Metabolomics and Dual RNA-Sequencing on Root Nodules Revealed New Cellular Functions Controlled by Paraburkholderia phymatum NifA

Abstract

Paraburkholderia phymatum STM815 is a nitrogen-fixing endosymbiont that nodulate the agriculturally important Phaseolus vulgaris and several other host plants. We previously showed that the nodules induced by a STM815 mutant of the gene encoding the master regulator of nitrogen fixation NifA showed no nitrogenase activity (Fix−) and increased in number compared to P. vulgaris plants infected with the wild-type strain. To further investigate the role of NifA during symbiosis, nodules from P. phymatum wild-type and nifA mutants were collected and analyzed by metabolomics and dual RNA-Sequencing, allowing us to investigate both host and symbiont transcriptome. Using this approach, several metabolites’ changes could be assigned to bacterial or plant responses. While the amount of the C4-dicarboxylic acid succinate and of several amino acids was lower in Fix− nodules, the level of indole-acetamide (IAM) and brassinosteroids increased. Transcriptome analysis identified P. phymatum genes involved in transport of C4-dicarboxylic acids, carbon metabolism, auxin metabolism and stress response to be differentially expressed in absence of NifA. Furthermore, P. vulgaris genes involved in autoregulation of nodulation (AON) are repressed in nodules in absence of NifA potentially explaining the hypernodulation phenotype of the nifA mutant. These results and additional validation experiments suggest that P. phymatum STM815 NifA is not only important to control expression of nitrogenase and related enzymes but is also involved in regulating its own auxin production and stress response. Finally, our data indicate that P. vulgaris does sanction the nifA nodules by depleting the local carbon allocation rather than by mounting a strong systemic immune response to the Fix− rhizobia.

A novel function of the key nitrogen-fixation activator NifA in beta-rhizobia: Repression of bacterial auxin synthesis during symbiosis

“…Out of all detected metabolites, we focused on the ion counts belonging to IAM and IAA. As we previously reported, plants inoculated with P. phymatum wild-type produced significantly higher amounts of IAM and IAA than non-inoculated plants (Bellés-Sancho et al, 2021a), while the levels of these two auxins in the nifA mutant were found elevated compared to the wild-type nodules (Figure 5; Bellés-Sancho et al, 2021a). The amount of IAM and IAA in nodules induced by the single iaaMH and double nifA-iaaMH mutant decreased to the same levels as observed in non-inoculated roots, suggesting that bacterial auxin production accounted for the main portion of IAA and IAM found in wild-type and nifA nodules (Figure 5).…”

“…In analogy to lateral root development, such sites might be genetically or hormonally primed to respond to internal auxin signaling. To confirm that the nodular levels of auxinics we previously measured (Bellés-Sancho et al, 2021a) originated from bacterial production under NifA-IaaMH regulation, we estimated the amount of indolics in our strains after planktonic growth (Figure 7F). In comparison to wild-type P. phymatum that produced traces of indolics, the nifA mutant synthetized considerable amounts (37.9 µg/ml).…”

“…Furthermore, a combined metabolomics and dual RNA-sequencing analysis of common bean nodules induced by a P. phymatum nifA mutant, revealed that NifA down-regulates the expression of P. phymatum ’s auxin genes iaaM and iaaH thus leading to IAM and IAA accumulation. In contrast to the above-mentioned studies on legume symbioses with different rhizobial nifA mutants, the loss of NifA in P. phymatum did not trigger an immune response against the mutant bacteria ( Bellés-Sancho et al, 2021a ).…”

“…The metabolite abundance in P. vulgaris root nodules inoculated with different P. phymatum auxin-mutant strains was compared. As previously described, approximately 30 mg of nodules or uninfected roots were immediately frozen with liquid nitrogen and metabolites were extracted using cold methanol ( Lardi et al, 2016 , 2018 ; Bellés-Sancho et al, 2021a , b ). Three independent biological inoculants were processed with one or two plants per inoculum.…”

“…The estimation of the amount of auxinic compounds secreted by P. phymatum wild-type, nifA mutant, iaaMH mutant and the corresponding iaaMH complemented strains, was performed as previously described ( Bellés-Sancho et al, 2021a ). Briefly, overnight cultures were routinely grown in LB-NaCl and cells were collected by centrifugation (5 mins at 5,000 rpm) and washed twice with LB-NaCl.…”