Nodulation Signaling Pathway 1 and 2 Modulate Vanadium Accumulation and Tolerance of Legumes

Abstract Vanadium (V) pollution potentially threatens human health. Here, it is found that nsp1 and nsp2, Rhizobium symbiosis defective mutants of Medicago truncatula, are sensitive to V. Concentrations of phosphorus (P), iron (Fe), and sulfur (S) with V are negatively correlated in the shoots of wild‐type R108, but not in mutant nsp1 and nsp2 shoots. Mutations in the P transporter PHT1, PHO1, and VPT families, Fe transporter IRT1, and S transporter SULTR1/3/4 family confer varying degrees of V tolerance on plants. Among these gene families, MtPT1, MtZIP6, MtZIP9, and MtSULTR1; 1 in R108 roots are significantly inhibited by V stress, while MtPHO1; 2, MtVPT2, and MtVPT3 are significantly induced. Overexpression of Arabidopsis thaliana VPT1 or M. truncatula MtVPT3 increases plant V tolerance. However, the response of these genes to V is weakened in nsp1 or nsp2 and influenced by soil microorganisms. Mutations in NSPs reduce rhizobacterial diversity under V stress and simplify the V‐responsive operational taxonomic unit modules in co‐occurrence networks. Furthermore, R108 recruits more beneficial rhizobacteria related to V, P, Fe, and S than does nsp1 or nsp2. Thus, NSPs can modulate the accumulation and tolerance of legumes to V through P, Fe, and S transporters, ion homeostasis, and rhizobacterial community responses.

Medicago truncatula seeds were sterilized with 75 % ethanol and washed five times using ddH 2 O.After carefully cutting the seed coat with a scalpel, the seeds were transferred to a double-layer filter paper for vernalization at 4 °C for 2 days, and then germinated at 22 °C in dark.The germinated seedlings were transferred to sponge bars and floated on ½-strength Hoagland's nutrient solution for further growth.The plants were cultured under long-day conditions (16 h of illumination (150 µmol m -2 sec -1 ) and 8 h of dark) at 22 °C and 50 % relative humidity in a growth chamber.For the analysis of rhizobacterial community, one-week-old seedlings with uniform growth were transferred to potting media containing a mixture of soil and vermiculite (2:1, v/v) as described by Durán et al. [9] .During growth, the plants were watered with ½-strength Hoagland nutrient solution twice and distilled water at other times.After nodulation, plants were treated with different concentrations of Na 3 VO 4 (0, 20, 200, and 2000 mg L -1 ) for one week, and then rhizosphere soil was obtained for microbiome determination.In order to analyze the dependence of vanadium (V) tolerance of wild-type R108 plants on soil microorganisms, autoclave and non-autoclave potting media were used.For strictly control the impact of microorganisms on plant V tolerance, 3-week-old wild-type (R108), nsp1 and nsp2 seedlings were cultured in a hydroponic device with an absorbent slope (HDAS) containing sterilized or unsterilized ½-strength Hoagland's nutrient solution formulated with soil extract. [10]After nodulation, plants were treated with 0 or 30 mg L -1 Na 3 VO 4 for one week.For investigating the effect of vacuolar phosphate transporter mutations on plant V tolerance, one-week-old of wild-type (R108) and mtvpt3-1 seedlings were transplanted into vermiculite media containing ½-strength Hoagland's nutrient solution and cultivated for 3 weeks before undergoing V treatment at concentrations of 0 and 1000 mg L -1 .
Arabidopsis thaliana was germinated according to the method described by Liu et al. [1a] .1b] For investigating the effect of V stress on the root growth of A. thaliana phosphorus (P), iron (Fe), and sulfur (S) transporter mutants, 4-day-old seedlings were transferred to ANS agar medium with or without 5 mg L -1 Na 3 VO 4 for 10 days.

