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Structural and biochemical characterization of the key components of an auxin degradation operon from the rhizosphere bacterium Variovorax [1]

['Yongjian Ma', 'National Key Laboratory Of Blood Science', 'Key Laboratory Of Immune Microenvironment', 'Disease', 'Ministry Of Education', 'The Province', 'Ministry Co-Sponsored Collaborative Innovation Center For Medical Epigenetics', 'Haihe Laboratory Of Cell Ecosystem', 'Department Of Biochemistry', 'Molecular Biology']

Date: 2023-07

Plant-associated bacteria play important regulatory roles in modulating plant hormone auxin levels, affecting the growth and yields of crops. A conserved auxin degradation (iad) operon was recently identified in the Variovorax genomes, which is responsible for root growth inhibition (RGI) reversion, promoting rhizosphere colonization and root growth. However, the molecular mechanism underlying auxin degradation by Variovorax remains unclear. Here, we systematically screened Variovorax iad operon products and identified 2 proteins, IadK2 and IadD, that directly associate with auxin indole-3-acetic acid (IAA). Further biochemical and structural studies revealed that IadK2 is a highly IAA-specific ATP-binding cassette (ABC) transporter solute-binding protein (SBP), likely involved in IAA uptake. IadD interacts with IadE to form a functional Rieske non-heme dioxygenase, which works in concert with a FMN-type reductase encoded by gene iadC to transform IAA into the biologically inactive 2-oxindole-3-acetic acid (oxIAA), representing a new bacterial pathway for IAA inactivation/degradation. Importantly, incorporation of a minimum set of iadC/D/E genes could enable IAA transformation by Escherichia coli, suggesting a promising strategy for repurposing the iad operon for IAA regulation. Together, our study identifies the key components and underlying mechanisms involved in IAA transformation by Variovorax and brings new insights into the bacterial turnover of plant hormones, which would provide the basis for potential applications in rhizosphere optimization and ecological agriculture.

Data Availability: The atomic coordinates of the structures have been deposited in the Protein Data Bank under accession codes 7YLT (IadK2), 7YLS (IadD/E), 7YLR (IadC) and 8H2T (IadD/E-IAA). The cryo-EM map of IadD/E in complex with IAA has been deposited to the Electron Microscopy Data Bank under the corresponding accession code EMD-34443. Source data are available in S1 Data .

In this study, we have focused on 10 genes around an overlapped region of 2 genomic fragments of iad operon ( Figs 1A and S1A ), which were found sufficient for enabling IAA degradation and/or RGI reversion when transformed to non-IAA-degrading bacteria [ 14 ]. Using biochemical and structural approaches, we have elucidated the molecular mechanism of IAA transformation by the Variovorax operon. Two proteins, IadK2 andIadD, were found to directly associate with IAA. IadK2, featured with a two-lobed structure, was identified as an ATP-binding cassette (ABC) transporter solute-binding protein (SBP) that binds specifically and strongly with IAA, most likely mediating IAA uptake from the environment. Combined biochemical, structural, and mass spectrometry studies revealed that IAA was transformed to the biologically inactive 2-oxindole-3-acetic acid (oxIAA) by the two-component IadC-IadD/IadE dioxygenase-reductase system encoded by the iad operon in Variovorax. This is consistent with a latest report published during the preparation of this manuscript [ 18 ]. Further, we demonstrated that such an IAA-degradation property of Variovrax could be transplanted to Escherichia coli by the transformation of a minimum gene set containing iadC/D/E. Together, our results uncover the major underlying molecular mechanism of IAA turnover by a new auxin degradation gene cluster in Variovorax, providing guidance for potential plant microbiota manipulation and optimization for ecological farming.

More recently, a new auxin degradation operon has been reported for strains of Variovorax, which actively guides IAA degradation and readily counteracts RGI induced by bacterially produced IAA in plant microbiomes [ 14 ], indicating distinguishing root growth promoting traits of the Variovorax strains. Variovorax is a gram-negative bacteria genus in the family Comamonadaceae, commonly found in the rhizosphere and regulating IAA levels [ 17 ]. The auxin IAA degradation (iad) operon is found highly conserved among strains of Variovorax and unique to the Variovorax genus [ 14 , 18 ]. However, transformation of non-IAA-degrading bacteria, such as Acidovorax root219, with genomic fragments of the iad operon could generate gain-of-function strains, capable of IAA degradation and rescuing RGI [ 14 ]. Notably, the iad operon shares limited homology with the iac and iaa gene clusters, representing a new auxin IAA-degradation pathway. Dissecting and defining the functional mechanism of auxin degradation by Variovorax is therefore of significant importance for potential applications in ecological and stress agricultures [ 11 ].

