FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2017507144> ?p ?o ?g. }

• W2017507144 endingPage "44" @default.
• W2017507144 startingPage "34" @default.
• W2017507144 abstract "Upon recognition of bacterial flagellin, the plant receptor FLS2 heterodimerizes with brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and activates plant defense responses. Because constitutive activation of defense responses is detrimental, plant resistance signaling pathways must be negatively controlled, although the mechanisms involved are unclear. We identified Arabidopsis BIR1 as a BAK1-interacting receptor-like kinase. Knocking out BIR1 leads to extensive cell death, activation of constitutive defense responses, and impairment in the activation of MPK4, a negative regulator of plant resistance (R) protein signaling, by flagellin. sobir1-1, a mutant obtained in a screen for suppressors of the bir1-1 phenotype, rescued cell death observed in bir1-1. SOBIR1 encodes another receptor-like kinase whose overexpression activates cell death and defense responses. Our data suggest that BIR1 negatively regulates multiple plant resistance signaling pathways, one of which is the SOBIR1-dependent pathway identified here. Upon recognition of bacterial flagellin, the plant receptor FLS2 heterodimerizes with brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and activates plant defense responses. Because constitutive activation of defense responses is detrimental, plant resistance signaling pathways must be negatively controlled, although the mechanisms involved are unclear. We identified Arabidopsis BIR1 as a BAK1-interacting receptor-like kinase. Knocking out BIR1 leads to extensive cell death, activation of constitutive defense responses, and impairment in the activation of MPK4, a negative regulator of plant resistance (R) protein signaling, by flagellin. sobir1-1, a mutant obtained in a screen for suppressors of the bir1-1 phenotype, rescued cell death observed in bir1-1. SOBIR1 encodes another receptor-like kinase whose overexpression activates cell death and defense responses. Our data suggest that BIR1 negatively regulates multiple plant resistance signaling pathways, one of which is the SOBIR1-dependent pathway identified here. Plants rely solely on innate immunity to combat pathogen infections, as they do not have adaptive immune systems like animals (Jones and Dangl, 2006Jones J.D. Dangl J.L. The plant immune system.Nature. 2006; 444: 323-329Crossref PubMed Scopus (7346) Google Scholar). There are two types of innate immune responses in plants. One is pathogen-associated molecular pattern (PAMP)-triggered immunity. PAMPs are conserved microbial components that elicit pathogen immune responses when recognized by the host receptors. The second type of innate immunity is effector-triggered immunity, which is evolved by plants to detect pathogen effectors and initiate defense responses to inhibit pathogen growth and restrict pathogen spread. Two well-studied plant receptors of PAMPs, FLS2 and EFR, are members of the receptor-like kinase (RLK) family (Gomez-Gomez and Boller, 2000Gomez-Gomez L. Boller T. FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis.Mol. Cell. 2000; 5: 1003-1011Abstract Full Text Full Text PDF PubMed Google Scholar, Zipfel et al., 2006Zipfel C. Kunze G. Chinchilla D. Caniard A. Jones J.D. Boller T. Felix G. Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.Cell. 2006; 125: 749-760Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar). RLK family is the largest group of protein kinases in plants. A typical RLK contains an extracellular ligand-binding sequence, a transmembrane region, and a C-terminal intracellular protein kinase domain. FLS2 recognizes the peptide flg22 from bacterial flagellin, while EFR recognizes a peptide derived from bacterial translation elongation factor EF-Tu. Recently, FLS2 was found to heterodimerize with brassinosteroid insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) in the perception of flagellin (Chinchilla et al., 2007Chinchilla D. Zipfel C. Robatzek S. Kemmerling B. Nürnberger T. Jones J.D. Felix G. Boller T. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence.Nature. 2007; 448: 497-500Crossref PubMed Scopus (1154) Google Scholar, Heese et al., 2007Heese A. Hann D.R. Gimenez-Ibanez S. Jones A.M. He K. Li J. Schroeder J.I. Peck S.C. Rathjen J.P. The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants.Proc. Natl. Acad. Sci. USA. 2007; 104: 12217-12222Crossref PubMed Scopus (715) Google Scholar). bak1 mutant plants exhibited reduced sensitivity to flagellin in growth assays and are impaired in responsiveness to flagellin. BAK1 was originally identified as a BRI1 interactor, and it forms a protein complex with BRI1 during perception of Brassinosteroids (BRs) (Li et al., 2002Li J. Wen J. Lease K.A. Doke J.T. Tax F.E. Walker J.C. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling.Cell. 2002; 110: 213-222Abstract Full Text Full Text PDF Scopus (941) Google Scholar, Nam and Li, 2002Nam K.H. Li J. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling.Cell. 2002; 110: 203-212Abstract Full Text Full Text PDF Scopus (799) Google Scholar). In addition, BAK1 was also found to negatively regulate a BR-independent cell-death pathway. bak1 mutant plants develop spreading necrosis upon pathogen infections (Kemmerling et al., 2007Kemmerling B. Schwedt A. Rodriguez P. Mazzotta S. Frank M. Qamar S.A. Mengiste T. Betsuyaku S. Parker J.E. Müssig C. et al.The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control.Curr. Biol. 2007; 17: 1116-1122Abstract Full Text Full Text PDF Scopus (274) Google Scholar). Double knockout mutant plants of bak1 and bak1-like 1 (bkk1) exhibit a seedling lethality phenotype and constitutive defense responses (He et al., 2007He K. Gou X. Yuan T. Lin H. Asami T. Yoshida S. Russell S.D. Li J. BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways.Curr. Biol. 2007; 17: 1109-1115Abstract Full Text Full Text PDF Scopus (297) Google Scholar). BAK1 was also shown to be a target of bacterial effectors AvrPto and AvrPtoB (Shan et al., 2008Shan L. He P. Li J. Heese A. Peck S.C. Nürnberger T. Martin G.B. Sheen J. Bacterial effectors target the common signaling partner BAK1 to disrupt multiple MAMP receptor-signaling complexes and impede plant immunity.Cell Host Microbe. 2008; 4: 17-27Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Effector-triggered immunity is mediated by the host resistance (R) proteins. Recognition of pathogen effectors by R proteins can be either direct or indirect. The guard hypothesis explains the indirect recognition (Van der Biezen and Jones, 1998Van der Biezen E.A. Jones J.D. Plant disease-resistance proteins and the gene-for-gene concept.Trends Biochem. Sci. 1998; 23: 454-456Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar), where R proteins bind to host components important for innate immunity. Modifications of these host components by pathogen effectors can be sensed by the R proteins, leading to activation of defense responses. Most R proteins belong to the NB-LRR (nucleotide-binding site leucine-rich repeats) group, which shares structural similarity to the NOD proteins in animals (Jones and Dangl, 2006Jones J.D. Dangl J.L. The plant immune system.Nature. 2006; 444: 323-329Crossref PubMed Scopus (7346) Google Scholar). Downstream of R proteins, NDR1 was shown to be required for the signaling of CC-NB-LRR type of R proteins containing a coiled-coil region at the N terminus (Century et al., 1995Century K.S. Holub E.B. Staskawicz B.J. NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.Proc. Natl. Acad. Sci. USA. 1995; 92: 6597-6601Crossref Scopus (317) Google Scholar), while EDS1, PAD4, and SAG101 are required for the signaling by the TIR-NB-LRR type R proteins carrying an N-terminal Toll interleukin receptor domain (Aarts et al., 1998Aarts N. Metz M. Holub E. Staskawicz B.J. Daniels M.J. Parker J.E. Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis.Proc. Natl. Acad. Sci. USA. 1998; 95: 10306-10311Crossref Scopus (588) Google Scholar, Feys et al., 2005Feys B.J. Wiermer M. Bhat R.A. Moisan L.J. Medina-Escobar N. Neu C. Cabral A. Parker J.E. Arabidopsis SENESCENCE-ASSOCIATED GENE101 stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex in plant innate immunity.Plant Cell. 2005; 17: 2601-2613Crossref Scopus (317) Google Scholar, Glazebrook et al., 1996Glazebrook J. Rogers E.E. Ausubel F.M. Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening.Genetics. 1996; 143: 973-982Crossref Google Scholar, Parker et al., 1996Parker J.E. Holub E.B. Frost L.N. Falk A. Gunn N.D. Daniels M.J. Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes.Plant Cell. 1996; 8: 2033-2046PubMed Google Scholar). R protein-mediated resistance is often accompanied by localized cell death, which may play important roles in the restriction of pathogen spread. Since constitutive activation of R proteins is detrimental to plant growth and development (Bendahmane et al., 2002Bendahmane A. Farnham G. Moffett P. Baulcombe D.C. Constitutive gain-of-function mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato.Plant J. 2002; 32: 195-204Crossref PubMed Scopus (257) Google Scholar, Mackey et al., 2003Mackey D. Belkhadir Y. Alonso J.M. Ecker J.R. Dangl J.L. Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance.Cell. 2003; 112: 379-389Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, Noutoshi et al., 2005Noutoshi Y. Ito T. Seki M. Nakashita H. Yoshida S. Marco Y. Shirasu K. Shinozaki K. A single amino acid insertion in the WRKY domain of the Arabidopsis TIR-NBS-LRR-WRKY-type disease resistance protein SLH1 (sensitive to low humidity 1) causes activation of defense responses and hypersensitive cell death.Plant J. 2005; 43: 873-888Crossref PubMed Scopus (136) Google Scholar, Shirano et al., 2002Shirano Y. Kachroo P. Shah J. Klessig D.F. A gain-of-function mutation in an Arabidopsis Toll Interleukin1 receptor-nucleotide binding site-leucine-rich repeat type R gene triggers defense responses and results in enhanced disease resistance.Plant Cell. 2002; 14: 3149-3162Crossref Scopus (244) Google Scholar, Yang and Hua, 2004Yang S. Hua J. A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growth control in Arabidopsis.Plant Cell. 2004; 16: 1060-1071Crossref Scopus (224) Google Scholar, Zhang et al., 2003aZhang Y. Goritschnig S. Dong X. Li X. A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1.Plant Cell. 2003; 15: 2636-2646Crossref PubMed Scopus (322) Google Scholar), R protein-mediated signaling pathways must be subject to tight negative regulation to avoid activation of defense responses without pathogen attack. Here, we report the identification of bir1-1, a mutant that constitutively activates defense responses similar to the autoactivated R gene mutants. BIR1 encodes an RLK interacting with BAK1 in vivo. We carried out a suppressor screen of bir1-1 and found that sobir1-1 (suppressor of bir1-1, 1) strongly suppresses cell death in bir1-1. Combining the sobir1 and pad4-1 mutations leads to suppression of all mutant phenotypes of bir1-1, suggesting that SOBIR1 functions in parallel with PAD4. SOBIR1 encodes an RLK, probably functioning as a key regulator in a signal transduction pathway activated in bir1-1 to promote cell death and disease resistance. To identify important regulators of plant innate immunity, we carried out systematic reverse genetics analysis of genes whose transcript levels increased significantly 48 hr after inoculation with the bacterial pathogen Pseudomonas syringae pv. maculicola (P.s.m.) ES4326 (OD600 = 0.001). From initial analysis of homozygous knockout mutants of 200 genes (Table S1), one mutant was found to have a seedling lethality phenotype when grown at 22°C (Figure 1A), and none of the other T-DNA mutants has similar phenotypes. The mutant contains a T-DNA insertion in the third exon of At5g48380, which encodes an RLK (Figure 1B). We focused our analysis on this mutant because it has dramatic phenotypes, and At5g48380 encodes an interesting protein most likely functioning in signal transduction. We later named it BAK1-interacting receptor-like kinase 1 (BIR1) due to its association with BAK1. BIR1 belongs to the LRRX group of RLKs (Figure S1) with 620 amino acids and 5 LRRs. RT-PCR analysis of BIR1 transcript in bir1-1 mutant plants showed that the full-length cDNA of BIR1 was no longer expressed (Figure S2A). Real-time RT-PCR analysis confirmed that BIR1 is indeed induced by bacterial infection (Figure S2B). To determine whether the mutant phenotypes in bir1-1 are caused by the T-DNA insertion, two additional T-DNA lines (SALK_008775 and WiscDsLox341G08) with insertions in the promoter region of At5g48380 were analyzed. The expression of At5g48380 is not affected by the T-DNA insertions, and no obvious mutant phenotype was observed in these two lines. So we transformed the wild-type BIR1 gene into the bir1-1 mutant to test whether it can complement the mutant phenotypes. As shown in Figure 1A, the wild-type BIR1 reverted bir1-1 completely back to wild-type, confirming that the seedling lethality phenotype was caused by the T-DNA insertion in BIR1. In addition, silencing At5g48380 in wild-type background using artificial miRNA also caused a seedling lethality phenotype (data not shown). Trypan blue staining was performed on mutant seedlings to test whether cell death occurred in bir1-1. Extensive cell death was found in both cotyledons and true leaves of the mutant plants, particularly along the veins (Figure S3A). We also performed 3, 3′-diaminobenzidine (DAB) staining on the bir1-1 mutant seedlings. Strong staining was observed in bir1-1 mutant plants (Figure S3B), indicating that the mutant seedlings accumulated high levels of H2O2 compared to the wild-type seedlings. To test whether defense responses were constitutively activated in bir1-1, salicylic acid (SA) levels in bir1-1 and wild-type plants were measured with high-performance liquid chromatography. bir1-1 plants accumulated about 6-fold more free SA and about 20-fold more total SA (free SA plus glucose-conjugated SA) than wild-type controls (Figure 1C). In addition, high levels of PR-1 and PR-2 were constitutively expressed in the mutant plants without induction (Figures 1D and 1E). To test whether bir1-1 mutant has enhanced pathogen resistance, bir1-1 seedlings were challenged with the virulent oomycete pathogen Hyaloperonospora parasitica Noco2 (H. p. Noco2). As shown in Figure 1F, bir1-1 seedlings displayed strong enhanced resistance against H. p. Noco2, suggesting that defense responses were constitutively activated in the bir1-1 mutant plants. Interestingly, the seedling lethality phenotype of bir1-1 can be partially suppressed by growing the plants at high temperatures. bir1-1 plants grown on soil at 28°C remain small, but can complete their life cycle and set seeds. When grown on Murashige and Skoog (MS) medium at 27°C, bir1-1 plants were significantly bigger than those grown at 22°C (Figure S4A). Constitutive expression of PR-1 and PR-2 was also reduced at 27°C (Figures S4B and S4C). To test whether all the bir1-1 mutant phenotypes are caused by the T-DNA insertion in BIR1, bir1-1 plants transformed with a genomic clone of BIR1 were further analyzed in regards to its defense phenotypes. The wild-type BIR1 complements the cell death phenotypes of bir1-1 and suppressed the H2O2 accumulation in bir1-1 (Figure S5A). The expression of PR-1 and PR-2 in the transgenic plants also reverted to wild-type levels (Figures S5B and S5C). In addition, the transgenic plants are no longer resistant to H. p. Noco2 (Figure S5D). Thus, all the mutant phenotypes observed in bir1-1 can be complemented by BIR1. To test whether BIR1 is an active protein kinase, the BIR1 cytoplasmic kinase domain was expressed in E. coli and tested for autophosphorylation activity. As shown in Figure S6A, the BIR1 kinase domain has autophosphorylation activity, indicating that BIR1 is an active protein kinase. To determine whether the kinase activity is important for its biological function, a mutant form of BIR1 kinase domain was created by mutating a conserved lysine (K331) to a glutamic acid (E331). This amino acid substitution was known to abolish kinase activity of other known RLKs (Horn and Walker, 1994Horn M.A. Walker J.C. Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana.Biochim. Biophys. Acta. 1994; 1208: 65-74Crossref Scopus (80) Google Scholar, Li et al., 2002Li J. Wen J. Lease K.A. Doke J.T. Tax F.E. Walker J.C. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling.Cell. 2002; 110: 213-222Abstract Full Text Full Text PDF Scopus (941) Google Scholar). The autophosphorylation activity of BIR1 kinase domain is dramatically reduced by the point mutation (Figure S6A). The K331E mutation was also introduced into a cDNA clone of BIR1. While wild-type BIR1 cDNA can fully complement the bir1-1 mutant phenotypes, the mutated gene can only partially complement the phenotypes of bir1-1 (Figure S6B), suggesting that the kinase activity of BIR1 is important for its function. To determine the localization of the BIR1, Arabidopsis mesophyll protoplasts were transfected with a construct expressing the BIR1-GFP fusion protein under the cauliflower mosaic virus 35S promoter. As shown in Figure 2A, the BIR1-GFP fusion protein was localized to the plasma membrane, suggesting that BIR1 is a plasma membrane protein. To identify proteins associated with BIR1, we generated transgenic plants expressing BIR1-3×FLAG fusion protein under its native promoter in the bir1-1 mutant background. bir1-1 plants expressing the BIR1-3×FLAG fusion protein displayed wild-type morphology (Figure S7A), suggesting that the fusion protein is as functional as wild-type BIR1. A quantitative proteomics approach was previously used to identify specific components of protein complexes using isotope-coded affinity tag reagents and mass spectrometry (Ranish et al., 2003Ranish J.A. Yi E.C. Leslie D.M. Purvine S.O. Goodlett D.R. Eng J. Aebersold R. The study of macromolecular complexes by quantitative proteomics.Nat. Genet. 2003; 33: 349-355Crossref Scopus (307) Google Scholar). To identify proteins specifically interacting with BIR1, we modified this method by labeling the protein in vivo using 15N medium (Dong et al., 2007Dong M.Q. Venable J.D. Au N. Xu T. Park S.K. Cociorva D. Johnson J.R. Dillin A. Yates 3rd, J.R. Quantitative mass spectrometry identifies insulin signaling targets in C. elegans.Science. 2007; 317: 660-663Crossref PubMed Scopus (250) Google Scholar). The transgenic plants were plated on 1/2 MS plates with 15N-labeled KNO3 and NH4NO3 as sole nitrogen source while wild-type control plants were grown on regular 1/2 MS plates with 14N KNO3 and NH4NO3. After the seedlings were harvested, equal amounts of tissue from the transgenic plants and wild-type controls were mixed together for membrane isolation. Total protein from the membrane fraction was incubated with anti-FLAG agarose beads to selectively immunoprecipitate the FLAG-tagged BIR1. Proteins coimmunoprecipitated with BIR1-3×FLAG were subsequently analyzed by mass spectrometry. As shown in Figure S8, one of the peptides (LADGTLVAVK) identified is enriched in the 15N fraction, suggesting that it is most likely from proteins specifically coimmunoprecipitated with BIR1-3×FLAG. Database search showed that this peptide is present in BAK1 and its close homologs SERK1 and SERK2. Probably due to low abundance of the BAK1 and SERK proteins, this peptide was the only one identified by mass spectrometry from these proteins. We did not find any other RLKs that specifically coimmunoprecipitate with BIR1-3×FLAG. Next, we utilized a bimolecular fluorescence complementation (BiFC) approach (Walter et al., 2004Walter M. Chaban C. Schütze K. Batistic O. Weckermann K. Nake C. Blazevic D. Grefen C. Schumacher K. Oecking C. et al.Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.Plant J. 2004; 40: 428-438Crossref PubMed Scopus (1173) Google Scholar) to test whether interactions between BAK1 and BIR1 can be visualized in vivo. In this experiment, BAK1 was fused to the N-terminal fragment of YFP (BAK1-YFPN) while BIR1 was fused to the C-terminal fragment of YFP (BIR1-YFPC). If BAK1 and BIR1 associate with each other, a fluorescent YFP complex would be formed. As shown in Figure 2B (top panel), YFP fluorescence was observed on the plasma membrane of Arabidopsis protoplasts cotransformed with the BAK1-YFPN and BIR1-YFPC constructs, suggesting that BAK1 and BIR1 interact with each other. We further tested the interactions between BIR1 and the BAK1 homologs, including BKK1, SERK1, and SERK2, using BiFC and found that BIR1 also interacts with these proteins on the plasma membrane. When the BIR1-YFPC construct was cotransformed with BRI1-YFPN, FLS2-YFPN, or CLV1-YFPN constructs, no YFP fluorescence was observed, suggesting that BIR1 does not interact with other RLKs like BRI1, FLS2, or CLV1. This is consistent with our observation that flg22-mediated growth inhibitions were not affected by the bir1-1 mutation (Figure S9A). bak1 and other BR mutants often have shorter