Molecular mechanism of Tsukamurella tyrosinosolvens strain P9 in response to root exudates of peanut

Tsukamurella tyrosinosolvens strain P9 is a rare actinomycete with plant growth-promoting properties and can improve the growth of peanut. We analyzed the differentially expressed genes (DEGs) of P9 under the influence of peanut root exudates from RNA-sequencing data and analyzed the effects of root exudates and their organic acid and amino acid components on the growth and growth-promoting effects of this strain to explore the molecular mechanism of the P9 response. The results showed that peanut root exudates promoted the growth and growth-promoting activity of P9. Transcriptome analysis revealed 126 DEGs in P9, comprising 81 up-regulated and 45 down-regulated genes. The DEGs were significantly enriched in 17 KEGG metabolic pathways, including arginine biosynthesis, butyric acid metabolism, fatty acid degradation, and tryptophan metabolism. Peanut root exudates induced up-regulation of nutrient transport, carbohydrate metabolism and energy production, siderophore and IAA biosynthesis, adhesion, and biofilm formation, and down-regulation of arginine biosynthesis and the urea cycle in P9. Organic acids and amino acids are the major components of peanut root exudates. Glycine, proline, and alanine promoted the growth and IAA secretion of P9. Proline, alanine (40 mM), and oxalic acid significantly enhanced siderophore biosynthesis, whereas citric acid, oxalic acid, and malic acid significantly promoted biofilm formation of P9. This study clarifies the response of T. tyrosinosolvens P9 to peanut root exudates at the molecular level, examining the molecular basis of the relationship between P9 and peanut, and provides a theoretical foundation for improved exertion of the growth-promoting properties of P9.


Introduction
Plant growth-promoting rhizobacteria (PGPR) are a group of beneficial bacteria that inhabit the rhizosphere soil and promote plant growth and stress tolerance. PGPR provide nutrients directly through phosphorus solubilization, nitrogen fixation, and potassium dissolution, and indirectly affect plant growth by producing phytohormones and secreting siderophores; in addition, the strong ability of PGPR to compete for nutrients and ecological niches and to induce systemic resistance in plants can enhance plant resistance to pathogens and tolerance to stress (Pahari et al. 2017). Thus, PGPR have strong potential for practical application to promote plant growth and improve crop yields.
The plant rhizosphere is a complex environment and, when PGPR enter the soil, root exudates directly affect the interaction between PGPR and the plant owing to the unique rhizosphere environment (Alawiye and Babalola  Prashar et al. 2014). As an important factor mediating plant-soil-microbe interactions (González-López et al. 2020), root exudates are the main source of rhizosphere nutrients and create ecological niches for rhizosphere microorganisms (Hassan et al. 2019). In addition, certain compounds in root exudates can act as signaling molecules to alter the gene expression patterns of microorganisms and influence their interactions with the host plant (Kandaswamy et al. 2019;Mark et al. 2005). Although root exudates play a crucial role in the interaction between plants and rhizosphere microorganisms (Badri and Vivanco 2009), the mechanisms and pathways of bacterial gene regulation in response to plant root exudates are poorly known and have been predominantly studied in Bacillus spp. and Pseudomonas spp. (Kandaswamy et al. 2019;Carvalhais 2010;Mavrodi et al. 2021). For example, the expression of genes associated with nutrient metabolism, cell motility, chemotaxis, and biofilm formation in Bacillus amyloliquefaciens SQR9 is significantly elevated under the influence of maize root exudates . Brachypodium root exudates alter the expression of metabolic, transport, regulatory, and stress response genes in Pseudomonas fluorescens (Mavrodi et al. 2021). However, few studies have been conducted on the response of other species of PGPR to root exudates, and the molecular mechanism of the response of strains of Tsukamurella spp. to root exudates has not been investigated to date.
Tsukamurella is a genus of rare actinomycetes with a broad ecological niche that degrade compounds such as cellulose and aromatics (Soler et al. 2018). In our previous study, we first isolated T. tyrosinosolvens strain P9, which has the ability for phosphorus solubilization, nitrogen fixation, indoleacetic acid (IAA) synthesis, and siderophore synthesis, from the rhizosphere soil of tea (Camellia sinensis) plants; this strain can effectively colonize the roots and stems of peanut and has significant growth-promoting effects on peanut (Zhang et al. 2021;Li et al. 2022a), but the effect of peanut roots on this bacterial strain is unknown. Therefore, in the present study, we used transcriptome analysis to examine the gene expression patterns of T. tyrosinosolvens P9 in response to peanut root exudates and examined the effects of root exudates and their components on the growthpromoting characteristics and biofilm-forming properties of this strain. The aim was to further resolve the response mechanism of P9 to peanut root exudates and to lay a foundation for elucidation of the interaction mechanism between P9 and peanut plants. The results will contribute to clarification of the molecular mechanism of the growth-promoting effects of Tsukamurella spp.

