Ntionl Key Fcility for Crop Gene Resources nd Genetic Improvement,Institute of Crop Sciences,Chinese Acdemy of Agriculturl Sciences,Beijing 100081,Chin

bJiangxi Academy of Forestry,Nanchang 330013,Jiangxi,China

cState Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,China

Keywords:

A B S T R A C T Barley yellow dwarf virus(BYDV)can infect wheat(Triticum aestivum L.),leading to yield loss.Among four BYDV strains(GAV,GPV,PAV,and RMV)identified in China,BYDV-GAV is the prevailing isolate.YW642,a wheat—Thinopyrum intermedium translocation line,is resistant to BYDV isolates at both seedling and adult stages.Zhong 8601 is the wheat recurrent parent of YW642 and is susceptible to BYDV.In this study,we investigated the adult-stage resistance mechanism of YW642,measured BYDV titer and hydrogen peroxide(H2O2)in adult-stage leaves of YW642 and Zhong 8601 inoculated with BYDV-GAV,and identified transcriptional differences between YW642 and Zhong 8601 using microarray-based comparative transcriptomics.Enzyme-linked immunosorbent assay and H2O2assay showed that both BYDV titer and H2O2content were markedly lower in YW642 than in Zhong 8601 at 21,28,35,and 40 days post-inoculation(dpi).The transcriptomic comparison revealed that many types of genes were significantly up-regulated at 35 dpi in adult-stage leaves of YW642 compared to Zhong 8601.The important up-regulated genes associated with the adult-stage resistance encoded 15 resistance-like proteins,pathogenesis-related proteins(such as defensin and lipid transferproteins),protein kinase homologs,transcription factors,reactive oxygen species scavenging-related proteins,and jasmonic acid and gibberellic acid biosynthesis enzymes.These results suggest that precise expression regulation of these proteins plays a crucial role in adult-stage resistance of YW642 against BYDV infection.

1.Introduction

Common wheat(Triticum aestivum L.)is an important staple food crop cultivated worldwide that provides 20%calorific intake of the global population[1].Yellow dwarf virus(YDV)disease,caused by barley yellow dwarf virus(BYDV),is a viral disease affecting cereal crops(including wheat)and grasses worldwide[2].Yield loss in BYDV-infected wheat ranges from 10%to 48%[3—7].Five(MAV,RMV,SGV,RPV,and PAV)and four(GAV,GPV,PAV,and RMV)of BYDV strains have been identified in the U.S.[8,9]and China[10,11],respectively.BYDV-GAV is the predominant strain of wheat YDV throughout the northern and northwestern regions of China[12].

To date,no effective BYDV-resistance genes have been identified in the primary or secondary gene pools of wheat[13].Several groups have identified＞10 resistant wild relatives in the tertiary gene pool [14—17],including Thinopyrum intermedium,which possesses resistance to BYDV.Using chromosome engineering techniques,three resistance genes in Th.intermedium have been introgressed into common wheat[17—22].Bdv2 on the distal long arm of 7Ai-1 has been transferred to the 7DL terminus of common wheat,and confirmed by genomic in situ hybridization and molecular marker analysis in the wheat—Th.intermedium translocation lines TC14 and YW642[21—23].At the adult stage,the BYDVGAV susceptible Zhong 8601 showed visible YDV symptoms(plant dwarfing and leaf yellowing)at28—40 days postinoculation(dpi)[24],whereas YW642 showed no YDV symptoms at 35—40 dpi.YDV resistance at the adult stage could be an important part of host resistance to BYDV infection.However,the BYDV-resistance mechanism of wheat at the adult stage has not been identified.

In defense against pathogen attack,numerous plant response components participate in pathogen perception,signal generation and transmission of defense responses,and activation of defense products,resulting in restriction of pathogen invasion[25].For example,disease resistance proteins recognize pathogen effectors and activate defense responses[26].The importance of salicylic acid,jasmonic acid(JA),and ethylene,which act as primary phytohormone signals in the regulation of plant immune responses,has been well established[27].More recently,other hormones,such as gibberellins(gibberellic acid,GA)and abscisic acid(ABA),have been implicated in defense responses in plants species[27].Additionally,reactive oxygen species(ROS),including hydrogen peroxide(H2O2),play crucial roles in many physiological processes,including defense response,senescence, photorespiration, photosynthesis, stomatal movement,abiotic stress tolerance,and development[28,29].An appropriate H2O2level not only can promote cell wall reinforcement and phytoalexin production,but also has a signaling role in mounting defense responses[28].However,excessive accumulation of H2O2in plants can damage membranes and even lead to cell death and chloroplast damage.A balance between generation and scavenging of H2O2is vital for plant defense and growth.ROS homeostasis of plants is maintained by ROS-scavenging enzymes,including ascorbate peroxidase(APX),glutathione peroxidase(GPX),catalase(CAT),superoxide dismutase(SOD),germin-like protein(GLP),and ROS-producing enzymes like NADPH oxidase,as well as antioxidants like glutaredoxin[29—32].

The plant transcriptome is reprogrammed in response to pathogen infection[33].The transcriptional levels of a subset of genes,including defense-associated,pathogenesis-related(PR),and ROS-related genes and those associated with hormone signaling pathways,increase upon attempted pathogen infection[26,34—36].Precise transcriptional regulation of a battery of genes encoding diverse molecules in these processes determines plant resistance or susceptibility[37,38].In recent years,microarray technology has been used to investigate seedling defense mechanisms in plants.The Affymetrix GeneChip Wheat Genome Array represents over 55,000 wheat transcripts from all chromosomes and ancestral genomes.Ithas been used to analyze whole-genome gene expression in young seedlings in response to infection of fungal pathogens or BYDV[24,39,40].Our previous transcriptomic comparison between YW642 and Zhong8601leaves at seedlingstage[24]revealed that transcriptional reprogramming of resistance-like proteins,kinases,ROS-related genes,and JA-signaling genes all participated in the early defense response to BYDV infection.However,little is known about transcriptome differences between adult plants of YW642 and Zhong 8601.

In this study,the causal basis of BYDV resistance conferred by Bdv2 at the adult stage of YW642 was investigated via integrative transcriptome and biochemical analyses of BYDV-inoculated or mock-inoculated adult plant leaves of YW642 and Zhong 8601.The experiments included enzyme-linked immunosorbent assay(ELISA)for BYDV content in the leaves,measurement of H2O2contents,microarray analysis,and confirmatory quantitative real-time RT-PCR(qRT-PCR)for transcriptomic comparison.

2.Materials and methods

2.1.Plant materials,BYDV isolation,and treatments

The wheat—Th.intermedium translocation line YW642(resistant to BYDV)with pedigree Zhong 8601*4/4/Zhong7902/3/CSph*2/L1//CSN5BT5D[21]and the wheat line Zhong 8601(susceptible to BYDV)were maintained in our laboratory.Both lines have spring growth habit.Zhong 8601 was the recurrent parent of YW642.The BYDV-GAV strain was isolated from wheat leaves with YDV symptoms in Shanxi province,China[11].

Four hundred plants per line were planted in February for two successive years in a field nursery in Beijing,China.The fields were managed in a manner normally accepted for agronomic practices of the region in north China.The trials were designed with 20 rows per line,20 seeds per row,with rows 1.2 m long and 25 cm apart.At the two-leaf stage,300 plants of each genotype were inoculated with viruliferous aphids carrying BYDV-GAV and the other 100 plants of the genotype were mock-inoculated with non-viruliferous aphids as controls.All inoculation was with～10 aphids per plant.Because our previous real time quantitative(RT-qPCR)analyses showed that BYDV-GAV accumulation in Zhong 8601 reached the highest level at 35 dpi[24],ten adult-stage leaves of YW642 and Zhong 8601,both mock and BYDV-GAV inoculated,were sampled at 35 dpi and used for microarray analysis.

2.2.ELISA for BYDV relative content in BYDV-responsive YW642 and Zhong 8601

Leaf sections near sites penetrated by the viruliferous or nonviruliferous aphids,or sections with YDV symptoms,were sampled from 10 individual plants of YW642 and Zhong 8601 at 21,28,35,and 40 dpi.The leaf tissues(0.1 g per plant and 10 plants in each treatment)were assayed by ELISA for virus titer.The virus titers of YW642 or Zhong 8601 inoculated with non-viruliferous aphids were used as control.ELISA was performed according to the manual for the BYDV ELISA reagent set(Cat.No.SRA26500,Agdia,USA)with polyclonal antibodies,and absorbance was measured at 405 nm within 30 min after beginning of color reaction.Three technical replicates for each treatment were assayed and the ELISA value for each sample was the mean of the three values.

2.3.RNA extraction,first-strand cDNA synthesis,and microarray analysis

Total RNA was extracted from leaf tissues using Trizol reagent(Invitrogen,USA)and subjected to RNase-free DNase treatment.First-strand cDNA was synthesized using the Superscript II First-Strand Synthesis Kit for real time quantitative PCR(RT-qPCR)(Invitrogen,USA).

RNAs were extracted from 10 biological replicate leaves of BYDV-and mock-inoculated YW642 and Zhong 8601 for 35 dpi.Those purified RNAs were used to generate labeled cRNAs according to Affymetrix manufacturer's protocol.The labeling cRNAs were hybridized to the Affymetrix GeneChip wheat genome array by ShanghaiBio Corporation,China.Differential expression probe sets were filtered out(fold change threshold≥2.0;FDR≤0.05;P≤0.001)and three replicates were assayed for each treatment.Putative-function annotation of differentially expressed genes and the removal of redundant probe sets were performed following reported methods[24,39,41].

2.4.RT-qPCR analyses

The expression patterns of 25 differentially expressed genes identified by microarray analysis were investigated by RT-qPCR.These 25 genes included disease resistance-like genes,defense-associated genes,signaling transduction factors,transcription factors,ROS-scavenging genes,photosynthesis related genes,and JA and GA biosynthesis-related genes(Table 1).Primer information and sequences are presented in Table 1.The RT-qPCR experiment was performed using the SYBR Green I Master Mix(TaKaRa)and analyzed with an ABI 7500 system(Applied Biosystems).The wheat actin gene(TaActin-F:5′-CACTGGAATGGTCAAGGCTG-3′;TaActin-R:5′-CTCCATGTCATCCCAGTTG-3′)was used as internal control.The 2?ΔΔCTmethod[42]was used to calculate the relative expression of tested genes.All reactions were repeated three times.

2.5.Measurement of H2O2content in YW642 and Zhong 8601

Leaves from BYDV-and mock-inoculated plants of YW642 and Zhong 8601 for 21,28,35,and 40 dpi were used to assay H2O2content according to a previously reported protocol[43].Mock-inoculated YW642 and Zhong 8601 samples were used as controls.Three replicate values for YW642 and Zhong 8601 at each time point were obtained and the samples for each replicate were taken from 10 plants.

3.Results

3.1.ELISA for BYDV content in YW642 and Zhong 8601

ELISA was used to estimate relative BYDV content in newly emerged leaves of mock-inoculated and BYDV-GAV-inoculated YW642 and Zhong 8601 at 21,28,35,and 40 dpi.The BYDV titers were markedly lower in YW642 than in Zhong 8601 at each BYDV-inoculation time point tested:23.52-and 25.17-fold lower in YW642 than in Zhong 8601 at 35 and 40 dpi,respectively.BYDV titers in BYDV-inoculated Zhong8601ranged from0.130to 1.529,with all values exceeding the 0.1(susceptible)threshold of a virus infection[44].The BYDV-GAV titers in the susceptible genotype Zhong 8601 were markedly elevated after virus infection(Table 2),and the titers at 35 dpi were 218.00-fold higher than that of mock-inoculated Zhong 8601.These observations indicated that BYDV-GAV could infect and replicate in infected Zhong 8601 plants.However,in resistant line YW642,BYDV-GAV titers remained relatively low(0.011—0.065)from21to 40 dpi and did not reach the 0.1 susceptibility threshold response to virus infection[44]at any BYDV-inoculation time point.These results suggested that the replication,movement,and accumulation of BYDV were suppressed in tissues of the resistant line YW642.The resistance picture derived from ELISA data was consistent with the phenotype of BYDG-GAV resistance by YW642 and BYDV susceptibility of Zhong 8601.

3.2.Globally differential transcripts between YW642 and Zhong 8601

RNAs extracted from leaf tissues of YW642(R)and Zhong 8601(S)at 35 dpi following BYDV-GAV inoculation(RI35 and SI35)and mock-inoculation(RM and SM)were subjected to microarray analyses with three pairwise comparisons:RI35 vs.SI35,RM vs.SM,and RI35 vs.SI35—RM vs.SM(Table S1).A total of 387 up-regulated and 229 down-regulated transcripts as determined by the third comparison,RI35 vs.SI35—RM vs.SM,were identified,designated as BYDV-responsive in adult-stage leaves of YW642,and further analyzed(Table S1,Fig.1-A,B).The 387 up-regulated transcripts included 42 resistance-like and defense-related(accounting for 10.8%),21 ROS-related(5.4%),25 chlorophyll biosynthesis and photosynthesis-related(6.5%),30 hormone signaling-related(7.8%),20 signal transduction(5.2%),30 metabolism-related(7.8%),33 transcription related(8.5%),17 abiotic stress-responsive(4.4%),16 transportrelated,28 with other functions(Table S2),and 133 with unknown/unclear functions(Tables 3—5 and Tables S1,S2,Fig.1-A).The 229 down-regulated transcripts included 46 defense-related(20.1%),nine WRKY transcription factor homologs(3.9%),three germin-like(1.3%),15 involving signal transduction (6.6%),two hormone signaling-related(0.9%),18 metabolism-related(7.9%),three ROS-related(1.3%),seven abiotic stress-related(3.1%),13 transport-related(5.7%),2 withother functions(Table S2),and 112 with unknown/unclear functions(Tables 3–5 and Table S1,Fig.1-B).

Table 2–ELISA values of YW642 and Zhong 8601 after BYDV-GAV inoculation.

Fig.1–Functional categories of differential transcripts in adult leaves of YW642(R)relative to Zhong8601(S)after BYDV inoculation and measured for 35 days(RI35 vs SI35–RM vs SM).A.Functional categories of up-regulated transcripts;B.Functional categories of down-regulated transcripts.

To verify the reliability of microarray values for the transcripts differentially expressed between YW642 and Zhong 8601,RT-qPCR was used to validate the transcript levels of 21 up-regulated and four down-regulated probe sets in adult-stage leaves of YW642 and Zhong 8601 inoculated with BYDV-GAV over 35 days.The tested genes were involved in defense,signal transduction,ROS homeostasis,photosynthesis,and JA or GA signal pathways.The experimental RT-qPCR analyses for transcriptional levels of the 25 selected genes showed trends similar to those obtained with the microarray analysis(Table 1),suggesting that the transcriptome analyses were reliable.

3.3.Important genes associated with adult-stage resistance in YW642 against BYDV

Among the up-regulated BYDV-responsive transcripts in adult-stage leaves of YW642 compared to those of Zhong 8601,42 resistance-like and defense-related transcripts included 15 resistance-like transcripts, nine lipid transfer protein (LTP)homologous transcripts, one defensin homologous transcript,and the seven other classes as listed in Table 3. Notably, among the down-regulated transcripts (Table 3), no resistance-like transcripts were identified. Also, following BYDV infection, two protein kinases, brassinosteroid insensitive1-associated receptor kinase 1 (BRI1) and calcium-dependent protein kinase (CDPK11),and three transcription factor homologs (MYB1, WRKY23, and bZIP46), were up-regulated in YW642 compared to Zhong 8601(Table S1).

Microarray analysis of the third comparison indicated that 21 ROS-related transcripts were significantly up-regulated in BYDV-responsive YW642 compared with those in Zhong 8601(Table 4).These ROS-related genes included APX homologous transcript(APX 1),other peroxidase gene homologs,SOD homologous gene(SOD 3.1),glutathione s-transferase gene(GST)homologs(Cla47,GST1,and GST37),GPX15Hv,glutaredoxin homologous gene(Glutaredoxin-C1),and germin-like homologous genes(GLP1 and OXO GF-2.8).Only three transcripts encoding germin-like proteins(GLP8-5)were down-regulated in the BYDV-responsive YW642 transcripts compared to Zhong 8601.These results suggest that elevated expression of the ROS related genes is very important for adult-stage BYDV resistance in YW642.

Gibberellin 20 oxidase(GA20ox)is a key enzyme catalyzing the penultimate step in GA biosynthesis[45].Lipoxygenase(LOX)is an important enzyme of JA biosynthesis[46].Among the adult-stage microarray results(Table 5),the transcripts encoding two GA20ox homologs(Ta.23667.1 and Ta.23668.1)and three lipoxygenase homologs(Ta.23763.1.S1_at,Ta.1967.1.S1_x_at,and TaAffx.104812.1.S1_s_at)were dramatically upregulated in the BYDV-responsive YW642 relative to the BYDV-responsive Zhong 8601(Table 5).Interestingly,transcripts associated with JA and GA biosynthesis were up-regulated only in the BYDV-responsive YW642 leaves.Other transcripts involved in ethylene and ABA biosynthesis or signaling pathways were identified(Table 5).

RT-qPCR was used to investigate the transcription profiles of seven up-regulated genes in BYDV-GAV-inoculated YW642 and Zhong 8601 adult-plant leaves sampled at 21,28,35,and 40 dpi,in which mock-inoculated YW642 was used as control.The seven genes encode one NBS-LRR,one CDPK11,MYB1,WRKY23,one NPR1,one PR,and one lipid transfer protein like(LTPL17)homologous proteins.The results revealed that from 21 dpi to 40 dpi,the transcriptional levels of these seven upregulated genes were significantly higher in YW642 than in Zhong 8601(Fig.2),further supporting the microarray results.These findings suggested that the elevated expression of these genes might promote resistance to BYDV infection.

3.4.H2O2concentration was differently modulated in BYDV infected YW642 and Zhong 8601

To determine whether the expression of ROS-related genes affects H2O2homeostasis in BYDV-infected wheat plants at adult stage,H2O2content in newly emerged leaves from both BYDV-inoculated YW642 and Zhong 8601sampled at 21,28,35,and 40 dpi was assayed,and H2O2in newly emerged leaves of mock-inoculated YW642 and Zhong 8601 was used ascontrols.In Zhong 8601 leaves,H2O2concentrations were markedly increased after BYDV infection during the tested time points:0.94 μmol g?1in the mock inoculation,5.53 μmol g?1at 21 dpi,6.72 μmol g?1at 35 dpi,increasing to 7.55 μmol·g?1at 40 dpi.Although the contents of H2O2in YW642 leaves were also elevated after BYDV-inoculation,they were 4—5-fold lower than those in Zhong 8601:1.26 μmol g?1at 21 dpi,1.37 μmol g?1at 35 dpi,1.45 μmol g?1at 40 dpi(Fig.3).The results suggested that at the adult stage,YW642 had higher ROS-scavenging activity,and the BYDV-resistance of YW642 might be positively associated with its ROS homeostasis after BYDV inoculation.

Table 3–Defense-related differential transcripts in YW642 relative to Zhong 8601(RI35 vs SI35–RM vs SM).

Table 3(continued)

Table 4–ROS-related differential transcripts in YW642 relative to Zhong 8601(RI35 vs SI35–RM vs SM).

Table 5–Hormone signaling-related differential transcripts in YW642 relative to Zhong 8601(RI35 vs SI35–RM vs SM).

4.Discussion

The degree of BYDV infection in wheat is directly correlated with yield loss in field plots[7,47].Reliable detection and quantification of BYDV are critical for identification of resistancesources,studying host resistance genetics,and managing yellow dwarf disease in wheat production.ELISA is a reliable method currently used for investigating relative virus content[47].In the present study,ELISA was used to estimate relative BYDV contents in BYDV-GAV-inoculated or mock-inoculated adult-stage leaves of resistant YW642 and susceptible Zhong 8601 at 21,28,35,and 40 dpi.The relative BYDV titers measured by ELISA accumulated to＞0.1 in adult leaves of BYDV-GAV infected Zhong 8601,exceeding the susceptible plant threshold after a virus infection[44].While BYDV titers remained quite low,＜0.1,in YW642 both with and without BYDV-GAV inoculation Thus,YW642 was further verified to be BYDV-resistant and Zhong 8601 BYDV-susceptible,results consistent with their phenotypes.After BYDV inoculation,the BYDV-GAV relative contents were markedly elevated in Zhong 8601relative to YW642:24-fold higher at 40 dpi.Our previous RT-qPCR and semi RT-qPCR investigations showed that both from 6 h post inoculation(hpi)to 35 dpi and from 12 hpi to 14 dpi,respectively,relative contents of BYDV in YW642 were always markedly lower than in Zhong 8601[24,48],and these findings were consistent with the ELISA results.Taken together,these results suggest that Bdv2-derived resistance response could suppress the replication,movement,and accumulation of BYDV in the resistant line YW642 at all growth stages.

In the present study,microarray and experimental RT-qPCR analyses revealed that in BYDV-GAV-inoculated adult-stage YW642 leaves,many genes were up-regulated in response to BYDV infection,which include resistance-like and certain PR genes,ROS-related genes,and genes in phytohormone(JA,GA,ethylene,and ABA)signaling pathways.These results supported the previous documents about plant defense mechanisms against viruses infection involved with the precious regulations on a series of genes[49,50].In YW642 relative to Zhong 8601 at adult stage leaves infected by BYDV-GAV,15 specific resistance-like transcriptswere up-regulated,whereas no resistance-like transcripts were down-regulated,suggesting that the elevated expression of these resistance-like genes might be important for adult stage resistance in YW642 against BYDV infection.This finding is further supported by and in accordance with the previous reports that resistance genes confer resistance to viruses,and could be detected as up-regulated upon infection of viruses in plants[50,51].The up-regulated expression of some PR genes,such as defensin and six LTPL homologs,and of BRI1,CDPK11,MYB1,WRKY23,and bZIP46 homologous genes,may also participate in the resistance of YW642 at adult stage.In contrast,the transcriptional levels of other PR genes,such as PR1A/1B,PR4,PR17d,chitinases II&IV,β-1,3-glucanase and wheat win 2,and mlo3 homologous genes were down-regulated in YW642 compared with those in Zhong 8601.These findings further support the previous report[38]that precise transcriptional regulation of a battery of genes encoding diverse proteins in these processes determines the resistance or susceptibility of plants.Comparing the up-regulated transcripts between seedling[24]and adult-stage YW642 after BYDV-GAV infection,we found that the expression of resistance-like and certain PR genes,ROS-related and JA-signaling genes was always elevated during whole stages but GA and ABA signaling genes were up-regulated only at adult stage.Detailed comparison of the expression patterns of the resistance-like and PR transcripts identified by the microarray assay showed that only three transcripts displayed expression patterns at the adult stage similar to those at the seedling stage.Comparing YW642 to Zhong 8601,one LTP gene(Ta.994.3.S1_at)and one Lr10 gene(TaAffx.43049.1.A1_at)showed up-regulated patterns in both the transcript levels of BYDV-GAV inoculation for 72 h and 35 days,and one PR4 gene(TaAffx.108556.1.S1_at)displayed similar down-regulated transcriptional patterns in transcript levels at both 72 hpi and 35 dpi.These results suggested that the expression patterns of the host major resistance response genes are precisely modulated and may change at different growth stages of BYDV-infected wheat plants.

Fig.2–RT-qPCR analysis of transcript levels of nine defense-related genes in BYDV-responsive YW642 and Zhong 8601.Relative transcript abundances of the nine tested genes were quantified relative to those in mock-inoculated YW642.Significant differences between YW642 and Zhong 8601 lines at the same time points were determined based on three replicates using Student's t-test.

Fig.3–H2O2contents in YW642 leaves after mock inoculation and BYDV-GAV inoculation and sampling at 21,28,35,and 40 days.Three replicates were averaged in YW642.Significant differences between BYDV-GAV inoculated YW642 and mock inoculated YW642 were identified by Student's t-test(*P＜0.05,difference).

The microarray and RT-qPCR analyses of BYDV-inoculated adult-plant leaves showed that transcriptional levels of ROS-related genes,including APX,GPX,peroxidase (POX),glutaredoxin,and GST,were significantly up-regulated in BYDV-responsive YW642 in comparison to BYDV-responsive Zhong 8601,suggesting that YW642 at the adult stage might possess higher ROS-scavenging activity.Consistently,the H2O2assay results showed that H2O2contents were 4—5-fold higher in Zhong 8601 than in YW642 adult-plant leave after BYDV-GAV inoculation,further supporting the deduction that YW642 adult-plant leaves had stronger ROS-scavenging activity and could maintain ROS homeostasis.Thus,the ROS homeostasis reaction may be a factor in adult-stage resistance of YW642 against BYDV infection.Detailed comparison of the expression of ROS-related transcripts identified by the seedling[24]and adult-stage microarray assays indicated that even though there were better ROS-scavenging ability in YW642 than in Zhong 8601 at both seedling and adult-stage upon BYDV-GAV infection,the ROS-scavenging system is regulated by different ROS-related transcripts at each plant growth stage.At seedling-stage in YW642 relative to Zhong 8601,the transcripts encoding superoxide dismutase(SOD)and APX were mainly up-regulated;while at adult-stage in YW642 relative to Zhong 8601,the up-regulated ROS-related transcripts encoded APX,POX,GPX,glutaredoxin,GLP,and GST.It was reported that the over expression of SOD,APX,POX,and GST could defend chloroplast nucleoids against oxidative stress and is essential for chloroplast development in plants[52—55].The fact may partially explain the non-yellowing symptom in YW642 upon BYDV infection and strongly suggest that different ROS-related genes contributing to the BYDV-resistance in YW642 at the seedling and adult stages were transcriptionally regulated.

Accumulating evidence[27,45,46,56]indicates that in plants,JA and GA signaling is implicated in developmental processes and signaling networks responding to biotic and abiotic stresses.GA20ox and lipoxygenase are key enzymes in GA and JA biosynthesis,respectively[45,46].In the present study,the comparative transcriptome analyses showed that in adultplant leaves after BYDV inoculation,the transcription levels of GA20ox and lipoxygenase encoding genes were dramatically upregulated in YW642 compared to Zhong 8601.Notably,transcripts related to JA and GA biosynthesis were up-regulated only in BYDV-responsive YW642 adult-plant leaves.This finding suggested that JA and GA biosynthesis might support the Bdv2-derived resistance response at the adult stage against BYDV infection.We previously showed that transcripts participating in the JA signaling pathway played important roles during the Bdv2-derived resistance response to BYDV at the seedling stage[24].Thus,the JA signaling pathway plays an important role in defense response during different growth stages in BYDV-resistant wheat lines,and the GA signaling pathway participates positively only in the adult-stage resistance response.The comparative transcriptomic data also suggest that transcriptional regulation of ethylene-and ABA-signaling pathway related genes might also be involved in the resistance response at the adult stage.

In summary,after resistance responses were triggered in BYDV-resistant wheat lines with Bdv2,the transcriptional levels of resistance-like,ROS-related,JA,GA,ABA,and ethylene signaling-related genes and defense genes were precisely regulated at different growth-stages.In these BYDV-resistant wheat-Th.intermedium translocation lines at the adult stage,the transcription of resistance-like, certain defense,ROS-scavenging,and JA and GA biosynthesis genes was markedly up-regulated and ROS homeostasis was maintained.Consequently,BYDV replication,movement,and accumulation were suppressed.This study sheds light on the resistance mechanism of BYDV-resistance wheat lines at the adult stage.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.02.002.

Acknowledgments

This study was supported by the National Key Research and Development Program of China(2016YFD0101802).The authors are very grateful to Mr.Liang Wang(Institute of Plant Protection,CAAS,Beijing,China)for his assistance in ELISA experiment.

R E F E R E N C E S

[1]X.Huang,M.S.R?der,Molecular mapping of powdery mildew resistance genes in wheat:a review,Euphytica 137(2004)203—223.

[2]C.J.D'Arcy,Symptomology and host range of barley yellow dwarf,in:C.J.D'Arcy,P.A.Burnett(Eds.),Barley Yellow Dwarf:40 Years of Progress,APS Press,St.Paul,Minnesota,USA 1995,pp.9—28.

[3]S.J.McKirdy,R.A.C.Jones,Effect of sowing time on barley yellow dwarf virus infection in wheat:virus incidence and grain yield losses,Aust.J.Agric.Res.12(1997)105—109.

[4]D.A.J.Herbert,E.L.Stromberg,G.F.Chappel,S.M.Malone,Reduction of yield components by barley yellow dwarf infection in susceptible winter wheat and winter barley in Virginia,J.Prod.Agric.12(1999)105—109.

[5]W.E.Riedell,R.W.Kieckhefer,S.D.Haley,M.A.C.Langham,P.D.Evenson,Winter wheat responses to bird cherry-oat aphids and barley yellow dwarf virus infection,Crop Sci.39(1999)158—163.

[6]K.L.Perry,F.L.Kolb,B.Sammons,C.Lawson,G.Cisar,H.Ohm,Yield effects of barley yellow dwarf virus in soft red winter wheat,Phytopathology 90(2000)1043—1048.

[7]P.M.Banks,J.L.Davidson,H.Bariana,P.J.Larkin,Effects of barley yellow dwarf virus on the yield of winter wheat,Aust.J.Agric.Res.46(1995)935—946.

[8]W.F.Rochow,Biological properties of four isolates of BYDV,Phytopathology 59(1969)1580—1589.

[9]W.F.Rochow,Comparative diagnosis of BYDV by serological and aphid transmission test,Plant Dis.Rep.63(1979)426—430.

[10]G.H.Zhou,S.X.Zhang,Y.T.Qian,Identification and application of four strains of wheat yellow dwarf virus,Sci.Agric.Sin.20(1987)7—12(in Chinese with English abstract).

[11]Y.Liu,B.Sun,X.F.Wang,C.L.Zheng,G.H.Zhou,Three digoxigenin-labeled cDNA probes for specific detection of the natural population of barley yellow dwarf viruses in China by dot-blot hybridization,J.Virol.Methods 145(2007)22—29.

[12]N.Li,Z.Chen,Y.Liu,Y.Liu,X.P.Zhou,J.X.Wu,Development of monoclonal antibodies and serological assays specific for barley yellow dwarf virus GAV strain,Virol.J.12(2015)136—144.

[13]L.Ayala,M.Van Ginkel,M.Khairallah,B.Keller,M.Henry,Expression of Thinopyrum intermedium-derived barley yellow dwarf virus resistance in elite bread wheat backgrounds,Phytopathology 91(2001)55—62.

[14]Z.Y.Zhang,Z.S.Lin,Z.Y.Xin,Research progress in BYDV resistance genes derived from wheat and its wild relatives,J.Genet.Genomics 36(2009)567—573.

[15]H.Sharma,B.Gill,J.Uyemoto,High levels of resistance in Agropyron species to barley yellow dwarf and wheat streak mosaic viruses,J.Phytopathol.110(1984)143—147.

[16]Z.Y.Xin,R.I.S.Brettell,Z.M.Cheng,R.Waterhouse,P.Appels,M.Banks,G.H.Zhou,X.Chen,P.J.Larkin,Characterization of a potential source of barley yellow dwarf virus resistance for wheat,Genome 30(1988)250—257.

[17]Z.Y.Xin,H.J.Xu,X.Chen,Z.S.Lin,G.H.Zhou,Y.T.Qian,Z.M.Chen,P.J.Larkin,P.M.Banks,R.Apples,B.Clarke,R.I.S.Brettell,Development of common wheat germplasm resistant to barley yellow dwarf virus by biotechnology,Sci.China B 34(1991)1055—1062.

[18]P.J.Larkin,P.M.Banks,E.S.Lagudah,R.Appels,X.Chen,Z.Y.Xin,H.W.Ohm,R.A.McIntosh,Disomic Thinopyrum intermedium addition lines in wheat with barley yellow dwarf virus resistance and with rust resistances,Genome 38(1995)385—394.

[19]H.Sharma,L.Ohm,R.Goulart,R.Lister,R.Appels,O.Benlhabib,Introgression and characterization of barley yellow dwarf virus resistance from Thinopyrum intermedium into wheat,Genome 38(1995)406—413.

[20]O.R.Crasta,M.G.Francki,D.B.Bucholtz,H.C.Sharma,J.Zhang,R.C.Wang,H.W.Ohm,J.M.Anderson,Identification and characterization of wheat-wheatgrass translocation lines and localization of barley yellow dwarf virus resistance,Genome 43(2000)698—706.

[21]P.M.Banks,P.J.Larkin,H.S.Bariana,E.S.Lagudah,R.Appels,P.M.Waterhouse,R.I.S.Brettel,X.Chen,H.J.Xu,Z.Y.Xin,Y.T.Qian,X.N.Zhou,Z.M.Cheng,G.H.Zhou,The use of cell culture for subchromosomal introgressions of barley yellow dwarf virus resistance from Thinopyrum intermedium to wheat,Genome 38(1995)395—405.

[22]Z.Y.Xin,Z.Y.Zhang,X.Chen,Z.S.Lin,Y.Z.Ma,H.J.Xu,P.M.Banks,P.J.Larkin,Development and characterization of common wheat—Thinopyrum intermedium translocation lines with resistance to barley yellow dwarf virus,Euphytica 119(2001)161—165.

[23]Z.Y.Zhang,Z.Y.Xin,Y.Z.Ma,X.Chen,Q.F.Xu,Z.S.Lin,Mapping of a BYDV resistance gene from Thinopyrum intermedium in wheat background by molecular markers,Sci.China C 42(1999)663—668.

[24]X.D.Wang,Y.Liu,L.Chen,D.Zhao,X.F.Wang,Z.Y.Zhang,Wheat resistome in response to barley yellow dwarf virus infection,Funct.Integr.Genomics 13(2013)155—165.

[25]M.A.López,G.Bannenberg,C.Castresana,Controlling hormone signaling is a plant and pathogen challenge for growth and survival,Curr.Opin.Plant Biol.11(2008)420—427.

[26]J.D.Jones,J.L.Dangl,The plant immune system,Nature 444(2006)323—329.

[27]C.M.Pieterse,A.Leon-Reyes,S.van der Ent,S.C.van Wees,Networking by small-molecule hormones in plant immunity,Nat.Chem.Biol.5(2009)308—316.

[28]R.Desikan,M.K.Cheng,A.Clarke,S.Golding,M.Sagi,R.Fluhr,Hydrogen peroxide is a common signal for darkness-and ABA-induced stomatal closure in Pisum sativum,Funct.Plant Biol.31(2004)913—920.

[29]L.J.Quan,B.Zhang,W.Shi,H.Y.Li,Hydrogen peroxide in plants:a versatile molecule of the reactive oxygen species network,Plant Biol.50(2008)2—18.

[30]D.H.Lee,C.B.Lee,Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber:in gel enzymes activity assays,Plant Sci.159(2000)75—85.

[31]G.Wingsle,J.E.Hallgren,Influence of SO2and NO2exposure on glutathione,superoxide dismutase and glutathione reductase activities in Scots pine needles,J.Exp.Bot.44(1993)463—470.

[32]J.S.Zhang,C.J.Li,J.Wei,M.B.Kirkham,Protoplasmic factors,antioxidants responses,and chilling resistance in maize,Plant Physiol.Biol.Chem.382(1995)1123—1131.

[33]K.Hahlborck,P.Bednarek,I.Ciolkowski,B.Hamberger,A.Heise,H.Liedgens,E.Logemann,T.Nürnberger,E.Schmelzer,I.E.Somssich,J.Tan,Non-self recognition,transcriptional reprogramming,and secondary metabolite accumulation during plant/pathogen interactions,Proc.Natl.Acad.Sci.U.S.A.100(2003)14569—14576.

[34]F.M.Ausubel,Are innate immune signaling pathways in plants and animals conserved,Nat.Immunol.6(2005)973—979.

[35]T.Boller,G.Felix,A renaissance of elicitors:perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors,Annu.Rev.Plant Biol.60(2009)379—406.

[36]Z.Q.Fu,X.Dong,Systemic acquired resistance:turning local infection into global defense,Annu.Rev.Plant Biol.64(2013)839—863.

[37]I.E.Somssich,K.Hahlbrock,Pathogen defence in plants-a paradigm of biological complexity,Trends Plant Sci.3(1998)86—90.

[38]Y.Zhu,C.M.Schluttenhoffer,P.Wang,F.Fu,J.Thimmapuram,J.K.Zhum,S.Y.Lee,D.J.Yun,T.Mengiste,Cyclin-dependent kinase8 differentially regulates plant immunity to fungal pathogens through kinase-dependent and-independent functions in Arabidopsis,Plant Cell 26(2014)4149—4170.

[39]M.D.Bolton,J.A.Kolmer,W.W.Xu,D.F.Garvin,Lr34-mediated leaf rust resistance in wheat:transcript profiling reveals a high energetic demand supported by transient recruitment of multiple metabolic pathways,Mol.Plant-Microbe Interact.21(2008)1515—1527.

[40]M.M.Xin,X.F.Wang,H.R.Peng,Y.Y.Yao,C.J.Xie,Y.Han,Z.F.Ni,Q.X.Sun,Transcriptome comparison of susceptible and resistant wheat in response to powdery mildew infection,Genomics Proteomic Bioinformatics 10(2012)94—106.

[41]M.Ashburner,C.A.Ball,J.A.Blake,D.Botstein,H.Butler,J.M.Cherry,A.P.Davis,K.Dolinski,S.S.Dwight,J.T.Eppig,M.A.Harris,D.P.Hill,L.Issel-Tarver,A.Kasarskis,S.Lewis,J.C.Matese,J.E.Richardson,M.Ringwald,G.M.Rubin,G.Sherlock,Gene ontology:tool for the unification of biology.The Gene Ontology Consortium,Nat.Genet.25(2000)25—29.

[42]K.J.Livak,T.D.Schmittgen,Analysis of relative gene expression data using real-time quantitative PCR and the 2?ΔΔCTmethod,Methods 25(2001)402—408.

[43]X.Hu,S.Neill,W.Cai,Z.Tang,Hydrogen peroxide and jasmonic acid mediate oligogalacturonic acid-induced saponin accumulation in suspension-cultured cells of Panax ginseng,Physiol.Plant.118(2003)414—421.

[44]C.Riedel,A.Habekuss,E.Schliephake,R.Niks,I.Broer,F.Ordon,Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of barley yellow dwarf virus-PAV(BYDV-PAV-ASL-1)leads to quantitative resistance against this isolate,Theor.Appl.Genet.123(2011)69—76.

[45]T.Oikawa,M.Koshioka,K.Kojima,H.Yoshida,M.Kawata,A role of OsGA20ox1,encoding an isoform of gibberellin 20-oxidase,for regulation of plant stature in rice,Plant Mol.Biol.55(2004)687—700.

[46]A.Schilmiller,G.A.Howe,Systemic signaling in the wound response,Curr.Opin.Cell Biol.8(2005)369—377.

[47]B.Balaji,D.B.Bucholtz,J.M.Anderson,Barley yellow dwarf virus and cereal yellow dwarf virus quantification by real-time polymerase chain reaction in resistant and susceptible plants,Phytopathology 93(2003)1386—1392.

[48]X.D.Liu,Z.Y.Zhang,Y.Liu,Z.Y.Xin,Molecular evidence of barley yellow dwarf virus replication/movement suppressed by the resistance gene Bdv2,Acta Genet.Sin.32(2005)942—947.

[49]V.I.Nicaise,Crop immunity against viruses:outcomes and future challenges,Front.Plant Sci.5(2014)660.

[50]J.L.M.Soosaar,T.M.Burch-Smith,S.P.Dinesh-Kumer,Mechanisms of plant resistance to viruses,Nat.Rev.Microbiol.3(2005)789—798.

[51]J.M.Andrew,C.Carole,I.B.Margaret,Sources of natural resistance to plant viruses:status and prospects,Mol.Plant Pathol.8(2007)223—231.

[52]A.Caverzan,A.Bonifacio,F.E.Carvalho,C.M.Andrade,G.Passaia,M.Schünemann,S.Maraschin Fdos,M.O.Martins,F.K.Teixeira,R.Rauber,R.Margis,J.A.Siveira,M.Margis-Pinheiro,The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice,Plant Sci.214(2014)74—87.

[53]F.Myouga,C.Hosoda,T.Unezawa,H.Jizumi,T.Kuromori,R.Motohashi,Y.Shono,N.Nagata,M.Ikeuchi,K.Shinozaki,A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis,Plant Cell 20(2008)3148—3162.

[54]M.H.Cui,S.H.Ok,K.S.Yoo,K.W.Jung,S.D.Yoo,J.S.Shin,An Arabidopsis cell growth defect factor-related protein,CRS,promotes plant senescence by increasing the production of hydrogen peroxide,Plant Cell Physiol.54(2013)155—167.

[55]B.Le Martret,M.Poage,K.Shiel,G.D.Nugent,P.J.Dix,Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase,glutathione reductase,and glutathione-S-transferase,exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance,Plant Biotechnol.J.9(2011)661—673.

[56]S.Van der Ent,S.C.Van Wees,C.M.Pieterse,Jasmonate signaling in plant interactions with resistance-inducing beneficial microbes,Phytochemistry 70(2009)581—588.