AvrRpm1 Functions as an ADP-Ribosyl Transferase to Modify NOI Domain-Containing Proteins, Including Arabidopsis and Soybean RPM1-Interacting Protein4

The Pseudomonas syringae effector protein AvrRpm1 activates the Arabidopsis (Arabidopsis thaliana) intracellular innate immune receptor protein RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) via modification of a second Arabidopsis protein, RPM1-INTERACTING PROTEIN4 (AtRIN4). Prior work has shown that AvrRpm1 induces phosphorylation of AtRIN4, but homology modeling indicated that AvrRpm1 may be an ADP-ribosyl transferase. Here, we show that AvrRpm1 induces ADP-ribosylation of RIN4 proteins from both Arabidopsis and soybean (Glycine max) within two highly conserved nitrate-induced (NOI) domains. It also ADP ribosylates at least 10 additional Arabidopsis NOI domain-containing proteins. The ADP-ribosylation activity of AvrRpm1 is required for subsequent phosphorylation on Thr-166 of AtRIN4, an event that is necessary and sufficient for RPM1 activation. We also show that the C-terminal NOI domain of AtRIN4 interacts with the exocyst subunits EXO70B1, EXO70E1, EXO70E2, and EXO70F1. Mutation of either EXO70B1 or EXO70E2 inhibited secretion of callose induced by the bacterial flagellin-derived peptide flg22. Substitution of RIN4 Thr-166 with Asp enhanced the association of AtRIN4 with EXO70E2, which we posit inhibits its callose deposition function. Collectively, these data indicate that AvrRpm1 ADP-ribosyl transferase activity contributes to virulence by promoting phosphorylation of RIN4 Thr-166, which inhibits the secretion of defense compounds by promoting the inhibitory association of RIN4 with EXO70 proteins

Arabidopsis EDR1 Protein Kinase Regulates the Association of EDS1 and PAD4 to Inhibit Cell Death

ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid–dependent and independent pathways. Here, we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss-of-function mutations in the protein kinase EDR1 (Enhanced Disease Resistance1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here, we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet-unknown PAD4 signaling function

The GH3 Acyl Adenylase Family Member PBS3 Regulates Salicylic Acid-Dependent Defense Responses in Arabidopsis1[W][OA]

The pbs3-1 mutant, identified in a screen for Arabidopsis (Arabidopsis thaliana) mutants exhibiting enhanced susceptibility to the avirulent Pseudomonas syringae pathogen DC3000 (avrPphB), also exhibits enhanced susceptibility to virulent P. syringae strains, suggesting it may impact basal disease resistance. Because induced salicylic acid (SA) is a critical mediator of basal resistance responses, free and glucose-conjugated SA levels were measured and expression of the SA-dependent pathogenesis-related (PR) marker, PR1, was assessed. Surprisingly, whereas accumulation of the SA glucoside and expression of PR1 were dramatically reduced in the pbs3-1 mutant in response to P. syringae (avrRpt2) infection, free SA was elevated. However, in response to exogenous SA, the conversion of free SA to SA glucoside and the induced expression of PR1 were similar in pbs3-1 and wild-type plants. Through positional cloning, complementation, and sequencing, we determined that the pbs3-1 mutant contains two point mutations in the C-terminal region of the protein encoded by At5g13320, resulting in nonconserved amino acid changes in highly conserved residues. Additional analyses with Arabidopsis containing T-DNA insertion (pbs3-2) and transposon insertion (pbs3-3) mutations in At5g13320 confirmed our findings with pbs3-1. PBS3 (also referred to as GH3.12) is a member of the GH3 family of acyl-adenylate/thioester-forming enzymes. Characterized GH3 family members, such as JAR1, act as phytohormone-amino acid synthetases. Thus, our results suggest that amino acid conjugation plays a critical role in SA metabolism and induced defense responses, with PBS3 acting upstream of SA, directly on SA, or on a competitive inhibitor of SA

AvrRpm1 Functions as an ADP-Ribosyl Transferase to Modify NOI Domain-Containing Proteins, Including Arabidopsis and Soybean RPM1-Interacting Protein4.

The Pseudomonas syringae effector protein AvrRpm1 activates the Arabidopsis (Arabidopsis thaliana) intracellular innate immune receptor protein RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) via modification of a second Arabidopsis protein, RPM1-INTERACTING PROTEIN4 (AtRIN4). Prior work has shown that AvrRpm1 induces phosphorylation of AtRIN4, but homology modeling indicated that AvrRpm1 may be an ADP-ribosyl transferase. Here, we show that AvrRpm1 induces ADP-ribosylation of RIN4 proteins from both Arabidopsis and soybean (Glycine max) within two highly conserved nitrate-induced (NOI) domains. It also ADP ribosylates at least 10 additional Arabidopsis NOI domain-containing proteins. The ADP-ribosylation activity of AvrRpm1 is required for subsequent phosphorylation on Thr-166 of AtRIN4, an event that is necessary and sufficient for RPM1 activation. We also show that the C-terminal NOI domain of AtRIN4 interacts with the exocyst subunits EXO70B1, EXO70E1, EXO70E2, and EXO70F1. Mutation of either EXO70B1 or EXO70E2 inhibited secretion of callose induced by the bacterial flagellin-derived peptide flg22. Substitution of RIN4 Thr-166 with Asp enhanced the association of AtRIN4 with EXO70E2, which we posit inhibits its callose deposition function. Collectively, these data indicate that AvrRpm1 ADP-ribosyl transferase activity contributes to virulence by promoting phosphorylation of RIN4 Thr-166, which inhibits the secretion of defense compounds by promoting the inhibitory association of RIN4 with EXO70 proteins