Bacterial strain and plant material
Tsukamurella tyrosinosolvens strain P9 was isolated from the rhizosphere soil of tea plants (Guizhou Province, China) (Zhang et al. 2021) and was conserved in the China Center for Type Culture Collection (Strain Collection No. CCTCC AA 2020052). The study plant was peanut (Arachis hypogaea) 'Fuyusilihong.'

Collection and analysis of peanut root exudates
Peanut seeds were sterilized with 20% hydrogen peroxide solution for 20 min, washed in sterile water and soaked for 8 h, then placed in petri dishes containing moistened sterile filter paper, and incubated at 28 °C for 2-3 days to germinate. Seeds of uniform germination were selected and transplanted to pots. Forty peanut seedlings were selected after growth for 30 days. The roots were washed with deionized water and placed in 200 mL sterile deionized water, and the seedlings were incubated at 28 °C under illumination in an incubator. The culture water containing root exudates was collected after 5 days and combined, then filtered, concentrated with a rotary evaporator at 35 °C, filter sterilized, and stored at − 80 °C.
The root exudates were analyzed by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) using an Agilent 7890 gas chromatograph and a Pegasus HT time-of-flight mass spectrometer. The mass spectrometry data were analyzed by peak exaction, baseline calibration, deconvolution analysis, peak integration, and peak alignment using ChromaTOF software (v.4.3x, LECO) and the LECO-Fiehn Rtx5 database.

Effect of peanut root exudates on growth of P9
Tsukamurella tyrosinosolvens P9 stored in 50% glycerol was inoculated into Luria-Bertani (LB) liquid medium and shaken at 150 rpm at 30 °C overnight for activation. The activated P9 culture was inoculated into LB medium for 24 h. Standard LB medium lacking root exudates was used as the control, and the treatments comprised different concentrations (1, 2, 3, and 4%) of root exudates added to LB medium. One milliliter of activated P9 culture was inoculated into 10 mL LB medium and incubated at 30 °C with shaking at 150 rpm for 22 h with three biological replicates. Samples were taken regularly to determine the number of viable bacteria using the plate count method.

Sample preparation and RNA-sequencing analysis
After incubation, the control group (P9_N) and root exudate treatment group (P9_RE) cultures were centrifuged at 4 °C at 3,500 g for 4 min. After removing the supernatant, the cell pellets were frozen in liquid nitrogen for 30 min and then submitted for cDNA library construction and sequencing with an Illumina HiSeq 2500 platform by Novogene Biotechnology Co., Ltd.
After removal of reads containing sequencing adapters, reads containing poly-N, and low-quality reads from the raw data, simultaneously, the GC content, Q20, and Q30 of the clean data were calculated. The resulting high-quality transcriptomic data were used for subsequent analysis.

