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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 bacterium Pseudomonas syringae strain Pto DC3000 ( Pto DC3000) and the model plant host, Arabidopsis thaliana . Previous work demonstrated that indole-3-acetaldehyde dehydrogenase A (AldA) of Pto DC3000 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 by Pto DC3000 does not contribute significantly to the increase in auxin levels in infected A. thaliana leaves. Using available arogenate dehydratase ( adt ) mutant lines of A. thaliana compromised 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 in A. thaliana influences the outcome of plant-microbial interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lee, Harper, Lee, Qi, Clay, Aoi, Jez, Kasahara, Blodgett and Kunkel.)

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. IMPORTANCE Polycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, including Streptomyces and Lysobacter spp . 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., Competing Interests: The authors declare no conflict of interest.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2-2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

Plant pathogens have evolved several strategies to manipulate the biology of their hosts to facilitate colonization, growth to high levels in plant tissue, and production of disease. One of the less well known of these strategies is the synthesis of plant hormones and hormone analogs, and there is growing evidence that modulation of host hormone signaling is important during pathogenesis. Several plant pathogens produce the auxin indole-3-acetic acid (IAA) and/or virulence factors that modulate host auxin signaling. Auxin is well known for being involved in many aspects of plant growth and development, but recent findings have revealed that elevated IAA levels or enhanced auxin signaling can also promote disease development in some plant-pathogen interactions. In addition to stimulating plant cell growth during infection by gall-forming bacteria, auxin and auxin signaling can antagonize plant defense responses. Auxin can also act as a microbial signaling molecule to impact the biology of some pathogens directly. In this review, we summarize recent progress towards elucidating the roles that auxin production, modification of host auxin signaling, and direct effects of auxin on pathogens play during pathogenesis, with emphasis on the impacts of auxin on interactions with bacterial pathogens., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The bacterial pathogen Pseudomonas syringae modulates plant hormone signaling to promote infection and disease development. P. syringae uses several strategies to manipulate auxin physiology in Arabidopsis thaliana to promote pathogenesis, including its synthesis of indole-3-acetic acid (IAA), the predominant form of auxin in plants, and production of virulence factors that alter auxin responses in the host; however, the role of pathogen-derived auxin in P. syringae pathogenesis is not well understood. Here we demonstrate that P. syringae strain DC3000 produces IAA via a previously uncharacterized pathway and identify a novel indole-3-acetaldehyde dehydrogenase, AldA, that functions in IAA biosynthesis by catalyzing the NAD-dependent formation of IAA from indole-3-acetaldehyde (IAAld). Biochemical analysis and solving of the 1.9 Å resolution x-ray crystal structure reveal key features of AldA for IAA synthesis, including the molecular basis of substrate specificity. Disruption of aldA and a close homolog, aldB, lead to reduced IAA production in culture and reduced virulence on A. thaliana. We use these mutants to explore the mechanism by which pathogen-derived auxin contributes to virulence and show that IAA produced by DC3000 suppresses salicylic acid-mediated defenses in A. thaliana. Thus, auxin is a DC3000 virulence factor that promotes pathogenicity by suppressing host defenses.