Engineered auxin degrading microbiota for plant growth promotion

Abstract

The present disclosure provides an engineered nucleic acid sequence comprising a gene cassette, the gene cassette comprising one or more regulator genes, the regulator genes including an iadR gene, and at least three catabolic pathway genes downstream from the one or more regulator genes, the at least three catabolic pathway genes including an iadC gene, an iadD gene, and an iadE gene, wherein the engineered nucleic acid sequence is free of an iadA gene and iadB gene. The disclosure also provides bacteria cells comprising the engineered nucleic acid sequence, wherein the bacteria cell is capable of degrading indole-3-acetic acid. Methods for degrading auxin, controlling auxin, and promoting plant growth and root development are provided, comprising providing the bacteria cell and allowing the bacteria cell to interact with roots of a plant.

Description

[0001]PRINCETON-103476 ENGINEERED AUXIN DEGRADING MICROBIOTA FOR PLANT GROWTH PROMOTION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. US 63 / 705,597 filed October 10, 2024, US 63 / 747,680 filed January 21, 2025, and US 63 / 796,730 filed April 29, 2025, each of which is hereby incorporated by reference in its entirety. FIELD OF INVENTION The present disclosure relates to engineered microorganisms for agricultural applications, and more particularly to genetically modified bacteria containing auxin degradation pathways that promote plant growth by regulating auxin homeostasis in the rhizosphere. BACKGROUND Plant growth and development are fundamentally regulated by complex interactions between plants and their associated microbial communities in the rhizosphere, the narrow zone of soil surrounding plant roots. Within this dynamic environment, numerous microorganisms produce and respond to various signaling molecules that can profoundly influence plant physiology and growth patterns. Auxins represent a class of plant hormones that play central roles in regulating root development, cell elongation, and overall plant architecture. Indole-3-acetic acid (IAA) is the most prevalent naturally occurring auxin and serves as a primary regulator of root growth and branching patterns. While plants maintain sophisticated mechanisms for auxin biosynthesis, transport, and degradation to achieve optimal hormone balance, this delicate equilibrium can be disrupted by external factors. Many soil and rhizosphere bacteria possess the metabolic capability to produce IAA through various biosynthetic pathways. When present in excessive concentrations, microbially- produced auxin can overwhelm plant regulatory systems and lead to root growth inhibition rather than promotion. This phenomenon has been observed across diverse plant species and can result in stunted root development, reduced nutrient uptake, and compromised plant health. The disruption of auxin homeostasis by microbial communities presents challenges for agricultural productivity and sustainable crop production. Traditional approaches to managing PRINCETON-103476 plant-microbe interactions often rely on broad-spectrum treatments that may disrupt beneficial microbial relationships while addressing problematic ones. More targeted approaches that can selectively modulate specific aspects of plant-microbe signaling while preserving beneficial interactions would offer advantages for agricultural applications. Recent advances in understanding microbial auxin metabolism have revealed that certain bacterial species possess enzymatic pathways capable of degrading excess auxin compounds. These natural auxin degradation systems represent potential tools for restoring auxin balance in plant root environments. However, the complexity of these metabolic pathways and the challenges associated with deploying them in agricultural settings have limited their practical application. The development of engineered microbial systems that can effectively regulate auxin levels in plant root environments while maintaining compatibility with existing agricultural practices represents an area of ongoing research interest. Such systems could potentially address auxin-related growth inhibition while supporting overall plant health and productivity. SUMMARY This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. According to an aspect of the present disclosure, an engineered nucleic acid sequence is provided. The engineered nucleic acid sequence comprises a gene cassette. The gene cassette comprises one or more regulator genes, the regulator genes including an iadR gene. The gene cassette may include a non-coding region that a regulator gene (such as iadR) can bind to. The gene cassette comprises at least three catabolic pathway genes downstream from the one or more regulator genes, the at least three catabolic pathway genes including an iadC gene, an iadD gene, and an iadE gene. The engineered nucleic acid sequence is free of an iadA gene and iadB gene. This engineered nucleic acid sequence provides a streamlined auxin degradation pathway that enables efficient breakdown of indole-3-acetic acid while eliminating unnecessary genetic components. The inclusion of the iadR regulator gene allows for responsive control of the degradation pathway, while the core iadCDE genes provide the catalytic machinery for auxin degradation. The absence of iadA and iadB genes reduces the genetic burden on host cells while maintaining functional auxin degradation capability. PRINCETON-103476 According to other aspects of the present disclosure, the engineered nucleic acid sequence may include one or more of the following features. The one or more regulator genes may consist of the iadR gene. This configuration provides precise regulatory control over the auxin degradation pathway through a single, well-characterized transcriptional regulator, simplifying the genetic architecture while maintaining effective pathway regulation. The at least three catabolic pathway genes may consist of the iadC gene, the iadD gene, and the iadE gene. This minimal gene set provides the core enzymatic functions for auxin degradation, reducing the complexity of the engineered system while maintaining the ability to initiate auxin breakdown and reverse root growth inhibition. The at least three catabolic pathway genes may include the iadC gene, the iadD gene, and the iadE gene, and at least one additional catabolic pathway gene. The inclusion of additional catabolic pathway genes enhances the completeness of auxin degradation, allowing for more thorough breakdown of auxin molecules and improved detoxification under high auxin concentrations. The at least one additional catabolic pathway gene may include an iadF gene. The iadF gene encodes an acyl-CoA synthase that facilitates the removal of the acetyl side chain from auxin degradation intermediates, contributing to the complete mineralization of auxin compounds. The sequence may include a transporter component. The transporter component may include an iadK2 gene. The iadK2 gene encodes a highly specific ATP-binding cassette transporter that enhances auxin uptake, improving the efficiency of auxin degradation by facilitating substrate availability to the degradation enzymes. The at least three catabolic pathway genes may consist of the iadC gene, the iadD gene, the iadE gene, an iadFgene, an iadG gene, an iadH gene, an iadI gene, an iadJ gene, and the sequence may include the iadK2 gene. This complete gene set provides the full auxin degradation pathway, enabling comprehensive breakdown of auxin from the initial substrate through all intermediates to the final product anthranilic acid, maximizing the effectiveness of auxin detoxification. A reporter gene may be located after the iadE gene, such as between the iadE gene and the at least one additional catabolic pathway gene. The strategic placement of a reporter gene within the pathway allows for real-time monitoring of pathway activity and auxin-responsive gene expression, providing a valuable tool for detecting auxin levels and pathway function. PRINCETON-103476 The reporter gene may be located between the iadE gene and an iadF gene. This specific positioning enables monitoring of the transition from the initial degradation steps to the downstream processing steps, providing insight into pathway progression and efficiency. The reporter gene may be a gene for a fluorescent protein. The use of a fluorescent protein reporter enables rapid, non-invasive detection of pathway activity through fluorescence measurements, facilitating high-throughput screening and real-time monitoring of auxin degradation. According to another aspect of the present disclosure, a method of detecting auxin generation is provided. The method comprises introducing an engineered nucleic acid sequence of any one of the preceding aspects to an auxin-generating system. The method comprises allowing the auxin-generating system to generate an auxin. The method comprises detecting a response of the at least one regulator gene by detecting a chemical product of the reporter gene (such as a reporter protein, or small molecules made by an enzyme expressed by the reported gene). This detection method provides a sensitive and specific approach for monitoring auxin production in biological systems, enabling researchers to track auxin dynamics and identify auxin-producing organisms or conditions. According to another aspect of the present disclosure, a bacteria cell is provided. The bacteria cell comprises an engineered nucleic acid sequence of any one of the preceding aspects. The bacteria cell is capable of degrading auxins, such as indole-3-acetic acid. The engineered bacteria cell provides a living system capable of actively degrading auxins in its environment, offering a biological solution for controlling auxin levels and promoting plant growth through auxin homeostasis. According to other aspects of the present disclosure, the bacteria cell may include one or more of the following features. The bacteria cell may be a plant-associated bacteria cell. Plant-associated bacteria naturally colonize plant root systems, providing an ideal platform for delivering auxin degradation capabilities directly to the rhizosphere where auxin regulation is most needed for plant growth promotion. The plant-associated bacteria may be a Proteobacteria species, an Actinobacteria species, a Bacteroidetesspecies, a Firmicutes species, or an Acidobacteria species. These bacterial phyla represent major groups commonly found in plant root microbiomes, ensuring compatibility with existing microbial communities and effective colonization of plant root systems. PRINCETON-103476 The plant-associated bacteria may be a Polarimonas species or a Paraburkholderia species. These bacterial genera have demonstrated effectiveness as chassis organisms for genetic engineering and show strong plant colonization capabilities, making them suitable platforms for delivering auxin degradation functions to plant root systems. The bacteria cell may be an amino acid auxotroph. Amino acid auxotrophy creates a dependency on plant-produced amino acids, promoting stronger associations between the engineered bacteria and plants, thereby enhancing the delivery of auxin degradation capabilities to the plant environment. According to another aspect of the present disclosure, a method for degrading auxins, controlling auxin, and promoting plant growth and root development is provided. The method comprises providing a bacteria cell of any one of the preceding aspects. The method comprises allowing the bacteria cell to interact with a plant, such as with the roots of a plant. This method provides a biological approach to auxin regulation that can restore auxin homeostasis in plant root systems, reversing growth inhibition caused by excess auxin and promoting healthy plant development. According to other aspects of the present disclosure, the method may include one or more of the following features. The method may further comprise applying the bacteria cell to soil containing the plant or to seeds prior to planting. Direct application to soil or seeds ensures effective delivery of the engineered bacteria to the plant environment, establishing auxin degradation capabilities early in plant development or throughout the growing period. Allowing the bacteria cell to interact with a plant may promote plant growth by degrading auxins, such as indole-3-acetic acid, produced by auxin-producing microorganisms in the rhizosphere of the plant. This mechanism addresses the root cause of auxin-induced growth inhibition by targeting excess auxin produced by harmful microorganisms, restoring the natural auxin balance needed for optimal plant growth and development. According to another aspect of the present disclosure, a viral vector is provided. The viral vector comprises one or more regulator genes, the regulator genes including an iadR gene. The viral vector comprises at least three catabolic pathway genes downstream from the one or more regulator genes, the at least three catabolic pathway genes including an iadC gene, an iadD gene, and an iadE gene. The viral vector is free of an iadA gene and iadB gene. The viral vector provides an alternative delivery system for introducing auxin degradation capabilities into target organisms, offering flexibility in deployment strategies and potentially enabling transformation of organisms that are difficult to engineer through conventional methods. PRINCETON-103476 The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive. The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive. BRIEF DESCRIPTION OF FIGURES Non-limiting and non-exhaustive examples are described with reference to the following figures. FIG.1 depicts a genomic organization of Hot Spot 33 in V. paradoxus CL014. Gene annotations are shown above as the final two digits of the IMG gene ID (26436136##) and below with assigned gene names. The MarR-family transcriptional regulator marR73 (also referred to as iadR) is shown in hashed lines; the core nine-gene IAA degradation locus is shown in white FIG. 2 illustrates a heatmap of Log2fold changes in metabolite abundance in iad pathway mutants relative to the wild type. Data represent the mean of n = 3 biological replicates. Figs.3A-3H show bar plots of relative abundance of anthranilic acid (3A), IAA (3B), and key metabolic intermediate (3C-3H) across wild-type and mutant strains. Data represent mean ± s.e.m (standard error of the mean) of n = 3 biological replicates. Figs. 3E-3H show bar plots of metabolic intermediate abundance across bacterial strains, according to aspects of the present disclosure. FIG. 4 depicts a schematic representation of the iad-mediated IAA degradation pathway in V. paradoxus CL014. FIGS. 5A-5D illustrate LC-MS analysis of [2H7]IAA catabolism identifying key pathway intermediates and their deuterium labeling profiles. Left, LC–MS peak intensities of major metabolites. Right, deuterium isotope distributions following incubation with unlabeled IAA or [²H₇]IAA. Data represent mean ± s.e.m (standard error of the mean) of n = 3 biological replicates. FIG. 6 shows H₂¹⁸O (20% v / v) tracing confirms the source of oxygen atoms incorporated during the two-step oxidation reactions. Data are shown for two biological replicates. PRINCETON-103476 FIG.7 depicts a revised mechanistic model of IAA degradation consistent with isotope tracing. FIG.8 illustrates a crystal structure of the IadDE complex from V. paradoxus CL014 resolved at 1.28 Å (PDB code: 9O71). FIG.9 shows a heterohexameric IadDE complex is formed by trimerization of IadDE heterodimers. The Rieske-type [2Fe–2S] cluster and mononuclear iron within each IadD subunit are shown in matching colors. Trimer assembly reduces the spatial distance between the electron donor (Rieske cluster) and acceptor (iron center), potentially enhancing catalytic electron transfer efficiency. FIG. 10 depicts in vitro IAA degradation by engineered strains. IAA concentrations were measured from culture supernatants of strains grown in M9 minimal medium supplemented with glucose and 0.1 mg / mL IAA. The Salkowski reagent reacts with IAA to generate a pink-to-red chromophore with an absorption maximum at 530 nm (OD₅₃₀). Data represent the mean of two biological replicates per sample. FIG.11 shows primary root length of Arabidopsis seedlings exposed to 100 nM IAA or the auxin-producing strain Arthrobacter CL028. Letters above boxplots indicate statistically significant differences as determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). Groups not sharing the same letter differ significantly. “NB” indicates the no-bacteria control. Sample sizes (left to right): n = 23, 29, 31, 31, 27, 34, 26, 34, 26, 25, 31, 16, 21, 21, 25. Box plots represent the median (center line), interquartile range (box), and 1.5× interquartile range (whiskers). FIG. 12 illustrates primary root length of Arabidopsis seedlings exposed to a 32- member synthetic community (SynCom32) in Arabidopsis and Medicago seedlings. Letters above boxplots indicate statistically significant differences as determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). Groups not sharing the same letter differ significantly. “NB” indicates the no-bacteria control. Sample sizes (left to right): n = 64, 81, 56, 52, 53, 43, 52, 78, 52, 71, 47, 48. Box plots represent the median (center line), interquartile range (box), and 1.5× interquartile range (whiskers). FIG.13 depicts primary root length of Arabidopsis seedlings exposed to elevated IAA concentrations (1 μM and 10 μM) in Arabidopsis. Letters above boxplots indicate statistically significant differences as determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). Groups not sharing the same letter differ significantly. “NB” indicates the no-bacteria PRINCETON-103476 control. Sample sizes (left to right): n = 19, 15, 18, 14, 9, 19, 17, 19, 19, 14. Box plots represent the median (center line), interquartile range (box), and 1.5× interquartile range (whiskers). FIG.14 shows engineered Polaromonas MF047 strains cultured in 50% TSB medium and ParaburkholderiaMF376 strains cultured in M9 medium efficiently degraded IAA. IAA levels were quantified from culture supernatants using the Salkowski reagent, which forms a pink-to-red chromophore upon reaction with IAA and is measured at OD₅₃₀. Data represent the mean of two biological replicates per sample. FIGS. 15A-15B illustrate engineered chromosomal knock-in strains reversed RGI, as indicated by enhanced primary root elongation in Arabidopsis seedlings treated with 100 nM IAA or the auxin-producing strain Arthrobacter CL028. Letters above boxplots indicate statistically significant differences as determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). Groups not sharing the same letter differ significantly. “NB” denotes the no- bacteria control. Sample sizes (left to right): n = 23, 42, 37, 31, 34, 26, 30, 27, 31, 26, 25, 51, 42, 44, 33, 39, 52, 49, 42, 26, 28, 35, 31, 21, 15, 21, 21, 23, 21, 17, 22, 22, 29. Box plots show the median (horizontal line), interquartile range (boxes), and whiskers extending to 1.5× the interquartile range. FIG. 16 shows engineered chromosomal knock-in strains reversed RGI, as indicated by enhanced primary root elongation in Arabidopsis and Medicago seedlings exposed to a 32- member synthetic bacterial community (SynCom32). Letters above boxplots indicate statistically significant differences as determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). Groups not sharing the same letter differ significantly. “NB” denotes the no- bacteria control. Sample sizes (left to right): n = 64, 81, 45, 41, 33, 28, 61, 36, 17, 56, 75, 89, 52, 78, 76, 58, 67, 74, 57, 68. Box plots show the median (horizontal line), interquartile range (boxes), and whiskers extending to 1.5× the interquartile range. FIG. 17 depicts a heatmap showing average primary root lengths of Arabidopsis seedlings inoculated with 23 previously identified RGI-inducing strains, either alone (self) or co-inoculated with V. paradoxus CL014 wild type or the iad-deficient mutant ΔHS33, to assess iad-dependent reversion. FIG.18 illustrates primary root length of Arabidopsis seedlings co-inoculated with iad- independent RGI-inducing strains and treated with V. paradoxus CL014 wild type, ΔHS33, Δacc (ACC deaminase gene deleted), or ΔHS33 Δacc (both iad and ACC deaminase genes deleted). Letters above boxplots indicate statistically significant differences as determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). Groups not sharing the same letter PRINCETON-103476 differ significantly. “NB” denotes the no-bacteria control. Sample sizes (left to right): n = 40, 56, 48, 41, 48, 49, 14, 17, 25, 25, 21, 16, 28, 17, 15, 14, 15, 15. Box plots show the median (center line), interquartile range (boxes), and whiskers extending to 1.5× the interquartile range. FIG. 19 shows primary root lengths of Arabidopsis seedlings inoculated with Pseudomonas MF048 alone or with Arthrobacter CL028, and co-inoculated with V. paradoxus CL014, or iad-engineered strains of Polaromonas MF047 or Paraburkholderia MF376. Letters above boxplots indicate statistically significant differences as determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). Groups not sharing the same letter differ significantly. “NB” denotes the no-bacteria control. Sample sizes (left to right): n = 42, 41, 41, 17, 25, 17, 29, 27, 29, 25, 26, 28, 22, 19, 30, 21, 30, 24, 25. Box plots show the median (center line), FIG.20 depicts primary root length of Arabidopsis seedlings treated with V. paradoxus CL014 at OD₆₀₀ = 2. Statistical significance was assessed using Welch’s two-tailed t-test (n = 19, 16). FIGS. 21A-21B illustrate relative abundance of bacterial genera in the root microbiomes of Arabidopsis and Medicagoseedlings treated with SynCom32 in combination with wild-type or iad-engineered strains of V. paradoxus CL014, Polaromonas MF047, and Paraburkholderia MF376. Sample sizes are indicated above each bar. FIG. 22 shows unconstrained principal coordinate analysis (PCoA) of Bray–Curtis dissimilarity showing root microbiome profiles in Arabidopsis and Medicago seedlings treated with SynCom32 alone or with engineered Paraburkholderia MF376 strains. Ellipses represent 68% confidence intervals. Statistical significance was determined by PERMANOVA (Adonis2). FIG. 23 depicts fresh weight of 33-day-old Arabidopsis plants grown in untreated natural soil inoculated individually with Paraburkholderia MF376 wild type and mar73 iadC- JK2 knock-in strains. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (n = 36, 32, 31). FIG. 24 is a graph showing GFP fluorescent reporter: monitoring ligand-regulator interactions in Variovorax CL014 in vivo, using a knock on a reporter between iadE and iadF. FIGS. 25A-25D are graphs showing that IAA induces GFP signal in Polaromonas MF047–related knock-in strains. FIGS. 26A and 26B are graphs showing that IAA induces GFP signal in Paraburkholderia MF376–related knock-in strains. PRINCETON-103476 FIGS.27A-27C are graphs showing growth curves of engineered V. paradoxus CL014 (27A) Paraburkholderia MF376 (27B), and Polaromonas MF047 (27C) in 50% TSB at 28 °C. OD₆₀₀ measured over time; data are mean ± s.d. (n = 3). FIG.28 is a schematic showing genomic insertion sites of iad constructs in MF047 (3 loci) and MF376 (1 locus), based on IMG annotations. Stars in the center mark insertion sites. FIG. 29 is a graph showing amino acid auxotrophy of MF047 assessed by growth in M9 + glucose + single amino acids (0.1 or 5 mM). Data are mean ± s.d. (n = 2). FIGS. 30A and 30B show nonconstrained principal coordinate analysis (PCoA) of Bray–Curtis dissimilarity showing root microbiome composition in Arabidopsis and Medicago seedlings treated with SynCom32 alone or co-inoculated with engineered strains of V. paradoxus CL014 (30A) and Polaromonas MF047 (30B). Ellipses indicate 68% confidence intervals for each treatment group. Statistical significance was evaluated using PERMANOVA (Adonis2). FIG.31 shows shoot fresh weight of Arabidopsis plants grown for 33 days in natural soil treated with wild-type or engineered strains of V. paradoxus CL014 and Polaromonas MF047. Statistical significance was assessed by one-way ANOVA, with different letters indicating statistically distinct groups based on Tukey’s post hoc test. Sample sizes: n = 36, 34, 35, 32, 37, 35. Data were obtained from two independent experiments. DETAILED DESCRIPTION The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein. Plant roots develop within complex microbial environments where interactions with soil and rhizosphere microorganisms substantially influence root architecture and function. A central regulator of root development may be indole-3-acetic acid (IAA), which represents the predominant form of auxin in plants. While plants may maintain auxin homeostasis through biosynthesis, transport, conjugation, and degradation processes, many rhizosphere bacteria also produce IAA, which may disrupt this balance and lead to root growth inhibition. Genomic surveys indicate that over 80% of soil- and plant-associated bacterial genomes encode complete or partial IAA biosynthetic pathways, highlighting the widespread microbial influence on plant hormone dynamics. When rhizosphere bacteria overproduce IAA, PRINCETON-103476 the resulting auxin imbalance may cause detrimental effects on plant growth and development. This microbial auxin overproduction represents a significant challenge for maintaining proper plant-microbe interactions in agricultural and natural ecosystems. Certain beneficial microorganisms may counteract these negative effects by degrading excess IAA through specialized catabolic pathways. The indole-3-acetic acid degradation (iad) pathway represents one such natural mechanism for maintaining auxin homeostasis in the rhizosphere. This pathway may convert IAA through a series of enzymatic reactions, ultimately producing metabolites that do not interfere with plant auxin signaling. The iad pathway may be regulated by transcriptional regulators and may involve multiple enzymatic steps that systematically break down IAA molecules. The pathway may initiate with oxidative modifications to the indole ring structure, followed by sequential processing of intermediates through various enzymatic reactions. These reactions may ultimately convert IAA to less bioactive compounds, thereby restoring auxin balance in the plant-microbe environment. The iad genes demonstrate conservation across all available genomes of Variovorax species, suggesting that these genetic elements may be well-accepted by microbial communities and soil environments. This conservation may indicate the ecological importance of auxin degradation capabilities in maintaining balanced plant-microbe interactions. The widespread presence of these genes across related bacterial species may also suggest compatibility with various microbial community structures. Beyond agricultural applications, the iad pathway may have utility in chemical remediation applications. The enzymatic machinery involved in IAA degradation may also process other aromatic compounds with structures similar to auxins. Such capabilities may extend to wastewater treatment applications where degradation of aromatic pollutants may be beneficial. The broad substrate specificity of certain iad pathway enzymes may enable processing of various aromatic molecules found in industrial waste streams or environmental contamination scenarios. The natural occurrence and conservation of iad pathway genes across bacterial species may provide a foundation for developing engineered systems that maintain auxin homeostasis in various applications. Understanding the biochemical mechanisms underlying IAA degradation may enable the development of biotechnological approaches for managing plant- microbe interactions and addressing environmental remediation challenges involving aromatic compound degradation. PRINCETON-103476 Engineered nucleic acid sequence Referring to FIG.1, an engineered nucleic acid sequence may comprise a gene cassette that includes one or more regulator genes and at least three catabolic pathway genes. The gene cassette may be designed to enable auxin degradation through a controlled expression system. The one or more regulator genes may include an iadR gene, which may function as a transcriptional regulator. In some cases, the one or more regulator genes consist of the iadR gene. The sequence may include a non-coding region that a regulator gene (such as iadR) can bind to. In some cases, the entire region ahead of the iadA gene, as shown in FIG. 1, may be utilized. In some implementation, only promoter / regulator region is utilized. In some implementations, the non-coding region may comprise or consist of the promoter region. In some implementations, only the portions of the promoter / regulator region where IadR can bind to is utilized. The engineered nucleic acid sequence may include a regulatory region upstream of the iadR gene that controls expression of the iad genes in response to auxin compounds. The regulatory region may contain binding sites for transcriptional regulators that respond to the presence of indole-3-acetic acid and related auxin molecules. In some cases, the MarR73 regulator serves as the primary repressor controlling expression of the iad locus genes, providing auxin-responsive regulation of the pathway. The at least three catabolic pathway genes may be positioned downstream from the one or more regulator genes and may include an iadC gene, an iadD gene, and an iadE gene. These three genes may form a functional unit for auxin degradation. The iadC gene may encode a reductase component, while the iadD and iadE genes may encode the large and small subunits of a Rieske non-heme dioxygenase complex. In some cases, the at least three catabolic pathway genes consist of the iadC gene, the iadD gene, and the iadE gene, forming a minimal functional pathway. As shown in FIG.1, the iad locus in Variovorax paradoxus CL014 may consist of 25 genes designated as Hot Spot 33 (HS33). The engineered nucleic acid sequence may be free of an iadA gene and iadB gene, which are not present in the native iad pathway and are not required for auxin degradation functionality. In some cases, the at least three catabolic pathway genes may include the iadC gene, the iadD gene, and the iadE gene, and at least one additional catabolic pathway gene. The at least one additional catabolic pathway gene may include genes such as iadF, iadG, iadH, iadI, PRINCETON-103476 and iadJ. which may participate in downstream steps of the auxin degradation pathway. These additional genes may enable complete conversion of auxin to anthranilic acid through sequential enzymatic reactions. The sequence may also include a transporter component, such as the iadK2 gene. Referring to FIG.4, the engineered nucleic acid sequence may enable a complete auxin degradation pathway that converts indole-3-acetic acid through various intermediates to produce anthranilic acid as the final product. The gene cassette may be configured to provide either a minimal pathway using the iadCDE genes or a complete pathway incorporating additional downstream genes for full auxin catabolism. In some cases, the at least one additional catabolic pathway gene may include an iadF gene. The iadF gene may encode an acyl-CoA synthase that functions as an AMP-forming enzyme. The acyl-CoA synthase encoded by the iadFgene may activate the carboxylic group in the acetyl side chain of auxin degradation intermediates, enabling subsequent processing steps in the catabolic pathway. The at least one additional catabolic pathway gene may include an iadK2 gene. The iadK2 gene may encode an ATP-binding cassette (ABC) transporter solute-binding protein that facilitates IAA uptake. The ABC transporter protein encoded by the iadK2 gene may enhance the transport of indole-3-acetic acid into bacterial cells, though the protein may not be required for auxin degradation functionality. In some cases, the at least three catabolic pathway genes may consist of the iadC gene, the iadD gene, the iadEgene, an iadF gene, an iadG gene, an iadH gene, an iadI gene, an iadJ gene, and the iadK2 gene. This complete gene set may enable full auxin catabolism through sequential enzymatic reactions that convert indole-3-acetic acid to anthranilic acid. The iadG gene may encode an acetyl-CoA acetyltransferase, also known as a ketothiolase. The acetyl-CoA acetyltransferase encoded by the iadG gene may function in conjunction with the acyl-CoA synthase to process CoA-bound intermediates during auxin degradation. The iadH gene may encode an alcohol dehydrogenase that may facilitate further processing of pathway intermediates. The iadF, iadG, and iadH genes may work together to mediate reductive removal of the acetyl side chain from auxin degradation intermediates. These enzymes may process intermediates through a chain of reactions involving CoA-bound compounds, resulting in the removal of a C2H2O2group from the molecular structure. PRINCETON-103476 The iadI gene may encode a kynurenine formamidase that may convert pathway intermediates in later stages of auxin catabolism. The kynurenine formamidase encoded by the iadI gene may process intermediates with molecular formula C8H7NO2to produce compounds with molecular formula C7H7NO. The iadJ gene may encode an enzyme that catalyzes a second oxidation step in the auxin degradation pathway. The enzyme encoded by the iadJ gene may function via a dehydrogenase mechanism and may incorporate oxygen from water during the oxidation reaction. The dehydrogenase activity of the iadJ enzyme may convert pathway intermediates with molecular formula C10H9NO3 to products with molecular formula C10H9NO4. The complete gene cassette containing all nine genes may provide enhanced auxin degradation capability compared to minimal pathway variants. The additional genes beyond iadCDE may enable more efficient processing of auxin compounds and complete conversion to anthranilic acid under various environmental conditions. A reporter gene may be located after the iadE gene. This may be, e.g,. between the iadE gene and the at least one additional catabolic pathway gene. The reporter gene may enable monitoring of gene expression and auxin-responsive activity within the engineered nucleic acid sequence. In some cases, the reporter gene may be positioned to provide real-time feedback on the transcriptional activity of the iad locus genes. The reporter gene may be located between the iadE gene and an iadF gene. This positioning may allow the reporter gene to be co-expressed with the catabolic pathway genes while maintaining the functional organization of the gene cassette. The placement between iadE and iadF may provide a convenient insertion site that does not disrupt the enzymatic functions of the pathway genes. The reporter gene may be a gene for a fluorescent protein. The fluorescent protein may enable visual detection and quantitative measurement of gene expression levels. In some cases, the reporter gene may be enhanced green fluorescent protein (eGFP) that enables fluorescence- based monitoring of gene expression. The eGFP reporter may produce detectable fluorescence signals that correlate with the expression levels of the iad pathway genes. The fluorescent protein reporter may include variants such as green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or red fluorescent protein (RFP). These fluorescent protein variants may provide different spectral properties for detection and may be selected based on the specific monitoring requirements of the application. PRINCETON-103476 The reporter gene may enable real-time monitoring of gene expression and auxin- responsive activity. The fluorescent output from the reporter protein may provide immediate feedback on the transcriptional state of the iad locus in response to auxin compounds. The real- time monitoring capability may allow for dynamic assessment of pathway activity under varying environmental conditions. The engineered strains containing the reporter gene may exhibit fluorescence in response to various auxin compounds. The auxin compounds may include indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and 1-naphthaleneacetic acid. The fluorescent response to these different auxin compounds may demonstrate the specificity and sensitivity of the reporter system for detecting auxin-related molecules. The fluorescent response may be dose-dependent, with increasing auxin concentrations producing proportionally higher fluorescence signals. This dose-response relationship may enable quantitative measurement of auxin levels in the environment surrounding the engineered bacterial strains. The sensitivity of the fluorescent reporter may allow detection of auxin concentrations at physiologically relevant levels. A knock-in of gfp may be inserted between iadE and iadF genes in Variovorax paradoxus CL014 to monitor locus expression. This genetic modification may create a reporter strain that produces fluorescent signals proportional to the activity of the iad pathway. The gfp insertion may be designed to maintain the reading frame and preserve the functionality of the downstream genes. The reporter gene integration may provide a straightforward method for monitoring interactions between ligands and regulators. The fluorescent measurement system may offer real-time detection capabilities that can be used to assess the response of the iadR regulator to various auxin compounds. The system may demonstrate high specificity for auxin-related molecules and may provide sensitive detection of regulatory interactions. The fluorescent reporter system may be extended to other ligand-mediated regulatory mechanisms beyond auxin degradation. The reporter gene approach may be applicable to monitoring various bacterial regulatory systems that respond to environmental signals or metabolic intermediates. The versatility of fluorescent protein reporters may enable adaptation to different regulatory contexts and detection requirements. FIGS.24-26B illustrate fluorescent protein reporter (GFP) and signal induction. PRINCETON-103476 Bacteria Cell A bacteria cell may comprise an engineered nucleic acid sequence as described above, wherein the bacteria cell may be capable of degrading auxins, such as indole-3-acetic acid. The bacteria cell may incorporate the gene cassette containing the regulator genes and catabolic pathway genes into the bacterial chromosome or may maintain the engineered nucleic acid sequence on a plasmid vector. The integration of the engineered nucleic acid sequence may confer auxin degradation capabilities to bacterial strains that do not naturally possess such functionality. The bacteria cells may have been modified to remove iad genes outside those disclosed herein, or may have all relevant genes deleted (e.g., all genes from FIG.1, all iad genes, etc.), and then the bacteria cells may genes added back in via specific gene cassettes. For example, any bacteria could be modified to remove all genes shown in FIG.1, and then add back in iadR, iadC, iadD, iadE, etc. The bacteria cell may be a plant-associated bacteria cell that naturally colonizes plant root environments. Plant-associated bacteria cells may establish beneficial relationships with plants through various mechanisms including nutrient