Gene expression analysis and screening DEGs
The sequencing reads were mapped to the Tsukamurella tyrosinosolvens MH1 (NCBI accession number NZ_ CP019066.1) reference genome using Bowtie2. Sequences with total mapped reads or fragments larger than 70% of the data were selected for subsequent analysis. Gene expression was calculated using the fragments per kilobase of exon model per million mapped fragments (FPKM) method. The threshold FPKM > 1 was used as the criterion for gene expression level.
Differentially expressed genes (DEGs) were analyzed using EdgeR software. The screening criteria for the DEGs were |log 2 (fold change)|> 1 and P value < 0.05. Genes with low expression level and high error rate were filtered out to obtain high-quality DEGs. A KEGG pathway enrichment analysis was performed using the online platform of Novogene Biotechnology Co., Ltd.

Quantitative real-time PCR validation
We selected 14 candidate DEGs for validation by quantitative real-time PCR (RT-qPCR) analysis (Supplementary Table 1). The cDNA was synthesized using the StarScript II RT Kit with gDNA Remover (GenStar). The 16S rRNA gene was used as the internal reference gene. The reaction protocol was as follows: 95 °C for 2 min, then 40 cycles of 95 °C for 15 s, annealing temperature for 30 s, and 72 °C for 30 s. The relative expression level of the selected genes was calculated using the 2 −△△Ct method.

Effect of peanut root exudates on secretion of IAA and siderophores by P9
The activated bacterial suspension was inoculated into LB medium containing 2% root exudates, and LB medium lacking root exudates was used as the control. The cultures were incubated for 24 h with shaking, and the supernatant was retained after centrifugation. The supernatant was mixed with an equal amount of Salkowski's reagent, incubated at 25 °C for 30 min in the dark, then the OD 530nm was measured, and the IAA concentration was calculated (Fierro-Coronado et al. 2014).
A single colony of the activated strain was inoculated into MKB liquid medium containing 2% root exudates, and MKB medium lacking root exudates was used as the control. After culture for 48 h under the same conditions, the supernatant was collected by centrifugation. The chrome azurol S assay was used to detect siderophore content (siderophore units, Su%) = (A r − A s )/A r × 100, where A r is the absorbance of the uninoculated medium supernatant at 630 nm and A s is the absorbance of the sample at 630 nm (Gupta and Gopal 2008).

Effect of peanut root exudate components on the growth and growth-promoting characteristics of P9
Using LB medium for the control group, LB medium containing 500 mg/L l-tryptophan, and LB and MKB medium supplemented with different types of amino acids (proline, glycine, and alanine) and organic acids (citric acid, oxalic acid, and malic acid) were established as the treatment groups under two concentrations (20 mM and 40 mM). After inoculation with activated P9 bacterial suspension and shaking for 24 h (48 h for MKB), the growth of P9 and the ability to secret IAA and siderophores were measured.

Effect of peanut root exudate components on the biofilm-forming properties of P9
The activated bacterial suspension was used to inoculate LB medium containing different concentrations of organic acids and incubated with shaking until OD 600nm = 1.0. The culture was diluted 100-fold and then added to 96-well plates and incubated for 4 days. The culture was discarded, and the planktonic bacteria were gently washed with sterile water, dried naturally, and then stained with 0.5% crystal violet dye for 20 min. After the dye was discarded, 95% ethanol was added, the biofilm was blown with a pipette until it was completely detached, and the OD 600nm was measured after standing for 30 min (Dhale et al. 2014). All experiments were performed with three biological replicates.

Statistical analysis
Data were analyzed using SPSS 20.0 software with Student's t test (two groups) and one-way ANOVA (multiple groups) followed by Tukey's and Waller-Duncan post hoc tests. The significance levels P < 0.05 for significant differences and P < 0.01 for highly significant differences were applied.

Extraction and analysis of peanut root exudate components
Peanut root exudates were collected by using hydroponic culture, filtration sterilization, and GC-TOF-MS to detect the components. The root exudates mainly included organic acids, amino acids, alcohols, sugars, amides, growth factors, esters, aldehydes, pyridines, amines, ketones, nucleotides, and phenolic compounds. The root exudate components with relative content of more than 10% were organic acids and amino acid substances; collectively, organic acids were the predominant components (Supplementary Table 2).

