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Biochemical and Functional Characterization of the GH3 Amino Acid-Conjugase PBS3 of Arabidopsis thaliana

  • Author(s): Okrent, Rachel Allegra
  • Advisor(s): Wildermuth, Mary C
  • et al.

Plants have evolved sophisticated mechanisms to evade disease caused by pathogens. These resistance responses require intricate signaling networks mediated by phytohormones. Synthesis and accumulation of these molecules are regulated at several levels, including covalent modification through esterification and conjugation to amino acids and sugars. Many enzymes responsible for these modifications have been identified in the model plant Arabidopsis thaliana. The PBS3gene encodes an enzyme belonging to an uncharacterized group of the GH3 family present in many plant species; previously characterized GH3 family members act as phytohormone-amino acid synthetases. The pbs3 mutants (avrPphB Suceptible 3) exhibit enhanced susceptibility to virulent and avirulent Pseudomonas syringae strains, suggesting PBS3 is involved in basal disease resistance. PBS3 was found to be crucial for accumulation of induced salicylic acid (SA), a critical mediator of basal resistance, as well as expression of the SA-dependent pathogenesis related marker gene PR1. Using a novel high throughput adenylation assay, 4-substituted benzoates 4-hydroxybenzoate (4-HBA) and para-aminobenzoate (pABA) were identified as preferred acyl substrates of PBS3; no observed activity was observed with 2-substituted benzoates like SA. Notably, SA was found to specifically and reversibly inhibit PBS3 activity, perhaps revealing a general mechanism for the rapid regulation of GH3 activity. In addition, PBS3 influences the elicited accumulation of several compounds, including a novel compound. My study implicates 4-HBA and pABA as potential signaling molecules in plant-pathogen interactions and reveals PBS3 as a mediator of cross-talk between signaling pathways involving chorismate-derived compounds. Investigation of the evolutionary history of the GH3 family reveals that PBS3 is part of an ancient group of genes. The underexplored GH3 Group III is enriched in genes that have appeared through tandem duplication and insertion, consistent with function in response to environmental stress. However, functional analysis indicates that PBS3 is unique within the family in regards to its critical role in basal and SA-mediated defense responses.

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