hypocotyls when grown in the dark. Unlike the bak1-4 seedlings, elongation of hypocotyls in bir1-1 pad4-1 is similar to that in wild-type plants (Figure S9B). Since cell death and defense responses were activated in both bir1-1 and bak1 bkk1 mutant plants and RLKs often function through heterodimerization, additional co-IP analysis was carried out to confirm the in vivo interactions between BIR1 and BAK1. First, a construct expressing BAK1-3×FLAG protein in pCambia1305 under its native promoter was transformed into plants homozygous for bkk1 and heterozygous for bak1. bak1 bkk1 homozygous plants carrying the transgene were identified in the T2 generation, and they were found to exhibit wild-type morphology, suggesting that the 3×FLAG tags did not affect BAK1 function. Co-IP using anti-FLAG agarose beads was subsequently performed on total membrane proteins of these transgenic plants expressing the BAK1-3×FLAG fusion protein. As shown in Figure 2C, BIR1 coimmunoprecipitated with 3×FLAG-tagged BAK1, indicating that BIR1 and BAK1 interact in vivo. As a negative control, immunoprecipitated proteins were also probed with an anti-FLS2 antibody, and no FLS2 was detected in the immunoprecipitated proteins (data not shown). We also performed co-IP analysis using Arabidopsis protoplasts cotransformed with epitope-tagged BIR1 and BAK1. As BIR1 proteins with C-terminal 3×FLAG tags or GFP tag can complement the bir1-1 mutant phenotypes (Figure S7), the tags were fused to the C terminus of BIR1. When total protein from Arabidopsis protoplasts cotransfected with constructs expressing HA-tagged BIR1 and FLAG-tagged BAK1 proteins was incubated with anti-HA Sepharose beads to selectively immunoprecipitate the HA-tagged BIR1, BAK1 coimmunoprecipitated with HA-tagged BIR1, while BAK1 was not immunoprecipitated by itself (Figure 2D). The total protein from protoplasts cotransfected with the constructs expressing HA-tagged BIR1 and FLAG-tagged BAK1 protein was also incubated with anti-FLAG beads to immunoprecipitate the FLAG-tagged BAK1. As shown in Figure 2E, BIR1 coimmunoprecipitated with FLAG-tagged BAK1 while BIR1 was not immunoprecipitated by itself, further confirming that BIR1 and BAK1 interact in vivo. To test whether R protein-mediated resistance responses are activated in the bir1-1 mutant, bir1-1 was crossed to mutants carrying mutations in positive regulators of R protein-mediated defense, including ndr1-1, eds1-1, and pad4-1. As shown in Figure 3A, bir1-1 pad4-1 and bir1-1 eds1-1 double mutant plants are much bigger than the bir1-1 single mutant plants, indicating that bir1-1 activates the EDS1 and PAD4-dependent resistance pathway. Interestingly, the bir1-1 ndr1-1 double mutant is also bigger than the bir1-1 single mutant (Figure 3A), indicating that the NDR1-dependent resistance pathway may also be activated in the bir1-1 mutant. The effect of eds1-1, pad4-1, and ndr1-1 on the size of bir1-1 was confirmed by determining the plant biomass of bir1-1 single mutant and the double mutants (Figure S10A). Although these double mutants are bigger than bir1-1, they still cannot grow to maturity and set seeds at 22°C. To determine whether cell death in bir1-1 is dependent on NDR1, EDS1, and PAD4, trypan blue staining was performed on the double mutants. As shown in Figure 3B (upper panel), the double mutants have significantly less cell death compared to the bir1-1 single mutant. In addition, accumulation of H2O2 is reduced in the double mutants (Figure 3B, lower panel). Constitutive expression of PR-1 and PR-2 in bir1-1 was also reduced by mutations in PAD4, EDS1, and NDR1 to different degrees (Figures 3C and 3D). Analysis of resistance to H. p. Noco2 in the double mutants indicates that constitutive resistance to H. p. Noco2 in bir1-1 was partially compromised by mutations in PAD4 and EDS1 but not by the mutation in NDR1 (Figure 3E). We conclude that knocking out BIR1 results in activation of multiple resistance pathways that are also activated during R protein-mediated resistance. SA is an important signal molecule downstream of R protein-mediated resistance responses and mutations in EDS5, and SID2 blocks pathogen-induced SA biosynthesis (Nawrath and Métraux, 1999Nawrath C. Métraux J.P. Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation.Plant Cell. 1999; 11: 1393-1404Google Scholar, Wildermuth et al., 2001Wildermuth M.C. Dewdney J. Wu G. Ausubel F.M. Isochorismate synthase is required to synthesize salicylic acid for plant defence.Nature. 2001; 414: 562-565Crossref PubMed Scopus (1480) Google Scholar). NPR1 is an essential positive regulator downstream of SA (Durrant and Dong, 2004Durrant W.E. Dong X. Systemic acquired resistance.Annu. Rev. Phytopathol. 2004; 42: 185-209Crossref PubMed Scopus (1898) Google Scholar). To test whether the bir1-1 mutant phenotypes are dependent on the SA pathway, bir1-1 was crossed with eds5-3, sid2-1, and npr1-3. Both eds5-3 and sid2-1 can partially rescue the bir1-1 mutant phenotypes (Figure S11A), suggesting that high levels of SA are partly responsible for the bir1-1 mutant phenotype. The effect of eds5-3, sid2-1, and npr1-3 on the size of bir1-1 was confirmed by determining the plant biomass of bir1-1 and the double mutants (Figure S10B). In addition, cell death in bir1-1 is partially blocked by the eds5-3, sid2-1, and npr1-3 mutations (Figure S11B). Accumulation of H2O2 is also reduced in the double mutants (Figure S11B). Analysis of PR gene expression in the double mutant plants showed that constitutive expression of PR-1 in bir1-1 was dramatically reduced by mutations in SID2, EDS5, and NPR1 (Figures S11C and S11E), but the expression of PR-2 was not affected by these mutations (Figures S11D and S11F). When the double mutant plants were tested for resistance to H. p. Noco2, no significant difference was observed between bir1-1 and the double mutants (Figure S11G). Like the bir1-1 mutant, knockout mutants of Arabidopsis MEKK1 that encode a MAP kinase kinase kinase have temperature-dependent cell death and constitutive activation of defense responses (Ichimura et al., 2006Ichimura K. Casais C. Peck S.C. Shinozaki K. Shirasu K. MEKK1 is required for MPK4 activation and regulates tissue-specific and temperature-dependent cell death in Arabidopsis.J. Biol. Chem. 2006; 281: 36969-36976Crossref PubMed Scopus (224) Google Scholar, Nakagami et al., 2006Nakagami H. Soukupová H. Schikora A. Zársky V. Hirt H. A Mitogen-activated protein kinase kinase kinase mediates reactive oxygen species homeostasis in Arabidopsis.J. Biol. Chem. 2006; 281: 38697-38704Crossref PubMed Scopus (268) Google Scholar, Suarez-Rodriguez et al., 2007Suarez-Rodriguez M.C. Adams-Phillips L. Liu Y. Wang H. Su S.H. Jester P.J. Zhang S. Bent A.F. Krysan P.J. MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants.Plant Physiol. 2007; 143: 661-669Crossref PubMed Scopus (246) Google Scholar). The mpk4 single mutants and mkk1 mkk2 double mutants were also shown to exhibit temperature-dependent cell death and constitutive activation of defense responses (Gao et al., 2008Gao M. Liu J. Bi D. Zhang Z. Cheng F. Chen S. Zhang Y. MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants.Cell Res. 2008; 18: 1190-1198Crossref PubMed Scopus (262) Google Scholar, Petersen et al., 2000Petersen M. Brodersen P. Naested H. Andreasson E. Lindhart U. Johansen B. Nielsen H.B. Lacy M. Austin M.J. Parker J.E. et al.Arabidopsis map kinase 4 negatively regulates systemic acquired resistance.Cell. 2000; 103: 1111-1120Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar, Qiu et al., 2008Qiu J.L. Zhou L. Yun B.W." @default.
• W2017507144 created "2016-06-24" @default.
• W2017507144 creator A5007421479 @default.
• W2017507144 creator A5007712763 @default.
• W2017507144 creator A5016043975 @default.
• W2017507144 creator A5031536762 @default.
• W2017507144 creator A5032499056 @default.
• W2017507144 creator A5035482519 @default.
• W2017507144 creator A5046131302 @default.
• W2017507144 creator A5049798132 @default.
• W2017507144 creator A5056246750 @default.
• W2017507144 creator A5057097136 @default.
• W2017507144 creator A5059792650 @default.
• W2017507144 date "2009-07-01" @default.
• W2017507144 modified "2023-10-15" @default.
• W2017507144 title "Regulation of Cell Death and Innate Immunity by Two Receptor-like Kinases in Arabidopsis" @default.
• W2017507144 cites W1562306981 @default.
• W2017507144 cites W1942665378 @default.
• W2017507144 cites W1973813211 @default.
• W2017507144 cites W1978179863 @default.
• W2017507144 cites W1992843897 @default.
• W2017507144 cites W1994481770 @default.
• W2017507144 cites W1994602890 @default.
• W2017507144 cites W1995444885 @default.
• W2017507144 cites W2000350495 @default.
• W2017507144 cites W2003912358 @default.
• W2017507144 cites W2015093208 @default.
• W2017507144 cites W2016032876 @default.
• W2017507144 cites W2017784972 @default.
• W2017507144 cites W2026238074 @default.
• W2017507144 cites W2033841056 @default.
• W2017507144 cites W2055125075 @default.
• W2017507144 cites W2059856607 @default.
• W2017507144 cites W2075068237 @default.
• W2017507144 cites W2089361961 @default.
• W2017507144 cites W2104525554 @default.
• W2017507144 cites W2106158330 @default.
• W2017507144 cites W2106187536 @default.
• W2017507144 cites W2114410821 @default.
• W2017507144 cites W2116122104 @default.
• W2017507144 cites W2123058210 @default.
• W2017507144 cites W2123679376 @default.
• W2017507144 cites W2123783559 @default.
• W2017507144 cites W2124439705 @default.
• W2017507144 cites W2128049752 @default.
• W2017507144 cites W2129499959 @default.
• W2017507144 cites W2129742189 @default.
• W2017507144 cites W2143868806 @default.
• W2017507144 cites W2146052432 @default.
• W2017507144 cites W2147945104 @default.
• W2017507144 cites W2150659027 @default.
• W2017507144 cites W2161053859 @default.
• W2017507144 cites W2162515800 @default.
• W2017507144 cites W2164158837 @default.
• W2017507144 cites W2168810887 @default.
• W2017507144 cites W22308802 @default.
• W2017507144 doi "https://doi.org/10.1016/j.chom.2009.05.019" @default.
• W2017507144 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19616764" @default.
• W2017507144 hasPublicationYear "2009" @default.
• W2017507144 type Work @default.
• W2017507144 sameAs 2017507144 @default.
• W2017507144 citedByCount "327" @default.
• W2017507144 countsByYear W20175071442012 @default.
• W2017507144 countsByYear W20175071442013 @default.
• W2017507144 countsByYear W20175071442014 @default.
• W2017507144 countsByYear W20175071442015 @default.
• W2017507144 countsByYear W20175071442016 @default.
• W2017507144 countsByYear W20175071442017 @default.
• W2017507144 countsByYear W20175071442018 @default.
• W2017507144 countsByYear W20175071442019 @default.
• W2017507144 countsByYear W20175071442020 @default.
• W2017507144 countsByYear W20175071442021 @default.
• W2017507144 countsByYear W20175071442022 @default.
• W2017507144 countsByYear W20175071442023 @default.
• W2017507144 crossrefType "journal-article" @default.
• W2017507144 hasAuthorship W2017507144A5007421479 @default.
• W2017507144 hasAuthorship W2017507144A5007712763 @default.
• W2017507144 hasAuthorship W2017507144A5016043975 @default.
• W2017507144 hasAuthorship W2017507144A5031536762 @default.
• W2017507144 hasAuthorship W2017507144A5032499056 @default.
• W2017507144 hasAuthorship W2017507144A5035482519 @default.
• W2017507144 hasAuthorship W2017507144A5046131302 @default.
• W2017507144 hasAuthorship W2017507144A5049798132 @default.
• W2017507144 hasAuthorship W2017507144A5056246750 @default.
• W2017507144 hasAuthorship W2017507144A5057097136 @default.
• W2017507144 hasAuthorship W2017507144A5059792650 @default.
• W2017507144 hasBestOaLocation W20175071441 @default.
• W2017507144 hasConcept C104317684 @default.
• W2017507144 hasConcept C136449434 @default.
• W2017507144 hasConcept C143065580 @default.
• W2017507144 hasConcept C170493617 @default.
• W2017507144 hasConcept C184235292 @default.
• W2017507144 hasConcept C190283241 @default.
• W2017507144 hasConcept C203014093 @default.
• W2017507144 hasConcept C2779341262 @default.
• W2017507144 hasConcept C2779491563 @default.
• W2017507144 hasConcept C31573885 @default.
• W2017507144 hasConcept C54355233 @default.

• next