Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells

The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana

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 NBLRR 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

Autoimmunity conferred by chs3-2D relies on CSA1, its adjacent TIR-NB-LRR encoding neighbour

Plant innate immunity depends on the function of a large number of intracellular immune receptor proteins, the majority of which are structurally similar to mammalian nucleotidebinding oligomerization domain (NOD)-like receptor (NLR) proteins. CHILLING SENSITIVE 3 (CHS3) encodes an atypical Toll/Interleukin 1 Receptor (TIR)-type NLR protein with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C-terminus. The gain-of-function mutant allele chs3-2D exhibits severe dwarfism and constitutively activated defense responses, including enhanced resistance to virulent pathogens, high defence marker gene expression, and salicylic acid accumulation. To search for novel regulators involved in CHS3-mediated immune signaling, we conducted suppressor screens in the chs3-2D and chs3-2D pad4-1 genetic backgrounds. Alleles of sag101 and eds1-90 were isolated as complete suppressors of chs3-2D, and alleles of sgt1b were isolated as partial suppressors of chs3-2D pad4-1. These mutants suggest that SAG101, EDS1-90, and SGT1b are all positive regulators of CHS3-mediated defense signaling. Additionally, the TIR-type NLR-encoding CSA1 locus located genomically adjacent to CHS3 was found to be fully required for chs3-2D-mediated autoimmunity. CSA1 is located 3.9kb upstream of CHS3 and is transcribed in the opposite direction. Altogether, these data illustrate the distinct genetic requirements for CHS3-mediated defense signaling

Protein-protein interactions in the RPS4/RRS1 immune receptor complex

Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation

Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca2+. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca2+ link between MAMP recognition and SA accumulation that is important for resistance to P. syringae