Identification and characterization of genetic and molecular components of Arabidopsis thaliana PBS3-mediated salicylic acid induction during defense against microbial pathogens

The plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) is essential for plant defense in response to biotrophic and hemibiotrophic microbial pathogens. The concentration of SA increases during infection, determining the extent of defense gene induction and cellular responses to the pathogen. In Arabidopsis thaliana, plants with non-functional avrPphB Susceptible 3 (PBS3) fail to accumulate significant induced SA and consequently lack induction of associated defense genes such as pathogenesis related 1 (PR1). These plants have increased susceptibility to pathogens such as the bacterial pathogen Pseudomonas syringae. PBS3 is a member of the GH3 family of enzymes, which conjugate small acyl substrates to amino acids. PBS3 conjugates 4-substituted hydroxybenzoic acids preferentially to glutamic acid, but this activity does not clarify its function in defense. Metabolic analyses showed that pbs3 mutants accumulate the product of another GH3 family enzyme, SA conjugated to aspartic acid (SA-Asp). The kinetics of the known SA-Asp synthetase, GH3.5, were investigated to better understand the formation of SA-Asp in vitro. GH3.5 is also active on the growth hormone indole-3-acetic acid (IAA) and has a higher affinity for IAA than SA under moderate to high concentrations of Asp. However, when the concentration of Asp decreased, the affinity of GH3.5 for SA increased. The concentration of Asp decreases in response to pathogens, likely as part of nitrogen reallocation during the transition from growth to defense. This suggests that acyl substrate preference amongst these promiscuous enzymes can be affected by the amino acid substrate, and that GH3.5 affinity for SA is greatest during pathogen challenge.The production of SA-Asp could serve to pull SA away from the pool used for defense in pbs3 mutants. Therefore, a pbs3gh3.5 double mutant line was created to see if the elimination of SA-Asp restored defense responses in the pbs3 background. SA-Asp was not significantly reduced in this line, so a multiplexed knockout line of likely SA-Asp synthetases was created to reduce genetic redundancy. This pbs3gh3.1gh3.3gh3.4gh3.5gh3.6 line, named gh6x, did eliminate induced SA-Asp in pbs3. However, gh6x failed to restore SA accumulation and pathogen resistance.A genetic suppressor screen was used to identify new components in PBS3-mediated defense. Over 5,000 M2 lines were screened and ultimately two lines out of six with restored SA accumulation were chosen for further characterization. To this point, candidate causal mutations PAD4S135F and RAP2.6A93V have been identified for these two lines. PAD4 is a well-known regulator of SA-induced defense responses but may also be involved in cross talk with the SA antagonist jasmonic acid (JA). RAP2.6 is a transcription factor associated with JA and ethylene responses. The identification of these genes as candidates suggests that PBS3 may have SA-independent roles as well. RNA-sequencing identified de-repression of many JA genes in induced pbs3 as compared to induced Col-0. Furthermore, exogenous application of SA failed to restore wild type susceptibility to Pseudomonas syringae in pbs3 mutants. Taken together, these data suggest that PBS3 is important not just for the accumulation of SA, but as a higher order regulator of the complex cross talk between the mutually antagonistic SA and necrotroph-induced jasmonic acid signaling pathways