Lights, X-rays, oxygen!

Photosystem II uses metal ions to oxidize water to form O2. Two recent papers employ the new technique of serial femtosecond crystallography utilizing X-ray free-electron lasers and nanocrystals to obtain initial structures of intermediate states of photosystem II catalysis at the site of oxygen production.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

Fidelity in plant hormone modifications catalyzed by Arabidopsis GH3 acyl acid amido synthetases.

GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds.

Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

Chemical inhibition of the auxin inactivation pathway uncovers the roles of metabolic turnover in auxin homeostasis.

The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites.

The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.

Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta.

l-Tyrosine is an essential amino acid for protein synthesis and is also used in plants to synthesize diverse natural products. Plants primarily synthesize tyrosine via TyrA arogenate dehydrogenase (TyrAa or ADH), which are typically strongly feedback inhibited by tyrosine. However, two plant lineages, Fabaceae (legumes) and Caryophyllales, have TyrA enzymes that exhibit relaxed sensitivity to tyrosine inhibition and are associated with elevated production of tyrosine-derived compounds, such as betalain pigments uniquely produced in core Caryophyllales. Although we previously showed that a single D222N substitution is primarily responsible for the deregulation of legume TyrAs, it is unknown when and how the deregulated Caryophyllales TyrA emerged. Here, through phylogeny-guided TyrA structure-function analysis, we found that functionally deregulated TyrAs evolved early in the core Caryophyllales before the origin of betalains, where the E208D amino acid substitution in the active site, which is at a different and opposite location from D222N found in legume TyrAs, played a key role in the TyrA functionalization. Unlike legumes, however, additional substitutions on non-active site residues further contributed to the deregulation of TyrAs in Caryophyllales. The introduction of a mutation analogous to E208D partially deregulated tyrosine-sensitive TyrAs, such as Arabidopsis TyrA2 (AtTyrA2). Moreover, the combined introduction of D222N and E208D additively deregulated AtTyrA2, for which the expression in Nicotiana benthamiana led to highly elevated accumulation of tyrosine in planta. The present study demonstrates that phylogeny-guided characterization of key residues underlying primary metabolic innovations can provide powerful tools to boost the production of essential plant natural products.

Orthology-based analysis helps map evolutionary diversification and predict substrate class use of BAHD acyltransferases.

Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast-evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG-based analysis enabled identification of signature motifs in anthocyanin-acylating BAHDs, whose importance was validated via molecular dynamic simulations, site-directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration-enabled approach for improved functional annotation of plant enzyme families.

Distribution and the evolutionary history of G-protein components in plant and algal lineages.

Heterotrimeric G-protein complexes comprising Gα-, Gß-, and Gγ-subunits and the regulator of G-protein signaling (RGS) are conserved across most eukaryotic lineages. Signaling pathways mediated by these proteins influence overall growth, development, and physiology. In plants, this protein complex has been characterized primarily from angiosperms with the exception of spreading-leaved earth moss (Physcomitrium patens) and Chara braunii (charophytic algae). Even within angiosperms, specific G-protein components are missing in certain species, whereas unique plant-specific variants-the extra-large Gα (XLGα) and the cysteine-rich Gγ proteins-also exist. The distribution and evolutionary history of G-proteins and their function in nonangiosperm lineages remain mostly unknown. We explored this using the wealth of available sequence data spanning algae to angiosperms representing extant species that diverged approximately 1,500 million years ago, using BLAST, synteny analysis, and custom-built Hidden Markov Model profile searches. We show that a minimal set of components forming the XLGαßγ trimer exists in the entire land plant lineage, but their presence is sporadic in algae. Additionally, individual components have distinct evolutionary histories. The XLGα exhibits many lineage-specific gene duplications, whereas Gα and RGS show several instances of gene loss. Similarly, Gß remained constant in both number and structure, but Gγ diverged before the emergence of land plants and underwent changes in protein domains, which led to three distinct subtypes. These results highlight the evolutionary oddities and summarize the phyletic patterns of this conserved signaling pathway in plants. They also provide a framework to formulate pertinent questions on plant G-protein signaling within an evolutionary context.

Potent, specific MEPicides for treatment of zoonotic staphylococci.

Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, that DXR represents a promising, druggable target for future development.

Molecular Basis of the Evolution of Methylthioalkylmalate Synthase and the Diversity of Methionine-Derived Glucosinolates.

The globally cultivated Brassica species possess diverse aliphatic glucosinolates, which are important for plant defense and animal nutrition. The committed step in the side chain elongation of methionine-derived aliphatic glucosinolates is catalyzed by methylthioalkylmalate synthase, which likely evolved from the isopropylmalate synthases of leucine biosynthesis. However, the molecular basis for the evolution of methylthioalkylmalate synthase and its generation of natural product diversity in Brassica is poorly understood. Here, we show that Brassica genomes encode multiple methylthioalkylmalate synthases that have differences in expression profiles and 2-oxo substrate preferences, which account for the diversity of aliphatic glucosinolates across Brassica accessions. Analysis of the 2.1 Å resolution x-ray crystal structure of Brassica juncea methylthioalkylmalate synthase identified key active site residues responsible for controlling the specificity for different 2-oxo substrates and the determinants of side chain length in aliphatic glucosinolates. Overall, these results provide the evolutionary and biochemical foundation for the diversification of glucosinolate profiles across globally cultivated Brassica species, which could be used with ongoing breeding strategies toward the manipulation of beneficial glucosinolate compounds for animal health and plant protection.

Biochemical and clinical studies of putative allergens to assess what distinguishes them from other non-allergenic proteins in the same family.

Many protein families have numerous members listed in databases as allergens; however, some allergen database entries, herein called "orphan allergens", are members of large families of which all other members are not allergens. These orphan allergens provide an opportunity to assess whether specific structural features render a protein allergenic. Three orphan allergens [Cladosporium herbarum aldehyde dehydrogenase (ChALDH), Alternaria alternata ALDH (AaALDH), and C. herbarum mannitol dehydrogenase (ChMDH)] were recombinantly produced and purified for structure characterization and for clinical skin prick testing (SPT) in mold allergic participants. Examination of the X-ray crystal structures of ChALDH and ChMDH and a homology structure model of AaALDH did not identify any discernable epitopes that distinguish these putative orphan allergens from their non-allergenic protein relatives. SPT results were aligned with ChMDH being an allergen, 53% of the participants were SPT (+). AaALDH did not elicit SPT reactivity above control proteins not in allergen databases (i.e., Psedomonas syringae indole-3-acetaldehyde dehydrogenase and Zea mays ALDH). Although published results showed consequential human IgE reactivity with ChALDH, no SPT reactivity was observed in this study. With only one of these three orphan allergens, ChMDH, eliciting SPT(+) reactions consistent with the protein being included in allergen databases, this underscores the complicated nature of how bioinformatics is used to assess the potential allergenicity of food proteins that could be newly added to human diets and, when needed, the subsequent clinical testing of that bioinformatic assessment.Trial registration number and date of registration AAC-2017-0467, approved as WIRB protocol #20172536 on 07DEC2017 by WIRB-Copernicus (OHRP/FDA Registration #: IRB00000533, organization #: IORG0000432).

Protecting P-type plasma membrane H+-ATPases from ROS.

P-type ATPase are ubiquitous transport proteins across all kingdoms of life. These proteins share a common mechanism involving phosphorylation of an invariant aspartate to facilitate movement of substrates from protons to phospholipids across cellular membranes. In this issue of the Biochemical Journal, Welle et al. identify a conserved cysteine near the functionally critical aspartate of P-type plasma membrane H+-ATPases that protects the protein from reactive oxygen species.

Molecular basis for branched steviol glucoside biosynthesis.

Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 from Stevia rebaudiana catalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3' position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.

Plants in the real world: An introduction to the JBC Reviews thematic series.

The deep relationship between plants and humans predates civilization, and our reliance on plants as sources of food, feed, fiber, fuels, and pharmaceuticals continues to increase. Understanding how plants grow and overcome challenges to their survival is critical for using these organisms to meet current and future demands for food and other plant-derived materials. This thematic review series on "plants in the real world" presents a set of eight reviews that highlight advances in understanding plant health, including the role of thiamine (vitamin B1), iron, and the plant immune system; how plants use ethylene and ubiquitin systems to control growth and development; and how new gene-editing approaches, the redesign of plant cell walls, and deciphering herbicide resistance evolution can lead to the next generation of crops.