exchange, growth promotion, and protection against pathogens. The plant-associated bacteria cells may be particularly suitable for applications involving plant-microbe interactions due to their natural ability to colonize and persist in the rhizosphere environment. The plant-associated bacteria cell may be any appropriate plant-associated bacteria cell, and may be, e.g., a free-living rhizobacteria (such as Pseudomonas, Bacillus, Azospirillum, Arthrobacter, or Streptomyces), a symbiotic nitrogen-fixer (such as Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium, or Frankia), an endophytic bacteria (such as Burkholderia, Enterobacter, Klebsiella, or Serratia), or a plant growth-promoting rhizobacteria (PGPR) (such as Paenibacillus, Micrococcus, Variovorax, or Acinetobacter). The plant-associated bacteria may be selected from various bacterial phyla that commonly inhabit plant root environments. The plant-associated bacteria may be a Proteobacteria species, which represents one of the most abundant bacterial phyla in soil and rhizosphere environments. Proteobacteria species may include genera such as Pseudomonas, Rhizobium, Agrobacterium, and Burkholderia, which are commonly found in association with plant roots. The plant-associated bacteria may be an Actinobacteria species, which may include genera such as Streptomyces, Arthrobacter, and Micrococcus. Actinobacteria species may be PRINCETON-103476 particularly abundant in soil environments and may contribute to nutrient cycling and plant growth promotion through various metabolic activities. The plant-associated bacteria may be a Bacteroidetes species, which may include genera that are commonly found in soil and rhizosphere environments. Bacteroidetes species may contribute to organic matter decomposition and may establish beneficial associations with plant roots through various mechanisms. The plant-associated bacteria may be a Firmicutes species, which may include genera such as Bacillus and Paenibacillus. Firmicutes species may be particularly resilient due to their ability to form spores and may provide plant growth promotion benefits through various metabolic pathways. The plant-associated bacteria may be an Acidobacteria species, which may be abundant in acidic soil environments. Acidobacteria species may contribute to soil ecosystem functioning and may establish associations with plant roots in various environmental conditions. The plant-associated bacteria may be a Polarimonas species or a Paraburkholderia species. These bacterial genera may serve as suitable chassis organisms for the insertion of the engineered nucleic acid sequence due to their natural root-association capabilities and genetic tractability. Polarimonas species may be amino acid auxotrophs that exhibit poor growth in the absence of specific amino acids. The Polarimonas MF047 strain may represent an example of an amino acid auxotroph that requires external amino acid sources for optimal growth. The amino acid auxotrophy of Polarimonas species may promote stronger associations with plants that produce the necessary amino acids, thereby enhancing the delivery of engineered gene products to these plants. Paraburkholderia species may demonstrate superior colonization characteristics compared to other bacterial genera. The engineered Paraburkholderia MF376 strain may exhibit superior root colonization capabilities, achieving relative abundance levels of 50.84% in root microbiome communities. This colonization efficiency may be substantially higher than that observed for other bacterial genera, such as Variovorax species which may achieve relative abundance levels of 3.40%, or Polarimonas species which may achieve relative abundance levels of 0.01%. The superior colonization characteristics of Paraburkholderia species may make these bacteria particularly suitable for applications requiring robust plant-microbe interactions. The PRINCETON-103476 high relative abundance achieved by Paraburkholderia strains in root environments may indicate strong competitive abilities and effective colonization strategies. These characteristics may enhance the effectiveness of engineered Paraburkholderia strains in delivering auxin degradation capabilities to plant root environments. The colonization efficiency differences between bacterial genera may influence the selection of chassis organisms for engineering applications. Paraburkholderia species may be preferred for applications requiring high colonization levels and strong plant-microbe interactions, while Polarimonas species may be suitable for applications where amino acid auxotrophy provides beneficial plant association characteristics. The bacteria cell may be an amino acid auxotroph that requires external sources of specific amino acids for growth. Amino acid auxotrophy may represent a metabolic dependency that can influence bacterial colonization patterns and plant-microbe interactions. The amino acid auxotroph bacteria may exhibit poor growth in the absence of specific amino acids in the growth medium, which may promote stronger associations with plants that produce the necessary amino acids. See FIG.29. The amino acid auxotrophy may enhance the delivery of engineered gene products to plants by promoting closer associations between the bacteria and plant roots. Plants may produce amino acids through root exudation, creating a nutritional environment that supports the growth of amino acid auxotroph bacteria. This nutritional dependency may result in more stable colonization patterns and enhanced persistence of the engineered bacteria in the rhizosphere. The bacteria cells comprising the engineered nucleic acid sequence may maintain stable expression of the auxin degradation genes through chromosomal integration. Chromosomal integration may provide more stable gene expression compared to plasmid- based systems and may reduce the risk of gene loss during bacterial replication. The chromosomal integration approach may enable the development of engineered bacterial strains suitable for long-term applications in plant-microbe interaction systems. Referring to FIG. 27A-27C, growth curves of various strains can be seen. It is clear there are no growth defects in the engineered strains. Engineering Methods and Insertion Sites Genomic integration of engineered nucleic acid sequences may be performed at multiple intergenic sites in bacterial chromosomes. In Polarimonas MF047 (IMG genome ID PRINCETON-103476 2636416056), three distinct intergenic regions may serve as insertion sites, located between Gene IDs 2639079279-80, 2639079819-20, and 2639080354-55. In Paraburkholderia MF376 (IMG genome ID 2521172625), an intergenic region between Gene IDs 2521671121-22 may serve as an insertion site. These intergenic regions may range from 400 to 900 base pairs in length, which may minimize disruption of native gene expression patterns. The engineered nucleic acid sequences may be introduced into bacterial cells through conjugation methods. Biparental mating may involve direct transfer between a donor strain and the recipient bacteria. Tri-parental mating may utilize an additional helper strain carrying the pRK2013 plasmid to facilitate conjugal transfer. The mating procedures may be conducted on growth medium containing appropriate selective antibiotics to isolate successful conjugants. Electroporation methods may provide an alternative approach for introducing engineered nucleic acid sequences into bacterial cells. For introducing the marR73 iadCDE construct, electroporation parameters may include 1,800 V, 25 μF capacitance, and 200 Ω resistance. The marR73 iadC-K2 construct may require modified parameters of 2,500 V, 25 μF capacitance, and 200 Ω resistance for efficient transformation. Construction of engineered strains may utilize the suicide vector pMo130 for chromosomal integration through homologous recombination. The pMo130 vector may carry sequences homologous to the target integration sites, enabling precise insertion of engineered nucleic acid sequences into bacterial chromosomes. Selection of integrants may involve antibiotic resistance markers and counter-selection with sucrose. The broad-host-range vector pBBR1MCS-2 may enable plasmid-based expression of engineered nucleic acid sequences. This vector may replicate in diverse bacterial species and may maintain stable copy numbers under selective conditions. The pBBR1MCS-2 vector may carry appropriate antibiotic resistance markers for selection of transformants. Bacterial culture conditions may include growth at 28°C with orbital shaking at 250 revolutions per minute. Growth media may include 50% strength tryptic soy broth (TSB) or M9 minimal medium supplemented with glucose. The M9 minimal medium may contain additional nutrients such as magnesium sulfate, calcium chloride, and iron sulfate to support bacterial growth. Growth curves may demonstrate that engineered strains maintain similar growth characteristics compared to wild-type bacteria under standard culture conditions. The engineered strains may exhibit stable expression of inserted genes without measurable impairment to bacterial fitness. Application of engineered strains at optical densities up to 2.0 PRINCETON-103476 at 600 nanometers wavelength may not adversely affect plant growth, indicating compatibility with high-density bacterial applications. While the gene cassettes may be placed anywhere appropriate, FIG.28 shows examples of various insertion sites. Method for Detecting Auxin Generation A method of detecting auxin generation may provide a rapid and sensitive approach for monitoring auxin production in various biological systems. The method may utilize an engineered nucleic acid sequence containing a reporter gene to enable real-time detection of auxin compounds through fluorescence-based measurements. The method may comprise introducing an engineered nucleic acid sequence to an auxin-generating system. The engineered nucleic acid sequence may include one or more regulator genes and at least three catabolic pathway genes, with a reporter gene positioned between the iadE gene and at least one additional catabolic pathway gene. The auxin- generating system may comprise bacterial cultures, plant tissues, or synthetic biological systems capable of producing indole-3-acetic acid or related auxin compounds. The engineered nucleic acid sequence may be introduced to the auxin-generating system through various transformation methods. In bacterial systems, the nucleic acid sequence may be introduced through electroporation, chemical transformation, or conjugation techniques. The transformation process may result in stable integration of the engineered sequence into the host organism's genome or maintenance as an episomal element. The method may further comprise allowing the auxin-generating system to generate an auxin. The auxin generation may occur through natural biosynthetic pathways present in the system or through induced expression of auxin biosynthetic genes. The auxin compounds produced may include indole-3-acetic acid, indole-3-butyric acid, or other naturally occurring auxin molecules. The auxin generation process may be monitored over time to assess the dynamics of auxin production. The temporal aspects of auxin generation may vary depending on the specific biological system and environmental conditions. In some cases, auxin production may occur constitutively, while in other systems, auxin generation may be induced by specific stimuli or growth conditions. The method may comprise detecting a response of the at least one regulator gene by detecting a chemical product of the reporter gene. This may include detecting a reporter protein PRINCETON-103476 expressed by the reporter gene. This may include having the reporter gene express an enzyme, and detecting small molecules formed by the enzyme. The regulator gene response may be mediated through the iadR gene, which may function as a transcriptional regulator that responds to the presence of auxin compounds. The binding of auxin molecules to the regulator protein may result in conformational changes that affect transcriptional activity. The reporter protein detection may be accomplished through fluorescence measurements when the reporter gene encodes a fluorescent protein. The fluorescent protein may produce detectable signals that correlate with the expression levels of the regulator gene and the presence of auxin compounds in the system. The fluorescence intensity may be proportional to the concentration of auxin molecules present in the environment. The detection process may utilize standard fluorescence detection equipment, including fluorescence microscopes, plate readers, or flow cytometry systems. The fluorescence measurements may be performed at specific wavelengths corresponding to the excitation and emission spectra of the reporter protein. The detection parameters may be optimized to maximize signal-to-noise ratios and detection sensitivity. The method may enable rapid detection of auxin generation without requiring complex sample preparation or chemical extraction procedures. The real-time nature of the fluorescence-based detection may allow for continuous monitoring of auxin production dynamics. The non-invasive detection approach may preserve the integrity of the auxin- generating system during measurement. The fluorescence measurements may enable detection of auxin levels with high specificity for auxin-related compounds. The specificity may be conferred by the regulatory properties of the iadR gene, which may respond selectively to indole-3-acetic acid and structurally related auxin molecules. The regulatory system may discriminate between auxin compounds and other aromatic molecules that do not function as plant hormones. The method may demonstrate sensitivity across various auxin compounds, including natural and synthetic auxin analogs. The detection system may respond to indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, and other auxin compounds with varying degrees of sensitivity. The broad responsiveness to different auxin molecules may enhance the utility of the detection method for diverse applications. The sensitivity of the detection method may enable measurement of auxin concentrations at physiologically relevant levels. The fluorescence response may be detectable at auxin concentrations in the nanomolar to micromolar range, which may correspond to PRINCETON-103476 biologically active levels in plant and microbial systems. The high sensitivity may allow detection of auxin production in systems with low baseline auxin levels. The method may provide quantitative measurements of auxin generation through dose- response relationships between auxin concentration and fluorescence intensity. The quantitative nature of the detection may enable comparative studies of auxin production across different systems or experimental conditions. Calibration curves may be established using known auxin concentrations to enable accurate quantification of auxin levels in unknown samples. The detection method may be applicable to monitoring auxin production in complex biological systems, including plant-microbe interactions and synthetic microbial communities. The method may enable assessment of auxin dynamics in rhizosphere environments where multiple organisms may contribute to auxin production and degradation. The real-time monitoring capability may provide insights into the temporal patterns of auxin generation in these complex systems. The method may be used to screen for auxin-producing organisms or to evaluate the effectiveness of genetic modifications designed to alter auxin biosynthesis. The rapid detection capability may facilitate high-throughput screening applications where large numbers of samples require auxin level assessment. The method may also be used to monitor the stability of auxin production in engineered biological systems over time. Method For Degrading Auxin A method for degrading auxin, controlling auxin, and promoting plant growth and root development may comprise providing a bacteria cell comprising an engineered nucleic acid sequence and allowing the bacteria cell to interact with a plant, preferably including interacting with the roots of a plant. The bacteria cell may be any of the engineered bacteria cells described herein, including plant-associated bacteria cells such as Proteobacteria species, Actinobacteria species, Bacteroidetes species, Firmicutes species, or Acidobacteria species. In some cases, the bacteria cell may be an amino acid auxotroph, such as a Polarimonas species or a Paraburkholderia species. The method may further comprise applying the bacteria cell to soil containing the plant or to seeds prior to planting. In some cases, bacteria cells may be applied to natural soil at an threshold optical density at 600 nm (OD₆₀₀), such as an OD600 of 0.01, 0.02, 0.03, 0.04, or 0.5, in water for soil inoculation (such as 1-2 L of sterile water). The bacteria cells may be PRINCETON-103476 suspended in sterile water and distributed evenly throughout the soil to establish contact with plant root systems. Seeds may be treated with the bacteria cells prior to planting to establish early colonization of the developing root system. The bacteria cells may interact with plant roots through colonization of the rhizosphere, the soil region immediately surrounding plant roots. In some cases, allowing the bacteria cell to interact with roots of a plant promotes plant growth by degrading indole-3-acetic acid produced by auxin-producing microorganisms in the rhizosphere of the plant. The engineered bacteria cells may express the iadR regulator gene and catabolic pathway genes including iadC, iadD, and iadE genes, which enable degradation of excess indole-3-acetic acid that would otherwise cause root growth inhibition. The engineered strains may be tested in gnotobiotic systems using an appropriate medium under short-day conditions. For example, half-strength Murashige and Skoog (MS) agar medium may be used. The testing conditions may include temperatures of 15 °C - 30 °C, that may vary between day and night conditions. In some implementations, the temperatures may vary by 2 °C - 5 °C between day and night conditions. In some implementations, the day temperature may be 20 °C- 23°C, and the night temperature may be 16 °C - 19 °C. The photoperiods may vary as desired. In some embodiments, the photoperiod may have each 24 hour period consisting of 8-16 hours of light, with the remainder of time in the dark. The relative humdity may vary as desired, and may be, e.g., 40% - 80% relative humidity. Light intensity may also vary as desired. In some implementations, the light intensity may have a value of 100 μmol m⁻² s⁻¹ to 300 μmol m⁻² s⁻¹. These controlled conditions allow for assessment of the bacteria cells' ability to promote plant growth and root development in the absence of competing microorganisms. The method may result in enhanced primary root elongation and increased shoot biomass in treated plants compared to untreated controls. The bacteria cells may counteract root growth inhibition caused by auxin-producing bacteria in complex microbial communities, thereby restoring auxin homeostasis in the plant rhizosphere. The degradation of excess indole- 3-acetic acid by the engineered bacteria cells may allow plants to maintain normal root architecture and growth patterns even in the presence of auxin-overproducing microorganisms. Viral Vector A viral vector may comprise one or more regulator genes and at least three catabolic pathway genes to provide an alternative delivery system for introducing auxin degradation PRINCETON-103476 capabilities into target organisms. The viral vector may offer