Inorganic phosphorus (Pi) determination
Inorganic phosphorus concentration was measured using the ascorbate-molybdateantimony method, as previously described. [11]Briefly, plant tissues were quickly ground into powder with liquid nitrogen.Pre-cooled 5 % perchloric acid (1 mL) was then added to the 0.1 g sample to extract Pi.The extract was centrifuged at 14000 g for 5 min, and then, the supernatant was used to determine Pi concentration.A reaction mixture containing 500 µL of sample and 10 mL of Pi detection agent (0.2 % ammonium molybdate, 0.005% potassium antimonyl tartrate, 2.2 % sulfuric acid, and 0.4 % ascorbate) was incubated at 36 °C for 15 min.Pi concentration was measured at 690 nm.

ROS determination
H 2 O 2 was detected by DAB (3,3-diaminobenzidine) staining, as described previously. [12]Plant samples were vacuum infiltrated with 1 mg/ml DAB (50 mM Tris-acetate, pH 3.8) solution containing 0.05% (v/v) Tween 20 and incubated at 25 °C for 13 h in the dark.Next, the samples were rinsed in 80% (vol/vol) ethanol for 10 min at 70 °C and mounted in lactic acid/phenol/water (1:1:1, vol/vol/vol) before they were photographed using a DSLR camera.Intracellular ROS were detected using 2′-7′ dichlorodihydrofluorescein diacetate (H 2 DCFDA, HY-D0940, MedChemExpress, USA).Briefly, the leaf samples were vacuum infiltrated with the staining solution (10 µM H 2 DCFDA, 10 mM Tris-HCl, pH 7.2) for 5 min at 60 KPa pressure and incubated in the dark at room temperature for 25 min.Next, the samples were washed in three changes of autoclaved ddH 2 O and mounted in 20% glycerol.Their fluorescence was then analyzed under a confocal microscope (Leica TCS SP8, Germany) at 488 nm excitation.ROS signals were detected at an emission of 500-550 nm.In addition, the chlorophyll autofluorescence was detected at 640-700 nm.

RT qPCR analysis
Eastep Super Total RNA Extraction Kit (Promega, Shanghai, China) was used to extract total RNA from the samples.HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) was used to synthesize first-strand cDNA with oligo (dT) primers.Real-time quantitative PCR (RT-qPCR) was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a LightCycler 480II (Roche, Basel, Switzerland).MtACTIN (MTR_3g095530) was used as the internal control.The primers used for qPCR are listed in Supplementary Table S1.

DNA extraction, PCR amplification and sequencing of rhizospheric bacteria
Total genome DNA from samples was extracted using CTAB/SDS method.DNA concentration and purity was monitored on 1 % agarose gels.According to the concentration, DNA was diluted to l ug/µL using sterile water.
16S rRNA genes of distinct regions (16S V4-V5) were amplified used specific and PERMANOVA was used to analyze significant differences.The construction of rhizobacterial co-occurrence networks involved filtering out low-frequency groups with a set screening threshold, standardized using counters per million, and performing Spearman correlation analysis between OTUs, thus identifying significant positive correlations (ρ > 0.7 and p < 0.05).V-responsive OTUs in the modules were calculated and visualized, and Mantle tests were performed on the levels of V, P, Fe, S, and Ca in plants.Biomarkers were identified using Random Forests, and Spearman correlation analysis was performed between the biomarkers and plant element concentration.

Statistical analysis
All the data were statistically analyzed with SPSS 19.0 statistical software (IBM, Inc., Armonk, NY, USA) using Duncan's multiple range test P < 0.05 and independent samples t-tests (* P < 0.05; ** P < 0.01; *** P < 0.001).(c) The change of primary root length under V stress.The primary root length under normal conditions (-V) was used as the control.The log2 of the ratio of each primary root length under V stress (+V) divided by the value of control was calculated.Data are presented as mean ± SD. n = 4. Asterisks denote statistically significant differences according to independent sample t-tests (* P < 0.05, ** P < 0.01).

Supporting Tables
Figure S1.The Rhizobium symbiosis is defective in nsp1 and nsp2 mutant plants.(a) Diagrams of Tnt1-insertions in the NSP1 and NSP2 genes of the mutant lines nsp1 (NF9220) and nsp2 (NF10950).The gray and black boxes represent the 5'-UTR and exon, respectively.The gray arrow indicates the 3'-UTR.(b) Phenotypes of shoots and root nodules of wild-type (R108), nsp1, and nsp2 plants.Three-week-old seedlings were inoculated with Rhizobium meliloti 1021 for one week.Bars, 2 cm.