Natural product biosynthesis: What's next? An introduction to the JBC Reviews Thematic Series.

The diversity of natural products not only fascinates us intellectually, but also provides an armamentarium against the microbes that threaten our health. The increased prevalence of pathogens that are resistant to one or more classes of available medicines continues to be a growing global threat. As drug-resistant pathogens erode the effectiveness of the current reserve of antibiotics and antifungals, methodological advances open additional avenues for discovery of new classes of drugs, as well as novel derivatives of existing (and proven) classes of compounds. In this Thematic Review Series, we aim to provide a snapshot of the current state of the art in natural product discovery. The reviews in this series encapsulate convergent approaches toward the identification of different classes of primary and specialized metabolites, including nonribosomal peptides, polyketides, and ribosomally synthesized and post-translationally modified peptides, from all kingdoms of life. Traction in unraveling new and diverse classes of molecules has come largely from the academic sector, which ensures availability of methods and data sets. Such knowledge is needed to thwart serious threats to human health and calls to mind the proverb praemonitus praemunitus (forewarned is forearmed).

The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase.

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.

A natural single-nucleotide polymorphism variant in sulfite reductase influences sulfur assimilation in maize.

Plants absorb sulfur from the environment and assimilate it into suitable forms for the biosynthesis of a broad range of molecules. Although the biochemical pathway of sulfur assimilation is known, how genetic differences contribute to natural variation in sulfur assimilation remains poorly understood. Here, using a genome-wide association study, we uncovered a single-nucleotide polymorphism (SNP) variant in the sulfite reductase (SiR) gene that was significantly associated with SiR protein abundance in a maize natural association population. We also demonstrated that the synonymous C to G base change at SNP69 may repress translational activity by altering messenger RNA secondary structure, which leads to reduction in ZmSiR protein abundance and sulfur assimilation activity. Population genetic analyses showed that the SNP69C allele was likely a variant occurring after the initial maize domestication and accumulated with the spread of maize cultivation from tropical to temperate regions. This study provides the first evidence that genetic polymorphisms in the exon of ZmSiR could influence the protein abundance through a posttranscriptional mechanism and in part contribute to natural variation in sulfur assimilation. These findings provide a prospective target to improve maize varieties with proper sulfur nutrient levels assisted by molecular breeding and engineering.

Brassicaceae-specific Gretchen Hagen 3 acyl acid amido synthetases conjugate amino acids to chorismate, a precursor of aromatic amino acids and salicylic acid.

To modulate responses to developmental or environmental cues, plants use Gretchen Hagen 3 (GH3) acyl acid amido synthetases to conjugate an amino acid to a plant hormone, a reaction that regulates free hormone concentration and downstream responses. The model plant Arabidopsis thaliana has 19 GH3 proteins, of which 8 have confirmed biochemical functions. One Brassicaceae-specific clade of GH3 proteins was predicted to use benzoate as a substrate and includes AtGH3.7 and AtGH3.12/PBS3. Previously identified as a 4-hydroxybenzoic acid-glutamate synthetase, AtGH3.12/PBS3 influences pathogen defense responses through salicylic acid. Recent work has shown that AtGH3.12/PBS3 uses isochorismate as a substrate, forming an isochorismate-glutamate conjugate that converts into salicylic acid. Here, we show that AtGH3.7 and AtGH3.12/PBS3 can also conjugate chorismate to cysteine and glutamate, which act as precursors to aromatic amino acids and salicylic acid, respectively. The X-ray crystal structure of AtGH3.12/PBS3 in complex with AMP and chorismate at 1.94 Å resolution, along with site-directed mutagenesis, revealed how the active site potentially accommodates this substrate. Examination of Arabidopsis knockout lines indicated that the gh3.7 mutants do not alter growth and showed no increased susceptibility to the pathogen Pseudomonas syringae, unlike gh3.12 mutants, which were more susceptible than WT plants, as was the gh3.7/gh3.12 double mutant. The findings of our study suggest that GH3 proteins can use metabolic precursors of aromatic amino acids as substrates.

Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC3000.

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.