flexibility in deployment strategies compared to bacterial transformation methods and may enable introduction of auxin degradation functionality into organisms that are difficult to transform through conventional approaches. The viral vector may include one or more regulator genes, wherein the regulator genes include an iadR gene. The iadR gene may function as a transcriptional regulator that responds to the presence of auxin compounds and controls expression of downstream catabolic pathway genes. In some cases, the one or more regulator genes may consist of the iadR gene, providing a streamlined regulatory system for auxin-responsive gene expression. The viral vector may contain regulatory sequences upstream of the iadR gene that enable appropriate expression levels in target host organisms. The regulatory sequences may include promoter elements, ribosome binding sites, and transcriptional control regions that function across diverse host species. The regulatory elements may be selected to provide constitutive or inducible expression patterns depending on the intended application requirements. The viral vector may comprise at least three catabolic pathway genes positioned downstream from the one or more regulator genes. The at least three catabolic pathway genes may include an iadC gene, an iadD gene, and an iadEgene, which together may form a functional unit for auxin degradation. These three genes may encode the minimal enzymatic machinery required for initiating auxin catabolism through oxidative modification of the indole ring structure. The iadC gene in the viral vector may encode a reductase component that transfers electrons from NADH to support the oxidative reactions catalyzed by the IadDE complex. The iadD and iadE genes may encode the large and small subunits of a Rieske non-heme dioxygenase complex that functions as a monooxygenase to initiate auxin degradation. The coordinated expression of these three genes from the viral vector may enable target organisms to acquire auxin degradation capabilities. The viral vector may be free of an iadA gene and iadB gene, which are not present in the native iad pathway and are not required for auxin degradation functionality. The absence of these genes may reduce the size of the viral vector construct and may eliminate potential complications from non-functional genetic elements. The streamlined gene content may enhance the efficiency of viral packaging and delivery processes. PRINCETON-103476 Viral vectors may provide advantages for introducing auxin degradation capabilities into organisms that are recalcitrant to transformation through bacterial conjugation or electroporation methods. The viral delivery system may enable transduction of the engineered nucleic acid sequence into target organisms with higher efficiency than conventional transformation approaches. The viral vector system may be particularly useful for introducing auxin degradation genes into organisms that lack natural competence for DNA uptake. The viral vector may be derived from bacteriophages that have broad host ranges, enabling delivery of the auxin degradation genes to diverse bacterial species. The host range characteristics of the viral vector may determine which target organisms can be successfully transduced with the engineered nucleic acid sequence. Selection of appropriate viral vector systems may enable targeting of specific bacterial genera or species within complex microbial communities. The viral vector may incorporate packaging signals and structural elements required for viral replication and assembly. The packaging signals may ensure efficient incorporation of the engineered nucleic acid sequence into viral particles during production. The structural elements may include genes encoding viral coat proteins, replication machinery, and assembly factors that enable formation of infectious viral particles. The viral vector may be designed as a replication-defective system that delivers the auxin degradation genes without enabling viral propagation in target organisms. The replication-defective design may enhance biosafety by preventing uncontrolled viral spread while maintaining the ability to deliver and express the engineered genetic elements. The delivered genes may integrate into the host chromosome or may be maintained as episomal elements depending on the specific viral vector design. The viral vector system may enable temporal control over the introduction of auxin degradation capabilities into target microbial communities. The timing of viral vector application may be coordinated with plant growth stages or environmental conditions to optimize the effectiveness of auxin degradation interventions. The viral delivery approach may allow for precise targeting of specific time points when auxin balance restoration would be most beneficial for plant growth. The viral vector may carry additional genetic elements beyond the core auxin degradation genes to enhance functionality or enable monitoring of transduction events. Selection markers may facilitate identification of successfully transduced organisms, while reporter genes may enable tracking of gene expression levels in target hosts. The additional PRINCETON-103476 genetic elements may be designed to minimize interference with the auxin degradation pathway while providing useful experimental capabilities. The flexibility of viral vector deployment may enable application strategies that are not feasible with bacterial inoculation approaches. The viral vectors may be applied to established microbial communities to introduce auxin degradation capabilities into existing bacterial populations. This approach may avoid the need to establish new bacterial colonization while still providing auxin balance restoration benefits. The viral vector system may be particularly suitable for applications in complex soil environments where direct bacterial inoculation may face competition from established microbial communities. The viral delivery mechanism may enable introduction of auxin degradation genes into indigenous bacterial populations that are already adapted to local environmental conditions. The transduced indigenous bacteria may have superior survival and persistence characteristics compared to introduced bacterial strains. The viral vector approach may enable delivery of auxin degradation capabilities to bacterial species that naturally colonize specific plant hosts or soil niches. The targeted delivery to well-adapted bacterial populations may result in more stable and effective auxin degradation activity compared to approaches that rely on establishing new bacterial colonization patterns. The viral vector system may thus provide a complementary strategy to direct bacterial inoculation for managing auxin homeostasis in plant-microbe systems. The engineered nucleic acid sequence, bacteria cells, detection methods, and application methods may function as an integrated system to achieve auxin degradation and plant growth promotion through coordinated molecular and ecological interactions. The system integration may enable precise control over auxin levels in plant-microbe environments while providing monitoring capabilities and therapeutic interventions for auxin imbalance conditions. The regulatory control mechanism may initiate through MarR73 responding to auxin presence in the bacterial cellular environment. The MarR73 transcriptional regulator may function as the primary repressor controlling expression of the iad locus genes under normal conditions. When indole-3-acetic acid or related auxin compounds accumulate in the rhizosphere environment, these molecules may bind to the MarR73 regulator protein and induce conformational changes that reduce repressor activity. The auxin-responsive derepression of MarR73 may result in activation of the catabolic pathway genes downstream of the regulatory region. The iadR gene may be co-expressed with the catabolic pathway genes, providing additional regulatory control over the auxin degradation PRINCETON-103476 system. The coordinated expression of regulator and catabolic genes may ensure that auxin degradation activity occurs proportionally to the auxin concentration present in the environment. The activation of catabolic pathway genes may lead to production of the enzymatic machinery required for auxin catabolism. The iadC gene may be transcribed and translated to produce a reductase enzyme that facilitates electron transfer reactions. The iadD and iadE genes may be expressed to produce the large and small subunits of the IadDE complex, which may assemble into a functional monooxygenase enzyme. Enzymatic processing may commence through the IadCDE monooxygenase complex, which may catalyze the initial oxidative modification of indole-3-acetic acid. The IadC reductase may transfer electrons from NADH to the IadDE complex, enabling the monooxygenase to activate molecular oxygen for the oxidation reaction. The IadDE monooxygenase may incorporate a single oxygen atom into the indole ring structure of IAA, producing a 2-oxindole intermediate with molecular formula C₁₀H₉NO₃. The monooxygenase reaction may proceed through an epoxide mechanism followed by hydration and subsequent dehydrogenation steps. The enzymatic process may convert the indole ring system to an oxindole structure while preserving the acetic acid side chain. The 2- oxindole intermediate may represent the first stable product in the auxin degradation pathway and may serve as the substrate for subsequent enzymatic reactions. Subsequent enzymes encoded by additional catabolic pathway genes may process the 2-oxindole intermediate through sequential reactions. The IadJ enzyme may catalyze a second oxidation step, converting the C₁₀H₉NO₃ intermediate to a C₁₀H₉NO₄ product through a dehydrogenase mechanism that incorporates oxygen from water. The IadF, IadG, and IadH enzymes may work together to mediate reductive removal of the acetyl side chain from pathway intermediates. The IadF acyl-CoA synthase may activate the carboxylic group in the acetyl side chain, enabling subsequent processing by the IadG acetyl-CoA acetyltransferase and IadH alcohol dehydrogenase. These enzymes may process CoA-bound intermediates through a chain of reactions that result in removal of the C₂H₂O₂ group from the molecular structure. The coordinated activity of these enzymes may produce intermediates with molecular formula C₈H₇NO₂. The IadI kynurenine formamidase may convert the C₈H₇NO₂ intermediates to products with molecular formula C₇H₇NO through hydrolytic cleavage reactions. The final oxidation PRINCETON-103476 step may convert the C₇H₇NO compounds to anthranilic acid (C₇H₇NO₂), which represents the ultimate product of the auxin degradation pathway. The anthranilic acid product may not interfere with plant auxin signaling and may be further metabolized through standard aromatic compound degradation pathways. Production of metabolic intermediates may occur in a sequential manner that prevents accumulation of potentially harmful compounds. The enzymatic reactions may be coupled to ensure efficient channeling of intermediates through the pathway without significant buildup of reactive species. The metabolic flux through the pathway may be regulated by the availability of cofactors such as NADH, CoA, and ATP, which may provide additional control over auxin degradation rates. The ultimate conversion to anthranilic acid may represent the completion of auxin detoxification and the restoration of auxin balance in the microbial environment. The anthranilic acid product may be readily metabolized through central aromatic compound degradation pathways or may be excreted from bacterial cells without adverse effects on plant growth. The conversion process may effectively remove bioactive auxin molecules from the rhizosphere environment. Bacterial colonization of plant roots may enable in situ auxin degradation in the rhizosphere through establishment of stable microbial populations that express the auxin degradation genes. The engineered bacteria cells may colonize root surfaces and plant- associated soil regions where auxin-producing microorganisms may also be present. The spatial proximity between auxin-producing and auxin-degrading bacteria may enable efficient processing of excess auxin compounds before these molecules can affect plant physiology. The colonization process may be enhanced by the amino acid auxotrophy characteristics of certain engineered bacterial strains. The nutritional dependency on amino acids may promote closer associations with plant roots that exude amino acids and other nutrients. The enhanced colonization may result in higher local concentrations of auxin- degrading bacteria in the immediate vicinity of plant roots where auxin balance is most important for plant health. The engineered bacteria cells may compete with native microorganisms for colonization sites while providing beneficial auxin degradation services. The competitive colonization may be particularly effective for bacterial strains such as Paraburkholderia species that demonstrate superior root colonization capabilities. The high relative abundance achieved PRINCETON-103476 by these strains may ensure adequate auxin degradation capacity even in complex microbial communities. In situ auxin degradation may occur continuously as auxin-producing bacteria generate excess indole-3-acetic acid in the rhizosphere environment. The auxin-responsive regulation of the iad pathway may ensure that degradation activity increases proportionally to auxin accumulation, providing dynamic balance control. The real-time response to auxin levels may prevent both auxin excess and auxin depletion conditions that could adversely affect plant growth. Restoration of auxin homeostasis may result from the coordinated activity of auxin degradation and natural plant auxin regulation mechanisms. The bacterial auxin degradation may complement plant-based auxin control systems by removing excess auxin produced by rhizosphere microorganisms. The restoration process may enable plants to maintain normal auxin signaling patterns despite the presence of auxin-overproducing bacteria in the root environment. The auxin homeostasis restoration may be particularly important in complex microbial communities where multiple bacterial species may contribute to auxin production. The engineered auxin-degrading bacteria may process auxin compounds produced by diverse microorganisms, providing broad-spectrum auxin balance control. The system may be effective against both high-level auxin producers and communities of moderate auxin producers that collectively generate excess auxin levels. Reversal of root growth inhibition may occur as auxin levels return to physiologically appropriate ranges through bacterial degradation activity. The root growth inhibition caused by auxin excess may be alleviated within days of introducing auxin-degrading bacteria to plant- microbe systems. The reversal process may restore normal root elongation patterns and enable plants to develop appropriate root architecture for nutrient and water uptake. The root growth inhibition reversal may be measurable through primary root length assessments and root branching pattern analysis. The engineered bacteria cells may enable plants to achieve root growth rates comparable to those observed in the absence of auxin- producing bacteria. The restoration of normal root growth may be sustained over extended periods as the engineered bacteria maintain auxin degradation activity. Enhancement of plant growth may result from the combined effects of restored root function and improved plant-microbe interactions. The normalized auxin levels may enable plants to allocate resources more effectively between root and shoot development. The PRINCETON-103476 enhanced root growth may improve nutrient uptake efficiency and water acquisition, leading to increased shoot biomass and overall plant vigor. The plant growth enhancement may be particularly pronounced in natural soil environments where complex microbial communities may generate significant auxin imbalances. The engineered bacteria cells may provide auxin balance control that enables plants to thrive in soil conditions that would otherwise limit growth due to microbial auxin overproduction. The growth enhancement may be sustained over plant development cycles as the engineered bacteria establish stable populations in the rhizosphere. The system integration may enable applications in diverse microbial environments ranging from simplified synthetic communities to complex natural soil ecosystems. The engineered bacteria cells may function effectively across different soil types, pH conditions, and nutrient availability scenarios. The robust performance in diverse environments may be attributed to the natural root-association capabilities of the chassis bacterial strains and the conservation of iad pathway genes across bacterial species. The integrated system may provide a platform for developing next-generation microbial bioinoculants that address auxin imbalance issues in agricultural and ecological settings. The combination of auxin degradation capabilities with strong root colonization characteristics may enable practical applications for crop production systems. The system may reduce reliance on chemical interventions for managing plant-microbe interactions while providing sustainable approaches for enhancing plant growth and productivity. The engineered strains may be utilized for chemical remediation applications beyond plant-microbe interactions. The enzymatic machinery involved in indole-3-acetic acid degradation may also process other aromatic compounds with structures similar to auxins. The substrate specificity of the iad pathway enzymes may extend to various aromatic molecules that share structural features with indole-3-acetic acid, including the indole ring system and carboxylic acid functional groups. Wastewater treatment applications may benefit from the aromatic compound degradation capabilities of the engineered bacterial strains. Industrial wastewater streams may contain aromatic pollutants that are structurally related to auxin compounds and may be processed by the iad pathway enzymes. The IadCDE monooxygenase complex may catalyze oxidative modifications of aromatic substrates beyond indole-3-acetic acid, potentially enabling degradation of indole derivatives, phenolic compounds, and other aromatic molecules present in contaminated water systems. PRINCETON-103476 The engineered strains may be applied to wastewater treatment systems as biological remediation agents that complement conventional treatment processes. The bacterial strains may be introduced to bioreactors or constructed wetland systems where they may establish populations capable of processing aromatic contaminants. The auxin-responsive regulation of the iad pathway may provide adaptive control over enzyme expression levels based on substrate availability in the treatment environment. Chemical spill remediation may represent another application area where the aromatic compound degradation capabilities may be beneficial. Environmental contamination events involving aromatic chemicals may be addressed through deployment of engineered bacterial strains capable of metabolizing the contaminant compounds. The broad substrate specificity of certain iad pathway enzymes may enable processing of diverse aromatic pollutants that accumulate in soil or water environments following industrial accidents or improper waste disposal. The metabolic versatility of the iad pathway may extend to degradation of pharmaceutical compounds that contain indole or related aromatic structures. Pharmaceutical