Three Genes Related to Trehalose Metabolism Affect Sclerotial Development of Rhizoctonia solani AG-1 IA,Causal Agent of Rice Sheath Blight

WANG Chenjiaozi,ZHAO Mei,SHU Canwei,ZHOU Erxun

(Guangdong Province Key Laboratory of Microbial Signals and Disease Control,Department of Plant Pathology,South China Agricultural University,Guangzhou 510642,China)

Abstract:Trehalose metabolism is related to the sclerotial development of Rhizoctonia solani AG-1 IA,the causal agent of rice sheath blight (RSB).Here,we further elucidated the functions of three genes Rstre,Rstps1 and Rstpp that encode three key enzymes trehalase (TRE),alpha,alpha-trehalose-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPP) in the sclerotial development of R.solani AG-1 IA.Due to the lack of a stable genetic transformation system for R.solani,the heterologous expression of these three genes in Pichia p astoris GS115 was performed.The results showed that reactive oxygen species (ROS) contents and enzyme activities in R.solani decreased significantly in the treatments of the fermentation broths of Rstps1 and Rstpp transformants,and that in the treatment of the fermentation broth of Rstre transformant visibly increased.Furthermore,the fermentation broths of the transformants of all the three genes were added to potato dextrose agar (PDA) medium for the cultivation of R.solani,as a result,the dry weight of sclerotia in each PDA plate containing the fermentation broths of Rstps1 and Rstpp transformants significantly increased compared with the control,and that of Rstre transformant obviously decreased.Finally,178 proteins were found to interact with RSTPS1,and 16 of them were associated with ROS.Taken together,the findings suggest that all these three genes related to trehalose metabolism play important roles in the sclerotial development of R.solani AG-1 IA,and can be used as new targets for the development of novel high-efficiency fungicides for the controlling of RSB.

Key words:Rhizoctonia solani AG-1 IA;rice sheath blight;reactive oxygen species;trehalose metabolism;gene functional analysis;sclerotial development

Rhizoctonia solaniis one of the most important soil-borne plant pathogenic fungi,which can infect a lot of plants and causes severe plant diseases such as rice sheath blight (RSB).It is mainly in the forms of hyphae and sclerotia under natural conditions (Domingo et al,2014).Sclerotia,which can survive in extreme environments for several years,are a hard structure made of tangled vegetative hyphae (Wibberg et al,2014;Feng et al,2017).Therefore,the residual sclerotia in the field are the preliminary infection source in the next year.Sclerotia play an important role in the disease cycle of RSB,and the inhibition of sclerotial development inR.solaniAG-1 IA is conducive to control this important rice disease.

The development of fungal sclerotia is a complex process.Georgiou et al (2006) proposed an important hypothesis that reactive oxygen species (ROS) induced sclerotial development.Eukaryotes produce ROS such as hydroxyl radical (·OH),superoxide anion (O2·-),singlet oxygen (1O2) and hydrogen peroxide (H2O2) during metabolism.As an important regulatory molecule in cell signaling pathway,ROS maintains at a low and beneficial concentration level in biological cells (Fang et al,2002;Lara-Ortíz et al,2003).However,the excessive accumulation of ROS will cause toxicity to fungal cells and induce sclerotial differentiation to resist toxic ROS to mycelia.On the contrary,low ROS concentrations in the environment (e.g.,treated with antioxidants) lead to sclerotial formation retardation (Georgiou et al,2000;Lu et al,2016).When biological cells respond to oxidative stress,antioxidant enzymes (such as catalase and superoxide dismutase) and nonenzymatic protective systems (such as glutathione and thioredoxin) can protect cells from ROS damage.Trehalose is one of ROS scavengers,which can eliminate ROS in fungal cells by increasing the activities of superoxide dismutase,catalase and peroxidase (Benaroudj et al,2001;Pan et al,2016).

Trehalase (TRE) is an enzyme that catalyzes the hydrolysis of trehalose.It can decompose trehalose into two glucose molecules specifically and directly (Hottiger et al,1987),while alpha,alpha-trehalose-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPP) are two enzymes that can catalyze trehalose biosynthesis.In fungi,TRE reduces trehalose content in fungal cells by decomposing trehalose,and the other two enzymes,TPS1 and TPP,increase trehalose content by inducing the synthesis of trehalose.As a result,these three enzymes can regulate the balance of trehalose content in fungal cells.Therefore,the functional analysis of the genestre,tps1andtppencoding these three enzymes can provide molecular targets for the development of novel fungicides with high efficiency to fungi and low risk to environment (Fan et al,2009).In our previous studies,it was found that the reduced trehalose weakens ROS scavenging ability,which leads to the accumulation of ROS in fungal cells,and excessive ROS induces the sclerotial development ofR.solani(Lu et al,2016;Wang et al,2018).

So far,the functional analysis of genes related to trehalose metabolism inR.solanihas not been reported.In previous studies,transcriptional analysis of sclerotial morphogenesis inR.solaniwas conducted by the RNA-Seq technique,and the results showed that the expression levels of three trehalase-encoding genesRstre,Rstps1andRstppare significantly different during different sclerotial development stages,which is further verified by qRT-PCR (Shu et al,2014,2015,2019).Therefore,we hypothesized that the trehalose metabolic pathway could play an important role during the sclerotial development in RSB pathogen.To verify the hypothesis,the exact functions of these three genesRstre,Rstps1andRstpprelated to trehalose metabolism and the proteins that interact with protein RSTPS1 during the sclerotial development ofR.solaniAG-1 IA were investigated in this study.The results will provide new targets for developing novel fungicides so as to control RSB efficiently.

RESULTS

Characterization and expression patterns of Rstre,Rstps1 and Rstpp genes

The genes related to trehalose metabolism are characterized (Fig.S1).The coding sequence lengths ofRstre,Rstps1andRstppare 2 277,2 850 and 3 924 bp,respectively.The signal peptide ofRstrewas predicted at the 18th amino acid site,and there was no signal peptide inRstps1andRstpp(Fig.S2-A).TMHMM Server v.2.0 was used to analyze the transmembrane domains of these proteins.The results showed that there were no transmembrane domains inRstreandRstpp,and there was one transmembrane domain inRstps1(Fig.S2-B).qRT-PCR results showed that the expression level ofRstregene was the highest during 3-5 d after inoculation,whereas the expression levels ofRstps1andRstppwere the lowest at the same stage (Fig.1-A).This stage is just the period of sclerotial formation,and the morphology of sclerotia changes greatly at this stage.Therefore,the accumulation of ROS was the highest at this stage,and the three genes related to trehalose metabolism responded to the development of sclerotia.Western blotting analysis was performed to evaluate the protein expression level during the sclerotial development stage.As shown in Fig.1-B,the expression levels of RSTPS1 and RSTPP significantly decreased at 5 d after inoculation,indicating that proteins encoded by the trehalose metabolism-related genes responded to the development of sclerotia.The activities of three enzymes RSTRE,RSTPS1 and RSTPP at different developmental stages of sclerotia were measured (Fig.1-C).The results showed that the RSTRE activity markedly increased during 60-120 h after inoculation,which was highly consistent with the expression ofRstregene revealed by qRT-PCR (Fig.1-A).The enzyme activities of RSTPS1 and RSTPP encoded byRstps1andRstppwere also highly consistent with the results of qRT-PCR,respectively.These findings further indicated that trehalose accumulated at the most severe oxidative stress stage,and its enzyme activity decreased at the later stage of sclerotial development due to the sclerotia mature (Fig.1-C).

Heterologous expression of Rstre,Rstps1 and Rstpp genes in Pichia pastoris resulted in significant changes in enzyme activities of transformants

Three transformants GS115-Rstre,GS115-Rstps1and GS115-Rstppwere obtained.The copy numbers of the three genes in different transformants were determined by the absolute quantification technique.The samples were diluted to four gradients,and the CT values obtained by fluorescence quantitative PCR showed good reproducibility.The amplification curve and the melting curve showed that the amplification efficiency was good (Fig.S3).The high-copy trans-formants were selected for further study.SDS-PAGE results showed that the proteins were accumulated inP.pastoristransformants (Fig.S4).

Besides the solvent peak,three other peaks can be clearly seen from the standard curve of liquid chromatogram in the fermentation broth of GS115 transformants (Fig.2).Compared with the standard peaks and their retention time,intracellular RSTPS1 and RSTPP in the GS115 transformants can produce trehalose by catalyzing maltose,and the enzyme activities were 3 124.7 and 3 318.3 U/mL,respectively.Intracellular RSTRE in the GS115 transformant can produce glucose by catalyzing trehalose with the enzyme activity of 3 672.3 U/mL.

Heterologous expression of Rstre,Rstps1 and Rstpp genes in P.pastoris affected ROS content of transformants

The contents of ROS in GS115 and its transformants were detected by a fluorescence microplate system.It was found that the ROS contents in the fermentation broths ofRstps1andRstpptransformants were significantly reduced,and the ROS contents can restore after the addition of trehalose into the fermentation broths.However,the ROS contents in theRstretransformants were significantly increased,and the ROS contents can restore after the addition of Validamycin (Jinggangmycin) (an inhibitor of trehalase) in the fermentation broths (Fig.3).

Fermentation broths of transformants affected the amount of sclerotia in R.solani

The fermentation broths of the three transformants were added to the potato dextrose agar (PDA) media to prepare fermentation broth-containing PDA plates for culturing the RSB pathogenR.solani.The results showed that the dry weight of sclerotia in each PDA plate containing the fermentation broths of the transformants GS115-Rstps1and GS115-Rstppwere significantly increased,and that of the transformant GS115-Rstrewas obviously decreased,indicating that the dry weights of sclerotia in all the treatments were significantly different compared with the control.These findings suggested that these trehalose metabolism-related genes inR.solanican regulate the development of sclerotia (Fig.4).

Isolation of RSTPS1 interacting proteins

Using GST-pull down assay of RSTPS1 (Fig.S5),178 proteins were found to interact with RSTPS1.The majority of interacting proteins belong to the following six functional categories:ion binding,oxidoreductase activity,biosynthetic process,small molecule metabolic process,translation and cellular amino acid metabolic process.Among them,16 proteins were associated with ROS (Table S1).The results suggested that RSTPS1 may play an important role during sclerotial development by interacting with these proteins (Figs.5 and S5).

DISCUSSION

Trehalose and sclerotial development in R.solani

This study revealed the role of trehalose metabolic pathway in the sclerotial development ofR.solani.According to our previous study,sclerotial development inR.solaniis regulated by ROS (Wang et al,2018).Trehalose responses to environmental stress in many organisms,and it is also an effective ROS scavenger.In recent years,some studies related to the effect of trehalose metabolism on fungal pathogenicity and host-pathogen interaction have been conducted.The gene deletion mutants cannot make good use of exogenous trehalose as only carbon source when thetregene inMetarsiana anisoensisis knocked out,and they display slow growth and weak virulence (Jin et al,2015).Another sample is that the knockout oftregene inCandida albicansleads to reduced virulence (Pedre?o et al,2007).Therefore,thetregene can be used as a potential target for screening novel antifungal substances.Thetregene knockout mutants ofAspergillus nigeronly produce a few active conidia,and they cann’t degrade intracellular trehalose during conidial germination.These studies indicate that thetregene is necessary for conidial development and germination ofA.niger(Svanstr?m and Melin,2013).Leite et al (2016) also reported that the increase of intracellular TRE inDekkera bruxellensisprevents the storage of trehalose.Zou et al (2017) found that wheat stripe rust pathogenPuccinia striiformisf.sp.tritici(Pst) genePsttps1is related to the cold adaptation ofPstand is also involved in the pathogenesis ofPstby limiting its hyphae expansion.However,the effects of trehalose metabolism-related genes on sclerotial development have not been reported so far.In this study,we preliminarily demonstrated that the three key enzymes RSTRE,RSTPS1 and RSTPP and their encoding genesin the trehalose metabolism pathway played important roles during sclerotial development ofR.solaniAG-1 IA through qRT-PCR,Western blot and enzyme activity assays.The results showed that the expression levels of the three genes were changed greatly at the critical stage during sclerotial development.Western blot and enzyme activity assays also verified this result.Interestingly,we foundRstreapparently decreased at 168 h after inoculation.As trehalose accumulates in response to various environmental stresses (Tournu et al,2013;Leite et al,2016),we speculated that reduced trehalase might be in response to stress environment after sclerotium formation.

Rstre,Rstps1 and Rstpp can regulate sclerotial development by inducing oxidative stress

In trehalose decomposition pathway,TRE specifically and directly decomposes trehalose into two glucose molecules as a key enzyme (Hottiger et al,1987).In trehalose synthesis pathway,TPS1 and TPP can catalyze trehalose synthesis from glucose.TRE reduces the trehalose content in fungal cells by decomposing trehalose,and the less content of trehalose can not remove excessive ROS effectively,and thus inevitably leading to the accumulation of ROS in fungal cells,and excessive ROS will then induce fungal differentiation to form sclerotia (Jin et al,2015).TPS1 and TPP can induce ROS scavenging ability and delay fungal differentiation by catalyzing the synthesis of trehalose.Regards to scavenging ability of trehalose to ROS,Theerakulpisut and Gunnula (2012) also found that the activity of ascorbate peroxidase increases and the accumulation of H2O2significantly decreases in rice treated with exogenous trehalose.Luo et al (2008) found that exogenous trehalose with a concentration of 50 mmol/L can eliminate ROS produced by wheat under high temperature stress.Yeast cells treated with 10% exogenous trehalose also improve their resistance to H2O2(da Costa Morato Nery et al,2008).These studies indicated that exogenous trehalose treatment is beneficial for organisms to scavenge intracellular ROS so as to resist oxidative stress environment.Many high efficient fungicides have been developed with genes (such astre) related to trehalose metabolism as molecular targets,including Validamycin (Jinggangmycin),a specific fungicide for controlling RSB (Xu et al,2015).The fungicide disrupts the glucose metabolism of the organism and eventually leads to the death of the organism by inhibiting the activity of TRE.As it is difficult to establish a genetic transformation system forR.solani,no research on function verification of sclerotial development-related genes has been performed inR.solaniso far.We just conducted research to elucidate the effect of the three genes on the production of ROS through gene heterologous expression.This is the first trial to indirectly verify gene function on sclerotial development by adding the fermentation broths ofP.pastoristransformants into the culture medium.The effects of trehalose metabolism pathway on sclerotial development were analyzed by the gene heterologous expression technique,and the results showed that the three key enzymes related to trehalose metabolism can regulate sclerotial development by inducing the oxidative stress.

Proteins associated with ROS interacted with RSTPS1 are worthy of further investigation

For further research,our interests were focused on the proteins interacted with RSTPS1.By using GST-pull down technique,178 proteins were found to interact with RSTPS1,including 16 proteins associated with ROS.These findings will provide new molecular targets for developing novel fungicides with high efficiency for controlling RSB and reduce the risk to the environment.These results also confirmed that trehalose metabolism-related genes may induce the sclerotial development by regulating ROS content inR.solani.These interacting proteins may also affect the sclerotial development inR.solani.In this way,we can obtain more valuable molecular targets for the development of highly effective fungicides for the control of RSB disease.

METHODS

Strains and plasmids

Fungal strain GD-118,a typical strain of RSB pathogenR.solaniAG-1 IA with strong virulence,was used.Escherichia coliJM109 was used to propagate plasmid DNA.YeastP.pastorisstrain GS115 and vector pPIC9K were purchased from Invitrogen (Carlsbad,CA,USA).E.coliJM109 cells with plasmid were grown aerobically in Luria-Bertani medium supplemented with 100 μg/mL ampicillin.The media and culture conditions for the expression of recombinant α-amylases were performed according to thePichiaExpression Kit manual and thePichiaFermentation Process Guidelines (Invitrogen,CA,USA).

Isolation of total RNA and synthesis of cDNA

For further study of gene expression profiling during sclerotial development,the mycelia of GD-118 were collected at seven different time points (36,48,60,72,120,168 and 336 h of strain culture) during the sclerotial development (Wang et al,2018).Total RNA extraction was performed using TaKaRa RNA Extraction Kit (TaKaRa,Shanghai,China) and treated with DNase-free water.First strand cDNA was synthesized using an oligonucleotide DT and Superscript II reverse transcriptase (Invitrogen,CA,USA),according to the manufacturer’s instructions.

qRT-PCR

Primers were designed to amplify the transcript of the three genes (Table S2).In each set of qRT-PCR experiments,the data were normalized to transcripts encodingGAPDH(Shu et al,2015).The relative expression levels of the interested genes were calculated using the standard curve method for relative quantification (Bookout et al,2006).The primers for qRT-PCR analyses designed using the Primer Premier 6.0 software (St.Andrews,New Brunswick,Canada) were listed in Table S2.

Western blotting

The soluble fraction,cell wall-enriched fraction and membrane-enriched fraction proteins from different samples ofR.solaniAG-1 IA were run on polyacrylamide gel,and then transferred to a nylon membrane and checked by Ponceau S (Sigma-Aldrich,Saint Louis,USA) to determine equal loading amount.To improve the quality of the samples,a clean-up kit (GE Healthcare,Marlborough,USA) that can reduce streaking,background staining and other artifacts was used.RSTPS1 and RSTPP were detected using anti-TPS1 and anti-TPP antibodies,respectively.After reaction with alkaline phosphate anti-rabbit immunoglobulin G,the reaction was developed with 5-bromo-4-chloro-3-indolyl-phosphate-nitroblue tetrazolium.For electrophoresis and Western blot experiments,the same protein amounts (100 mg) from soluble fraction,cell wall-enriched fraction and membrane-enriched fraction were used.However,we did not perform the Western blot of RSTRE due to the failure in the preparation of RSTRE antibody.

Enzyme activity assays of RSTRE,RSTPS1 and RSTPP

To determine enzyme activity,0.3 g of mycelia samples were homogenized in 5.0 mL of 50 mmol/L potassium phosphate buffer (pH 7.0) containing 1 mmol/L EDTA and 1% polyvinyl-pyrrolidone.The homogenate was centrifuged at 15 000 r/min for 20 min at 4 °C,and supernatant was immediately used in the assay of enzyme activity.Protein content was determined according to the Bradford method,using bovine serum albumin as the standard.

To test RSTRE activity,we used the 3,5-dinitrosalicylic acid (DNS) colorimetry.The enzyme activity was assayed in 25 mmol/L citrate-sodium phosphate (pH 6.0) containing 7 mmol/L trehalose at 30 °C so that the product formation was proportional to sample protein concentration and to assay time (initial velocity).Controls without enzyme or without substrate were included.One unit of enzyme (U) is defined as the amount that hydrolyses 1 μmol of substrate per min.

RSTPS1 activity was measured by the method of Hottiger et al (1987).Assay mixture contained tricine (K+) (50 mmol/L,pH 7.0),glucose 6-phosphate (G-6-P,10 mmol/L),UDP-glucose (5 mmol/L),MgCl2(12.5 mmol/L),enzyme sample and water in a total volume of 0.4 mL.The reaction was stopped by the addition of 0.2 mL of 1 mol/L ice-cold perchloric acid.Because G-6-P and glucose uridine diphosphate can be converted to trehalose 6-phosphate by TPS1 in fungi (Richards et al,2002),the decrease in absorbance at 340 nm was recorded and used to calculate the concentration of UDP-glucose.One unit of enzyme (U) is defined as the amount that hydrolyses 1 μmol of UDP-glucose per min.The assay was run at 35 °C.

Assays for RSTPP activity were carried out in 1 mL of reaction mixture containing the following components:480 μL of 100 μmol/L Tris-HCl (pH 7.2),250 μL of 20 μmol/L MgCl2·6H2O,250 μL of 4 μmol/L trehalose-6-phosphate and an appropriate amount of enzyme.After incubation at 37 °C for 30 min,these mixtures were boiled for 5 min to stop the reaction.The amount of trehalose produced was determined by phosphate:300 μL of the above mixture was added with 900 μL of 100 mmol/L zinc acetate and 15 mmol/L ammonium molybdate solution (pH 5.0),ice bath for 1 min.Flurescence intensity at OD350was determined by a spectrophotometer (U-2910,Hitachi,Japan).

Cloning of Rstre,Rstps1 and Rstpp genes and generation of expression construction

Three pairs of primers were designed to amplify the transcript of the three genes (Table S2).The PCR reaction mixture (50 μL) containedR.solanicDNA,0.25 μmol/L of each primer,200 μmol/L of dNTPs and 1.25 U of PlatinumTaqDNA Polymerase High Fidelity (Invitrogen,Carlsbad,USA).Thermocycling consisted of 30 cycles at 94 °C for 30 s,57 °C for 30 s and 68 °C for 90 s.The resulting 2.85 kb ofRstre,3.60 kb ofRstps1and 3.90 kb ofRstppcDNA fragments were cloned into the pPIC9K cloning vector (Invitrogen,Carlsbad,USA).The pPIC9K cloning vectors containing the signal peptides were treated with restriction enzymesXhoI andXbaI.The cDNA fragments and the vector containing a fragment encoding the hygromycin B resistance gene (TaKaRa,Kyoto,Japan) were mixed and ligated with T4 DNA ligase (Sigma-Aldrich,Saint Louis,USA).The expression plasmids encoding the whole transcripts were named as pPIC9K-Rstps1,pPIC9K-Rstppand pPIC9K-Rstre,respectively.Moreover,the NCBI online comparison tool ORF Finder was used to predict the open reading frame of the genes,and the DNAMAN software was used for protein translation.SingaIP 5.0 was used to predict the signal peptides of proteins.

Heterologous expression of Rstre,Rstps1 and Rstpp genes in P.pastoris

Transformation into P.pastoris

After verification byPstI digestion,the recombinant plasmids were linearized and electrotransformed intoP.pastorisGS115 according to thePichiaExpression Kit Manual (Invitrogen,Carlsbad,USA).Mut+transformants were initially selected by minimal dextrose (MD) medium (13.4 g/L yeast nitrogen base,0.4 mg/L biotin,20 g/L dextrose).After incubation at 30 °C for 3 d,50 large colonies from MD plates were selected,and pointed onto yeast extract peptone dextrose (YPD) plates containing 0.5 mg/mL of G418 and grown at 30 °C for 2-3 d.The grown clones were then moved to YPD plates supplemented with 2 mg/mL of G418.

Gene expression of Rstre,Rstps1 and Rstpp

The clones from the YPD plates containing 2 mg/mL of G418 were selected and cultivated in a shaking flask.The clones were inoculated into 25 mL of buffered glycerol-complex medium at 30 °C on a rotary shaker at 220 r/min until the culture reached the logarithmic phase (OD600=2.0-6.0).The cells were harvested by centrifugation and resuspended in 50 mL of buffered methanol-complex medium and incubated at 30 °C and 200 r/min for 5 d with 0.5% methanol added daily.At the end of fermentation,the culture solution was centrifuged at 10 000 r/min for 10 min.The supernatant was collected and dialyzed against 0.5 mmol/L NaAc-HAc buffer (pH 5.0) overnight and lyophilized for further tests.

SDS-PAGE

Samples were subjected to SDS-PAGE,and proteins were electro-transferred to nitrocellulose membranes and immunodetected as described by Harlow and Lane (1988) with the antibody described above and reacted with a goat anti-rabbit secondary antibody labeled with horseradish peroxidase (Invitrogen,Carlsbad,USA).Lumi-Light Western Blotting Substrate Solution (Roche,Basel,Switzerland) was used as a substrate.

High performance liquid chromatography (HPLC)

The enzyme activities of RSTRE,RSTPS1 and RSTPP inP.pastorisGS115 and their transformants were measured with HPLC as described previously with a slight modification (Suzuki et al,2013).HPLC systems (Shimadzu LC-20A,Japan) were as follows:the chromatographic column was amino bonded phase column (5 μm,4.6 mm × 250 mm);the mobile phase was acetonitrile :water=75 :25 for GS115-Rstreand GS115-Rstps1and 80 :20 for GS115-Rstpp;the flow rate was 1.0 mL/min for GS115-Rstreand GS115-Rstps1and 1.4 mL/min for GS115-Rstpp;the column temperature was 40 °C;the detector was a differential refractive detector,and the sample volume was 10 μL.Taking the consistence of trehalose or maltose as theX-axis and the peak area as theY-axis,the standard curve was drawn.According to the standard curve,the content of trehalose or maltose in the sample can be calculated.Quantification of enzyme activity was performed by the final concentration of trehalose or glucose.The concentration of trehalose or glucose converted by maltose or trehalose per hour was indicated as an enzyme activity (U).

Detection of ROS

ROS measurements were performed by the fluorescence assay.Mycelia were stained with 2′,7′-dichloro-dihydro-fluorescein diacetate for 20 min and visualized under a fluorescence microscope (Eclipse 90i,Nikon,Japan) to assess the presence of H2O2.The H2O2content in liquid mycelia was quantitatively measured using a commercial Hydrogen Peroxide Assay Kit (Nanjing Jiancheng Bioengineering Institute,Nanjing,China).Briefly,H2O2binds to molybdenic acid to form a complex that can be measured at 405 nm,from which the H2O2content can then be calculated.

Detection of sclerotial development

Fermentation broth of each transformant was added to PDB medium.A mycelium block was taken along the edge of PDA medium and placed it in the center of PDB medium dish.Dry weights of sclerotia were measured at 14 d later.Three replicates were performed for each transformant,and the experiment was repeated three times.Sclerotial differentiation (SDIF) was defined as percent of dry weight of sclerotia (in respect to 100% SDIF of the control) per dish.

GST-pull down

The fusion expression of GST and GST-RSTPS1 inE.coliBL21 was transfected with the lysedR.solaniAG-1 IA.After incubation for 24 h,the cells were harvested and suspended in buffer A (50 mmol/L Tris-HCl at pH 7.5,1 mmol/L EDTA,2 mmol/L dithiothreitol,150 mmol/L NaCl,1% NP40) containing 1× protease inhibitor mixture (Roche,Basel,Switzerland).The cell extract was applied to GST-RSTPS1 immobilized on 10 μL of glutathione-sepharose beads (for GST-tag) (GE healthcare,Marlborough,USA).The beads were washed four times with buffer A,and the proteins bound to the beads were released by eluting with 1× SDS loading dye and boiling for 10 min.The samples were resolved on 8% SDS-PAGE gels and immuno-blotted with anti-GST.β-ACTIN was used as an internal control.

Statistical analysis

In each experiment,three replicates were used at each sampling point.Replicate values in each experiment and among three experiments were tested using the analysis of variance and the Student’st-test.The mean value and standard error were calculated for the dry weights of sclerotia measured from six independent experiments.All other values from each of the three experiments were combined and used to determine the mean value and the standard error.Resultswere considered to be statistically significant atP＜ 0.05.

ACKNOWLEDGEMENTS

This study was supported by the Major Program of Guangdong Basic and Applied Basic Research (Grant No.2019B030302006) and the National Natural Science Foundation of China (Grant No.31801677).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science;http://www.ricescience.org.

Fig.S1.Gene structures ofRstre,Rstps1andRstpp.

Fig.S2.Secondary structures and hydropathy profiles of RSTRE,RSTPS1 and RSTPP.

Fig.S3.Detection of transgenic copy number ofPichiapastorisGS115 with fluorescence quantitative PCR technique.

Fig.S4.Verification ofPichia pastorisGS115 transformants ofRstre,Rstps1andRstpp.

Fig.S5.Screening of proteins interacted with RSTPS1 inR.solaniby using GST-pull down technique.

Table S1.Annotation of interacting proteins associated with reactive oxygen species.

Table S2.Primer sequences used for gene cloning and qRT-PCR.