Project description:Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector. Five weeks old Arabidopsis (Ws-2 and rrs1-1) leaves were infiltrated with Pf0-1 carrying PopP2 or PopP2C321A in pEDV6 and samples were collected at 2, 4, 6 and 8 hours post infiltration (hpi); mRNA profiles were generated in triplet, by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol.
Project description:Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.
Project description:Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.
Project description:Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific M-bM-^@M-^\avirulentM-bM-^@M-^] pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector. Arabidopsis No-0 and slh1 plants were grown for 4 weeks at 28M-BM-0C after germination on MS plate. Plants were transferred to 19M-BM-0C growth chamber at the beginning of the light cycle and samples were harvested at 0 h, 9 h, 12 h, 16 h and 24 h after transfer for total RNA extraction. mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol. Please note that all 16 samples submitted for this study were sequenced in one lane of Illumina GAIIx and the "No-0_slh1_tempshift_nobarcode.fq" (linked to 'unassigned reads' sample) contains unassigned sequence reads, once sample de-multiplexing has been carried out based on barcode.
Project description:Innate immune responses of plant cells confer the first line of defence against pathogens. Signals generated by activated receptors are integrated inside the cell and converge on transcriptional programmes in the nucleus. The Arabidopsis Toll-related intracellular receptor RPS4 operates inside nuclei to trigger resistance to Pseudomonas bacteria expressing AvrRps4 and defence gene reprogramming through the stress response regulator, EDS1. In this immune response, RPS4 cooperates genetically with RRS1 encoding a nuclear TIR-NB-LRR receptor with an additional C-terminal ‘WRKY’ DNA-binding domain. Using transgenic Arabidopsis plants constitutively expressing RPS4 (35S:RPS4), an EDS1-dependent immune response can be turned on rapidly and synchronously in leaf cells after a switch from high (28°C) to moderate (19°C) temperature. In order to determine the relative contributions of RRS1 and EDS1 to temperature-conditioned 35S:RPS4-HS transcriptional reprogramming, we performed gene expression microarray analysis of 35S:RPS4-HS, 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 leaf mRNAs before and after temperature shift. We used transgenic Arabidopsis plants over-expressing RPS4 in EDS1 WT, eds1-2 or rrs1-11 mutant backgrounds. 35S:RPS4, 35S:RPS4 eds1-2 and 35S:RPS4 rrs1-11 plants were grown at 28°C for 3.5 weeks, and subsequently shifted to 19°C. Samples were collected before shift (0h) and 2, 8 and 24h after shift, in triplicates.
Project description:Innate immune responses of plant cells confer the first line of defence against pathogens. Signals generated by activated receptors are integrated inside the cell and converge on transcriptional programmes in the nucleus. The Arabidopsis Toll-related intracellular receptor RPS4 operates inside nuclei to trigger resistance to Pseudomonas bacteria expressing AvrRps4 and defence gene reprogramming through the stress response regulator, EDS1. In this immune response, RPS4 cooperates genetically with RRS1 encoding a nuclear TIR-NB-LRR receptor with an additional C-terminal ‘WRKY’ DNA-binding domain. Using transgenic Arabidopsis plants constitutively expressing RPS4 (35S:RPS4), an EDS1-dependent immune response can be turned on rapidly and synchronously in leaf cells after a switch from high (28°C) to moderate (19°C) temperature. In order to determine the relative contributions of RRS1 and EDS1 to temperature-conditioned 35S:RPS4-HS transcriptional reprogramming, we performed gene expression microarray analysis of 35S:RPS4-HS, 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 leaf mRNAs before and after temperature shift.
Project description:Innate immune responses of plant cells confer the first line of defence against pathogens. Signals generated by activated receptors are integrated inside the cell and converge on transcriptional programmes in the nucleus. The Arabidopsis Toll-related intracellular receptor RPS4 operates inside nuclei to trigger resistance and defence gene reprogramming through the stress response regulator, EDS1. Using a combinaison of ChIP-seq and gene expression microarray profiling, we found that RPS4 associates with the genomic coding sequence of (mainly) defense-associated genes whose expression is subsequently strongly upregulated. Using RNA-seq, we tested wether the association pattern of RPS4 to its targets correlates with alternative splicing of similar sets of genes.
Project description:The trade-off between growth and defense is a critical aspect of plant immunity. Therefore, the plant immune response needs to be tightly regulated. Salicylic acid (SA) is an important plant hormone regulating defense against biotrophic pathogens. Recently, N-hydroxy-pipecolic acid (NHP) was identified as anotherregulator for plant innate immunity and systemic acquired resistance. Although the biosynthetic pathway leading to NHP formation has already been identified, how NHP is further metabolized was unclear. Here, we present UGT76B1 as a UDP-dependent glycosyltransferase that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid (NHP-OGlc) in Arabidopsis thaliana. Analysis of T-DNA and CRISPR knock-out mutant lines of UGT76B1 by targeted and non-targeted UHPLC-HRMS underlined NHP and SA as endogenous substrates of this enzyme in response to Pseudomonas infection and UV treatment. ugt76b1 mutant plants have a dwarf phenotype and constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme Flavin-dependent monooxygenase 1 (FMO1). This suggests that elevated accumulation of NHP contributes to the enhanced disease resistance in ugt76b1. Externally applied NHP can move to distal tissue in ugt76b1 mutant plants. Although glycosylation is not required for the long-distance movement of NHP during systemic acquired resistance, it is crucial to balance growth and defense.
Project description:This study investigates extent and functional significance of alternative splicing in Arabidopsis thaliana defense against the bacterial pathogen Pseudomonas syringae pv tomato (Pst). We have provided a detailed characterization of the Arabidopsis thaliana transcriptional response to Pseudomonas syringae infection in both susceptible and resistant hosts. We carried out two independent inoculation experiments (biological replicates) for each treatment. Col-0 is susceptible to virulent Pst DC3000 but has a functional RPS4 resistance gene effective against DC3000 expressing AvrRps4