manufacturing wastewater may contain residual drug compounds or synthetic intermediates that share structural features with auxin molecules. The engineered bacterial strains may provide biological treatment options for removing these pharmaceutical contaminants from wastewater streams before discharge to natural water bodies. Metabolomic analysis may be performed using liquid chromatography-mass spectrometry (LC-MS) platforms to characterize the degradation products and metabolic intermediates produced during aromatic compound processing. A Waters XBridge BEH Amide column may provide chromatographic separation of polar metabolites generated during the degradation process. The amide-based stationary phase may enable effective separation of compounds with varying polarities and functional group compositions. A Vanquish UHPLC system may be coupled to an Orbitrap Exploris 480 mass spectrometer to provide high-resolution mass spectrometric analysis of metabolic products. The ultra-high-performance liquid chromatography system may deliver precise separation of complex metabolite mixtures with enhanced resolution and reduced analysis times compared to conventional HPLC systems. The coupling of chromatographic separation with mass spectrometric detection may enable identification and quantification of degradation products with high specificity and sensitivity. PRINCETON-103476 The Orbitrap Exploris 480 mass spectrometer may provide accurate mass measurements and high-resolution mass spectra that enable structural characterization of unknown metabolites. The orbitrap mass analyzer may deliver mass accuracy within parts-per- million ranges, facilitating molecular formula determination and structural elucidation of degradation products. The high-resolution capabilities may enable discrimination between closely related compounds and may provide confidence in metabolite identification. The LC-MS platform may operate in both positive and negative ionization modes to maximize detection coverage for diverse metabolite classes. The electrospray ionization source may generate gas-phase ions from liquid-phase samples with high efficiency across a broad range of compound polarities. The dual-polarity capability may ensure comprehensive detection of both acidic and basic metabolites produced during aromatic compound degradation. Full-scan mass spectrometry acquisition may enable untargeted metabolomic profiling that captures the complete range of metabolic products without prior knowledge of specific compounds. The full-scan approach may facilitate discovery of unexpected degradation pathways or novel metabolic intermediates that may not be detected through targeted analysis methods. The comprehensive metabolite coverage may provide insights into the mechanistic details of aromatic compound processing by the iad pathway enzymes. Tandem mass spectrometry (MS / MS) capabilities may enable structural characterization of metabolites through fragmentation pattern analysis. The collision-induced dissociation of molecular ions may generate characteristic fragment ions that provide structural information about functional groups and molecular connectivity. The MS / MS data may support metabolite identification through comparison with spectral databases or through de novo structural interpretation. The engineered strains may be combined with additional genes for other plant-growth promoting traits to create multi-functional bacterial inoculants. The auxin degradation capabilities may be complemented by genes encoding enzymes for nitrogen fixation, phosphate solubilization, or production of plant growth hormones other than auxin. The multi-trait engineering approach may provide comprehensive plant growth promotion benefits that address multiple nutritional and physiological requirements. Genes for enhanced microbiome invasion may be incorporated into the engineered strains to improve their competitive abilities in complex microbial communities. The microbiome invasion genes may encode antimicrobial compounds, competitive exclusion PRINCETON-103476 factors, or metabolic capabilities that provide advantages over indigenous microorganisms. The enhanced competitive abilities may enable the engineered strains to establish stable populations in environments with high microbial diversity and competition for resources. Plant colonization genes may be added to improve the root-association capabilities of the engineered bacterial strains. The colonization genes may encode adhesion factors, biofilm formation proteins, or chemotaxis systems that enhance bacterial attachment to root surfaces. The improved colonization may result in higher bacterial densities in the rhizosphere and more effective delivery of beneficial traits to plant hosts. Persistence genes may be incorporated to enhance the survival and stability of engineered bacterial populations under environmental stress conditions. The persistence genes may encode stress response systems, DNA repair mechanisms, or metabolic adaptations that enable survival under adverse conditions such as drought, temperature extremes, or nutrient limitation. The enhanced persistence may ensure long-term effectiveness of the bacterial inoculants across variable environmental conditions. Product formulation stability genes may be added to improve the shelf-life and storage characteristics of bacterial inoculant products. The formulation stability genes may encode protective compounds, stress tolerance factors, or metabolic adjustments that enable bacterial survival during product manufacturing, storage, and distribution processes. The improved formulation stability may enable development of commercial bacterial products with extended shelf-life and consistent performance characteristics. The multi-trait engineering approach may utilize modular genetic constructs that enable flexible combination of different beneficial traits. The modular design may allow for customization of bacterial strains for specific applications or environmental conditions. The genetic modules may be designed with compatible regulatory systems that enable coordinated expression of multiple trait genes without interference between different functional pathways. The integration of multiple beneficial traits may require careful consideration of metabolic burden and resource allocation within the engineered bacterial cells. The expression of additional genes may compete for cellular resources such as ribosomes, amino acids, and energy molecules. The multi-trait engineering may incorporate regulatory mechanisms that balance the expression levels of different trait genes to optimize overall bacterial performance and trait delivery. The combined trait systems may provide synergistic benefits that exceed the sum of individual trait contributions. The auxin degradation capabilities may enhance the effectiveness PRINCETON-103476 of other plant growth promotion traits by creating more favorable conditions for plant-microbe interactions. The improved plant health resulting from auxin balance restoration may enhance the plant's responsiveness to other beneficial microbial activities such as nutrient provision or pathogen protection. The multi-trait engineered strains may be particularly valuable for applications in challenging agricultural environments where multiple stress factors may limit plant growth. The comprehensive trait portfolio may address diverse plant needs simultaneously, providing more robust and reliable plant growth promotion compared to single-trait approaches. The integrated system may enable sustainable agricultural practices that reduce dependence on chemical inputs while maintaining or improving crop productivity. Examples Here, the iad-mediated IAA degradation pathway in V. paradoxus CL014 is systematically dissected using a combination of genetic, metabolomics, and isotope tracing. A nine-gene region (iadCDEFGHIJK2) has been identified within the iad locus that is responsible for IAA catabolism. V. paradoxus CL014 initiates IAA degradation through an unreported two-step oxidative mechanism. In the first step, the IadCDE complex functions as a monooxygenase that incorporates one oxygen atom from molecular oxygen into IAA. Although IadCDE is structurally homologous to canonical dioxygenase complexes, metabolomic and isotopic analyses demonstrates that it functions as a monooxygenase, revealing a striking divergence between structure and catalytic mechanism. The second oxidative step is mediated by the dehydrogenase IadJ following hydrolysis, leading to oxygen incorporated from water. Notably, IadCDE product is identified to be an uncharacterized compound (C₁₀H₉NO₃), rather than the commonly known intermediate, 2-oxindole-3-acetic acid (oxIAA, C₁₀H₉NO₃), which is supportive of an epoxide-forming mechanism by IadCDE. To explore the broader applicability of this pathway, the iad pathway genes were genomically integrated, under control of the MarR73 regulator, into two additional plant-associated bacteria, Polaromonas MF047 and Paraburkholderia MF376, and their IAA-degrading activity and ability to mitigate RGI was evaluated in both simplified and complex bacterial community treatments, using Arabidopsis thaliana and Medicago truncatula as host plants. Engineered Paraburkholderia MF376 emerged as a promising chassis for agricultural applications, as it effectively reversed RGI caused by strains that V. paradoxus CL014 did not fully counteract, enhanced Arabidopsis growth in natural soil over long-term incubation, and exhibited robust PRINCETON-103476 colonization when introduced into new microbial communities. These findings advance our understanding of microbial auxin metabolism and its relevance to restoring rhizosphere auxin balance and enhancing plant growth. Identification of functional genes involved in each step of IAA degradation Previous studies indicated that V. paradoxus CL014 degrades IAA through a pathway similar to that of Bradyrhizobium japonicum, producing intermediates such as dioxindole, isatin, isatinic acid, and anthranilic acid—an intermediate in tryptophan biosynthesis. However, the specific genes responsible for each step remained unidentified. The iad locus (Hot Spot 33, HS33), consisting of 25 genes (see FIG. 1) was identified as responsible for IAA degradation via genomic deletion. To pinpoint key genes, different segments of the iad locus were cloned into the broad-host vector pBBR1 and introduced into an iad locus deletion mutant (ΔHS33). Metabolomic analysis revealed that mutants carrying the iadC-K2 region restored the level of intermediates and the final product, anthranilic acid, indicating that this nine-gene region encodes the complete IAA degradation pathway. To identify specific substrates and products of iad genes, liquid chromatography was performed in tandem with high-resolution mass spectrometry (LC-MS), focusing on metabolites previously associated with IAA degradation in V. paradoxus CL014. It was reasoned that knocking out a gene downstream of a metabolite or overexpressing a gene upstream would lead to metabolite accumulation or depletion, respectively. Specifically, knockout mutants were created in the wild-type background and overexpression mutants in the ΔHS33 background (see FIG.2). Based on gene annotations and structural data, iadD and iadE encode the large and small subunits of a Rieske non-heme dioxygenase, while iadC encodes a reductase, forming a two-component dioxygenase system (see FIG. 4). Accordingly, iadCDE was treated as a single functional unit for mutant construction. The analysis revealed that deletion of iadCDE, iadF, iadG, iadH, iadI, or iadJ significantly reduced anthranilic acid (C₇H₇NO₂) production (see FIG. 3A), confirming their essential roles in IAA degradation. Complementation of the ΔHS33 mutant with the full iadC– K2 gene region fully restored anthranilic acid levels (see FIG. 3A). Notably, the presence of iadCDE in these segments was able to rapidly degrade IAA. This confirms that IadCDE directly uses IAA as its substrate and catalyzes the initial step of IAA degradation (see FIG. 3B). Metabolite accumulation in the ΔHS33::iadCDE strain was examined, and two compounds were identified, C₁₀H₉NO₃ and likely its hydration product C₁₀H₁₁NO₄, suggesting PRINCETON-103476 C₁₀H₉NO₃ to be the product of IadCDE (see FIGS.3C-3D). Interestingly, iadJ deficiency also led to the accumulation of C₁₀H₉NO₃ and C₁₀H₁₁NO₄, indicating that IadJ likely consumes one of these intermediates (see FIG. 3C). Similarly, it was found that C₁₀H₉NO₄ is depleted with iadJ deletion while it accumulates in ΔHS33::iadCDEJK2, suggesting C₁₀H₉NO₄ to be the product of IadJ (see FIGS.3E-3F). Deletion of iadF, iadG, iadH and iadI all lead to C₁₀H₉NO₄ accumulation, among which ΔiadF shows the strongest accumulation suggesting functional proximity of IadF to IadCDEJ (see FIG. 3E). Other intermediates were not detected. Thus, IadFGHI likely carries out a chain of reactions, with most intermediates quickly channeled through the enzymes. Therefore, this example resorted to annotated enzyme function to infer reactions carried out by these genes. Annotated as an acyl-CoA synthase (AMP-forming), IadF likely activates the carboxylic group in the acetyl side chain, enabling its subsequent removal by IadG, an acetyl-CoA acetyltransferase (ketothiolase, EC 2.3.1.16) (see FIG.4). This reaction yields CoA-bound intermediates that are membrane-impermeable, ensuring their retention within bacterial cells and enabling efficient recognition and processing. Meanwhile, IadH, annotated as an alcohol dehydrogenase, likely facilitates further processing (see FIG. 4). Together, IadF, IadG, and IadH mediate the reductive removal of the acetyl side chain (- C₂H₂O₂), producing C₈H₇NO₂ (see FIG. 3G). Indeed, C₈H₇NO₂ accumulates in ΔHS33::iadCDEFGH_JK2 expression strain as well as the iadI deletion strain (see FIG. 3G), suggesting that IadI acts downstream of C₈H₇NO₂. C₇H₇NO and C₈H₇NO₃ were also detected, which strongly accumulate only in strains overexpressing the whole iadC-K2 gene locus, suggesting that they are likely intermediates between the IadI product and the final product anthranilic acid (C₇H₇NO₂) (see FIG. 2 and FIG. 3H). Although iadI is annotated as a kynurenine formamidase, our MS / MS analysis confirmed that C₈H₇NO₃ is not N- formylanthranilic acid—the expected formylated derivative of anthranilic acid—indicating that IadI may instead function as a broad-specificity amidase. Lastly, IadK2 was previously identified as a highly IAA-specific ATP-binding cassette (ABC) transporter solute-binding protein involved in IAA uptake. However, it is not essential, as ΔHS33::iadCDE was still capable of taking up and utilizing IAA, suggesting alternative uptake mechanisms. In summary, a nine-gene region (iadC-K2) was identified within the iad locus responsible for IAA degradation in V. paradoxus CL014 (see FIG.1). By integrating metabolite abundance changes with gene functional annotations, putative reaction steps were assigned to each gene and reconstructed the degradation pathway (see FIG.4). This pathway links specific PRINCETON-103476 functional genes to corresponding intermediates, providing a framework for understanding IAA metabolism in this microorganism. Isotope tracing reveals new intermediates, revising the iad auxin degradation pathway Previously, IAA degradation via the iad pathway was proposed to proceed through monooxygenation to generate 2-oxindole-3-acetic acid (oxIAA, C₁₀H₉NO₃), followed by conversion to dioxindole. The metabolomic analysis from this example, however, revealed two distinct C₁₀H₉NO₃ peaks in wild-type extracts: a major peak at 4 min and a minor peak at 7 min. Using an oxIAA standard, it was confirmed that the minor peak corresponds to oxIAA; however, this compound is not depleted in the ΔHS33 mutant, making it unlikely to be the product of IadCDE. By contrast, the major C₁₀H₉NO₃ peak is depleted in the ΔHS33 mutant and accumulates in iadCDE overexpressing strains, indicating that it represents the product of IadCDE. These findings indicate that the observed C₁₀H₉NO₃ is not oxIAA but rather a previously uncharacterized intermediate, suggesting that IAA degradation in V. paradoxus CL014 proceeds via a mechanism distinct from the canonical oxIAA pathway. To elucidate the mechanism of IAA degradation, isotope tracing and LC-MS / MS analysis were performed of both labeled and unlabeled pathway intermediates (see FIGS.5-7). Specifically, uniformly deuterium-labeled IAA ([²H₇]IAA) was employed to monitor hydrogen rearrangements during catabolism, providing greater mechanistic resolution than the previously used [¹³C₆]IAA. Tracing with [²H₇]IAA revealed distinct mass shifts in pathway intermediates relative to unlabeled controls, indicating the number of retained deuterium atoms. The first intermediate, C₁₀H₉NO₃, retained all seven deuterium atoms, suggesting no isotope loss during the initial oxidation step (see FIGS.5A-5D). This observation is inconsistent with mechanisms involving oxIAA or its enol form, 2-hydroxy-IAA, or a radical intermediate from hydrogen atom abstraction. Indeed, the minor C₁₀H₉NO₃ peak corresponding to oxIAA retained only six deuterium atoms. The subsequent intermediate, C₁₀H₉NO₄, showed the loss of one deuterium, whereas all downstream intermediates consistently retained four deuterium atoms on the aromatic ring (see FIGS.5A-5D). To identify the source of oxygen in the formation of C₁₀H₉NO₃, C₁₀H₁₁NO₄, and C₁₀H₉NO₄, respectively, ¹⁸O-labeled water (H₂¹⁸O) tracing was performed in bacterial culture (see FIG.6). The IadCDE monooxygenase complex uses molecular oxygen (O₂) and therefore will not generate labeled product (C₁₀H₉NO₃) from H₂¹⁸O. On the contrary, the IadJ PRINCETON-103476 dehydrogenase mechanism leads to oxidized product that can be labeled by H₂¹⁸O. The minor C₁₀H₉NO₃ peak corresponding to oxIAA is significantly labeled by H₂¹⁸O, further confirming oxIAA is not formed by IadCDE. In contrast, no labeling was detected in the major C₁₀H₉NO₃ peak in either the wild type or iadCDE overexpressing strains, consistent with it being the product of IadCDE (see FIG. 6). In contrast, C₁₀H₁₁NO₄ incorporated ¹⁸O in the same strains, supporting its formation via hydration of C₁₀H₉NO₃ in the cells before being processed by IadJ through a dehydrogenase mechanism (see FIG. 6). Further MS / MS analysis of C₁₀H₉NO₄ revealed that the incorporated ¹⁸O resides either on the carboxylate or the ketone moiety. Interestingly, C₁₀H₉NO₄ remained almost unlabeled in the wild type, which may be due to the rapid hydrolysis of C₁₀H₉NO₃ within the IadCDE catalytic center, where H₂O is derived from catalytic reduction of molecular oxygen possibly by IadC. Collectively, the data support a revised IAA degradation pathway in V. paradoxus CL014 (see FIG.7). Specifically, the initial oxidation of IAA proceeds through two sequential steps (C₁₀H₉NO₂ → C₁₀H₉NO₃ → C₁₀H₉NO₄). IadCDE functions as a monooxygenase complex that carries out the first oxidation, incorporating oxygen derived from molecular oxygen, whereas IadJ acts as a dehydrogenase that completes the second oxidation, incorporating the oxygen atom from H₂O. The data reveal that the intermediate C₁₀H₉NO₃ is distinct from commonly believed pathway intermediate, oxIAA, although they share the same molecular formula. Following the observed deuterium labeling and mechanisms of known heme- dependent tryptophan or indole dioxygenases, it is proposed that oxidation by IadCDE proceeds via a 2,3-epoxide intermediate. This is followed by spontaneous ring opening via hydrolysis and IadJ catalyzed dehydrogenation to produce dioxindole-3-acetic acid (C₁₀H₉NO₄). This revised pathway