Plants are associated with millions of microorganisms. The plant–microbiota interaction serves as an important layer of auxin gradient regulation, tuning plant development, phenotypes, and fitness [ 7 , 8 ]. For instance, most rhizobacteria are found capable of producing IAA [ 7 , 9 ], which works in conjunction with IAA synthesized in plants to stimulate the growth of primary and lateral roots within an optimal concentration range but cause root growth inhibition (RGI) at higher concentrations [ 7 , 10 ]. Apart from the IAA-synthesizing bacteria, some plant-associated bacteria could also degrade IAA [ 7 , 11 ], which could serve as a down-regulating scheme of both bacterially produced and endogenous IAA to fine tune the IAA concentration gradients. Elucidating the underlying mechanism of bacterial IAA regulation would help to maximize the beneficial effects of auxin production or degradation for ecological agriculture [ 12 – 14 ]. Two gene clusters, iac and iaa, were previously discovered responsible for aerobic and anaerobic degradation of IAA, respectively [ 15 , 16 ].

Auxin is a vital phytohormone in plants, affecting almost all the aspects of plant’s life. In the meristems, auxin functions as a regulator of cell division, elongation, and differentiation, determining the growth and architectures of shoots and roots [ 1 ]. Indole-3-acetic acid (IAA) is a highly abundant, natively synthesized auxin in plants. IAA is not uniformly distributed but instead locally synthesized and transported directionally via the polar distributed PIN and PIN-like transporters in plants [ 2 , 3 ]. The resultant IAA concentration gradients instruct plant development and are therefore dedicatedly regulated in synthesis, inactivation, and degradation [ 4 – 6 ].

Results

IadK2 directly associates with IAA To understand the molecular mechanism of IAA turnover by Variovorax iad operon, we first set out to identify the components responsible for IAA binding (Fig 1A and 1B). SBPs located in the periplasm work in concert with ABC transporters to transport a variety of compounds into bacteria [19,20]. The iad operon is predicted to encode 2 SBPs, IadK2 and IadK3 (Figs 1A and S1A), which could potentially mediate the uptake of IAA in the rhizosphere. To determine whether IadK2 and IadK3 could bind with IAA, we expressed and purified the proteins for isothermal titration calorimetry (ITC) measurements. IadK2 could engage IAA with nanomolar affinity, whereas no obvious binding was detected for IadK3 and IAA (Figs 1C and S1B). To further understand the specificity of IadK2, we also tested the binding of IadK2 with other endogenous and synthetic auxins including 4-chloroindole-3-acetic acid (4-Cl-IAA), indole-3-butyric acid (IBA), indole-3-propionic acid (IPA), 1-naphthylacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) (Fig 1B). IadK2 could bind with most bicyclic analogs but not the single aromatic ring 2,4-D, indicating a preference for bicyclic compounds (Fig 1D). To test this hypothesis, we further measured the binding between IadK2 and phenylacetic acid (PAA) composed of a single aromatic ring and found indeed no binding between IadK2 and PAA (S2A Fig). Additionally, IadK2 associates with the bicyclic auxins and analogs with substantially reduced affinities in comparison to IAA (Fig 1D), suggesting the specificity of IadK2 for IAA. This is further supported by the results of the thermal shift assay (TSA), where IAA displayed the most significant stabilization effect on IadK2 (Fig 1E). Interestingly, neither IAA nor the auxin analogs could bind to IadK3 (S1B and 1C and S2B Figs), suggesting a distinct substrate specificity for IadK3. Together, these data suggest that IadK2 is an IAA-specific SBP in Variovorax. The high affinity of IadK2 for IAA may facilitate efficient IAA uptake by Variovorax from the environment [21–23]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. IadK2 encoded by the Variovorax iad operon selectively binds with auxin IAA. (A) Schematics for the auxin IAA-degradation (iad) operon in Variovorax paradoxus CL14. Ten genes ranging from IMG gene ids 2643613661–2643613670 are illustrated. (B) Chemical structures of IAA and auxin analogs. IAA is the most dominant natural auxin in most plants. Other endogenous or synthetic auxins or analogs such as 4-Cl-IAA, IBA, IPA, NAA, and 2,4-D are also displayed. (C) ITC measurement of IAA binding to iadk2. The binding isotherm for iadk2-IAA is biphasic, revealing 2 dissociation constants. Three independent measurements were performed and the Kd values are shown as mean ± SEM. (D) ITC measurements of auxin analogs to iadk2. The binding of other auxins and analogs to iadk2 is distinct from IAA with a single-phase isotherm and substantially reduced binding affinity. ND: no binding detected. Kd values are shown as mean ± SEM from 3 or more independent measurements. (E) TSA for iadk2 with IAA and analogs. The orthogonal TSA experiments were performed for iadk2 with different auxins and analogs to further determine the binding specificity. Three replicates of each TSA experiments were performed. Error bars represent the standard deviations. Source data for C–E can be found in S1 Data. IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; IPA, indole-3-propionic acid; ITC, isothermal titration calorimetry; NAA, 1-naphthylacetic acid; TSA, thermal shift assay; 4-Cl-IAA, 4-chloroindole-3-acetic acid. https://doi.org/10.1371/journal.pbio.3002189.g001