Effect of different concentrations of peanut root exudates on the growth of P9
Dynamic measurement of the growth of P9 revealed that, although the addition of different concentrations of root exudates promoted growth, 2% root exudates significantly increased the viable count of the P9 strain, especially at 22 h in the logarithmic growth phase (Fig. 1). Thus, this concentration was selected for transcriptome sample preparation for the P9 strain. In addition, the initial pH of the medium, as well as the pH of the P9 culture in the early logarithmic growth phase, were slightly higher than that of LB medium after the addition of 2% root exudates, but was essentially identical at 22 h (Table 1).

Illumina sequencing data and DEGs
High-throughput sequencing yielded 14.5 G clean reads of the P9 transcriptome under treatment with root exudates (P9_RE) and the P9 transcriptome in the absence of root exudates (P9_N) (Supplementary Table 3). The proportions of the total sequences matched to the T. tyrosinosolvens MH1 reference genome were 88.44% and 92.12% for P9_RE and P9_N, respectively, indicating that the reference genome was appropriately selected and the data quality was acceptable. Analysis of DEGs showed that there were 126 DEGs under the influence of peanut root exudates, of which 81 genes were up-regulated and 45 genes were down-regulated ( Fig. 2), accounting for 64.29% and 35.71% of the total DEGs, respectively.

KEGG pathway analysis
The KEGG pathway analysis revealed that the DEGs of P9 exposed to root exudates were enriched in 43 KEGG pathways of which 17 were differentially significant pathways (Fig. 3, Supplementary Table 4). The differentially significant pathways were mainly annotated in three categories: (i) substance degradation pathways, including fatty acid degradation, caprolactam degradation, lysine degradation, benzoate degradation, valine, leucine, and isoleucine degradation, aminobenzoate degradation, and limonene and pinene degradation; (ii) substance metabolism pathways, including butanoate metabolism, tryptophan metabolism,

Verification of DEGs by RT-qPCR analysis
Based on the results of the transcriptome analysis, we selected 14 DEGs that affected the metabolism and growthpromoting properties of P9 under root exudate treatment. Among these DEGs, four genes involved in energy production (aceA, alkM, gcl, and echA), five genes associated with siderophores (nfeF, yqjH, fagD, fepB, and fepD), two genes associated with transport (smvA and ABC), and one gene associated with virulence (ahpC) were up-regulated, whereas two genes involved in the urea cycle (argG and argH) were down-regulated. The RT-qPCR results showed consistent up-and down-regulation trends for the 14 DEGs, and their expression patterns were consistent with the transcriptome sequencing results ( Supplementary Fig. 1).

Effect of peanut root exudates on the ability of P9 to secrete IAA and siderophores
The addition of 2% peanut root exudates to the medium significantly increased the IAA and siderophore contents of the P9 strain compared with those of the control (Table 2). In the absence of exogenous tryptophan addition, the IAA secreted by the P9 strain increased by 70.53% and the relative content of siderophores increased by 13.87%. Thus, root exudates were indicated to promote IAA biosynthesis by P9 and the tryptophan component in root exudates may directly provide precursors for IAA synthesis.

Effect of peanut root exudate components on the growth, biofilm formation, and growth-promoting properties of P9
We analyzed the effects of different types of organic acids and amino acid components in the root exudates on the growth of P9. The addition of 20 or 40 mM citric acid, oxalic acid, and malic acid had no significant effect on the growth and IAA synthesis of P9 compared with the control, but significantly promoted biofilm formation, and oxalic acid significantly increased the capacity for siderophore biosynthesis (Fig. 4). Proline, glycine, and alanine significantly promoted the growth and IAA biosynthesis of P9, whereas proline and 40 mM alanine significantly enhanced siderophore biosynthesis (P < 0.05).