Figure S2 .
Figure S2.NSP1 and NSP2 mutations increase the sensitivity of chlorophyll fluorescence to vanadium (V) stress.(a-c) Images of F0 (a), Fm (b), and Fv/Fm ratios (c) in the leaves of wild type (R108) and mutant nsp1 and nsp2 Medicago truncatula plants.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with (+V) or without (-V) 100 mg L -1 V for 16 h.Then, leaves were obtained for chlorophyll fluorescence imaging.(d-f) Statistical analysis of F0 (d), Fm (e), and Fv/Fm ratios (f) in (a-c).Data are expressed as mean ± SD values (n = 4).Statistical significance is denoted by asterisks based on independent sample t-tests (** P < 0.01, *** P < 0.001).

Figure S3 .
Figure S3.NSP1 and NSP2 mutations reverse the increase in the ratio of nutrient elements to vanadium (V) caused by V stress increase.The P/V (a), Fe/V (b), S/V (c), and Ca/V (d) ratios in the shoots of 5-week-old wild-type (R108), nsp1, and nsp2 Medicago truncatula plants treated with 20 mg L -1 (V20) or 200 mg L -1 (V200) for one week.Data were calculated from the ion concentrations in Figure 2. Data are expressed as mean ± SD values (n = 4).Statistical significance is denoted by asterisks based on independent sample t-tests (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure S4 .
Figure S4.Effects of different phosphorus (P) conditions on vanadium (V) tolerance and ion accumulation in plants.(a) Growth phenotypes of wild-type (R108) and mutant nsp1 and nsp2 Medicago truncatula plants induced by V stress under low or high P conditions.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with 30 mg L -1 V under low P (LP, 1 μM PO 4 3-) or high P (HP, 1 mM PO 4 3-) conditions for 5 or 7 days.Arrows indicate plant leaves that curl or fall after death.Bars, 2 cm.(b,c) Concentrations of V (b) and P (c) in the shoots of wild-type (R108) and mutant nsp1 and nsp2 plants after 5 days of V treatment described in (a).(d) The P/V ratio in the shoots of R108, nsp1, and nsp2 plants.Data were calculated from P and V concentrations in (b,c).Data are expressed as mean ± SD values (n = 4).Statistical significance is denoted by asterisks based on independent sample t-tests (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure S5 .
Figure S5.ROS accumulation in plants exposed to vanadium (V) stress under different phosphorus (P) conditions.ROS accumulation in the leaves (a) and roots (b) of wild-type (R108) and mutant nsp1 and nsp2 Medicago truncatula plants.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with 30 mg L -1 V under low P (LP, 1 μM PO 4 3-) or high P (HP, 1 mM PO 4 3-) conditions for 5 days.Then, leaves and roots were obtained for DAB staining.

Figure S6 .
Figure S6.NSP1 and NSP2 mutations increase low phosphorus (P) tolerance of Medicago truncatula.(a) Growth phenotypes of wild-type (R108) and mutant nsp1 and nsp2 plants under high or low P conditions.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with high P (HP, 1 mM PO 4 3-) or low P (LP, 1 μM PO 4 3-) for 7 days.Arrows indicate dead and yellow leaves.Bars, 2 cm.(b,c) Shoot (b) and root (c) biomass of R108, nsp1, and nsp2 plants under high or low P conditions.Plants were treated as described in (a).Data are expressed as mean ± SD values (n = 4).Different letters above the bars indicate significant differences at P < 0.05 (Duncan's test).

Figure S7 .
Figure S7.Effects of different iron (Fe) conditions on vanadium (V) tolerance and ion accumulation in plants.(a) Growth phenotypes of wild-type (R108) and mutant nsp1 and nsp2 Medicago truncatula plants induced by V stress under low or high Fe conditions.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with 30 mg L -1 V under low Fe (L-Fe, 1 μM Fe 2+ ) or high Fe (H-Fe, 89.6 μM Fe 2+ ) conditions for 5 or 7 days.Arrows indicate plant leaves that curl or fall after death.Bars, 2 cm.(b) The V concentration in the shoots and roots of wild-type (R108) and mutant nsp1 and nsp2 plants after 5 days of V treatment described in (a).(c) The translocation factor of V from roots to shoots of R108, nsp1, and nsp2 plants after 5 days of V treatment described in (a).Data were calculated from shoot-to-root ratios of V concentrations in (b).(d,e) Concentrations of P (d) and Fe (e) in the shoots and roots of wild-type (R108) and mutant nsp1 and nsp2 plants after 5 days of V treatment described in (a).(f) The Fe/V and P/V ratios in the shoots of R108, nsp1, and nsp2 plants.Data were calculated from P, Fe, and V concentrations in (b,d,e).Data are expressed as mean ± SD values (n = 4).Statistical significance is denoted by asterisks based on independent sample t-tests (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure S8 .
Figure S8.ROS accumulation in plants exposed to vanadium (V) stress under different iron (Fe) conditions.ROS accumulation in the leaves (a) and roots (b) of wild-type (R108) and mutant nsp1 and nsp2 Medicago truncatula plants.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with 30 mg L -1 V under low Fe (L-Fe, 1 μM Fe 2+ ) or high Fe (H-Fe, 89.6 μM Fe 2+ ) conditions for 5 days.Then, leaves and roots were obtained for DAB staining.

Figure S9 .
Figure S9.Effect of NSP1 and NSP2 mutations on iron (Fe) adaptation of Medicago truncatula.(a) Growth phenotypes of wild-type (R108) and mutant nsp1 and nsp2 plants under high or low Fe conditions.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with high Fe (H-Fe, 89.6 μM Fe 2+ ) or low Fe (L-Fe, 1 μM Fe 2+ ) for 7 days.Bars, 2 cm.(b,c) Shoot (b) and root (c) biomass of R108, nsp1, and nsp2 plants under high or low Fe conditions.Plants were treated as described in (a).Data are expressed as mean ± SD values (n = 4).Different letters above the bars indicate significant differences at P < 0.05 (Duncan's test).

Figure S10 .
Figure S10.Effects of different sulfur (S) conditions on vanadium (V) tolerance and ion accumulation in plants.(a) Growth phenotypes of wild-type (R108) and mutant nsp1 and nsp2 Medicago truncatula plants induced by V stress under low or high S conditions.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with 30 mg L -1 V under low S (LP, 1 μM SO 4 2-) or high S (HP, 5 mM SO 4 2-) conditions for 5 or 7 days.Bars, 2 cm.(b,c) Concentrations of V (b) and S (c) in the shoots of wild-type (R108) and mutant nsp1 and nsp2 plants after 5 days of V treatment described in (a).(d) The S/V ratio in the shoots of R108, nsp1, and nsp2 plants.Data were calculated from S and V concentrations in (b,c).Data are expressed as mean ± SD values (n = 4).Statistical significance is denoted by asterisks based on independent sample t-tests (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure S11 .
Figure S11.ROS accumulation in plants exposed to vanadium (V) stress under different sulfur (S) conditions.ROS accumulation in the leaves (a) and roots (b) of wild-type (R108) and mutant nsp1 and nsp2 Medicago truncatula plants.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with 30 mg L -1 V under low S (LS, 1 μM SO 4 2-) or high S (HS, 5 mM SO 4 2-) conditions for 5 days.Then, leaves and roots were obtained for DAB staining.

Figure S12 .
Figure S12.Effect of NSP1 and NSP2 mutations on sulfur (S) adaptation of Medicago truncatula.(a) Growth phenotypes of wild-type (R108) and mutant nsp1 and nsp2 plants under high or low S conditions.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with soil extract.After nodulation of R108, plants were treated with high S (HS, 5 mM SO 4 2-) or low S (LS, 1 μM SO 4 2-) for 7 days.Bars, 2 cm.(b,c) Shoot (b) and root (c) biomass of R108, nsp1, and nsp2 plants under high or low S conditions.Plants were treated as described in (a).Data are expressed as mean ± SD values (n = 4).Different letters above the bars indicate significant differences at P < 0.05 (Duncan's test).

Figure S14 .
Figure S14.Effects of mutation and overexpression of MtVPT3 on element accumulation in plants under vanadium (V) stress.(a) Growth phenotypes of Medicago truncatula wild-type (R108) plants and the vacuolar phosphate transporter mutant mtvpt3 plants under V stress.Five-week-old seedlings were treated with 1000 mg L -1 V for five days.(b) ROS accumulation in the leaves of wild-type (R108) and mtvpt3 mutant plants under V stress.Plants were treated as described in (a).Then, leaves were obtained for DAB staining.(c-e) Concentrations of total V (c), P, S, Fe, and Ca (d) and inorganic P (Pi) (e) in shoots of wild-type (R108) and mtvpt3 mutant plants described in (a).(f,g)Concentrations of total V (f), P, S, Fe, and Ca (g) in roots of wild-type (R108) and mtvpt3 mutant plants described in (a).(h) The P/V ratio in the roots of wild-type (R108) and mtvpt3 mutant plants described in (a).Data were calculated from P and V concentrations in (f,g).(i) Growth phenotypes of Arabidopsis thaliana wild-type (Col-0) and MtVPT3-overexpressing plants (MtVPT3-OE) under V stress.Three-week-old seedlings were treated with 1000 mg L -1 V for 10 days.(j,k) Concentrations of total V (j), P, S, Fe, and Ca (k) in shoots of wild-type (Col-0) and MtVPT3-OE plants described in (a).Data are expressed as mean ± SD values (n = 4).Statistical significance is denoted by asterisks based on independent sample t-tests (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure S15 .
Figure S15.The IRT1 mutation alters the Inorganic phosphorus (Pi) concentrations in the shoot of Arabidopsis thaliana.Three-week-old seedlings grown in vermiculite media were treated with (+V) or without (-V) 1000 mg L -1 VO 43-for one week.Data are presented as mean ± SD. n = 5.Asterisks denote statistically significant differences according to independent sample t-tests (** P < 0.01).

Figure S16 .
Figure S16.The impact of vanadium (V) stress on the root growth of Arabidopsis thaliana phosphate transporter mutants.(a) Growth phenotype of two-week-old wild-type (Col-0), pht1;1, pht1;4, pht1;9, vpt1, and pho1 mutant plants under V stress.Seedlings were cultured on ANS agar medium with (+V) or without (-V) 5 mg L -1 VO 4 3-.Bars, 1 cm.(b) Primary root length of wild-type (Col-0) and phosphate transporter mutant plants as described in (a).The data are expressed as mean ± SD. n = 4.(c) The change of primary root length under V stress.The primary root length under normal conditions (-V) was used as the control.The log2 of the ratio of each primary root length under V stress (+V) divided by the value of control was calculated.Data are presented as mean ± SD. n = 4. Different letters above the bars represent significant differences at P < 0.05 (Duncan's test).

Figure S17 .
Figure S17.Growth phenotype of the phosphorus (P) transporter mutant vpt1 under vanadium (V) stress.(a) Growth phenotype of 2-week-old wild-type (Col-0) and vpt1 mutant plants under different P and V conditions.Seedlings were cultured on ANS agar medium containing 260 or 26 µM PO 4 3-(Pi), and 0 (-V) or 5 mg L -1 VO 4 3-(+V).(b) Primary root length of wild-type (Col-0) and vpt1 seedlings under different P and V conditions as described in (a).The data are expressed as mean ± SD. n = 4.

Figure S18 .
Figure S18.Growth phenotypes of Arabidopsis thaliana iron (Fe) transporter mutant irt1 under different concentrations of vanadium (V) and Fe.(a) Growth phenotype of 2-week-old wild-type (Col-0) and irt1 mutant plants under different V and Fe conditions.Seedlings were cultured on ANS agar medium with (+V) or without (-V) 5mg L -1 VO 4 3-, accompanied by low iron (L-Fe, 8.96 µM Fe 2+ ), normal iron (N-Fe, 44.8 µM Fe 2+ ), and high iron (H-Fe, 89.6 µM Fe 2+ ) concentrations.(b) Primary root length of wild-type (Col-0) and irt1 seedlings under different P and V conditions as described in (a).The data are expressed as mean ± SD. n = 4. Different letters above the bars represent significant differences at P < 0.05 (Duncan's test).

Figure S20 .
Figure S20.Soil autoclave was not able to completely eliminate the impact of microorganisms on plant vanadium (V) tolerance.(a)Some wild-type (R108) plants was still more tolerant to V stress than nsp1 and nsp2 mutants under autoclaved soil growth conditions.Three-week-old seedlings were treated with (+V) or without (-V) 500 mg L -1 VO 4 3-for one week.Bars, 2 cm.(b)Wild-type (R108) plants with nodules had higher V tolerance than nsp1, nsp2, and R108 without nodules.Seedlings were treated as described in (a).The plants with nodules circled by red boxes in the middle and right images were the same plants.Bars, 2 cm.

Figure S21 .
Figure S21.Root phenotypes of wild-type (R108) and nsp1 and nsp2 mutant Medicago truncatula plants under vanadium (V) stress in the presence and absence of soil microorganisms.Three-week-old seedlings were cultured with ½-strength Hoagland nutrient solution prepared with non-sterile (Natural) or sterile soil extract (Sterilized Soil Extract Nutrient Solution).After nodulation of R108, plants were treated with (+V) or without (-V) 30 mg L -1 VO 4 3-for one week.The roots in images correspond to the shoots shown in Figure 5. Bars, 2 cm.

Figure S30 .
Figure S30.Species-differentiated operational taxonomic units (SdOTUs) distribution of rhizobacteria from wild-type (R108), nps1, and nsp2 plants under the same vanadium concentration treatment.(a) The rhizobacterial co-occurrence network and modules containing SdOTUs under 0 mg L -1 VO 4 3-treatment.Low frequency operational taxonomic units (OTUs) with frequencies less than 83% were removed from all samples.SdOTUs were highlighted and colored.The edges of the co-occurrence network were selected based on Spearman's rho > 0.7 and P-value < 0.001.The bar chart displays the cumulative relative abundance of SdOTUs (in millions) in R108 (pink), nsp1 (baby blue), and nsp2 (green).The cumulative relative abundance reflects the significant influence of plant materials on the SdOTUs.(b) The rhizobacterial co-occurrence network and modules containing SdOTUs under 20 mg L -1 VO 4 3-treatment.Data were analyzed as described in (a).(c) The rhizobacterial co-occurrence network and modules containing SdOTUs under 200 mg L -1 VO 4 3-treatment.Data were analyzed as described in (a).(d) The rhizobacterial co-occurrence network and modules containing SdOTUs under 2000 mg L -1 VO 4 3-treatment.Data were analyzed as described in (a).

Figure S31 .
Figure S31.Relative abundance accumulation of rhizobacterial biomarkers in wild-type (R108), nsp1, and nsp2 plants under vanadium (V) stress.(a) The relative abundance accumulation of rhizobacterial biomarkers in wild-type (R108) plants under different V conditions.Four-week-old seedlings were treated with 0 mg L -1 (T0), 20 mg L -1 (T20), 200 mg L -1 (T200), and 2000 mg L -1 (T2000) VO 4 3for one week.(b) The relative abundance accumulation of rhizobacterial biomarkers in nsp1 plants under different V conditions.Seedlings were treated as described in (a).(c) The relative abundance accumulation of rhizobacterial biomarkers in nsp2 plants under different V conditions.Seedlings were treated as described in (a).

. Bioinformatics analysis on 16S rRNA gene profiling of rhizospheric bacteria
primer (515F-806R) with the barcode.All PCR reactions were carried out with 15 µL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs); 0.2 µM of forward and reverse primers, and about 10 ng template DNA.Thermal cycling