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Several plant-associated microbes synthesize the auxinic plant growth regulator phenylacetic acid (PAA) in culture; however, the role of PAA in plant-pathogen interactions is not well understood. In this study, we investigated the role of PAA during interactions between the phytopathogenic bacteriumPseudomonas syringaestrainPtoDC3000 (PtoDC3000) and the model plant host,Arabidopsis thaliana. Previous work demonstrated that indole-3-acetaldehyde dehydrogenase A (AldA) ofPtoDC3000 converts indole-3-acetaldehyde (IAAld) to the auxin indole-3-acetic acid (IAA). Here, we further demonstrated the biochemical versatility of AldA by conducting substrate screening and steady-state kinetic analyses, and showed that AldA can use both IAAld and phenylacetaldehyde as substrates to produce IAA and PAA, respectively. Quantification of auxin in infected plant tissue showed that AldA-dependent synthesis of either IAA or PAA byPtoDC3000 does not contribute significantly to the increase in auxin levels in infectedA. thalianaleaves. Using availablearogenate dehydratase(adt) mutant lines ofA. thalianacompromised for PAA synthesis, we observed that a reduction in PAA-Asp and PAA-Glu is correlated with elevated levels of IAA and increased susceptibility. These results provide evidence that PAA/IAA homeostasis inA. thalianainfluences the outcome of plant-microbial interactions. 

ABSTRACT Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. To address the origins of this complexity and understand its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery.<bold>IMPORTANCE</bold>Polycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, includingStreptomycesandLysobacterspp.Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggest new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize the practical limitations of genomics-based discovery by exposing hidden complexity.