ABSTRACT
In both plants and animals, intracellular nucleotide-binding leucine-rich repeat proteins (NLRs; or Nod-like receptors) serve as immune receptors to recognize pathogen-derived molecules and mount effective immune responses against microbial infections. Plant NLRs often guard the presence or activity of other host proteins, which are the direct virulence targets of pathogen effectors. These guardees are sometimes immune-promoting components such as those in a mitogen-activated protein kinase cascade. Plant E3 ligases serve many roles in immune regulation, but it is unclear whether they can also be guarded by NLRs. Here, we report on an immune-regulating E3 ligase SAUL1, whose homeostasis is monitored by a Toll interleukin 1 receptor (TIR)-type NLR (TNL), SOC3. SOC3 can associate with SAUL1, and either loss or overexpression of SAUL1 triggers autoimmunity mediated by SOC3. By contrast, SAUL1 functions redundantly with its close homolog PUB43 to promote PAMP-triggered immunity (PTI). Taken together, the E3 ligase SAUL1 serves as a positive regulator of PTI and its homeostasis is monitored by the TNL SOC3.
Subject(s)
Arabidopsis Proteins/metabolism , Homeostasis , NLR Proteins/metabolism , Pathogen-Associated Molecular Pattern Molecules/metabolism , Plant Immunity , Ubiquitin-Protein Ligases/metabolism , Arabidopsis Proteins/chemistry , Autoimmunity , Cloning, Molecular , Conserved Sequence , Cysteine/metabolism , Gene Knockout Techniques , Green Fluorescent Proteins/metabolism , Models, Biological , Mutation/genetics , NLR Proteins/chemistry , Phenotype , Plants, Genetically Modified , Protein Binding , Subcellular Fractions/metabolism , Suppression, Genetic , Nicotiana/metabolism , Ubiquitin-Protein Ligases/chemistryABSTRACT
Post-translational protein modification plays a pivotal role in the regulation and specific turnover of proteins. One of these important modifications is the ubiquitination of target proteins, which can occur at distinct cellular compartments. At the plasma membrane, ubiquitination regulates the internalization and thus trafficking of membrane proteins such as receptors and channels. The Arabidopsis plant U-box (PUB) armadillo repeat (PUB-ARM) ubiquitin ligase SAUL1 (SENESCENCE-ASSOCIATED UBIQUITIN LIGASE1) is part of the ubiquitination machinery at the plasma membrane. In contrast to most other PUB-ARM proteins, SAUL1 carries additional C-terminal ARM repeats responsible for plasma membrane-association. Here, we demonstrated that the C-terminal ARM repeat domain is also essential and sufficient to mediate plasma membrane-association of the closest Arabidopis paralog AtPUB43. We investigated targeting of PUB-ARM ubiquitin ligases of different plant species to find out whether plasma membrane-association of SAUL1-type PUB-ARM proteins is conserved. Phylogenetic analysis identified orthologs of SAUL1 in these plant species. Intracellular localization of transiently expressed GFP fusion proteins revealed that indeed plasma membrane-association due to additional C-terminal ARM repeats represents a conserved feature of SAUL1-type proteins. Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects. Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.
ABSTRACT
Age-dependent leaf senescence and cell death in Arabidopsis (Arabidopsis thaliana) requires activation of the transcription factor ORESARA1 (ORE1) and is not initiated prior to a leaf age of 28 d. Here, we investigate the conditional execution of events that regulate early senescence and cell death in senescence-associated ubiquitin ligase1 (saul1) mutants, deficient in the PLANT U-BOX-ARMADILLO E3 ubiquitin ligase SAUL1. In saul1 mutants challenged with low light, the switch of age-dependent cell death was turned on prematurely, as indicated by the accumulation of ORE1 transcripts, induction of the senescence marker gene SENESCENCE-ASSOCIATED GENE12, and cell death. However, ORE1 accumulation by itself was not sufficient to cause saul1 phenotypes, as demonstrated by double mutant analysis. Exposure of saul1 mutants to low light for only 24 h did not result in visible symptoms of senescence; however, the senescence-promoting transcription factor genes WRKY53, WRKY6, and NAC-LIKE ACTIVATED BY AP3/PI were up-regulated, indicating that senescence in saul1 seedlings was already initiated. To resolve the time course of gene expression, microarray experiments were performed at narrow intervals. Differential expression of the genes involved in salicylic acid and defense mechanisms were the earliest events detected, suggesting a central role for salicylic acid in saul1 senescence and cell death. The salicylic acid content increased in low-light-treated saul1 mutants, and application of exogenous salicylic acid was indeed sufficient to trigger saul1 senescence in permissive light conditions. Double mutant analyses showed that PHYTOALEXIN DEFICIENT4 (PAD4) but not NONEXPRESSER OF PR GENES1 (NPR1) is essential for saul1 phenotypes. Our results indicate that saul1 senescence depends on the PAD4-dependent salicylic acid pathway but does not require NPR1 signaling.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/cytology , Arabidopsis/growth & development , Carboxylic Ester Hydrolases/metabolism , Mutation/genetics , Salicylic Acid/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Carboxylic Ester Hydrolases/genetics , Cell Death/drug effects , Cell Death/genetics , Cell Death/radiation effects , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Light , Oligonucleotide Array Sequence Analysis , Phenotype , Salinity , Seedlings/cytology , Seedlings/drug effects , Seedlings/growth & development , Seedlings/radiation effects , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/radiation effects , Sodium Chloride/pharmacology , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Ubiquitination plays important roles in plant growth and development. Whereas ubiquitin-dependent protein degradation and modulation in the cytoplasm and nucleus are well established in plants, ubiquitination events mediated by E3 ubiquitin ligases at the plasma membrane are largely unknown. Here, it is demonstrated that the suppressor of premature senescence and cell death SENESCENCE-ASSOCIATED UBIQUITIN LIGASE 1 (SAUL1), a plant U-box armadillo repeat (PUB-ARM) E3 ubiquitin ligase, localizes at the plasma membrane. Among the members of the PUB-ARM protein family, this localization is unique to SAUL1 and its two closest homologues. A novel armadillo repeat domain was identified at the SAUL1 C-terminus that directs specific association with the plasma membrane and is crucial for SAUL1 function in vivo. The data suggest that a small subgroup of PUB-ARM proteins including SAUL1 have functions at the plasma membrane probably by modifying target proteins by ubiquitination.
