The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis

Copines are calcium-dependent membrane-binding proteins that are highly conserved among protozoa, plants, nematodes and mammals. Although they are implicated in membrane trafficking and signal transduction, the functions of these proteins are not well understood. The Arabidopsis copine gene BON1/CPN1 was previously shown to negatively regulate a disease resistance (R) gene SNC1. Here we report that in Arabidopsis, as in other organisms, there is a family of copine genes, BON1, 2 and 3. Using double and triple mutant combinations we show that these three copine genes have overlapping functions essential for the viability of plants. The loss of function of BON1 combined with that of BON2 or BON3 leads to extensive cell death phenotypes resembling the hypersensitive response (HR) in defense responses. The resulting lethality can be suppressed by mutations in PAD4 or EDS1 which are required for R gene signaling and cell death control. Accession-dependent phenotypes of the mutant combinations suggest that the BON/CPN genes may together repress several R genes other than SNC1. Moreover, the mutant combinations exhibit developmental defects when R-gene-mediated defense responses are largely suppressed in pad4 and eds1 mutants. Thus, the copine family in Arabidopsis may have effects in promoting growth and development in addition to repressing cell death, and these two processes might be intricately intertwined.     

Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7

Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. EDS1 is also needed for basal resistance to invasive pathogens by restricting the progression of disease. In both responses, EDS1, assisted by its interacting partner, PHYTOALEXIN-DEFICIENT4 (PAD4), regulates accumulation of the phenolic defense molecule salicylic acid (SA) and other as yet unidentified signal intermediates. An Arabidopsis whole genome microarray experiment was designed to identify genes whose expression depends on EDS1 and PAD4, irrespective of local SA accumulation, and potential candidates of an SA-independent branch of EDS1 defense were found. We define two new immune regulators through analysis of corresponding Arabidopsis loss-of-function insertion mutants. FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) positively regulates the EDS1 pathway, and one member (NUDT7) of a family of cytosolic Nudix hydrolases exerts negative control of EDS1 signaling. Analysis of fmo1 and nudt7 mutants alone or in combination with sid2-1, a mutation that severely depletes pathogen-induced SA production, points to SA-independent functions of FMO1 and NUDT7 in EDS1-conditioned disease resistance and cell death. We find instead that SA antagonizes initiation of cell death and stunting of growth in nudt7 mutants.     

The disease resistance signaling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis

Specific recognition of pathogens is mediated by plant disease resistance (R) genes and translated into a successful defense response. The extent of associated hypersensitive cell death varies from none to an area encompassing cells surrounding an infection site, depending on the R gene activated. We constructed double mutants in Arabidopsis between positive regulators of R function and a negative regulator of cell death, LSD1, to address whether genes required for normal R function also regulate the runaway cell death observed in lsd1 mutants. We report here that EDS1 and PAD4, two signaling genes that mediate some but not all R responses, also are required for runaway cell death in the lsd1 mutant. Importantly, this novel function of EDS1 and PAD4 is operative when runaway cell death in lsd1 is initiated through an R gene that does not require EDS1 or PAD4 for disease resistance. NDR1, another component of R signaling, also contributes to the control of plant cell death. The roles of EDS1 and PAD4 in regulating lsd1 runaway cell death are related to the interpretation of reactive oxygen intermediate-derived signals at infection sites. We further demonstrate that the fate of superoxide at infection sites is different from that observed at the leading margins of runaway cell death lesions in lsd1 mutants.     

Requirement of Calcium Binding,Myristoylation, and Protein-Protein Interaction for the Copine BON1 Function in Arabidopsis

Copines are highly conserved proteins with lipid-binding activities found in animals, plants, and protists. They contain two calcium-dependent phospholipid binding C2 domains at the amino terminus and a VWA domain at the carboxyl terminus. The biological roles of most copines are not understood and the biochemical properties required for their functions are largely unknown. The Arabidopsis copine gene BON1/CPN1 is a negative regulator of cell death and defense responses. Here we probed the potential biochemical activities of BON1 through mutagenic studies. We found that mutations of aspartates in the C2 domains did not alter plasma membrane localization but compromised BON1 activity. Mutation at putative myristoylation residue glycine 2 altered plasma membrane localization of BON1 and rendered BON1 inactive. Mass spectrometry analysis of BON1 further suggests that the N-peptide of BON1 is modified. Furthermore, mutations that affect the interaction between BON1 and its functional partner BAP1 abolished BON1 function. This analysis reveals an unanticipated regulation of copine protein localization and function by calcium and lipid modification and suggests an important role in protein-protein interaction for the VWA domain of copines.     

A SNARE-protein has opposing functions in penetration resistance and defence signalling pathways

Penetration resistance is often the first line of defence against fungal pathogens. Subsequently induced defences are mediated by the programmed cell death (PCD) reaction pathway and the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways. We previously demonstrated that full penetration resistance in Arabidopsis against the non-host barley powdery mildew fungus (Blumeria graminis f.sp. hordei) requires the syntaxin SYP121 (PEN1). Here we report that SYP121, together with SYP122, functions as a negative regulator of subsequently induced defence pathways. The SA level in the syntaxin double mutant syp121-1 syp122-1 is dramatically elevated, resulting in necrosis and dwarfism. This phenotype is partially rescued by introducing the SA-signalling mutations eds1-2, eds5-3, sid2-1 and npr1-1 as well as the NahG transgene. These partially rescued triple mutants have an unknown defence to Pseudomonas syringae pv. tomato, and have increased HR-like responses to non-host and host powdery mildew fungi. The HR-like responses cause efficient resistance to the latter. These defence pathways are SA-independent. Furthermore, the JA/ET signalling marker, PDF1.2, is highly upregulated in the triple mutants. Thus SYP121 and SYP122 are negative regulators of PCD, SA, JA and ET pathways through a molecular function distinct from that of SYP121 in penetration resistance. Our data suggest that individual cells preferentially express either penetration resistance or the subsequently induced defences.     

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.     

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.     

Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?

The identification of several lesion mimic mutants (LMM) that misregulate cell death constitutes a powerful tool to unravel programmed cell death (PCD) pathways in plants, particularly the hypersensitive response (HR), a form of PCD associated with resistance to pathogens. Recently, the characterization of novel LMM has enabled genes that might regulate cell death programmes to be identified as well as the dissection of defense signaling pathways and of crosstalk between multiple pathways in ways that might not be possible by studying the responses of wild-type plants to pathogens.     

Bacterial lipopolysaccharides induce defense responses associated with programmed cell death in rice cells

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.     

Dynamic intracellular reorganization of cytoskeletons and the vacuole in defense responses and hypersensitive cell death in plants

Plants have evolved various means for controlled and organized cell destruction, known as programmed cell death (PCD). In plant immune responses against microbial infection, hypersensitive cell death as a form of PCD is a crucial event to prevent the spread of biotrophic pathogens. Recent live cell imaging techniques have revealed dynamic features and significant roles of cytoskeletons and the vacuole during defense responses and the PCD. Actin microfilaments (MFs) focus on the infection sites and function as tracks for the polar transport of antimicrobial materials. To accomplish hypersensitive cell death, further dynamic changes in cytoskeletons are induced. MFs play a role in the structural and functional regulation of the vacuole, leading to execution of the PCD. We here overview spatiotemporal dynamic changes in the cytoskeletons and the vacuoles triggered by signals from pathogens, and propose a hypothetical model for MF-regulated vacuole-mediated PCD in plant immunity.     

ZmPep1, an ortholog of Arabidopsis elicitor peptide 1, regulates maize innate immunity and enhances disease resistance

ZmPep1 is a bioactive peptide encoded by a previously uncharacterized maize (Zea mays) gene, ZmPROPEP1. ZmPROPEP1 was identified by sequence similarity as an ortholog of the Arabidopsis (Arabidopsis thaliana) AtPROPEP1 gene, which encodes the precursor protein of elicitor peptide 1 (AtPep1). Together with its receptors, AtPEPR1 and AtPEPR2, AtPep1 functions to activate and amplify innate immune responses in Arabidopsis and enhances resistance to both Pythium irregulare and Pseudomonas syringae. Candidate orthologs to the AtPROPEP1 gene have been identified from a variety of crop species; however, prior to this study, activities of the respective peptides encoded by these orthologs were unknown. Expression of the ZmPROPEP1 gene is induced by fungal infection and treatment with jasmonic acid or ZmPep1. ZmPep1 activates de novo synthesis of the hormones jasmonic acid and ethylene and induces the expression of genes encoding the defense proteins endochitinase A, PR-4, PRms, and SerPIN. ZmPep1 also stimulates the expression of Benzoxazineless1, a gene required for the biosynthesis of benzoxazinoid defenses, and the accumulation of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside in leaves. To ascertain whether ZmPep1-induced defenses affect resistance, maize plants were pretreated with the peptide prior to infection with fungal pathogens. Based on cell death and lesion severity, ZmPep1 pretreatment was found to enhance resistance to both southern leaf blight and anthracnose stalk rot caused by Cochliobolis heterostrophus and Colletotrichum graminicola, respectively. We present evidence that peptides belonging to the Pep family have a conserved function across plant species as endogenous regulators of innate immunity and may have potential for enhancing disease resistance in crops.     

Identification, expression analysis and characterization of defense and signaling genes in Vitis vinifera.

The reduction of phytochemicals applied to grapevine relies on the development of alternative strategies involving activation of the plant's own defense system. The aim of this work was to study the signaling of defense responses to pathogens in Vitis vinifera. We identified in V. vinifera cv. Chardonnay two putative regulatory elements, VvNHL1 and VvEDS1, with similarity to Arabidopsis defense regulators NDR1 and EDS1. Expression studies of these putative signaling genes together with other known grape defense genes show that they are differentially regulated by salicylic acid and jasmonate-ethylene treatments, as well as by inoculation with different types of pathogens. The expression of VvEDS1 was stimulated by salicylic acid treatment, Botrytis cinerea and Plasmopara viticola inoculation, whereas VvNHL1 was repressed by B. cinerea. VvNHL1 overexpression introduced in Arabidopsis ndr1 mutant did not complement the mutation in terms of sensitivity to avirulent Pseudomonas syringae pv. tomato. Moreover, we observed a weakened resistance to B. cinerea of ndr1 mutants overexpressing VvNHL1, which may be related to cell death enhancement. Together, our results identify two new pathogen-responsive regulatory elements in Vitis vinifera, with potential roles in pathogen defense.     

Evidence that the BONZAI1/COPINE1 protein is a calcium- and pathogen-responsive defense suppressor

Copines are calcium-responsive, phospholipid-binding proteins involved in cellular signaling. The Arabidopsis BONZAI1/COPINE1 (BON1/CPN1) gene is a suppressor of defense responses controlled by the disease resistance (R) gene homolog SNC1. The BON1/CPN1 null mutant cpn1-1 has a recessive, temperature- and humidity-dependent, lesion mimic phenotype that includes activation of Pathogenesis-Related (PR) gene expression. Here, we demonstrated that the accumulation of BON1/CPN1 protein in wild-type plants was up-regulated by bacterial pathogen inoculation and by the activation of defense signaling responses controlled by two R genes, SNC1 and RPS2. Interestingly, however, over-accumulation of BON1/CPN1 in two BON1/CPN1 promoter T-DNA insertion mutants did not affect resistance to a bacterial pathogen. Promoter deletion analysis identified a 280 bp segment of the BON1/CPN1 promoter as being required for pathogen-induced gene expression; the same promoter region was also required for calcium ionophore-induced gene expression. Leaf infiltration with calcium ionophore triggered high-level PR gene expression specifically in cpn1-1 plants grown under permissive conditions, while co-infiltration of the calcium chelator EGTA attenuated this effect. These results explain the conditional nature of the cpn1-1 phenotype and are consistent with BON1/CPN1 being a calcium- and pathogen-responsive plant defense suppressor.     

The F-box protein CPR1/CPR30 negatively regulates R protein SNC1 accumulation

Disease resistance (R) proteins, as central regulators of plant immunity, are tightly regulated for effective defense responses and to prevent constitutive defense activation under non-pathogenic conditions. Here we report the identification of an F-box protein CPR1/CPR30 as a negative regulator of an R protein SNC1 likely through SCF (Skp1-cullin-F-box) mediated protein degradation. The cpr1-2 (cpr30-1) loss-of-function mutant has constitutive defense responses, and it interacts synergistically with a gain-of function mutant snc1-1 and a bon1-1 mutant where SNC1 is upregulated. The loss of SNC1 function suppresses the mutant phenotypes of cpr1-2 and cpr1-2 bon1-1, while overexpression of CPR1 rescues mutant phenotypes of both bon1-1 and snc1-1. Furthermore, the amount of SNC1 protein is upregulated in the cpr1-2 mutant and down-regulated when CPR1 is overexpressed. The regulation of SNC1 by CPR1 is dependent on the 26S proteosome as a protease inhibitor MG132 stabilizes SNC1 and reverses the effect of CPR1 on SNC1. Interestingly, CPR1 is induced after infection of both virulent and avirulent pathogens similarly to the other negative defense regulator BON1. Thus, this study reveals a new mechanism in R protein regulation likely through protein degradation and suggests negative regulation as a critical component in fine control of plant immunity.     

ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis

The molecular interactions between Arabidopsis and the pathogenic powdery mildew Golovinomyces cichoracearum were studied by characterizing a disease-resistant Arabidopsis mutant atg2-2. The atg2-2 mutant showed enhanced resistance to powdery mildew and dramatic mildew-induced cell death as well as early senescence phenotypes in the absence of pathogens. Defense-related genes were constitutively activated in atg2-2. In atg2-2 mutants, spontaneous cell death, early senescence and disease resistance required the salicylic acid (SA) pathway, but interestingly, mildew-induced cell death was not fully suppressed by inactivation of SA signaling. Thus, cell death could be uncoupled from disease resistance, suggesting that cell death is not sufficient for resistance to powdery mildew. ATG2 encodes autophagy-related 2, a protein known to be involved in the early steps of autophagosome biogenesis. The atg2-2 mutant exhibited typical autophagy defects in autophagosome formation. Furthermore, mutations in several other ATG genes, including ATG5, ATG7 and ATG10, exhibited similar powdery mildew resistance and mildew-induced cell death phenotypes. Taken together, our findings provide insights into the role of autophagy in cell death and disease resistance, and may indicate general links between autophagy, senescence, programmed cell death and defense responses in plants.