provides a more accurate framework for understanding IAA degradation in V. paradoxus CL014. By homology, it suggests that the iad-like pathway represents a conserved and unique auxin-catabolic strategy among certain plant-associated commensals. This work also lays the foundation for further exploration of iad-like pathways in these plant-associated bacteria in genera such as Bradyrhizobium, Alcaligenes, and Achromobacter. Functional and structural insights into the IadCDE monooxygenase system and IAA degradation in V. paradoxus CL014 Oxygenases play a crucial role in aerobic bacteria by incorporating oxygen into chemically stable aromatic compounds, enabling their degradation into metabolically PRINCETON-103476 accessible substrates. To date, the most extensively studied indole oxygenases fall into two major families: the tryptophan dioxygenase (TDO) superfamily, which catalyzes dioxygenation or monooxygenation via an Fe / heme-dependent mechanism; and the flavin-dependent monooxygenases (FMOs), which use a flavin cofactor to mediate oxygen transfer. Rieske-type dioxygenases may function divergently as dioxygenase, monooxygenase, or hydroxylase. The metabolomics and isotope tracing discussed herein reveal a monooxygenation-driven mechanism, identifying IadCDE in V. paradoxus CL014 as a two-component indole monooxygenase, essential for initiating IAA degradation, rather than a dioxygenase as previously annotated. The proposed reaction mechanism closely resembles that of MarE, a heme-dependent aromatic monooxygenase involved in the maremycin biosynthetic pathway of Streptomyces sp. B9173. While MarE belongs to the TDO superfamily and relies on a heme cofactor, IadDE is structurally similar to Rieske non-heme dioxygenases. See FIGS.8-9. To investigate the molecular basis of IadCDE function, the crystal structure of the IadDE complex (PDB code: 9O71) from V. paradoxus CL014 was determined at 1.28 Å resolution, providing a high-resolution view of the complete holoenzyme. While a homologous structure was previously resolved by cryo-EM at 1.8 Å resolution (with 98.4% and 100% protein sequence identity for IadD and IadE, respectively), the improved resolution of this X- ray structure allows for more precise visualization of overall structural features. Structurally, IadDE adopts the canonical fold of Rieske-type non-heme dioxygenases. IadD and IadE assemble into a heterodimeric α₃β₃ quaternary complex with threefold symmetry and a characteristic mushroom-shaped morphology (see FIG. 9). In the IadCDE complex, IadD contains both a Rieske-type [2Fe–2S] cluster and a mononuclear iron center and serves as the catalytic subunit, while IadE provides structural support. Trimerization of IadDE heterodimers brings the Rieske iron-sulfur cluster of IadD close to the iron center of an adjacent IadD, potentially facilitating electron transfer during catalysis (see FIG. 9). IadC is annotated as a reductase and is proposed to mediate electron transfer from NADH to IadD to activate molecular oxygen. Phylogenetic analysis revealed that IadD forms a distinct subclade within the Rieske dioxygenase and indole oxygenase family, showing close evolutionary relationships to the phthalate family of Rieske non-heme dioxygenases, as well as to members of the TDO and FMO superfamilies. This positioning may reflect shared functional features among distinct oxygenase lineages. PRINCETON-103476 To determine whether iadCDE alone is sufficient to reverse RGI in the context of complex microbial communities, we genomically integrated the native repressor marR73 alongside either the minimal (iadCDE) or full (iadC-K2) degradation pathway into the Variovorax ΔHS33 strain. This enabled direct functional comparison of the oxygenase module with the complete pathway under IAA-rich conditions. In M9 minimal medium supplemented with glucose and 0.1 mg / mL IAA, both engineered strains showed significantly enhanced IAA degradation relative to the wild type, independent of bacterial growth (see FIG. 10). In a gnotobiotic system, engineered strains fully rescued RGI in Arabidopsis seedlings challenged with 100 nM exogenous IAA, the auxin-producing strain Arthrobacter CL028, or a 32-member synthetic community (SynCom32), with comparable effects observed in Medicago (see FIGS. 11-12). The SynCom32 community includes Streptomycetaceae Streptomyces (4 different strains), Burkholderiaceae Ralstonia, Micrococcaoeae Arthrobacter (3 different strains), Moraxellaceae Acinetobacter, Rhizobiaoeae Rhizobium, Flavobacteriaceae Chryseobacterium, Bacillaoeae Bacillus, Nocardiaceae Rhodococcus, Rhizobiaoeae Agrobacterium, Flavobacteriaceae Flavobacterium, Bacillaoeae Bacillus, Pseudomonadaceae Pseudomonas, Xanthomonadaceae Dyella, Bacillaoeae Bacillus, Microbacteriaoeae Leifsonia, Bacillaoeae Bacillus, Rhodanobacteraceae Luleibacter, Paenibacillaceae Paenibacillus, lntrasporangiaceae Terracoccus, Phyllobacteriaceae Phyllobacterium, Microbacteriaceae Curtobacterium, Promicromonosporaceae Promicromonospora, Nocardiaceae Rhodococcus, Nocardioidaceae Nocardioides, Mycobacteriaceae Mycobacterium, Brucellaceae Ochrobactrum, and Caulobacteraceae Brevundimonas. These results demonstrate that iadCDE alone is sufficient to reverse RGI caused by both exogenous IAA and auxin-producing microbes. However, under elevated IAA concentrations (1 μM or 10 μM), the strain harboring the full degradation pathway outperformed the minimal mutant, suggesting that downstream enzymatic steps confer an advantage in detoxifying excess IAA (see FIG.13). Engineering iad genes into plant-associated bacteria confers IAA degradation and alleviates RGI To evaluate whether plant-associated bacteria can acquire IAA-degrading activity through genetic engineering, iad genes were introduced into two plant-associated strains: Polaromonas MF047 and Paraburkholderia MF376. Both strains, like Variovorax, are PRINCETON-103476 Betaproteobacteria and do not impair host growth when co-inoculated with Arabidopsis seedlings (see FIGS. 15A-15B). Neither strain produced IAA when cultured in M9 minimal medium supplemented with tryptophan and glucose, nor were they able to reverse RGI triggered by exogenous IAA or by the auxin-producing strain Arthrobacter CL028 (see FIGS. 15A-15B). Additionally, as an amino acid auxotroph, Polaromonas MF047 exhibited no growth in M9 minimal medium unless supplemented with amino acids. To confer IAA-degrading capacity, marR73 was genomically integrated together with either the minimal (iadCDE) or complete (iadC-K2) pathway into three intergenic sites in Polaromonas MF047 and one site in Paraburkholderia MF376. To minimize potential disruption of native gene expression, insertion sites were located within non-coding intergenic regions ranging from 400 to 900 bp in length. All engineered strains degraded IAA within 24 hours, and Paraburkholderia MF376 knock-ins achieved complete IAA removal within four hours (see FIG. 14). This rapid degradation alleviated RGI in Arabidopsis seedlings exposed to 100 nM exogenous IAA (see FIGS.15A-15B). Growth curves in 50% TSB medium revealed no significant differences between wild-type and engineered strains, indicating that chromosomal integration did not impair bacterial fitness. To assess their function in plant-microbe interactions, engineered strains were co- inoculated with Arthrobacter CL028 or SynCom32 on 7-day-old Arabidopsis seedlings in a gnotobiotic system. After 7 days, all engineered strains fully rescued RGI triggered by Arthrobacter CL028, restoring primary root length to control levels (see FIGS. 15A-15B). In parallel, they significantly alleviated RGI induced by SynCom32, with Paraburkholderia MF376 consistently showing greater efficacy than Polaromonas MF047 (see FIG.16). To test cross-species efficacy, we repeated the SynCom32 experiment using Medicago seedlings. In this context, strains carrying the full iad pathway promoted stronger root growth than those expressing only the minimal iadCDE module, highlighting the importance of a complete IAA degradation pathway for effective function in complex microbial environments (see FIG. 16). These results demonstrate for the first time that stable genomic integration and expression of iad genes in plant-associated bacteria is able to restore root growth under microbial auxin stress. The engineered strains are effective across microbial contexts and plant hosts, underscoring their potential as tools for microbiome-based modulation of plant development. The role of V. paradoxus CL014 and engineered strains in mitigating RGI induced by individual bacterial isolates PRINCETON-103476 To assess the contribution of the iad pathway to RGI mitigation, several previously identified RGI-inducing strains were selected from nine bacterial genera, including:Moraxellaceae Acinetobacte, Rhizobiaoeae Agrobacterium (2 strains), Micrococcaceae Arthrobacter (8 strains), Bacillaoeae Bacillus (3 strains), Flavobacteriaceae Chryseobacterium, Burkholderiaceae Burkholderia, Rhodanobacteraceae Luleibacter, Paenibacillaceae Paenibacillus, Pseudomonadaceae Pseudomonas (6 strains), and Nocardiaceae Rhodococcus. Each strain was co-inoculated with either wild-type V. paradoxus CL014 or the iad deletion mutant ΔHS33 on 7-day-old Arabidopsis seedlings. After 7 days, root length measurements revealed that 22 strains induced RGI (see FIGS.17-18). Among these, wild-type V. paradoxus CL014 more effectively reversed RGI than ΔHS33 in 15 cases, indicating iad- dependent mitigation. However, in three cases—Agrobacterium MF224, Arthrobacter MF135, and Pseudomonas MF051—both the wild-type and ΔHS33 strains restored root growth to similar levels, suggesting an iad-independent mechanism (see FIG.18). Genome analysis of V. paradoxus CL014 revealed a gene encoding 1- aminocyclopropane-1-carboxylic acid (ACC) deaminase, an enzyme that degrades ACC, the ethylene precursor. Ethylene, a plant hormone, can inhibit primary root elongation at high levels. Prior studies show that RGI induced by Arthrobacter CL028 and a synthetic microbial community requires both auxin and ethylene signaling in the host. While plants produce ethylene endogenously, certain soil and rhizosphere microbes also contribute to ethylene levels. Notably, some Agrobacterium, Arthrobacter, and Pseudomonas strains have been reported to produce ethylene, which can suppress root elongation and alter root architecture. Based on these observations, we hypothesized that the three iad-independent strains may produce both auxin and ethylene, with IAA degradation mediated by the iad pathway and ethylene detoxification potentially supported by ACC deaminase activity in V. paradoxus CL014. However, deletion of the ACC deaminase gene in both wild-type and ΔHS33 backgrounds did not impair their ability to reverse RGI, ruling out ethylene degradation through this ACC deaminase as the primary mechanism (see FIG.18). These findings suggest that while the iad pathway is a central mechanism for suppressing RGI caused by many plant-associated strains, V. paradoxus CL014 also employs iad-independent strategies to mitigate RGI from certain microbes. Despite its broad effectiveness, V. paradoxus CL014 was unable to fully rescue RGI induced by Pseudomonas MF048. To test whether iad-engineered strains could provide PRINCETON-103476 enhanced reversion, Polaromonas MF047 and Paraburkholderia MF376 engineered with the iad pathway were evaluated. These strains were co-inoculated with Pseudomonas MF048 alone or in combination with Arthrobacter CL028 on Arabidopsis seedlings. Root length analysis showed that Paraburkholderia MF376 knock-in strains significantly outperformed both V. paradoxus CL014 and engineered Polaromonas MF047 under both conditions (see FIG. 19). Collectively, these results demonstrate that engineering Paraburkholderia MF376 with the iad pathway enhances its ability to mitigate complex RGI interactions, highlighting its potential as a robust and versatile chassis for promoting plant growth in diverse microbial environments. Impact of V. paradoxus CL014 and engineered strains on the root microbiome and plant growth V. paradoxus CL014 can degrade IAA without harming plant growth at normal treatment levels (OD₆₀₀ = 0.05). To test whether this effect holds under high bacterial load, V. paradoxus CL014 at OD₆₀₀ = 2 were applied to Arabidopsis seedlings in a gnotobiotic system. No significant changes in primary root length were observed, indicating that even at high densities, V. paradoxus CL014 does not adversely affect plant growth or disturb auxin balance (see FIG.20). These findings suggest that its IAA-degrading function may primarily influence microbe–microbe interactions rather than directly altering plant development. To assess the impact of V. paradoxus CL014, Polaromonas MF047, Paraburkholderia MF376, and their iad-gene-containing engineered strains on the root microbiome, Arabidopsis and Medicago seedlings were treated with SynCom32 in the presence or absence of each strain. After 9 days, plant-associated communities were profiled by 16S rRNA amplicon sequencing. Despite receiving the same SynCom32 inoculum, Arabidopsis and Medicago developed distinct root microbiomes, highlighting the influence of host genotype (see FIGS. 21A-21B). Among the three introduced genera, Paraburkholderia exhibited the highest relative abundance in roots (50.84%), followed by Variovorax (3.40%), whereas Polaromonas (0.01%) showed minimal colonization (see FIGS. 21A-21B). Due to its robust root colonization, Paraburkholderia MF376—both wild-type and engineered—substantially altered root microbiome composition, explaining 52% and 33.8% of the community variation in Arabidopsis and Medicago, respectively, as shown by unconstrained principal coordinate analysis (PCoA) using Bray–Curtis distances (P = 0.001; see FIG. 22). In contrast, V. paradoxus CL014 and Polaromonas MF047 had more modest effects (17.1-28.3%, P = 0.05), consistent with their lower colonization (see FIGS.21A-21B). See also FIGS.30A-30B. PRINCETON-103476 Notably, no major differences were observed between wild-type and knock-in strains of V. paradoxus CL014, Paraburkholderia MF376 and Polaromonas MF047, suggesting that IAA degradation pathway did not markedly alter their interactions within the microbiome (see FIG. 22). Log2fold-change analysis revealed no significant differences in the relative abundance of any genera between wild-type and engineered strain inoculated conditions, further suggesting that microbiome shifts were primarily driven by colonization capacity rather than IAA degradation activity. To evaluate potential benefits for plant performance, untreated natural soil was inoculated with each strain and fresh shoot weight was measured after 33 days. Arabidopsis grown in soil supplemented with Paraburkholderia MF376 carrying the complete iad pathway (marR73 iadC-K2) exhibited a significant increase in shoot biomass compared to uninoculated controls and those treated with the wild-type strain (see FIG.23). Similarly, FIG.31 includes plant data on engineered Variovorax and Polaromonas vs. wild types. These findings indicate that combining a strong plant colonizer chassis strain with the full IAA degradation pathway can enhance plant growth in natural soil, offering a promising strategy for microbiome-based agricultural interventions. For all steps discussed in this example, the following methods were used. Knock-out mutant construction. Gene deletions in V. paradoxus CL014 were generated using the suicide vector pMo130, following previously established methods. The vector backbone was PCR amplified and treated with DpnI to remove the template DNA. Upstream and downstream flanking regions of target genes were amplified from V. paradoxus CL014 genomic DNA using Platinum SuperFi II PCR Master Mix (Thermo Fisher Scientific). PCR products were purified using the DNA Clean & Concentrator Kit (Zymo Research) and assembled into pMo130 using HiFi Gibson Assembly Master Mix (New England Biolabs). Assembled plasmids were transformed into E. coli NEB 5-alpha (New England Biolabs), selected on Lysogeny Broth (LB, Thermo Fisher Scientific, BP1427) agar (2% w / v) supplemented with kanamycin (50 μg / mL), and verified by Sanger sequencing (Genewiz). Sequence-confirmed plasmids were transferred into the diaminopimelic acid (DAP) auxotrophic E. coli WM3064 for conjugation. Transformants were selected on LB agar containing kanamycin (50 μg / mL) and DAP (0.3 mM), and grown at 37 °C for 24 h. For biparental mating, donor E. coli WM3064 and recipient V. paradoxus CL014 (pre-grown in PRINCETON-103476 50% TSB (Tryptic Soy Broth, Thermo Fisher Scientific, CM0129) agar (2% w / v) with ampicillin (100 μg / mL)) were washed twice with 50% TSB, mixed at a 1:1 volume ratio, pelleted (5,000 × g, 5 min), resuspended in 1 / 10 volume of 50% TSB, and spotted onto 50% TSB agar containing DAP (0.3 mM). After overnight incubation at 28 °C, exconjugants were selected on 50% TSB agar containing ampicillin and kanamycin without DAP, and incubated for 3–4 days. Resulting colonies were re-streaked onto fresh antibiotic 50% TSB agar to ensure clonality and remove residual donor cells. Single colonies were screened by colony PCR to confirm single-crossover integration. Verified integrants were cultured in 50% TSB with ampicillin and Isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM). Cultures were plated on sucrose counter-selection agar (10 g / L tryptone, 5 g / L yeast extract, 10% w / v sucrose, 2% w / v agar, 100 μg / mL ampicillin, 1 mM IPTG) and incubated at 28 °C for 3-4 days. Resulting colonies were passaged in the same liquid medium, and deletion events were verified by PCR. Final deletion strains were re-streaked on 50% TSB with ampicillin and confirmed by diagnostic PCR using one primer located outside the deletion region and one within the deleted gene to ensure complete excision and strain purity. DNA templates for all PCRs were prepared using the following rapid lysis protocol40. A single colony or 6 μl of bacterial culture was mixed with 10 μl of alkaline lysis buffer (25 mM NaOH, 0.2 mM Na₂-EDTA, pH 12), incubated at 95 °C for 30 min, and neutralized with 10 μl Tris-HCl (40 mM, pH 7.5). The resulting material was used directly as PCR template (1:10, template:total PCR reaction volume). Overexpression mutant construction. V. paradoxus CL014 mar73 iadCDE and marR73 iadC-K2 were cloned into the broad-host-range vector pBBR1MCS-2 as known in the art. Briefly, genomic fragments were amplified using Platinum SuperFi II PCR Master Mix and assembled into pBBR1MCS-2 via Gibson assembly using HiFi Gibson Assembly Master Mix (New England Biolabs). Circular template DNA was digested with DpnI, and assembled plasmids were transformed into E. coli NEB 10-beta (New England Biolabs). Transformants were selected on LB agar containing kanamycin (50 μg / mL), and plasmids were extracted (ZR Plasmid Miniprep Kit, Zymo Research) and verified by Sanger sequencing. Verified constructs were introduced into V. paradoxus CL014 ΔHS33 by tri-parental mating, using E. coli pRK2013 as a helper strain. Donor and helper strains were grown in LB with kanamycin (50 μg / mL) at 37 °C, and the recipient strain was cultured in 50% TSB with ampicillin (100 μg / mL) at 28 °C. All strains were pelleted (5,000 × g, 5 min), washed in 50% TSB twice, mixed at equal volumes, and spotted onto 50% TSB agar for overnight conjugation at 28 °C. Exconjugants PRINCETON-103476 were selected on 50% TSB agar supplemented with kanamycin (50 μg / mL) and ampicillin (100 μg / mL), confirming successful plasmid transfer into V. paradoxus CL014 ΔHS33. Knock-in mutant construction. Two gene combinations, marR73 iadCDE and marR73 iadC-K2, were