the most divergence in the plant genome. Their great diversity is assumed to allow MAPKKKs to regulate many specific signaling pathways in plants despite the relatively limited number of MAPKKs and MAPKs. Although some plant MAPKKKs, including the MAPKKKalpha of Nicotiana benthamiana (NbMAPKKKalpha), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. Here, we performed a comparative functional analysis of MAPKKKs to investigate the signaling pathway leading to the defense response. RESULTS: We cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKepsilon2. Transient overexpression of full-length NbMAPKKKbeta or NbMAPKKKgamma or their kinase domains in N. benthamiana leaves induced hypersensitive response (HR)-like cell death associated with hydrogen peroxide production. This activity was dependent on the kinase activity of the overexpressed MAPKKK. In addition, virus-induced silencing of NbMAPKKKbeta or NbMAPKKKgamma expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKalpha (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKKalpha suppressed cell death induced by the overexpression of the NbMAPKKKbeta kinase domain or of NbMAPKKKgamma, but silencing of NbMAPKKKbeta failed to suppress cell death induced by the overexpression of NbMAPKKKalpha or NbMAPKKKgamma. Silencing of NbMAPKKKgamma suppressed cell death induced by the NbMAPKKKbeta kinase domain but not that induced by NbMAPKKKalpha. CONCLUSIONS: These results demonstrate that in addition to NbMAPKKKalpha, NbMAPKKKbeta and NbMAPKKKgamma also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKKbeta to NbMAPKKKgamma to NbMAPKKKalpha.     

Tomato MAPKKKε is a positive regulator of cell-death signaling networks associated with plant immunity

Mitogen-activated protein (MAP) kinase cascades are fundamental components of the signaling pathways associated with plant immunity. Despite the large number of MAP kinase kinase kinases (MAPKKK) encoded in the plant genome, only very few of them have an assigned function. Here, we identified MAPKKK gene of tomato (Solanum lycopersicum), SIMAPKKKε, which is required for hypersensitive response cell death and disease resistance against Gram-negative bacterial pathogens. Silencing of SIMAPKKKε compromised tomato resistance to Xanthomonas campestris and Pseudomonas syringae strains, resulting in the appearance of disease symptoms and enhanced bacterial growth. In addition, silencing of NbMAPKKKε in Nicotiana benthamiana plants significantly inhibited the cell death triggered by expression of different R gene/effector gene pairs. Conversely, overexpression of either the full-length SIMAPKKKε gene or its kinase domain in N. benthamiana leaves caused pathogen-independent activation of cell death that required an intact kinase catalytic domain. Moreover, by suppressing the expression of various MAPKK and MAPK genes and overexpressing the SIMAPKKKε kinase domain, we identified a signaling cascade acting downstream of SIMAPKKKε that includes MEK2, WIPK and SIPK. Additional epistasis experiments revealed that SIPKK functions as a negative regulator of SIMAPKKKε-mediated cell death. Our results provide evidence that SIMAPKKKε is a signaling molecule that positively regulates cell death networks associated with plant immunity.     

Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins

Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction.     

Role of caspases and mitochondria in the steroid-induced programmed cell death of a motoneuron during metamorphosis

Accessory planta retractor (APR) motoneurons of the hawk moth, Manduca sexta, undergo a segment-specific pattern of programmed cell death (PCD) 24 to 48 h after pupal ecdysis (PE). Cell culture experiments show that the PCD of APRs in abdominal segment 6 [APR(6)s] is a cell-autonomous response to the steroid hormone 20-hydroxyecdysone (20E) and involves mitochondrial demise and cell shrinkage. Twenty-four hours before PE, at stage W3-noon, APR(6)s require further 20E exposure and protein synthesis (as tested with cycloheximide) to undergo PCD, and death can be blocked by a broad-spectrum caspase inhibitor. By PE, death is 20E- and protein synthesis-independent and the caspase inhibitor blocks cell shrinkage but not loss of mitochondrial function. Thus, the commitment to mitochondrial demise precedes the commitment to execution events. The phenotype of necrotic cell death induced by a mitochondrial electron transfer inhibitor differs unambiguously from 20E-induced PCD. By inducing PCD pharmacologically, the readiness of APR(6)s to execute PCD was found to increase during the final larval instar. These data suggest that the 20E-induced PCD of APR(6)s includes a premitochondrial phase which includes 20E-induced synthetic events and apical caspase activity, a mitochondrial phase which culminates in loss of mitochondrial function, and a postmitochondrial phase during which effector caspases are activated and APR(6) is destroyed.     

Function of endoplasmic reticulum calcium ATPase in innate immunity‐mediated programmed cell death

Programmed cell death (PCD) initiated at the pathogen‐infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER‐localized type IIB Ca²⁺‐ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N‐ and fungal‐immune receptor Cf9‐mediated PCD, as well as non‐host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein‐induced cell death. The accelerated PCD rescues loss‐of‐resistance phenotype of Rar1, HSP90‐silenced plants, but not SGT1‐silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N‐immune receptor‐mediated PCD. Our results indicate that ER‐Ca²⁺‐ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response.     

Pseudomonas Type III effector AvrPto suppresses the programmed cell death induced by two nonhost pathogens in Nicotiana benthamiana and tomato

Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell. Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive. Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions. Pseudomonas syringae pv. tomato T1 causes an HR-like cell death on Nicotiana benthamiana. This rapid cell death was delayed significantly in plants inoculated with P. syringae pv. tomato expressing avrPto. In addition, P. syringae pv. tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines. Transient expression of avrPto in both N. benthamiana and tomato prf3 plants also was able to suppress nonhost HR. Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens. AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions. Experiments with avrPto mutants revealed several residues important for the suppression effects. AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and 196T enhanced suppression of cell death in N. benthamiana. These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion.