WU Fan-lin,QU De-hui,TlAN Wei,WANG Meng-yun,CHEN Fei-yan,Ll Ke-ke,SUN Ya-dong,SU Ying-hua,YANG Li-na,SU Hong-yan,WANG Lei

1 Key Laboratory of Molecular Module-Based Breeding of High Yield and Abiotic Resistant Plants in Universities of Shandong,School of Agriculture,Ludong University,Yantai 264025,P.R.China

2 College of Life Sciences,Ludong University,Yantai 264025,P.R.China

3 State Key Laboratory of Microbial Metabolism and School of Life Sciences &Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,P.R.China

4 State Key Laboratory of Crop Biology,College of Life Sciences,Shandong Agricultural University,Tai’an 271018,P.R.China

5 Department of Genetics and Cell Biology,Basic Medical College,Qingdao University,Qingdao 266071,P.R.China

Abstract Sweet cherry is one of the most popular fresh fruits in the world.Previously,we isolated a soilborne dark septate endophyte (DSE) strain,S16,which promoted the growth of Gisela 5 sweet cherry rootstock.However,little is known about the molecular mechanism of the effect of S16 on the growth of sweet cherry.In this study,the physiological parameters and transcript profiles of sweet cherry roots were analyzed under S16 treatment compared with a control to elucidate the molecular mechanisms of the effect of this strain on sweet cherry growth.After inoculation with S16,sweet cherry seedlings exhibited more vigorous growth.Moreover,we identified 4 249 differentially expressed genes (DEGs)between S16-treated plants and the control.Many of the DEGs are involved in pathways related to plant growth,such as cellular metabolic and plant hormone pathways.Additionally,some genes involved in nitrate regulation were also enriched;and these genes may be involved in the regulation of nitrate uptake in plants.Physiological index detection demonstrated that S16 could improve the nitrate assimilation of sweet cherry via NO3– transporters.This RNA-seq dataset provides comprehensive insight into the transcriptomic landscape to reveal the molecular mechanisms whereby the DSE influences the growth of sweet cherry.

Keywords:sweet cherry,dark septate endophyte,RNA-seq,growth,NO3– transporters

1.lntroduction

Sweet cherry (Prunus avium) is one of the most popular fresh fruits in the world,and it is commonly appreciated for its bright color,delicious taste and high nutritional value (Ballistreriet al.2013).There is a close connection between the productivity of sweet cherry and its rootstock.The production of sweet cherry requires the late fruiting of trees,which significantly lengthens the investment period for rootstock growth.To accelerate tree growth,a variety of mineral fertilizers,which are harmful to the soil,are used.Hence,to reduce the environmental and economic problems associated with fertilizer use,biofertilization combined with soil microorganisms has been considered as an economically positive strategy for sweet cherry production and soil fertility improvement.

Symbiotic fungi play important roles in ecological environmental processes,which could contribute to nutrient uptake and disease resistance in host plants.These fungi in turn require carbon compounds from host plants for their growth (Harrison 2005).Dark septate endophytes (DSEs),a type of dematiaceous,septate fungal hyphae,are usually found in host plant roots in the ecosystem (Zhanget al.2008).DSEs play important roles in improving plant vitality and reducing heavy metal toxicity by regulating plant physiological characteristics.For example,the DSE strainPiriformospora indicacan enhance cadmium (Cd) tolerance by increasing antioxidant enzyme activities inNicotiana tabacumleaves (Huiet al.2015).In addition,Suaeda salsaimproves rice growth by increasing antioxidant activity and photosynthesis under moderate lead (Pb) stress (Liet al.2012).Hence,DSEs play significant roles in improving host plant physical properties under heavy metal stress.In addition,several DSE fungi have been reported to benefit host plant growth by facilitating P,N and C uptake (Newsham 2011),releasing metabolites (Waqaset al.2012),and protecting plants against drought (Barrow 2003) or high salinity(Baltruschatet al.2008).Therefore,it is important to understand the specific mechanisms by which DSEs can promote plant growth through nutrient assimilation,which may play a key role in ecosystem nutrient cycling and protect plants from environmental stress.

Most of the relevant studies have reported that mycorrhizal colonization can improve nitrate absorption by plant roots.The roles of arbuscular mycorrhizal (AM)fungi in contributing to nitrogen nutrition absorption by host plants have been studied most frequently.The ammonium transporters (AMTs) of plants have been considered receptors for the uptake of external ammonium(Parniske 2008).In addition,NO3–transporters (NRTs),the other transporters of plants used for the uptake of nitrate,are mainly encoded by the NRT1 and NRT2 gene families.The NRT1 family is responsible for lowaffinity nitrate transportation,while NRT2 is responsible for the high-affinity transportation system (Wittgensteinet al.2014).There is a close connection between the two pathways for nitrate acquisition of plant uptake and for mycorrhiza-mediated uptake.Therefore,a better understanding of the mechanism through which DSE symbiosis enhances nitrate uptake in plants is important for improving the production of some economically important horticultural crops,such as sweet cherry.

This study was designed to explore the symbiosis between sweet cherry woody plants and DSEs,and to elucidate the mechanisms of nitrate assimilation.Transcriptome analysis of DSE strain S16 colonizing sweet cherry roots was conducted against an uncolonized control.A total of 4 249 differentially expressed genes(DEGs) were identified,and most of the DEGs were involved in important metabolic pathways.Moreover,nitrate assimilation related genes were highly enriched,and nitrogen related physiological index studies demonstrated that S16 probably promotes plant growth through nitrate uptake.Therefore,the results of this study may clarify the molecular mechanisms of DSEs associated with sweet cherry host plants and reveal how symbiosis improves plant growth and nitrate uptake.

2.Data and methods

2.1.lsolation of mycorrhizal fungi

Sweet cherry plants were identified from a plantation in Shandong Province,China.Three sweet cherry root samples were dug up from the soil.The soil that adhered to the surface of the roots was removed by shaking.Then,the roots were placed in a container and stored at 4°C in the laboratory until use.

The fungi were isolated from the hair roots of sweet cherry as previously described (Zhanget al.2009).Briefly,the roots were placed in water and washed three times to remove the surface soil.Then,the hair roots were subjected to surface sterilization with 72%ethanol,after which they were immersed in 10% sodium hypochlorite and washed with sterile distilled water three times.Next,the roots were cut into 0.5 cm pieces,placed on potato dextrose agar (PDA) medium,and incubated at 28°C for two weeks.Thereafter,a total of 20 fungal strains were screened for further morphological identification,and five strains were selected for evaluation of their ability to form mycorrhizae.After the preliminary evaluation,one fungal strain (S16) that may show the ability to form mycorrhizae with sweet cherry roots and improve plant growth was isolated.

2.2.Morphological and molecular identification of S16

Morphological characteristics of S16,including its colony diameter,thickness,texture,and color,were examined after culture on PDA medium for 14 days at 28°C in the dark.The S16 strain isolate was grown in potato dextrose broth at 120 r min–1at 28°C for two weeks.The genomic DNA of S16 was extracted with the cetyltrimethylammonium bromide (CTAB) method.The internal transcribed spacer (ITS) region was amplified using the ITS1 and ITS4 primers.Amplifications were performed in a 50-μL reaction volume containing 5 μL of reaction buffer,5 μL of dNTPs,1 μL of each primer,50 ng of genomic DNA,and 1 U of Taq polymerase.The mixture was incubated at 95°C for 2 min,followed by 30 cycles of 94°C for 30 s,60°C for 30 s,and 72°C for 60 s,and a final extension for 5 min at 72°C.The PCR products were cloned into the T vector and sequenced.The sequences that closely matched those of S16 were acquired from the NCBI database by using BLAST.Alignments were carried out with Clustal X and analyzed with MEGA 5.0 Software.

2.3.Plant materials and treatments

Gisela 5 sweet cherry seedlings were purchased from the Yantai Academy of Agricultural Sciences.S16 was cultured on PDA medium at 28°C for two weeks.The mature spores were selected by growth in PD liquid medium for another two weeks at 28°C on a rotary shaker(120 r min–1).The broth was centrifuged at 6 000×g for 5 min at room temperature (RT),after which the hyphal pellet was collected.Gisela 5 sweet cherry seedlings were cultured in regeneration medium consisting woody plant medium (WPM) supplemented with 1.0 mg L–1IBA,2.0 mg L–1BA,3% sucrose and 0.6% agar at 28°C for four weeks.

Soil and vermiculite were mixed at a ratio of 1:1 (v:v).S16 hyphae were added to the soil at a ratio of 1:10 (w:w).The same treatment conditions without colonization were applied as a control.The plants were grown in soil for two months.Thereafter,the height,weight and number of leaves of the Gisela 5 seedlings were recorded.The experiments were conducted with 20 Gisela 5 seedlings colonized with S16,and 20 seedlings without S16 treatment were used as a control.

2.4.RNA extraction and RNA-Seq

The root materials from seedlings with S16 colonization and the control were prepared for RNA extraction.Four weeks after inoculation,the roots harvested from S16 and control were quickly transferred into the liquid nitrogen and the total RNA was isolated with the kit for RNA extraction of plants (Tiangen,Shanghai,China).The integrity of RNA was analyzed by a 2100 Bioanalyzer(Agilent).The cDNA library products were sequenced on the Illumina HiSeq 2000 platform (Majorbio,Shanghai,China).

2.5.Validation of RNA-seq data through qRT-PCR analysis

To verify the RNA-seq results,we randomly selected 12 DEGs and genes related to N uptake for quantitative real-time PCR (qRT-PCR) testing.The sweet cherry housekeeping gene β-tubulin was used as an internal standard.The qRT-PCR mixture consisted of 2× SYBR Green PCR buffer (10 μL),10 μmol L–1forward (0.5 μL)and reverse primers (0.5 μL),cDNA (1 μL) and ddH2O to 20 μL.The amplification conditions were as follows:95°C for 10 min;40 cycles of 95°C for 15 s,60°C for 30 s,and 72°C for 45 s.Data analysis was conducted using the Bio-Rad quantitative PCR application system.

2.6.Detection of the contents of ,,and

The contents of NH4+in the roots and leaves were detected using a previously described method (Brautigamet al.2007).Briefly,frozen powdered root and leaf samples (0.1 g) were extracted with extraction buffer.After shaking for 15 min at 4°C,the samples were centrifuged at 10 000×g for 10 min.Then,the aqueous phase was transferred to a new 2 mL tube,and 50 mg of activated charcoal was added.After mixing,the samples were centrifuged at 10 000×g for 5 min.The NH4+concentration was detected by absorption at 620 nm using a spectrophotometer.

The contents of NO3−in the roots and leaves were determined using the method of Pattersonet al.(2010).Specifically,the frozen fine powder (0.1 g) was extracted with 1 mL of ddH2O at 45°C for 1 h.Thereafter,the samples were centrifuged at 5 000×g for 15 min at RT,and 0.2 mL of the supernatant was removed and mixed with 5% salicylic acid (0.8 mL).After incubation for 20 min at RT,NaOH (2 mol L–1,19 mL) was added to adjust the pH to above 12.After cooling to RT,the concentration of NO3−was detected by absorption at 410 nm.

The contents of NO2−in the roots and leaves were determined using methods described previously (Ogawaet al.1999).Briefly,the frozen powdered samples (0.1 g)were added to extraction buffer (50 mmol L–1Tris-HCl,pH 8,5 mmol L–1cysteine,and 2 mmol L–1EDTA).Thereafter,centrifugation was conducted at 10 000×g for 20 min,and the supernatant (500 μL) was mixed with 1%sulphanilamide (250 μL) and 0.02% N-(1-naphtyl)-ethylenediamine dihydrochloride (250 μL).The concentration of NO2−was detected by absorption at 540 nm.

2.7.Measuring the activities of N-related enzymes

The assay for detecting the activity of NO3−reductase (NR)in the roots and leaves was performedviathe method by Högberget al.(1986).The activity of nitrite reductase(NiR) was measured using the method of Seithet al.(1994).The activity of glutamine synthetase (GS) was analyzed according to the method of Wanget al.(2008).

2.8.Statistical analysis

All data are presented as the mean±standard error of at least three biological replicate samples.Student’st-test was used to examine the statistical significance of differences between the two groups,which was defined according to aP-value＜0.05,as indicated.

3.Results

3.1.Morphological and molecular characteristics of S16

In the previous screening assay,we obtained a series of endophytic fungi from the roots of sweet cherry.Based on the results of the inoculation experiments,a fungal isolate designated S16,which exerted obvious beneficial effects on the growth of Gisela 5 sweet cherry,was screened.As shown in Fig.1-A,S16 formed white-to-gray colonies after incubation on PDA medium at 25°C in the dark,and the diameter of the colonies ranged from 1–2 cm.A series of septa in the hyphae of S16 could be observed,but no conidia or reproductive structures were observed under the microscope (Fig.1-B).To determine the classification of the strain,molecular analysis of the ITS rDNA sequence was performed.The results of BLAST searches in the NCBI database showed that the sequence was 99% identical to the sequence of theFungal sp.2563 YZ-2011 strain,which was isolated from the mycelial cortices of naturalOphiocordyceps sinensisspecies in the Tibetan Plateau (Zhanget al.2010).Thus,the strain was placed in the cluster withFungal sp.2563 YZ-2011,which is supported by a bootstrap of 100% (Fig.1-C).

3.2.Effect of S16 on the growth of sweet cherry

To better characterize the effects of DSE S16 on the growth of sweet cherry,some phenotypic indices were determined.The growth tendency of sweet cherry colonized with S16 was increased over that of the control after two months of cultivation,and the plants were much larger with more roots (Fig.2-A).Specifically,the height and weight of sweet cherry treated with S16 were significantly greater than those of the controls.Similarly,the leaf number of the plants treated with S16 was 1.5 times that of the controls (Fig.2-B).To further confirm that the observed biomass increase was related to S16 colonization,light microscopy observations were performed.Hyphae were present in the epidermal cells of sweet cherry roots (Fig.2-C),and some root cortical cells were filled with sclerotium structures following S16 inoculation (Fig.2-D).Therefore,these results indicated that S16 could promote the growth of sweet cherry with mutualistic symbiosis.

3.3.Functional categorization of S16-regulated genes in roots

To compare the transcriptome of sweet cherry roots colonized with S16 with that of uncolonized roots,de novo-assembled sweet cherry root transcriptomes were generated.In total,46.1 million and 49.3 million clean reads were generated after filtration for the S16-treated and control samples,respectively (Appendix A).In particular,more than 92 and 93% of the bases in the clean reads exhibited aQ-value ≥30 (with error probabilities of 0.0265 and 0.0254%,respectively).A total of 55 826 transcripts were generated by Trinity Software (Appendix A).Among them were 16 161,13 988,34 123,34 962,28 779 and 30 684 transcripts with significant similarity to sequences in the GO,KEGG,COG,NR,SwissProt and Pfam databases,respectively (Appendix A).

To further elucidate the molecular mechanism of the effect of S16 treatment on sweet cherry growth,DEGs were identified between the S16-treated and control samples through the comparison of the FPKM values(Appendix B).In total,4 249 genes were found to be differentially expressed,including 1 749 upregulated and 2 500 downregulated genes (Appendix B).GO analysis was performed to assign functional information to the DEGs.The DEGs were assigned to 34 level-2 GO terms,which were summarized under the three main GO categories of biological processes (BP),cellular components (CC) and molecular functions (MF).In the BP category,the DEGs were clustered into 14 classifications.The most abundant genes were clustered under metabolic processes,single organism processes,cellular processes,and biological regulation,which contained 1 210,848,732 and 262 DEGs,respectively.In the CC category,they were clustered into 10 classifications,with the highest abundances in the membrane,cell,and organelle clusters,which contained 755,674,and 449 DEGs,respectively.In the MF category,the unigenes were clustered into 10 classifications,the largest of which were associated with catalytic activity,binding and transporter activity,containing 1 112,950 and 109 DEGs,respectively(Fig.3).

Fig.1 Isolation of dark separate endophyte S16 from the soil of a sweet cherry orchard.A,colony of S16 cultured on PDA medium for 14 days.B,the intervals of the hyphae were measured with dark septate chambers under a microscope.C,neighbor-joining phylogenetic tree based on ITS rDNA sequence data of S16,along with selected fungal species which have high sequence similarity in GenBank.

To further systematically evaluate the metabolic pathways involving the DEGs,KEGG analysis was performed (Fig.3).The most enriched pathways were related to metabolism,including amino acid metabolism(79),biosynthesis of other secondary metabolism (112),carbohydrate metabolism (268),energy metabolism (97),lipid metabolism (67),cofactor and vitamin metabolism(51),terpenoid and polyketide metabolism (49),and nucleotide metabolism (21).The second most enriched category of pathways was genetic information processes,including folding,sorting and degradation (140),transcription (109),translation (45),and replication and repair (10).The last group of pathways was related to environmental information processing,including signal transduction (87),transport and catabolism (137),and environmental adaptation (163).These results suggested that DSE colonization regulated important cellular metabolic pathways that control sweet cherry growth.

3.4.Validation of RNA-seq data

To further validate the reliability of the DEGs based on RNA-seq,we randomly selected approximately 12 genes that were up-or down-regulated by S16 treatment for validation by qRT-PCR.The variation tendency of the genes validated by qRT-PCR was similar to that shown by the RNA-seq data,and the correlation reached 0.75(Fig.4).Therefore,these results indicated that the data identified through RNA-seq analysis are reliable.

Fig.2 Effects of S16 on Gisela 5 sweet cherry seedlings.A,photograph of 2-month-old plants.Left,S16-treated plants (S16);right,controls (CK).B,height of stem,fresh weight of roots,fresh weight of stem and leaves,and leaf numbers measured in S16 and CK plants.Data are mean±SD (n≥20 seedlings per group).＊,P＜0.05 by Student’s t-test.C and D,microscopic observation of the roots of sweet cherry infected with S16 hyphae after inoculation.

3.5.Network construction associated with S16 colonization

To systematically elucidate the functions of the DEGs,STRING analysis was conducted for further bioinformatic analysis,and the results were displayed with CYTOSCAPE.In the resultant map,red represents upregulated genes,while green represents downregulated genes (Fig.5).From this analysis,we found that the DEGs regulated by S16 play significant roles in various metabolic processes,which could provide a good start for identifying the functions of the DSE strain related to the growth of the host plants.

Fig.3 GO and KEGG analyses of the differentially expressed genes (DEGs).

Fig.4 Validation of RNA-seq results by qRT-PCR.The expression levels of 12 differentially expressed genes (DEGs) selected in this study were detected by RT-qPCR.R2 represents the correlation coefficient between the two platforms.The numbers on the scale bar represent RPKM values according to RNA-seq and ΔΔCt in qRT-PCR,which were used to evaluate the correlation (R2).

According to this network,many DEGs were involved in hormone responses,such as ABI family genes (ABI1andABI5) for the abscisic acid (ABA) signaling pathway and auxin response genes such asAXR3,ETT,IAA16andMP,andLOX2andLOX3for the jasmonic acid response pathway,which is consistent with the notion that DSEs play key roles in regulating plant growth.Additionally,some DEGs that respond to stress,such asMPK3,IRX1,HSP7CandCTP38,were also enriched in these networks.Furthermore,this analysis identified more than 25 transcription-related genes,including auxin signaling pathway transcription genes (ARF6,MP,ETT,andFLP),that were upregulated under S16 treatment.In contrast,Ubl conjugation-related genes,such asPIL5,ABI5,ICE1andJAZ8,were downregulated by S16 treatment.Differentiation-related transcript genes such asSEP3,AGandFVEwere also affected.Furthermore,some phosphorus metabolic process-related genes were enriched in our DEG data,which included the phosphatase activity-related genesPEPC1andPFADSP3and kinase activity-related genes such asAK3,APK,ADK2,PRK,MPK3andPPDK.These results indicated that DSEs may be involved in posttranslational modification.Some antioxidant enzymes were also found among the DSEs,such as the APX enzyme family geneAPX3and the GPX enzyme family genesGPX1andGPX6,which may play a significant role in tolerance to abiotic stressors.

In summary,this network highlighted the significant role of DSEs in symbiosis with sweet cherry by regulating cell metabolism,growth,and oxidation resistance pathways,extending our understanding of the critical cellular functions related to plant growth in which DSEs are involved.

3.6.ldentification of genes related to nitrogen transportation

Fig.5 A variety of biological functions enriched according to GO analysis based on the differentially expressed genes (DEG)interaction map.Some DEG proteins with clear functional annotations were selected for GO and network analysis.The results of the network analysis were then further analyzed and visualized using CYTOSCAPE.

The DSE inoculation of plants can reportedly affect plant growth and nitrogen availability;therefore,we sought to explore the molecular changes associated with DEGs related to nitrogen uptake and metabolism.The results of screening the RNA-seq data showed that some N-related metabolic pathway genes were enriched,such as NO3–uptake-related transporters (NRT2.1,NRT2.5 and NRT3.1),NH4+uptake-related transporters (AMT1.1,AMT2.1,AMT3.1 and AMT4.1),N metabolism enzymes(NR,GS,GD1 and GD4),and glutamate receptors (GR1.3,GR2.8,GR3.1,GR3.2,GR3.3 and GR3.4) (Fig.6-A).We also found that,in response to S16 treatment,most of the identified genes were upregulated,especially the nitrate transporter family genes.To further validate the reliability of the selected N-related genes,qRT-PCR was performed.The transcript abundances of NRT family genes (NRT2.1,NRT2.5 and NRT3.1) in the roots and leaves were much higher under S16 treatment than in the control,although the expression level in the leaves was lower than that in the roots (Fig.6-B).Similarly,there were significant differences between AMT family genes(AMT1.1,AMT2.1 and AMT3.1) in the roots and leaves under S16 treatment compared with the control (Fig.6-C).However,the expression level of the AMT genes in leaves was higher than the expression level of the NRT genes,which indicated that NRTs were specifically expressed in roots.N metabolic enzymes (NR,GS and GGAT2)were also regulated by S16 treatment:GS showed a significant difference between the S16 treatment and the control both in the roots and leaves.GGAT2 showed a slight difference in the roots but not in the leaves,and NR showed no difference in the roots or leaves (Fig.6-D).Therefore,all these results indicated that DSE may promote the growth of sweet cherry through colonization by increasing nitrogen absorption.

3.7.The effects of S16 colonization on nitrate accumulation and related enzyme activities

To further qualitatively and quantitatively detect the influence on nitrate in sweet cherry,the contents of NO3−,NH4+and NO2−were detected in the roots and leaves after four weeks of S16 colonization.S16 significantly increased the contents of NO3−in the roots and leaves(Fig.7-A).The contents of NH4+also increased under S16 treatment in both the roots and leaves (Fig.7-B).However,the NO2−concentration was only affected by S16 treatment in the roots,but not in the leaves (Fig.7-C).

After the uptake of NH4+and NO3−into the roots,enzymes play a significant role in nitrate reduction and assimilation.We found that the activity of NR was significantly higher in the S16-treated roots,while there was no significant change in the leaves compared with the control (Fig.7-D).Similarly,NiR activity was significantly affected in the roots but not in the leaves under S16 treatment (Fig.7-E).Along with the increasing N levels under S16 colonization,GS activity also increased in the roots and leaves (Fig.7-F).These results indicated that colonization by DSEs significantly promotes nitrate assimilation-related metabolism in sweet cherry.

4.Discussion

Depending on the host response,the interaction between DSEs and plant roots ranges from pathogenic to mutualistic (Mandyam and Jumpponen 2014).However,the effects of DSEs are usually beneficial to host plants under various stresses (Johnsonet al.2014).In the present study,we found that the effects of S16 on the growth of sweet cherry were positive.Although the ITS rDNA of S16 shared 99% identity with that of theFungalsp.2563YZ-2011 strain isolated from the mycelial cortices of naturalO.sinensisspecimens,reports on the function of theFungalsp.2563YZ-2011 have not been available(Zhanget al.2010).Thus,we speculated that S16 and 2563YZ-2011 belong to the same species,but whether they are two biovars or not needs further definition.Using high-throughput sequencing technologies,we conducted an in-depth exploration of the molecular basis of sweet cherry growth in response to DSEs.The results showed that genes related to metabolic processes,genetic information processes,and responses to environmental signals were enriched under S16 colonization (Fig.3).Furthermore,nitrogen-related genes,most of which showed upregulation upon S16 treatment,were also enriched.In addition,the nitrogen-related physiological indices indicated that S16 probably promotes plant growth by increasing nitrate uptake.

Nitrogen is an important nutrient for plant growth,and the nitrogen content accounts for 2% of plant dry weight(Boudsocqet al.2012).RNA-seq data analysis showed that the increased transcript levels of sweet cherry NRTs and AMTs may be partly responsible for the increased uptake of NO3–and NH4+under S16 treatment (Fig.6).In addition to the NRT and AMT genes that were upregulated under S16 treatment according to the RNA-seq analysis,we found that the gene coding nitrate reductase was also enriched in the DEGs.Nitrate reductase can reduce NO3−to nitrite when NO3−is taken up by plants.Furthermore,it has been previously reported that nitrate reductase activity is generally higher in plants colonized with fungi than in control plants without mycorrhizae in both roots and shoots.In the plants without mycorrhizae,NO3−reduction primarily occurs in the leaves,while in plants with mycorrhizae,NO3−is predominantly reduced in the roots,which is in accord with the results shown in Fig.7.In the next step,nitrite reductase converts nitrite to NH4+.Due to the ability of plants to perform uncoupled respiration,excess N in its reduced form of NH4+is toxic to cells,so NH4+is rapidly assimilated into amino acids (Templeet al.1998).Hence,two pathways,the NAD(P)-glutamate dehydrogenase (GDH) and glutamine synthetase-glutamate synthase (GS-GOGAT) pathways,are involved in NH4+assimilation by fungi.In the GSGOGAT pathway,glutamine synthetase converts Glu and NH4+to Gln,and Glu is then produced from Gln and 2-oxoglutarate by glutamate synthase (Govindarajuluet al.2005).Studies have reported that the activities of GS and GOGAT are significantly higher in the roots and shoots of fungus-colonized plants (Fatureet al.1998),while our results showed that the gene encoding GS was upregulated under S16 colonization (Fig.6) and that GS activity was higher in the roots and leaves under S16 treatment than in the control (Fig.7).

Fig.6 Nitrate uptake-related genes enriched under S16 treatment.A,heatmap analysis of the nitrate-related genes among the DEGs between the S16 treatment and the control.B–D,validation of the RNA-seq results of nitrate-related genes by qRT-PCR in the roots and leaves.Data are mean±SD (n=3).＊,＊＊,and ＊＊＊,P＜0.05,P＜0.01,and P＜0.001 by Student’s t-test.

Fig.7 Nitrate-related physiological indices measured in the roots and leaves.Concentrations of NO3− (A),NH4+ (B),and NO2− (C)in roots and leaves of sweet cherry plants under S16 treatment.Activities of nitrate reductase (NR) (D),nitrite reductase (NiR)(E) and glutamine synthetase (GS) (F) in the roots and leaves of sweet cherry plants under S16 treatment.Data are mean±SD(n=3).＊,P＜0.05 by Student’s t-test.

In addition to promoting nitrate assimilation under S16 colonization,some phytohormone-related genes were also enriched in the network associated with the DSE interaction with sweet cherry (Fig.5).Phytohormones play a significant role in regulating plant growth as active signals.Among phytohormones,IAA and GA play important roles in plant growth and development and are considered essential endogenous growth regulators in plants (Gray 2004).In addition,endophyte colonization regulates the contents of phytohormones in the host plant and increases host plant tolerance under heavy metal stress (Khan and Lee 2013).Additionally,endophytic fungi have the ability to produce phytohormones (IAA/GA),which could effectively promote the growth of plant roots and improve nutrient uptake (Sukumaret al.2013).To gain further insight into DSE-related growth promotion,we analyzed the ability of the fungal-colonized plants to secrete IAA at the transcriptional level.The results showed that S16 increased the levels of IAA biosynthesis pathway-regulated genes in sweet cherry (Fig.5).This phenomenon suggested that IAA synthesis regulated by DSEs may play key roles in plant development,increasing the root surface absorption area and resulting in increased water and nutrient uptake,thereby promoting root colonization by other fungi.

According to our RNA-Seq data,some genes encoding antioxidant enzymes were also enriched.As previously reported,symbiotic fungi can promote the activation of antioxidant enzymes in plants to scavenge ROS (Baltruschatet al.2008).Increased activities of antioxidant enzymes,including CAT (catalase),APX(ascorbate peroxidase),DHAR (dehydroascorbate reductase),and GR (glutathione reductase),all play significant roles in tolerance to abiotic stressors.Furthermore,a gene related to the HSP family,HSP70,was upregulated in our interaction network.Most HSP family genes are important in maintaining the homeostasis of protein folding as a function of molecular chaperones.It has been reported that HSP overexpression could enhance the tolerance of plants under various stresses(Sunet al.2001).In our study,ROS scavenging system-related genes were also upregulated under S16 colonization (Appendix C).These results indicated that DSEs may induce the ability to scavenge ROS and to accumulate HSPs,which may be a good strategy for promoting the growth of sweet cherry in association with DSE colonization.

5.Conclusion

In this study,we found that the S16 DSE strain could promote the growth of sweet cherry seedlings and improve nitrate assimilation.RNA-seq analysis showed that some plant hormone-related genes,such as indoleacetic acid,abscisic acid,and salicylic acid,were also enriched under S16 colonization.Hence,RNA-seq data may be an important resource for future studies of the symbiosis of DSEs with host plants.Further genetic and molecular analyses will be critical for elucidating the detailed roles of these genes in the growth of sweet cherry and understanding plant–fungus mutualistic interactions.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (31570649),the Key Research and Development Program of Yantai City,Shandong Province,China (2019XDHZ089),the Natural Science Foundation of Shandong Province,China (ZR2019PC052 and 2017C03),and the Key Research and Development Program of Shandong Province,China (2019GSF107091,2019GSF109114,2018YYSP004 and 2018GNC110028).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm