BON1 interacts with the protein kinases BIR1 and BAK1 in modulation of temperature-dependent plant growth and cell death in Arabidopsis

The Arabidopsis copine gene BON1 encodes a calcium-dependent phospholipid-binding protein involved in plant growth homeostasis and disease resistance. However, the biochemical and molecular mechanisms by which BON1 modulates plant growth and defense responses are not well understood. Here, we show that BON1 interacts physically with the leucine-rich-repeat receptor-like kinases BIR1 (BAK1-interacting receptor-like kinase 1) and pathogen-associated molecular pattern (PAMP) receptor regulator BAK1 in vitro and in vivo. Additionally, bon1 and bir1 mutants exhibit synergistic interaction. While a bir1 null mutant has similar growth and cell-death defects compared with bon1, a bir1 bon1 double mutant displays more severe phenotypes than does the single mutants. The bon1-1 and bir1-1 phenotypes are partially suppressed by overexpression of BIR1 and BON1, respectively. Furthermore, the bir1 phenotype is attenuated by a loss-of-function mutation in the resistance (R) gene SNC1 (Suppressor of npr1-1, constitutive 1), which mediates defense responses in bon1. Intriguingly, BON1 and BIR1 can be phosphorylated by BAK1 in vitro. Our findings suggest that BIR1 functions as a negative regulator of plant resistance and that BON1 and BIR1 might modulate both PAMP- and R protein-triggered immune responses.     

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.     

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 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.     

Abscisic acid deficiency antagonizes high-temperature inhibition of disease resistance through enhancing nuclear accumulation of resistance proteins SNC1 and RPS4 in Arabidopsis

Plant defense responses to pathogens are influenced by abiotic factors, including temperature. Elevated temperatures often inhibit the activities of disease resistance proteins and the defense responses they mediate. A mutant screen with an Arabidopsis thaliana temperature-sensitive autoimmune mutant bonzai1 revealed that the abscisic acid (ABA)-deficient mutant aba2 enhances resistance mediated by the resistance (R) gene suppressor of npr1-1 constitutive1 (SNC1) at high temperature. ABA deficiency promoted nuclear accumulation of SNC1, which was essential for it to function at low and high temperatures. Furthermore, the effect of ABA deficiency on SNC1 protein accumulation is independent of salicylic acid, whose effects are often antagonized by ABA. ABA deficiency also promotes the activity and nuclear localization of R protein resistance to Pseudomonas syringae4 at higher temperature, suggesting that the effect of ABA on R protein localization and nuclear activity is rather broad. By contrast, mutations that confer ABA insensitivity did not promote defense responses at high temperature, suggesting either tissue specificity of ABA signaling or a role of ABA in defense regulation independent of the core ABA signaling machinery. Taken together, this study reveals a new intersection between ABA and disease resistance through R protein localization and provides further evidence of antagonism between abiotic and biotic responses.     

The Arabidopsis BAP1 and BAP2 genes are general inhibitors of programmed cell death

Here we identify the BAP1 and BAP2 genes of Arabidopsis (Arabidopsis thaliana) as general inhibitors of programmed cell death (PCD) across the kingdoms. These two homologous genes encode small proteins containing a calcium-dependent phospholipid-binding C2 domain. BAP1 and its functional partner BON1 have been shown to negatively regulate defense responses and a disease resistance gene SNC1. Genetic studies here reveal an overlapping function of the BAP1 and BAP2 genes in cell death control. The loss of BAP2 function induces accelerated hypersensitive responses but does not compromise plant growth or confer enhanced resistance to virulent bacterial or oomycete pathogens. The loss of both BAP1 and BAP2 confers seedling lethality mediated by PAD4 and EDS1, two regulators of cell death and defense responses. Overexpression of BAP1 or BAP2 with their partner BON1 inhibits PCD induced by pathogens, the proapoptotic gene BAX, and superoxide-generating paraquat in Arabidopsis or Nicotiana benthamiana. Moreover, expressing BAP1 or BAP2 in yeast (Saccharomyces cerevisiae) alleviates cell death induced by hydrogen peroxide. Thus, the BAP genes function as general negative regulators of PCD induced by biotic and abiotic stimuli including reactive oxygen species. The dual roles of BAP and BON genes in repressing defense responses mediated by disease resistance genes and in inhibiting general PCD has implications in understanding the evolution of plant innate immunity.     

Temperature Modulates Plant Defense Responses through NB-LRR Proteins

An elevated growth temperature often inhibits plant defense responses and renders plants more susceptible to pathogens. However, the molecular mechanisms underlying this modulation are unknown. To genetically dissect this regulation, we isolated mutants that retain disease resistance at a higher growth temperature in Arabidopsis. One such heat-stable mutant results from a point mutation in SNC1, a NB-LRR encoding gene similar to disease resistance (R) genes. Similar mutations introduced into a tobacco R gene, N, confer defense responses at elevated temperature. Thus R genes or R-like genes involved in recognition of pathogen effectors are likely the causal temperature-sensitive component in defense responses. This is further supported by snc1 intragenic suppressors that regained temperature sensitivity in defense responses. In addition, the SNC1 and N proteins had a reduction of nuclear accumulation at elevated temperature, which likely contributes to the inhibition of defense responses. These findings identify a plant temperature sensitive component in disease resistance and provide a potential means to generate plants adapting to a broader temperature range.     

Transgenic expression of the von Willebrand A domain of the BONZAI 1/COPINE 1 protein triggers a lesion-mimic phenotype in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

The copines are a newly identified, widely distributed class of Ca²⁺-dependent, phospholipid-binding proteins that may be involved in cellular signaling. The copines have a characteristic domain structure: two C2 domains in the N-terminal region and a von Willebrand A (VWA) domain in the C-terminal region. Studies suggest that copines interact with target protein(s) via their VWA domain and recruit the proteins to a membrane location through the activity of the C2 domains. Arabidopsis thaliana (L.) Heynh. plants with loss-of-function mutations in the BONZAI 1/COPINE 1 (BON1/CPN1) gene display aberrant regulation of defense responses, including development of a lesion-mimic phenotype, an accelerated hypersensitive response, and increased resistance to a bacterial and an oomycetous pathogen. The phenotype of mutants in BON1/CPN1 is both humidity- and temperature-sensitive. In this study, we generated transgenic plants expressing either the VWA or the C2 portions of BON1/CPN1 in the wild-type Columbia-0 (Col-0) genetic background. Transgenic plants expressing the BON1/CPN1 C2 domain portion appeared like wild-type plants. However, transgenic plants expressing the BON1/CPN1 VWA domain exhibited a lesion-mimic phenotype that partially phenocopied bon1/cpn1 mutant plants. Our data suggest that BON1/CPN1 VWA domain fragments may interfere with the function of the full-length endogenous BON1/CPN1 protein, possibly by competing with the full-length BON1/CPN1 protein for association with target proteins normally bound to the full-length BON1/CPN1 protein.     

SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity

Plant defense responses need to be tightly regulated to prevent auto-immunity, which is detrimental to growth and development. To identify negative regulators of Resistance (R) protein-mediated resistance, we screened for mutants with constitutive defense responses in the npr1-1 background. Map-based cloning revealed that one of the mutant genes encodes a conserved TPR domain-containing protein previously known as SRFR1 (SUPPRESSOR OF rps4-RLD). The constitutive defense responses in the srfr1 mutants in Col-0 background are suppressed by mutations in SNC1, which encodes a TIR-NB-LRR (Toll Interleukin1 Receptor-Nucleotide Binding-Leu-Rich Repeat) R protein. Yeast two-hybrid screens identified SGT1a and SGT1b as interacting proteins of SRFR1. The interactions between SGT1 and SRFR1 were further confirmed by co-immunoprecipitation analysis. In srfr1 mutants, levels of multiple NB-LRR R proteins including SNC1, RPS2 and RPS4 are increased. Increased accumulation of SNC1 is also observed in the sgt1b mutant. Our data suggest that SRFR1 functions together with SGT1 to negatively regulate R protein accumulation, which is required for preventing auto-activation of plant immunity.     

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.     

Receptor-mediated autocrine growth-stimulatory effect of 5-hydroxytryptamine on cultured human pancreatic carcinoid cells.

5-hydroxytryptamine (5-HT) is a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5-HT. In this study, therefore, we examined the effect of 5-HT on growth of BON cells. Furthermore, by use of selective 5-HT receptor antagonists, we examined receptor and post-receptor mechanisms by which 5-HT-induced responses were produced. 5-HT stimulated growth of BON cells. 5-HT stimulated phosphatidylinositol (PI) hydrolysis in a dose-dependent fashion and inhibited cyclic AMP production in a dose-dependent fashion. The 5-HT1A/1B receptor antagonist, SDZ 21-009, prevented the reduction of cyclic AMP production evoked by 5-HT and inhibited the mitogenic action of 5-HT. The 5-HT1C/2 receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5-HT. The mitogenic action of 5-HT and the reduction of cyclic AMP production evoked by 5-HT were also inhibited by pertussis toxin. These results suggest that 5-HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5-HT involves receptor-mediated inhibition of the production of cyclic AMP which may be linked to pertussis toxin-sensitive GTP binding protein. 8-bromo-cyclic AMP inhibited growth of BON cells whereas 8-bromo-cyclic GMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a cAMP-dependent protein kinase antagonist (Rp-cAMPS) could stimulate BON cell growth.     

MOS2, a protein containing G-patch and KOW motifs, is essential for innate immunity in Arabidopsis thaliana

Innate immunity is critical for sensing and defending against microbial infections in multicellular organisms. In plants, disease resistance genes (R genes) play central roles in recognizing pathogens and initiating downstream defense cascades. Arabidopsis SNC1 encodes a TIR-NBS-LRR-type R protein with a similar structure to nucleotide binding oligomerization domain (Nod) proteins in animals. A point mutation in the region between the NBS and LRR of SNC1 results in constitutive activation of defense responses in the snc1 mutant. Here, we report the identification and characterization of mos2-1, a mutant suppressing the constitutive defense responses in snc1. Analysis of mos2 single mutants indicated that it is not only required for resistance specified by multiple R genes, but also for basal resistance. Map-based cloning of MOS2 revealed that it encodes a novel nuclear protein that contains one G-patch and two KOW domains and has homologs across the animal kingdom. The presence of both G-patch and KOW domains in the MOS2 protein suggests that it probably functions as an RNA binding protein critical for plant innate immunity. Our discovery on the biological functions of MOS2 will shed light on functions of the MOS2 homologs in animals, where they may also play important roles in innate immunity.     

The Ankyrin-Repeat Transmembrane Protein BDA1 Functions Downstream of the Receptor-Like Protein SNC2 to Regulate Plant Immunity

Plants utilize a large number of immune receptors to recognize pathogens and activate defense responses. A small number of these receptors belong to the receptor-like protein family. Previously, we showed that a gain-of-function mutation in the receptor-like protein SNC2 (for Suppressor of NPR1, Constitutive2) leads to constitutive activation of defense responses in snc2-1D mutant plants. To identify defense signaling components downstream of SNC2, we carried out a suppressor screen in the snc2-1D mutant background of Arabidopsis (Arabidopsis thaliana). Map-based cloning of one of the suppressor genes, BDA1 (for bian da; "becoming big" in Chinese), showed that it encodes a protein with amino-terminal ankyrin repeats and carboxyl-terminal transmembrane domains. Loss-of-function mutations in BDA1 suppress the dwarf morphology and constitutive defense responses in snc2-1D npr1-1 (for nonexpressor of pathogenesis-related genes1,1) and also result in enhanced susceptibility to bacterial pathogens. In contrast, a gain-of-function allele of bda1 isolated from a separate genetic screen to search for mutants with enhanced pathogen resistance was found to constitutively activate cell death and defense responses. These data suggest that BDA1 is a critical signaling component that functions downstream of SNC2 to regulate plant immunity.     

IBR5 Modulates Temperature-Dependent,R Protein CHS3-Mediated Defense Responses in Arabidopsis

Plant responses to low temperature are tightly associated with defense responses. We previously characterized the chilling-sensitive mutant chs3-1 resulting from the activation of the Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR)-type resistance (R) protein harboring a C-terminal LIM (Lin-11, Isl-1 and Mec-3 domains) domain. Here we report the identification of a suppressor of chs3, ibr5-7 (indole-3-butyric acid response 5), which largely suppresses chilling-activated defense responses. IBR5 encodes a putative dual-specificity protein phosphatase. The accumulation of CHS3 protein at chilling temperatures is inhibited by the IBR5 mutation. Moreover, chs3-conferred defense phenotypes were synergistically suppressed by mutations in HSP90 and IBR5. Further analysis showed that IBR5, with holdase activity, physically associates with CHS3, HSP90 and SGT1b (Suppressor of the G2 allele of skp1) to form a complex that protects CHS3. In addition to the positive role of IBR5 in regulating CHS3, IBR5 is also involved in defense responses mediated by R genes, including SNC1 (Suppressor of npr1-1, Constitutive 1), RPS4 (Resistance to P. syringae 4) and RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1). Thus, the results of the present study reveal a role for IBR5 in the regulation of multiple R protein-mediated defense responses.     

MAP KINASE PHOSPHATASE1 and PROTEIN TYROSINE PHOSPHATASE1 Are Repressors of Salicylic Acid Synthesis and SNC1-Mediated Responses in Arabidopsis

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1.