The GH3 Acyl Adenylase Family Member PBS3 Regulates Salicylic Acid-Dependent Defense Responses in Arabidopsis

Abstract

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.

Systemic acquired resistance: the elusive signal(s)

“…It should be noted that in an independent study, over expression of GH3.5 led to elevated SA levels and PR-1 transcripts and suppression of IAA levels [49]. Another member of the GH3 family, GH3.12, is required for SA-mediated disease resistance; mutations in this gene ( pbs3, gdg1, win3) appear to suppress SA and/or SA-glucoside accumulation and confer enhanced susceptibility to avirulent and virulent Pseudomonas, and/or suppress SAR, although not all of the results are consistent among these studies [46][47][48]. Identifying the substrates of defense-related GH3 acyl adenylases, including GH3.5 and GH3.12, might shed light on the mechanism(s) through which auxin and SA signaling perturb each other to establish either susceptibility or resistance.…”

“…Accumulating evidence suggests that SA and auxin perform mutually antagonistic roles in disease resistance [44,45 ], and repression of auxin-related genes was observed in the systemic tissue of SAR-induced Arabidopsis [45 ]. Recently, members of the GH3 family of acyl-adenylate/thioester-forming enzymes involved in the amino acid conjugation of, for example the auxin indole-3-acetic acid (IAA), were implicated in the regulation of basal and R gene-mediated resistance as well as SAR [46][47][48]49,50 ]. GH3.5 can conjugate both SA and IAA [51], and both signaling pathways were upregulated in plants over expressing GH3.5 after pathogen infection [50 ].…”

Neonicotinoid insecticides induce salicylate-associated plant defense responses

“…3D; method described in SI Appendix) or HPLC analysis with fluorescence detection (Fig. 3E) [method previously described (45)]. Statistical analysis of treatments compared with the control used an unpaired Student's t test.…”

“…Frozen CPA-treated or control Arabidopsis leaves (∼500 mg fresh weight) were extracted as for SA above (45). CPA-treated and control plant extracts were HPLC-fractionated with Data are mean ± SD (n = 3).…”

Modulating plant hormones by enzyme action

“…42 The only characterized group III GH3 member is AtGH3-12. [43][44][45] Three pbs3 mutants display increased disease susceptibility to virulent and avirulent Pseudomonas syringae. 43 The gdg1 mutant is allelic to pbs3.…”