Metabolite identification of IAA degraded by the IadD/E-IadC dioxygenase system Next, we set out to identify the product of IAA transformed by this new dioxygenase system. To this end, we performed the high-performance liquid chromatography (HPLC) and the high-resolution mass spectrometry (HRMS) analyses. IAA was treated in vitro with the IadD/E-IadC enzymes till complete consumption of IAA. The reacted mixture was first denatured and analyzed using HPLC. A new peak with a retention time of 5.4 min was observed accompanied with the disappearance of the IAA peak (Figs 5A and S9). Fractions from this potential product peak were then collected and subjected to the HRMS analysis (Fig 5B). Two possible products were proposed based on the HRMS spectra, including the 5-hydroxy indole-3-acetic acid (5-HIAA) and the oxIAA. IAA develops the pink color with the Salkowski reagent [31], which turned colorless after transformation by IadD/E-IadC (Fig 4B), indicating a product unreactive with Salkowski reagent. It therefore ruled out 5-HIAA as the potential product as that a dark gray color was observed after mixing 5-HIAA with the Salkowski reagent and 5-HIAA could be further degraded by IadD/E-IadC (Figs 5C and S8). By contrast, oxIAA was found unable to react with the Salkowski reagent (Fig 5C). Furthermore, the product peak overlapped with that of oxIAA and the peak intensity increased following the addition of oxIAA to the reacted mixture in the HPLC assays (S9 Fig); the adsorption spectrum of the reacted mixture also overlaid with that of oxIAA (Fig 5D), both supporting oxIAA as the product. PPT PowerPoint slide

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TIFF original image Download: Fig 5. The IadD/E-IadCdioxygenase-reductase system converted IAA to oxIAA. (A) HPLC analysis of IAA before (in pink) and after transformation (in blue) by iadd/E-iadc. (B) HRMS analysis of the transformed products of IAA by iadd/E-iadc. The elution for potential product peak in HPLC was collected and analyzed by HRMS. Characteristic peaks for oxiaa were identified, which agreed with the calculated mass. Source data for A and B can be found in S1 Data. (C) Color development of IAA and analogs in Salkowski reagent. The listed compounds (0.5 mm) developed different colors after mixing with the Salkowski reagent. (D) The absorbance spectra of IAA, oxiaa, and the products by iadd/E-iadc. The spectrum scan for IAA, oxiaa, and transformed IAA by the iadd/E-iadcafter mixing with the Salkowski reagent was performed. (E) The mechanism of IAA transformation by iadd/E-iadc. The iadd/E-iadcdioxygenase system catalyzes the incorporation of oxygen atoms to IAA from O 2 , leading to the formation of 2-hydroxyindole-3-acetic acid, which potentially primarily exist as the stable “keto” form, oxiaa. HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; IAA, indole-3-acetic acid. https://doi.org/10.1371/journal.pbio.3002189.g005 Domains of IadD/E and IadC would reconstitute the electron transport chain of Rieske dioxygenase, where electrons from NADH in NAD are passed to the [2Fe-2S] cluster by FMN bound to FAD of IadC followed by transporting to the active site of IadD through the [2Fe-2S] clusters of IadD/E, catalyzing the incorporation of oxygen atoms of O 2 into IAA [28–30,32]. Indeed, in the isotopic experiment with 95% H 2 O18 and 16O 2 , we found most oxIAA with m/z = 192.0655 containing 16O-bearing amide, indicating the oxygen was from 16O 2 (S10 Fig). Together, these data suggest that IAA was converted to oxIAA by the dioxygenase system composed of the IadD/E dioxygenase and the IadC reductase (Fig 5E). Interestingly, a recent study also reported the catalyzed conversion of IAA to 2-hydroxyindole-3-acetic acid intermediate by the iad operon in bacteria [18], which is chemically unstable and would form the stable “keto” form, oxIAA, as shown in our study.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002189

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