integrated into the iad locus of V. paradoxus CL014 ΔHS33, as well as three intergenic genomic sites in Polaromonas MF047 (IMG genome ID 2636416056 with insertion positions between Gene IDs: 2639079279–80, 2639079819–20, and 2639080354–55) and one site in Paraburkholderia MF376 (IMG genome ID 2521172625 with insertion position between Gene IDs 2521671121–22), using the pMo130 suicide vector. Gene amplification, vector assembly, and verification followed the same procedure as knockout mutant construction, except that plasmids were initially propagated in E. coli NEB 10-beta. Sequence- verified plasmids were electroporated into V. paradoxus CL014 ΔHS33, Polaromonas MF047, and Paraburkholderia MF376, as described below. Strains were cultured in 50% TSB at 28 °C with shaking (250 rpm) for 2 days, followed by 24 h incubation at 4 °C. Cells were harvested (5,000 × g, 10 min, 4 °C), washed twice with ice-cold sterile water, and resuspended in 10% sterile glycerol for electroporation. For each reaction, 100 ng of plasmid DNA was electroporated into 100 μl of competent cells using a 0.1 cm gap cuvette using the following conditions: 1,800 V, 25 μF, 200 Ω for marR73 iadCDE, and 2,500 V, 25 μF, 200 Ω for marR73 iadC-K2. After electroporation, cells were recovered in SOC medium (New England Biolabs, B9020) at 28 °C (250 rpm) for 3 h and plated on 50% TSB agar with selective antibiotics: ampicillin (100 μg / mL) + kanamycin (200 μg / mL) for Polaromonas MF047 and kanamycin (50 μg / mL) for Paraburkholderia MF376. After 4–5 days of incubation, single colonies were screened by colony PCR using crude DNA extracted with alkaline lysis buffer. PCR-confirmed integrants were grown overnight in 50% TSB with 1 mM IPTG, supplemented with ampicillin (100 μg / mL) for Polaromonas MF047 and without antibiotics for Paraburkholderia MF376. Cultures were then diluted 1,000-fold and plated on sucrose counter-selection agar to induce second recombination (5% w / v sucrose for Polaromonas MF047; 20% w / v sucrose for Paraburkholderia MF376). Double-crossover mutants were confirmed by PCR and Sanger sequencing. Liquid chromatography -mass spectroscopy (LC–MS) metabolomics. Sample preparation. V. paradoxus strains were streaked from glycerol stocks onto 50% TSB agar plates supplemented with appropriate antibiotics: ampicillin (100 μg / mL) for PRINCETON-103476 knockout mutants and wildtype, and ampicillin (100 μg / mL) plus kanamycin (50 μg / mL) for overexpression mutants. Plates were incubated at 28 °C for 3 days. Single colonies were inoculated into 5 mL of 50% TSB containing the appropriate antibiotics and cultured at 28 °C with shaking at 250 rpm for 48 h. Bacterial cultures were harvested and washed following the root growth inhibition assay protocol, then resuspended in 5 mL of modified M9 medium supplemented with 15 mM succinic acid to a final OD₆₀₀ of 0.05. Cultures were incubated at 28 °C, 250 rpm for 15 h, after which IAA or deuterium-labeled IAA ([2H7]IAA, DLM-8040- 0.1, Cambridge Isotope Laboratories) was added to a final concentration of 0.1 mg / mL. Cultures were incubated for an additional 4 h. Cells were collected at a total biomass of OD₆₀₀ × volume (mL) = 2, centrifuged at 5,000 × g for 10 min, and pellets were resuspended in 400 μl of cold quenching solvent (acetonitrile : methanol : water, 40:40:20, v / v / v). Samples were stored at −80 °C prior to metabolite extraction and LC–MS / MS analysis. LC-MS analysis. LC-MS analysis was performed on a Vanquish UHPLC system (Themo Fisher Scientific) coupled to a quadrupole Orbitrap Exploris 480 mass spectrometer. LC separation of polar metabolites was achieved using a Waters XBridge BEH Amide column (2.1 mm × 150 mm, 2.5-μm particle size, 130-Å pore size). The LC method has a 25-min solvent gradient at a flow rate of 150 μL / min, with the following gradient parameters: 0 min, 90% B; 2 min, 90% B; 3 min, 75%; 7 min, 75% B; 8 min, 70%, 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 14 min, 25% B; 16 min, 0% B, 20.5 min, 0% B; 21 min, 90% B; 25 min, 90% B, where Solvent A was 95:5 water : acetonitrile with 20 mM ammonium hydroxide and 20 mM ammonium acetate (pH 9.4) and solvent B was acetonitrile. The autosampler temperature was 4 °C, the column temperature was 25 °C, and the injection volume was 10 μl. The Exploris 480 mass spectrometer was operated in full scan mode in negative polarity on MS1 level, which allows the relative quantitation of the metabolite across by ion count. Following parameters are used for the full scan: resolution, 120,000; scan range, m / z 70-1000 (negative mode); AGC target, 1e6; ITmax, 500 ms. Other instrument parameters are spray voltage 3000 V, sheath gas 35 (Arb), aux gas 10 (Arb), sweep gas 0.5 (Arb), ion transfer tube temperature 300 °C, vaporizer temperature 35◦C, internal mass calibration on, RF lens 50. The MS2 spectra were collected in targeted mode using the parallel reaction monitoring (PRM) function at higher energy C-trap dissociation (HCD) energy of 20eV, and other instrument settings as following: resolution 30,000, AGC target 1e6, maximum injection time 250 ms, and isolation window 1.0 m / z. For the MS1 data analysis, raw LC–MS data were converted to mzXML format using ProteoWizard. Peak picking was performed with EL-Maven PRINCETON-103476 (v0.12.1-beta; Elucidata) for unlabeled and2H-labeled compounds, and MAVEN (v2.10.14c) for18O-labeled compounds. Relative abundance changes of each metabolite were quantified using relative peak area tops in the chromatogram. For2H-labeled data analysis, natural isotope abundance was corrected using the AccuCor R package (accessible at github.com / lparsons / accucor). For18O-labeled data analysis, isotope correction was performed using the Iso-Autocorr package (accessible at github.com / xxing9703 / Iso-Autocorr). For the MS2 data, raw LC–MS files were processed and peaks were extracted with the built-in Xcalibur Qual Browser (Thermo Scientific, v4.4). IadDE protein expression, purification and crystallization Protein expression. The iadD and iadE genes (IMG gene IDs: 2643613669, 2643613668) from V. paradoxus CL014 (IMG genome ID: 2643221508) were cloned into the pET28b expression vector with an N-terminal His-tag and transformed into E. coli BL21(DE3) for recombinant protein expression. Transformants were plated on LB agar supplemented with kanamycin (50 μg / mL) and chloramphenicol (33 μg / mL) and incubated at 37 °C for 24 h. Individual colonies were picked and grown overnight in LB medium at 37 °C with shaking at 250 rpm. Overnight cultures were diluted 1:1000 into autoinduction (AI) medium (ZYM- 5052)44containing the same antibiotics, and incubated at 37 °C, 250 rpm for 22 h. Cells were harvested by centrifugation at 5,000 × g for 20 min at 4 °C, and pellets were collected and stored at –80 °C for subsequent protein purification. Protein purification. Cell pellets were resuspended in IMAC Buffer A (20 mM NaH₂PO₄, 500 mM NaCl, pH 7.4) at a ratio of 1 g pellet per 10 mL buffer. To reduce viscosity, 0.1 μL benzonase nuclease (Sigma-Aldrich, 70746) was added per gram of cell pellet. Cells were lysed using an EmulsiFlex-C5 high-pressure homogenizer (three passes), and the lysate was clarified by centrifugation at 25,000 × g for 30 min at 4 °C. The supernatant was filtered through a 0.22 μm syringe filter and loaded onto a 5 mL Ni-charged Nuvia IMAC column (Bio- Rad) using a fast protein liquid chromatography (FPLC) system (Bio-Rad). The column was washed with IMAC Buffer A containing 15 mM imidazole, and bound proteins were eluted with IMAC Buffer B (20 mM NaH₂PO₄, 500 mM NaCl, 500 mM imidazole, pH 7.4). Eluted fractions were analyzed by SDS–PAGE (Bio-Rad Stain-Free gels), pooled, concentrated, and buffer-exchanged into 50 mM Tris-HCl (pH 7.0), 150 mM NaCl using 10 kDa MWCO concentrators (Pierce, Thermo Fisher). Purified protein was stored at 4 °C, and concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). PRINCETON-103476 Protein crystallography. Purified IadDE was concentrated to 10 mg / mL in buffer supplemented with 1 mM dithiothreitol (DTT) and crystallized using the sitting-drop vapor diffusion method at 20 °C. Crystallization drops were prepared by mixing 1.5 μl of protein solution with 1.5 μl of reservoir solution in 24-well sitting-drop plates. The reservoir solution contained 9.6–10.6% (w / v) PEG 3350 and 0.1 M sodium citrate tribasic dihydrate (pH 5.5). Crystals were cryoprotected by brief soaking in the reservoir solution supplemented with 30% (v / v) ethylene glycol and subsequently flash-cooled in liquid nitrogen for data collection. Diffraction data were collected at beamlines 17-ID1 (AMX) and 17-ID2 (FMX) at Brookhaven National Laboratory to a maximum resolution of 1.28 Å. Crystals grew in space group H3 (hexagonal setting of R3) with typical cell dimensions a=b=130.5 Å c=100.7 Å α=β=90oγ=120owith one complex per asymmetric unit. Data were processed with XDS45and scaled with AIMLESS. The structure was determined by the method of molecular replacement using the program PHASER utilizing sequential placement of the two subunits with the models derived from AlphaFold models. The structure was rebuilt in COOT, incorporating a [2Fe–2S] cluster and a mononuclear iron-binding site, and subsequently refined using PHENIX.REFINE. The final model showed good agreement with the experimental data and displayed excellent geometry. The final model and associated X-ray data have been deposited with the Protein Data Bank with code 9O71. Measurement of IAA degradation. IAA degradation by bacterial strains was assessed using a spike-in approach. Bacterial preparation followed the same procedure as the root growth inhibition assay, including streaking from glycerol stocks, cultivation, washing, and OD₆₀₀ measurement. Washed cells were inoculated into M9 medium (3 g / L KH₂PO₄, 0.5 g / L NaCl, 6.78 g / L Na₂HPO₄, and 1 g / L NH₄Cl), supplemented with 2 mM MgSO₄, 0.1 mM CaCl₂, 10 μM FeSO₄, and 5 g / L glucose, to a final OD₆₀₀ of 0.05. Cultures were incubated at 28 °C with shaking (250 rpm) for 15 h before IAA was spiked-in to a final concentration of 0.1 mg / mL. Aliquots (300 μl) were collected at 2 h or 4 h intervals, centrifuged at 5,000 × g for 10 min, and 50 μl of the supernatant was mixed with 100 μl of freshly prepared Salkowski reagent (10 mM FeCl₃ and 35% perchloric acid). After incubation for 40 min at room temperature, absorbance was measured at 530 nm using a BioTek Synergy H1 microplate reader. As Polaromonas MF047 is an amino acid auxotroph and cannot grow in minimal medium, this strain and its engineered strains were assayed in 50% TSB medium instead of M9. All strains were tested in three biological replicates to ensure reproducibility. PRINCETON-103476 Bacterial growth curve. Bacterial preparation followed the same protocol as the root growth inhibition assay. Washed cells were inoculated into 200 μl of either M9 minimal medium or 50% TSB medium at a final OD₆₀₀ of 0.05. Each strain was tested in three biological replicates to ensure reproducibility. Cultures were grown in sterile 96-well cell culture plates, sealed with Breathe-Easy gas-permeable film (Diversified Biotech), and incubated at 28 °C with continuous linear shaking. Optical density at 600 nm (OD₆₀₀) was recorded every 2 h over a 24-hour period using a BioTek Synergy H1 microplate reader to monitor bacterial growth dynamics. Root growth inhibition assay Seedling and seed preparation. Arabidopsis thaliana Col-0 seeds were surface- sterilized by vortexing in 70% bleach containing 0.2% Tween-20 for 10 min, followed by five washes with sterile distilled water. Seeds were sown on half-strength Murashige and Skoog (MS) agar medium (2.22 g / L MS basal medium with Gamborg vitamins (PhytoTech Labs, M- 404), 0.5 g / L MES, 5 g / L sucrose, 10 g / L agar, pH 5.7 adjusted with 3 M NaOH) in 12 × 12 cm square plates and grown vertically under short-day conditions (21 °C day / 18 °C night, 10 h light / 14 h dark, 70% relative humidity, 170 μmol m⁻² s⁻¹ light intensity) for 7 days. Medicago seeds were sterilized following published protocols50,51. Briefly, seeds were treated with concentrated sulfuric acid for 10 min with agitation, rinsed once with sterile water, then treated with 70% bleach for 3 min. After five additional washes, seeds were imbibed in sterile water for 2–6 h at room temperature prior to use. IAA-containing plate preparation. Half-strength MS agar medium was autoclaved and cooled until warm to the touch. IAA stock solutions (1 mM or 100 mM in 100% ethanol) were added to final concentrations of 100 nM, 1 μM, or 10 μM. Stocks were stored at –20 °C for up to two months. Bacterial and SynCom32 preparation. Bacterial strains were revived from 20% glycerol stocks by streaking onto 50% TSB agar plates (15 g / L tryptic soy broth, 20 g / L agar) and incubated at 28 °C for 3–4 days. Single colonies were inoculated into 5 mL 50% TSB and grown for 2 days at 28 °C with shaking (250 rpm). Cultures were pelleted at 5,000 × g for 10 min, washed twice with 3 mL of sterile 10 mM MgCl₂, and resuspended in 750 μl of the same buffer. The optical density at 600 nm (OD₆₀₀) was measured using a NanoDrop One C with semi-micro cuvettes and adjusted to 0.05. For SynCom32 assembly, each strain’s OD₆₀₀ PRINCETON-103476 was measured individually. To ensure equal biomass contribution, volumes were calculated using the equation: OD₆₀₀ × volume (μl) = 300, and combined accordingly. The pooled culture was washed and resuspended as described above. Bacterial inoculation. For plant-microbe co-inoculation assays, 100 μl of strain (OD₆₀₀ = 0.05) was evenly spread onto the surface of half-strength MS agar plates. For SynCom32, 100 μl of the pooled culture (OD₆₀₀ = 0.05) was applied, followed by 100 μl of V. paradoxus CL014, Polaromonas MF047, Paraburkholderia MF376, or their engineered strains at OD₆₀₀ = 0.005. Plates were incubated overnight at room temperature prior to seedling transfer. Plant growth and measurement. Seven-day-old Arabidopsis seedlings or sterilized Medicago seeds were transferred onto pre-inoculated plates. Plates were sealed with 3M Micropore tape and incubated vertically in a growth chamber under short-day conditions for 7 days. For 16S rRNA amplicon sequencing, plants were grown for 9 days to allow sufficient biomass for DNA extraction. Plates were imaged using a digital camera, and primary root elongation was measured as the distance from the initial to final root tip position using the freehand line tool in ImageJ. Microbiome analysis Sample collection. After nine days of co-incubation with bacteria, roots from Arabidopsis and Medicago were harvested for microbiome profiling. Each sample consisted of 5–10 roots pooled per plate, with 3–5 biological replicates per treatment. Roots were transferred to 15 mL Falcon tubes containing 7 mL sterile water and washed by vigorous vortexing. Excess water was removed using sterile filter paper, and dried roots were transferred to Lysing Matrix E tubes (MP Biomedicals) and stored at –80 °C until DNA extraction. For SynCom32 input controls, bacterial cultures were pelleted at 5,000 × g for 10 min, the supernatant was discarded, and pellets were stored at –80 °C. DNA extraction. Samples were homogenized using a FASTPREP-24™ 5G instrument (MP Biomedicals) with two 40 s cycles at 6.0 m / s for root samples, and one cycle for SynCom32 input controls, with 2 min on ice between runs to prevent overheating. DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals), eluted in 55 μl DES buffer (provided in the kit), quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher), and diluted to 3.5 ng / μl with DES buffer. 16S rRNA library preparation and sequencing. The V5–V7 region of the 16S rRNA gene was amplified using a two-step dual-indexed PCR approach. The first PCR was performed PRINCETON-103476 using Platinum SuperFi II PCR Master Mix (Thermo Fisher) in a 25 μl reaction containing: 12.5 μl Master Mix, 7 μl nuclease-free water (Qiagen), 2 μl of 5 μM forward primer, 1 μl of 10 μM reverse primer, and 2.5 μl of 3.5 ng / μl DNA template. Thermal cycling conditions were: 98 °C for 30 s, followed by 30 cycles of 98 °C for 10 s, 55 °C for 10 s, and 72 °C for 15 s, with a final extension at 72 °C for 5 min. Three technical PCR replicates were pooled per sample to minimize amplification bias. Products were verified by 1.2% agarose gel electrophoresis. For gel purification, 25 μl of each PCR product (two samples per lane) were pooled with 10 μl 6× loading dye, run on a 1.2% agarose gel, and ~400 bp bands were excised and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). DNA concentration was assessed by PicoGreen, and 100 ng of each sample was pooled for library construction and cleaned with 0.9× AMPure XP beads (Beckman Coulter). A second PCR was performed to add Illumina sequencing adapters. Final libraries were sequenced on an Illumina MiSeq platform (2 × 300 bp paired-end reads). After quality filtering, a total of 14,907,735 high-quality sequences were obtained from 175 samples, with an average of 85,187 reads per sample. Amplicon data processing. Raw reads were demultiplexed in QIIME2 (v2024.10) using qiime cutadapt demux-paired, and primer sequences were trimmed with qiime cutadapt trim- paired. Denoising, quality filtering, and chimera removal were performed with DADA2 (qiime dada2 denoise-paired). After testing multiple truncation lengths, forward reads were truncated at 220 bp and reverse reads at 180 bp to optimize read retention and ASV diversity. ASVs were taxonomically classified using a naïve bayes classifier trained on a custom database of plant- associated bacterial sequences via qiime feature-classifier classify-sklearn. Assignments were filtered based on confidence scores, prevalence, and relative abundance. Relative abundance tables were generated using qiime feature-table relative-frequency and merged with taxonomy and metadata for visualization in R using ggplot2. Beta diversity was calculated using Bray– Curtis dissimilarity (R package vegan, vegdist), followed by principal coordinate analysis (PCoA; cmdscale). Differences in community composition were assessed by PERMANOVA using adonis2. Differential abundance analysis was conducted using the Mann–Whitney U test with FDR correction. Log2fold changes were calculated, and heatmaps were visualized using pheatmap (R v4.4.2). Plant growth promotion assay in natural soil. Soil was collected from the Stony Ford Research Station (Princeton, NJ, USA), where no chemical fertilizers, pesticides, or plants had been applied or grown in recent years. The soil was sieved twice to remove rocks and plant PRINCETON-103476 debris, then distributed into pots placed within 9 × 13-inch aluminum foil trays. Pots were saturated overnight with 1.2 L of sterile water containing bacterial inoculum. Bacterial preparation followed the same protocol as the root growth inhibition assay, including streaking, cultivation, washing, and OD₆₀₀ measurement. Individual bacterial strains were suspended in 1.2 L of sterile water to a final OD₆₀₀ of 0.03 prior to soil application. Each treatment included four biological replicates (pots). Arabidopsis Col-0 seeds were surface sterilized as described above and placed directly onto the surface of inoculated soil. Pots were maintained under short- day conditions in a growth chamber and watered with 600–800 mL of distilled water every 4 days. After 33 days, above-ground tissues were harvested and shoot fresh weight was measured using an analytical balance. Referring to FIG.27A-27C, growth curves of various strains can be seen. It is clear there are no growth defects in the engineered strains. A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

Claims

PRINCETON-103476 CLAIMS 1. An engineered nucleic acid sequence comprising a gene cassette, the gene cassette comprising: one or more regulator genes, the one or more regulator genes including an iadR gene; and at least three catabolic pathway genes downstream from the one or more regulator genes, the at least three catabolic pathway genes including an iadC gene, an iadD gene, and an iadE gene; wherein the engineered nucleic acid sequence is free of an iadA gene and iadB gene.

2. The engineered nucleic acid sequence of claim 1, wherein the one or more regulator genes consist of the iadR gene.

3. The engineered nucleic acid sequence of claim 1 or 2, wherein the at least three catabolic pathway genes consist of the iadC gene, the iadD gene, and the iadE gene.

4. The engineered nucleic acid sequence of claim 1, wherein the at least three catabolic pathway genes include the iadC gene, the iadD gene, and the iadE gene, and at least one additional catabolic pathway gene.

5. The engineered nucleic acid sequence of claim 4, wherein the at least one additional catabolic pathway gene includes an iadF gene, an iadG gene, an iadH gene, and iadI gene, and / or an iadJ gene.

6. The engineered nucleic acid sequence of claim 4, further comprising a transporter component, the transporter component including an iadK2 gene.

7. The engineered nucleic acid sequence of claim 4, wherein the at least three catabolic pathway genes consist of the iadC gene, the iadD gene, the iadE gene, an iadF gene, an iadG gene, an iadH gene, an iadI gene, and an iadJ gene, and wherein the engineered nucleic acid sequence further comprising a transporter component, the transporter component including an iadK2 gene.PRINCETON-103476 8. The engineered nucleic acid sequence of claim 4, wherein a reporter gene is located after the iadE gene.

9. The engineered nucleic acid sequence of claim 8, wherein the reporter gene is located between the iadE gene and an iadF gene.

10. The engineered nucleic acid sequence of claim 8, wherein the reporter gene is a gene for a fluorescent protein.

11. The engineered nucleic acid sequence of claim 1, wherein the gene cassette further comprises a non-coding region IadR binds to.

12. A method of detecting auxin generation, comprising: introducing an engineered nucleic acid sequence of any one of claims 8 to 10 to an auxin- generating system; allowing the auxin-generating system to generate an auxin; and detecting a response of the at least one regulator gene by detecting a chemical product of the reporter gene reporter protein expressed by the reporter gene.

13. A bacteria cell comprising an engineered nucleic acid sequence of any one of claims 1 to 11, wherein the bacteria cell is capable of degrading auxins.

14. The bacteria cell of claim 13, wherein the bacteria cell is a plant-associated bacteria cell.

15. The bacteria cell of claim 14, wherein the plant-associated bacteria is a Proteobacteria species, an Actinobacteria species, a Bacteroidetes species, a Firmicutes species, an Acidobacteria species, a Polarimonas species, or a Paraburkholderia species.

16. The bacteria cell of claim 13, wherein the bacteria cell is an amino acid auxotroph.

17. The bacteria cell of claim 13, wherein the bacteria cell is free of all iad genes except those in the engineered nucleic acid sequence.PRINCETON-103476 18. A method for degrading auxin, controlling auxin, and promoting plant growth and root development, comprising: providing a bacteria cell of any one of claim 13 to 17; and allowing the bacteria cell to interact with a plant.

19. The method of claims 18, further comprising applying the bacteria cell to soil containing the plant or to seeds prior to planting.

20. The method of claim 18 or 19, wherein allowing the bacteria cell to interact with the plant promotes plant growth by degrading auxins produced by auxin-producing microorganisms in the rhizosphere of the plant.

21. A viral vector, comprising: one or more regulator genes, the regulator genes including an iadR gene; and at least three catabolic pathway genes downstream from the one or more regulator genes, the at least three catabolic pathway genes including an iadC gene, an iadD gene, and an iadE gene; wherein the viral vector is free of an iadA gene and iadB gene.

22. The viral vector of claim 21, further comprising a non-coding region IadR binds to.

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