Effect of peanut root exudates on substance transport and energy metabolism of P9
Microbe-plant interactions involve consumption of a large amount of energy. Fan et al. reported that maize root exudates promote significant up-regulation of the glycolytic pathway and tricarboxylic acid (TCA) cycle-related genes in Bacillus amyloliquefaciens FBZ42 (Fan et al. 2012). In the present study, the expression of genes associated with glycolysis, TCA cycle, and pentose phosphate pathways were up-regulated in response to peanut root exudates, and the P9 strain adopted a more efficient and productive approach for biosynthesis and metabolism. The up-regulated genes included fruK encoding 1-phosphofructokinase in the glycolytic pathway, aceA encoding isocitrate lyase (ICL) in the TCA cycle, garR encoding the NAD-binding domain of 6-phosphogluconate dehydrogenase in the pentose phosphate pathway, and gcl encoding glyoxylate carboligase that catalyzes decarboxylation of glyoxylate. High concentrations of acetyl coenzyme A (enoyl-CoA) are an   (Krivoruchko et al. 2015). The enoyl-CoA hydratase gene echA and the enoyl-CoA acetyltransferase gene ACAT were up-regulated in P9; these enzymes together catalyze β-oxidation of fatty acids to synthesize enoyl-CoA. The alanine dehydrogenase gene ald was up-regulated and encodes a protein that catalyzes the production of pyruvate from alanine, which can be further oxidized to acetyl CoA and participates in the TCA cycle (Dave and Kadeppagari 2019). In addition, the alkane 1-monooxygenase gene alkM, which encodes a protein that oxidizes alkanes to alcohols and further oxidizes alcohols and aldehydes to fatty acids (Tan et al. 2021), and ppk2, a polyphosphate kinase 2 gene involved in the oxidative phosphorylation pathway, were up-regulated and may lead to enhanced synthesis of GTP. Thus, root exudates of peanut were indicated to promote synthesis of enoyl-CoA, ATP, and GTP in P9, as well as increased regeneration and storage of fatty acids, which may produce sufficient energy for enhanced metabolism and substance synthesis. The corresponding up-regulation of the major facilitator superfamily (MFS) transporter protein gene smvA and ATP-binding cassette (ABC) transporter genes in P9 resulted in enhanced uptake and transport of exogenous nutrients, which was similar to the increased uptake of hexose by Burkholderia cepacia Q208 induced by sugarcane root exudates (Paungfoo-Lonhienne et al. 2016). The expression of bldC, a member of the BldC family of transcription regulators, was significantly up-regulated, whereas iclR, a gene of the IclR transcription regulator family associated with spore formation, was down-regulated. Up-regulated expression of bldC and down-regulated expression of iclR can prevent premature development of actinomycete spores and are important for maintenance of normal development during the vegetative growth period (Bush et al. 2019;Wezel et al. 2000). Potato root exudates cause differential expression of certain genes encoding membrane proteins and genes associated with spore formation or germination in Mycobacterium mycoides (Yi et al. 2018). Thus, we speculate that peanut root exudates may also be involved in mediating the normal biosynthesis and development of bacterial membranes and spores.

Effects of peanut root exudates on the stress tolerance and growth-promoting characteristics of P9 strain
Siderophores are chelating agents synthesized by microorganisms in a low iron environment that can specifically chelate Fe 3+ . The siderophores produced by PGPR can compete with plant rhizosphere pathogens for limited iron ions, and inhibit their growth and reproduction (Pahari et al. 2017;Li et al. 2022b). Klonowska et al. reported that Mimosa pundica root exudates promoted significant up-regulation of the siderophore biosynthetic pathway in symbiotic nitrogen-fixing bacteria (Klonowska et al. 2018). In the current study, after the addition of peanut root exudates, all seven genes associated with siderophore synthesis and utilization were up-regulated in P9, specifically, mbtH (MbtH protein family gene), fagD (iron siderophore-binding protein gene), fepB (encoding iron enterobacterin-binding protein), fepD (encoding iron siderophore transport system permease protein), yqjH (encoding NADPH-dependent ferric siderophore reductase/siderophore-interacting protein, SIP), and hpaC (flavin reductase gene). The MbtH proteins are associated with the biosynthesis of hydroxamate-type and catecholtype siderophores in many actinomycetes (Cruz-Morales et al. 2017), and up-regulation of mbtH is consistent with root exudates causing a marked increase in siderophore content in P9 (Table 1). Of interest, proteins encoded by fagD, fepB, and fepD are normally membrane proteins involved in the uptake transport of Fe 3+ -siderophores by Gram-positive bacteria, a class of periplasmic-binding proteins that bind Fe 3+ to siderophores and transport them into the cytoplasm via the siderophore-permease(s)-ATPase system (Fukushima et al. 2013); significant up-regulation of these three genes significantly improved the transport efficiency of Fe 3+ -siderophores by P9. NADPH-dependent ferric siderophore reductase, known as SIP, is a flavin protein containing a specific siderophore-binding site that reduces and releases Fe 3+ obtained from siderophores into highly soluble Fe 2+ (Cain and Smith 2021). Up-regulated expression of yqjH and hpaC promotes efficient utilization of Fe 3+ . Thus, peanut root exudates can induce P9 to improve the biosynthesis and utilization of siderophores and the uptake of Fe 3+ , which can not only promote the growth of peanut but also enhance the resistance of peanut to pathogens and play an important role in the growth-promoting function of P9.
Root exudates are a carbon and energy source for PGPR and provide a precursor library for biotransformation of PGPR (Alshaal et al. 2017). After adding peanut root exudates and components of proline, alanine, and glycine, the ability of P9 to secrete IAA was significantly improved. In addition to tryptophan as a precursor for IAA biosynthesis, other amino acid components in peanut root exudates also promoted IAA biosynthesis as a carbon source (Fig. 4).
Tyrosine, phenylalanine, and aspartate acid have been reported to be carbon sources to promote IAA biosynthesis in some bacteria to varying degrees (Sergeeva et al. 2007).
In B. amyloliquefaciens FBZ42, certain genes involved in antibiotic biosynthesis are significantly up-regulated under the influence of maize root exudates (Fan et al. 2012). In the present study, up-regulated expression of mbtH is associated with the biosynthesis of peptide antibiotics (Wolpert et al. 2007). The expression of the histidine kinase gene ASU32_16205 was up-regulated, which can act as a sensor to induce antibiotic production (Li et al. 2016). The lipid transfer protein encoded by the up-regulated gene SCP2 is considered to be a protein that inhibits and kills pathogens (Wong et al. 2019).

Effect of peanut root exudates on biofilm formation and colonization by P9
When PGPR enter the soil, their ability to colonize and interact with plants are crucial to their growth-promoting properties, and biofilm-related pathways and protein adhesion to roots are associated with rhizosphere colonization (Santoyo et al. 2021). In the present study, ata, a gene that encodes a trimeric autotransporter adhesin (TAA), was significantly up-regulated (9.29-fold, the highest). TAA proteins mediate Gram-negative bacterial adhesion to host tissues (Kiessling et al. 2020). We hypothesized that up-regulated expression of ata also enhanced the adhesion of P9, which laid the foundation for its effective colonization. Lin et al. observed that a ferric ABC transporter positively regulates biofilm formation (Lin et al. 2012). In addition to significant up-regulation of iron ABC transporter genes (fagD, fepB, and fepD), the MCE protein family gene mlaD and polyphosphate kinase 2 gene ppk2 were significantly up-regulated under the influence of peanut root exudates. The encoded product of mlaD has a MCE domain that binds lipids, and ppk2 is associated with polyphosphate homeostasis in phospholipid synthesis (Pina-Mimbela et al. 2015), all of which suggest that peanut root exudates promote the biofilm formation of P9. As components of root exudates, oxalic acid, citric acid, and malic acid promoted biofilm formation of P9, which is consistent with the reported biofilm-inducing effects on strains of Bacillus spp. (Yuan et al. 2015).
The intrinsic antibiotic resistance of PGPR is critical for the adaptation and survival of strains in the presence of other aggressive microorganisms (Kenawy et al. 2019). The MarR and WhiB transcription regulator families, and ABC transporters are associated with the development of bacterial antibiotic resistance (Burian et al. 2012;Perera and Grove 2010). The expression of marR, whiB1_2_3_4, ABC, and ASU32_16205 genes were all up-regulated in P9 under the influence of peanut root exudates, giving the strain a stronger competitive ecological niche. Genes lacking crucial virulence or virulence-related factors have been detected in some PGPR, thus ensuring a mutually beneficial relationship between PGPR and plants (Chen et al. 2015). In the current study, the expression of the IclR transcription regulator gene iclR and branched-chain amino acid ABC transporter gene liv were down-regulated; the former is associated with certain bacterial virulence and plant pathogenicity (Molina-Henares et al. 2006), whereas the latter is associated with virulence integrity (Kaiser and Heinrichs 2018). It is speculated that down-regulation of these genes may be more conducive to establishment of interaction between P9 and peanut roots.

Effects on arginine biosynthesis pathway and urea cycle
The arginine biosynthesis pathway was the only significantly down-regulated pathway in the present investigation. Three genes associated with the urea cycle, namely argG (encoding argininosuccinate synthase), argH (encoding argininosuccinate lyase), and argF (encoding ornithine carbamoyl transferase), and an allophanate hydrolase gene (atzF) and urease accessory protein gene (ureD) involved in urea decomposition, were significantly down-regulated in P9. The pH was lower in the initial LB medium supplemented with root exudates and at the beginning of the logarithmic growth phase of P9 compared with that of the control, whereas the pH of the two cultures all increased to the same level at 22 h (Table 1). We tentatively speculated that the control P9 group without the addition of root exudates required the enhancement of this pathway to decompose a greater amount of urea, and the ammonia produced led to the increase in pH of the culture, whereas the urea in the root exudate was directly supplied to P9 in the treated group, but this hypothesis needs to be studied further. In addition, the expression of genes involved in the urea cycle and decomposition was down-regulated overall, indicating a decrease in the biosynthesis of the metabolites citrulline and arginine. Previous studies have reported that citrulline performs roles such as scavenging hydroxyl groups and protecting DNA from oxidative reactions, and arginine is a source of polyamines involved in scavenging reactive oxygen species (ROS) and protecting membranes from lipid peroxidation via the arginine deiminase pathway (Cheng et al. 2017), which indirectly suggests that ROS contents of the P9 strain are lower under the influence of peanut root exudates.

Conclusions
The root exudates of peanut promote the growth and growthpromoting function of Tsukamurella tyrosinosolvens strain P9. The present study analyzed the response mechanism of T. tyrosinosolvens P9 to peanut root exudates. Transcriptome analysis showed that peanut root exudates induce upregulation of genes involved in nutrient transport, carbohydrate metabolism and energy production, siderophore and IAA biosynthesis, and adhesion and biofilm formation, and down-regulation of genes associated with the arginine biosynthesis pathway and urea cycle in P9. Organic acids and amino acids are the main components of peanut root exudates. Amino acids (glycine, proline, and alanine) promote the growth and IAA secretion of P9, and proline, alanine (40 mM), and oxalic acid significantly improve siderophore synthesis by P9. Organic acids (citric acid, oxalic acid, and malic acid) significantly promote biofilm formation by the strain. The results provide a theoretical basis for establishment of the interaction between T. tyrosinosolvens P9 and peanut, and lay a foundation for improved exertion of the growth-promoting function of P9.