Why mosaic?Gene expression profiling of African cassava mosaic virus-infected cassava reveals the effect of chlorophyll degradation on symptom development

Jiao Liu,Jun Yang,Huiping Bi and Peng Zhang＊

1Shanghai Chenshan Plant Science Research Center,the Chinese Academy of Sciences,Chenshan Botanical Garden,Shanghai 201602,China,2National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research(Shanghai),Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences,the Chinese Academy of Science,Shanghai 200032,China,3Key Laboratory of Systems Microbial Biotechnology,Tianjin Institute of Industrial Biotechnology,the Chinese Academy of Sciences,Tianjin Airport Economic Park,Tianjin 300308,China.＊Correspondence:zhangpeng@sibs.ac.cn

INTRODUCTION

Cassava(Manihot esculenta Crantz)is an important economic crop in tropical and subtropical regions,and a major source of carbohydrates(Cock 1982;Ceballos et al.2004).The most important constraint limiting cassava production is viral disease.Cassava mosaic disease(CMD),caused by geminiviruses,is the greatest constraint to cassava production(Varma and Malathi 2003;Sayre et al.2011).The viruses are transmitted by the whitefly(Bemisia tabaci)and perpetuated through cuttings(Fargette et al.1996).Cassava mosaic disease is characterized by discoloration and distortion of the leaves.Consequently,the storage roots are reduced in size and number so the crop yield is reduced.

African cassava mosaic begomoviruses cause the most serious disease of cassava in Africa(Thresh et al.1994;Fargette et al.1996).In sub-Saharan Africa,more than 30 million tons of fresh cassava roots are lost yearly as a result of CMD(Legg and Thresh 2000;Legg et al.2006;Sayre et al.2011).Outside Africa,a distinct species of begomovirus,Indian cassava mosaic virus,causes a similar disease of cassava in India(Fregene et al.2004).

So far,African cassava mosaic virus(ACMV)research has mainly focused on resistance breeding by inter-specific hybridization or genetic engineering(Zhang et al.2003;Vanderschuren et al.2007,2009).However,the pathogenesis of ACMV is poorly understood.Diminished chlorophyll content(Ayanru and Sharma 1982)and distorted chloroplasts(Chant and Beck 1959;Chant et al.1971)appear on CMV-infected cassava leaves.Infection with other geminiviruses also causes reduced chlorophyll content,for example,tomato plants infected with Tomato yellow mosaic virus(Leal and Lastra 1984)and Tomato leaf curl virus(TLCV)infection of the wild plant Eupatorium makinoi(Funayama et al.1997b).In the latter case,decreased maximum quantum yield of photosynthesis was also detected,and the leaf cells possessed less light-harvesting chlorophyll a/b-binding proteins(LHCP)(Funayama et al.1997a,1997b;Funayama and Terashima 1999;Funayama-Noguchi and Terashima 2006).In Abutilon mosaic virus-infected Abutilon,the ultrastructure of the chloroplasts was completely eliminated by inhibition and disorganization in the thylakoid system during the spring and summer(Schuchalter-Eicke and Jeske 1983).Besides geminiviruses,there are also some studies on photosynthesis-related parameters of virus-infected crops and cultivated plants(Matthews 1991;Balachandran et al.1994;Almási et al.2001).An improved molecular understanding of this pathogenesis would benefit both efforts to breed resistant crops and develop therapeutic tools.

Lehto et al.(2003)found that in mature tobacco leaves infected by the flavum strain of Tobacco mosaic virus(TMV),the induction of chlorotic symptoms was related to the accumulation of viral coat protein inside chloroplasts and depletion of the PSII core complexes.In some reports,mosaic symptoms were linked to the oxygen-evolving complex(OEC)in thylakoid membranes.The replicase protein of TMV has a high affinity for the 33-kDa protein of the OEC,and TMV infection also reduced the mRNA of the 33-kDa protein in Nicotiana benthamiana(Abbink et al.2002).Also,decreased OEC 23 and 16 kDa protein levels may result in the chlorosis of N.tabacum leaves infected with TMV(Sui et al.2006).In Cucumber mosaic virus strain Y-infected tobacco plants,the observed chlorotic symptoms have been associated with a decrease in the amount of 22 and 23 kDa polypeptides of OEC(Takahashi et al.1991).These studies suggest that the changes of host-specific gene expression may lead to mosaic symptoms.

For the bipartite begomoviruses,two movement proteins(MP and NSP)are involved in development of symptoms and systemic infection(Rojas et al.2005),but recently expression of ACMV genes in Nicotiana benthamiana suggested that symptom development could not be entirely attributed to MP and NSP(Amin et al.2011).Some plant host factors are known to interact with viral proteins to induce the disease symptoms,but the host factors involved in this process of cassava are still unknown.In order to provide candidate genes for further study,a high throughput investigation is necessary.In this study,digital gene expression(DGE)profiling based on Illumina Solexa sequencing technology was used to investigate the global transcriptional response of cassava to ACMV infection.We identified several photosynthesis-related genes that were regulated in virus-infected cassava,resulting in chlorophyll b(Chl b)and light-harvesting complex II(LHCII)deficiencies.Furthermore,we observed fewer grana lamellae in infected leaves,which demonstrated the chlorotic phenotype.We established the connection between expression levels of genes and CMD phenotype for the first time.To our knowledge,this is the first report to study the direct effect of a geminivirus on the global gene expression profile of its plant host using a high-throughput sequencing method.Also this is the first report of the molecular mechanisms of cassava symptom development.

aPercentage of matched tags/total tags.bPercentage of matched genes/total reference genes.

RESULTS

Virus inoculation and symptom development

After the tips of the shoot axis were inoculated with ACMV-NOg in the greenhouse,chlorotic patterns were visible at 20 d postinoculation.The new leaves of ACMV-infected cassava were mosaic,stunted,and distorted(Figure S1A,B).Subsequently,symptoms of systemic infection appeared(Figure S1C,D).

Tag identification and quantification

A total of 3,762,501 and 3,566,501 tags were sequenced in ACMV-infected(INF)and Control(MOCK)libraries,respectively(Table 1).After low quality tags(tags containing “N,” adaptors and single-copy tags)were filtered out,3,703,523 and 3,504,407 clean tags remained in INF and MOCK libraries,respectively.Of these,104,005(INF)and 98,632(MOCK)unique tags were obtained.Tags with copy number smaller than five accounted for the majority of the unique clean tags,and only a small portion of the transcripts were highly expressed in the conditions tested.However,there were 5,373 more unique tags in the INF library than the MOCK library,possibly representing genes related to pathogenesis and host–virus interactions.

Annotation analysis of the unique tags

To understand the molecular events through DGE,we mapped the unique tags to a reference database of cassava expressed sequence tags(ESTs)collected based on the methods described by Yang et al.(2011).This database contained 37,438 unigenes including 32,914 sequences with CATG site.The reference database contains 159,114 unambiguous reference tags in total.Among the unique tags from the Solexa sequencing,41.23%(INF)and 35.15%(MOCK)of all unique tags were mapped to one gene in the reference database.Meanwhile,15,725(42.00%)putative genes were identified in the INF library and 13,745(36.71%)in the MOCK library(Table 2).Saturation analysis was performed to determine the depth of sequencing.Results showed that when the total number of tags reached 2 million or higher,the number of mapped genes almost ceased to increase in both libraries(Figure S2).It means that a complete assessment of all transcripts existing in the libraries was performed.

Figure 1.Comparison of gene expression levels between the two librariesTo compare gene expression levels between the two libraries,each library was normalized to TPM(transcripts per million clean tags).Red dots represent more prevalent transcripts and green dots represent transcripts less prevalent in the African cassava mosaic virus(ACMV)-infected library.Blue dots indicate transcripts of equivalent levels in both libraries.The parameters “FDR≤0.001” and “|Log2(INF/MOCK)|≥1”were used as the threshold to judge the significance of gene expression difference.

Comparison of gene expression levels between the two libraries

Tags mapping to a single sequence are the most critical,so the number of these unambiguous tags for each gene was calculated and normalized to TPM(transcripts per million clean tags).The values of TPM for each gene in the INF and MOCK libraries were used to estimate gene expression levels in response to ACMV infection.The gene expression levels in the two libraries are compared in Figure 1(FDR＜0.001 and|Log2(INF/MOCK)|≥1).Two thousand two hundred and seventytwo transcripts were found to be more prevalent in the INF library,and 938 more prevalent in the MOCK library.The level of 12,889 transcripts differed by less than twofold or FDR≥0.001 between the two libraries,so these transcripts were designated as “no difference in expression.”

Of the differentially expressed genes(DEGs),57%were expressed at fourfold higher levels in one library compared to the other.Four hundred and eighty-two genes were expressed only in INF library,and 95 genes only in MOCK.These genes were predicted to be involved in many biological processes,including protein binding,ATP binding,transcription,and defense(Table S1).

Gene ontology enrichment analysis for DEGs

To determine the main biological function of the DEGs,all DEGs were mapped to the gene ontology(GO)terms.Among the 3,210 DEGs,659 genes were mapped to 110 terms from the component ontology and five terms were significantly enriched in DEGs(Table S2).Consistent with the chlorotic symptom,the “Chloroplast” component was most affected(Figure 2),confirming the effect of ACMV infection on the photosynthetic system.

We also mapped DEGs to two additional ontologies(process and function).Four hundred and one genes were mapped to 403 terms from the process ontology(Table S3)and 518 genes were mapped to 231 terms from the function ontology(Table S4).In the two ontologies,only one term“oxidoreductase activity”was significantly enriched,reflecting that ACMV infection may affect the enzymatic reaction of host.

Pathway enrichment analysis of DEGs

We used pathway analysis to better understand the biological function of DEGs.One thousand six hundred and fifty-seven DEGs were mapped to 120 pathways in the Kyoto Encyclopedia of Genes and Genomes(KEGG)database(Table S5).The pathway enrichment analysis revealed that the“Porphyrin and chlorophyll metabolism”pathway was most significantly implicated in DEGs.Of the top 10 enriched pathways,three are involved in photosynthesis(Figure 3);“Porphyrin and chlorophyll metabolism” (ko00860), “Carbon fixation in photosynthetic organisms” (ko00710),and “Photosynthesisantenna proteins”(ko00196).

Figure 2.First 10 enriched gene ontology(GO)terms for cellular component in INF libraryThe “y”-axis represents enriched GO terms in the INF library.The “x”-axis represents the P-value(-Log2).Among these,the top five terms from the component ontology were significantly enriched(P-value≤0.05).

Figure 3.First 10 enriched Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways in INF libraryThe “y”-axis represents enriched pathways in the INF library.The “x”-axis represents the P-value(-Log2).Among these,the top six pathways were significantly enriched(Q value≤0.05).

All data are shown Log2(INF/MOCK),no detected fold changes are indicated as“–”.Values of qRT-PCR analysis showing in bold font indicate inconsistent data compared with Solexa sequencing.“Contig” represents for Riken cassava flcDNA.fa.Contig,“.valid”represents for predicted transcript by Phytozome geno`me.

Real-time PCR analysis

Photosynthesis-related pathways were significantly enriched in DEGs,and disease symptoms suggest a deficiency in the photosynthesis pathway.We chose to measure the expression level of 22 key genes in photosynthesis-related pathways to validate the DGE data.The genes identified were similar to those detected by Solexa sequencing(Table 3).We found that 18 of these genes(encoding magnesium-chelatase,protochlorophyllide reductase,chlorophyll synthetase,chlorophyllase,chlorophyll b reductase,pheophorbide a oxygenase,chlorophyllide a oxygenase,pheophytinase,hydroxymethyl chlorophyll a reductase(HCAR),and STAYGREEN)were upregulated in infected leaves,and four genes(psb W and Lhcb1,2,3)were downregulated(Table 3).Compared to MOCK-infected plants,we found higher expression levels of genes encoding proteins implicated in chlorophyll degradation(chlorophyllase,pheophytinase,and pheophorbide a oxygenase),the last two enzymes in the chlorophyll synthesis process(protochlorophyllide reductase and chlorophyll synthetase)and proteins involved in the chlorophyll cycle(Table 3,Figure 5).

Except for the Chl catabolic enzyme genes,the nuclear gene STAYGREEN(SGR),encoding a family of novel chloroplastlocated proteins,has been noted over the past few years.Two candidate orthologs of SGR from cassava showed enhanced expression in INF(Table 3),especially SGR1,by qPCR,indicating the SGRs are involved in the development of chlorosis phenotype.We also found reduced expression of Lhcb1–3 and psb W genes.Lhcb1,Lhcb2,and Lhcb3 are the major apoproteins of LHCII(Schmid 2008),and the psb W protein is responsible for stabilizing the photosystem II supercomplex(Garcia-Cerdan et al.2011).These results suggest that ACMV-infection affects photosystem II especially host LHCII.

Decreased chlorophyll content in ACMV-infected cassava

The mosaic phenotype suggests that the major chlorophyll pigment levels may be reduced in ACMV infection.We measured the concentration of chlorophylls in the leaves of INF and MOCK samples.The total chlorophyll content in INF leaves was approximately 30%lower than in MOCK leaves.The level of Chl b was most significantly reduced(at approximately 60%MOCK),increasing the ratio of Chl a to b from 1.078 in MOCK to 2.680 in INF samples(Table 4).The reduction of chlorophyll coincided with both the upregulation of chlorophyll degradation-related genes,and upregulation of key genes in the chlorophyll synthesis pathway.We hypothesize that increased degradation of chlorophyll initiates a feedback mechanism that increases transcription of genes responsible for chlorophyll synthesis.

The photosynthetic performance was assessed by measuring the quantum efficiency of PSII(Fv/Fm)in INF(Krause and Weis 1991).The Fv/Fm value of leaves was 0.78±0.02 in MOCK and 0.72±0.02 in INF(Table 4),indicating a significant impact on the photochemical efficiency of PSII in ACMV infection.

Abnormal grana in ACMV-infected cassava

Previous studies found that leaf coloration was related to chloroplast development(Reiter et al.1994;Sundberg et al.1997).In our study,the chlorophyll content and genes encoding LHCII apoproteins were affected by virus infection,as was the photochemical efficiency of PSII.PSII and LHCII are mainly located in the appressed grana.We analyzed the chloroplast ultrastructure of INF leaf by transmission electron microscopy to determine whether the virus infection resulted in defective chloroplast ultrastructure.In the mature chloroplasts of control plants,the internal membranes were present as either stroma thylakoids or stacked grana thylakoids(Figure 4A,B).ACMV-infected cells contained smaller chloroplasts and fewer grana lamellae(Figure 4C,D).These results indicated that the virus infection had indeed affected the formation of stacked grana.

Figure 5.Molecular model of cassava chlorotic phenotype development by African cassava mosaic virus(ACMV)signalsThe details of the model are explained in the text.Upregulated genes are shown in red and downregulated genes are shown in green.Key intermediates are shown in bold.Green and box show the reduced pigment component.The thick gray arrow shows that chlorophyll b and LHCII levels impact upon each other.CAO,chlorophyllide a oxygenase;Chl G,chlorophyll synthetase;Chlase,chlorophyllase;Chl H,D and I,H,D and I subunit of Mg chelatase;HCAR,hydroxymethyl chlorophyll a reductase;LHC,lightharvesting complex;MP,Mg-protoporphyrin IX;MPM,Mg-protoporphyrin IX monomethyl ester;NYC1,NOL,chlorophyll(ide)b reductase;PaO,pheophorbide a oxygenase;Pchlide,proto-chlorophyllide;POR,protochlorophyllide reductase;PPH,pheophytinase;Proto IX,protoporphyrin IX.

DISCUSSION

In this report,we used a high throughput sequencing approach to estimate genome-wide gene expression profiles of ACMV-infected cassava leaves by the application of Illumina Solexa sequencing technology.We found that the expression levels of photosystem-related genes in ACMV-infected cassava leaves were consistent with chlorotic symptoms and reduced chlorophyll content.These results represent the first largescale investigation of the impact of geminivirus infection on gene expression in crops by direct sequencing.We found that the reduced levels of chlorophyll were likely a result of ACMV induced upregulation of genes responsible for chlorophyll degradation.We believe that this study will provide a road map for future investigations into the pathology of infectious disease of plants.

In Arabidopsis,CAO is the only enzyme implicated in Chl b biosynthesis(Oster et al.2000).Since we observed increased CAO mRNA in infected leaves(Table 3),we hypothesize that the increased CBR and HCAR levels trigger degradation of Chl b,complementing Chl a reduced by PPH and PaO,and the reduction of Chl b sends a feedback signal to upregulate CAO mRNA.

Chl a exists in all chlorophyll–protein complexes while Chl b is present only in LHC(Grossman et al.1995),so Chl a is more important than Chl b in higher plants.We believe that when viruses infect cassava,PPH and PaO will be activated to degrade Chl a.Plants then compensate for the loss of Chl a by regulating the chlorophyll cycle.Alternatively,viruses may transform Chl b to Chl a to make sure the host can survive.

SGR,a critical regulator,can regulate Chl degradation by inducing chlorophyll-protein complex dismantling (Park et al.2007;Barry 2009).Mecey et al.(2011)revealed that SGR was critical for the development of disease symptom caused by the bacterial pathogen Pseudomonas syringe pv tomato DC3000 in Arabidopsis.The increase of SGR in INF is associated with Chl breakdown and deficient LHC.SGR may play an important role in cassava chlorosis symptom development,similar to the situation in DC3000 infection.By silencing SGR expression in Arabidopsis pao1 mutant,Aubry et al.(2008)suggested that SGR acts upstream of PAO during chlorophyll breakdown.These results show an interesting network for the further study.

We have found that virus infection causes the chlorophyll degradation and deficient LHC.However,we have not determined whether chlorophyll reduction leads to LHC deficiencies or vice versa.There is a general agreement that Chl b is responsible for the stability of LHCP especially those of LHCII(Terao and Katoh 1989;Plumley and Schmidt 1995).The quantity of LHCII apoproteins increased with the increasing Chl b levels(Shimada et al.1990).Degradation of Chl b may trigger the degradation of LHCII and grana stacks in stay-green mutant models of leaf senescence(Hortensteiner 2006;Kusaba et al.2007;Horie et al.2009).

Additionally,both Chl a and Chl b are thought to be synthesized in association with LHCP(Plumley and Schmidt 1995).Chlorophyll accumulation could be inhibited by treatments that block LHCP synthesis(Plumley and Schmidt 1995).For instance,cycloheximide inhibits LHCP synthesis and causes a reduction in the accumulation of chlorophyll(Maloney et al.1989).Fradkin et al.(1981)suggested that the LHCP could be directly involved in the synthesis of chlorophyll.By analyzing chlorophyll synthesis in isolated chloroplasts,Huang and Hoffman(1990)suggested that synthesis of Chl b may take place on LHCP,and Plumley and Schmidt(1995)suggested LHCP could be the site of chlorophyll synthesis.We conclude that chlorophyll and LHC depend upon one another,and perceive the question of whether chlorophyll degradation causes LCH deficiencies,or vice versa,similar to the“chicken and the egg”problem.

Funayama et al.(1997a)hypothesized that the reduction of LHCII in TLCV-infected leaves may be caused by the suppression of chlorophyll synthesis.In contrast,we conclude that chlorophyll degradation is responsible for this process in cassava;although the expression levels of LHCII genes are also important.These two perspectives are not mutually exclusive,and the effect of ACMV on the cassava photosystem could result from cross-talking.

There is also evidence that the expression of nuclear encoding photosynthesis-related genes could be regulated by signals from chloroplasts.Chlorophyll precursors may generate a signal that causes transcriptional repression of photosynthesis-related genes(Mochizuki et al.2001;Gray et al.2003).Oster et al.(1996)found that increased levels of Mg-protoporphyrin IX monomethyl ester were followed by decreased transcription of Lhcb1,Lhcb2,and Lhca1 in barley seedlings treated with amitrole,a carotenoid biosynthesis inhibitor.Johanningmeier and Howell(1984)reported inhibition of Lhcb mRNA accumulation in Chlamydomonas when Mgprotoporphyrin IX(MgProto)and Mg-protoporphyrin IX monomethyl ester were accumulated by alpha,alpha-dipyridyl treatment.Together,MgProto and its methyl ester may negatively regulate LHC genes(Gray 2003;Gray et al.2003).Besides,the effect of the H-subunit of Mg chelatase(ChlH)on the regulation of LHC genes has also been proposed.By the research on Arabidopsis gun5 mutant,Mochizuki et al.(2001)proposed that ChlH may repress the transcription of nucleus localized genes that encode photosynthesis-related proteins.For the Chlamydomonas brs-1 mutant,decreased light induction of Lhcb1 may be the result of a frameshift mutation in the ChlH gene (Johanningmeier and Howell 1984;Chekounova et al.2001).These tetrapyrrole biosynthetic genes(ChlH,ChlD,and ChlI)were upregulated in INF(Table 3),and may have modulated the levels of chlorophyll precursors,suggesting that the effects of chloroplasts signals on LHC genes should also be included in the network.

采用SPSS 20.0软件对数据进行分析处理，计量资料以（均数±标准差）表示，采用t检验；计数资料以（n，%）表示，采用χ2检验，以P＜0.05表示差异具有统计学意义。

Here,we propose a model to explain the chlorosis caused by ACMV infection(Figure 5).When the virus invades the cell,some unknown signal pathways may be activated,and many nuclear encoding genes could be impacted.Light-harvesting complex II-related genes Lhcb1–3 are repressed and chlorophyll degradation-related genes(Chlase,PPH,and PaO)are activated.The cumulative effect is disruption of pigment biosynthesis,the reduction of Chl b levels,and LHCII deficiencies,followed by deficient thylakoid development as these protein complexes require LHC and pigments coordination(Pogson and Albrecht 2011).In this process,signals from chloroplasts such as intermediates Mg protoporphyrin play important roles too.Meanwhile Chl b and LHCII levels impact upon each other.Since photosystem II is the dominating component of the grana stacks(Albertsson 1995),ACMV-infected leaves posses fewer grana lamellae,producing the chlorotic phenotype,and limiting the photochemical efficiency of PSII.

Funayama et al.(1997a)suggested that because LHCII is the only photosynthetic component that can be lost without lethal effects,it is possible that the virus limits photosynthetic production to impair plant performance.When plant performance is impaired,the energy for anti-viral defense should be reduced.We believe that this non-lethal inhibition of photosystem II is a result of long-term co-evolution,and may explain why many plant pathogens induce this chlorotic phenotype.

In conclusion,using a high throughput method we identified photosynthesis-related genes that may contribute to the development of cassava disease symptoms,and revealed the important roles of chlorophyll degradation and LHCII inhibition during ACMV attack.This study could be applied to further exploration of the molecular mechanisms of viral pathogenesis.

MATERIALS AND METHODS

Plants material and virus infection

The present study used cassava variety TMS30555(a CMD-susceptible line),which were planted in a greenhouse condition under 25±2°C and 16 h/8 h photoperiod.Clones of A.tumefaciens strain LBA4404 containing the infectious clones of ACMV-NOg DNA-A and DNA-B(Briddon et al.1998)were separately cultured on YEB plates supplemented with Streptomycin(100 mg/L),Rifampicin(25 mg/L),and Kanamycin(50 mg/L)at 28°C for 48 h.Then equal amounts of each culture were mixed together.

One-month-old cassava plantlets with 5–6 leaves were needle-punctured 4–6 times with the mixed agrobacteria suspension at the tip of the shoot axis using a syringe needle(Ø 0.5 mm)(Vanderschuren et al.2009).When mosaic symptoms(Zhang et al.2005)appeared at 20 d post-inoculation,the first two expanded leaves from the shoot apex were harvested.Three independent replicates were collected for an ACMV-infected sample.Control samples were harvested from empty agrobacteria-treated leaves incubated under the same conditions.

Preparation of digital gene expression libraries and sequencing

Samples from three independent replicate plants were pooled for RNA isolation and library construction.Control leaves were treated in parallel.Total RNA was isolated from the leaf mixture using the RNAprep pure Plant Kit(Tiangen Biotech,Beijing,China).Of total RNA,6 μg were used for sequencing tag preparation with Digital Gene Expression Sample Prep Kit(Illumina,San Diego,CA,USA)according to the manufacturer’s protocol.Using Oligo(dT)-conjugated magnetic beads,mRNA was purified from each of the two samples,then Oligo(dT)was used as primer to synthesize cDNA.Bead-bound cDNA was subsequently digested with restriction enzyme NlaIII,which recognizes and removes the CATG sites.5′cDNA fragments were washed away and the Illumina adaptor 1 was ligated to the 5′end of the bead-bound cDNA fragments.The junction of Illumina adaptor 1 and CATG site is the recognition site of MmeI,and this enzyme sliced 17 bp downstream of the CATG site to produce tags with adaptor 1.After removing the 3′fragments by precipitation of magnetic bead,Illumina adaptor 2 was ligated to the 3′ends of tags.Tags acquired with different adaptors at both ends formed a tag library.After 15 cycles of linear PCR amplification,95 bp fragments were purified by 6%TBE PAGE gel electrophoresis.After denaturation,the singlechain molecules were fixed onto the Illumina Sequencing Chip.Each molecule grew into a single-molecule cluster sequencing template through in situ amplification and was sequenced with the method of sequencing by synthesis.Each tunnel generates millions of raw reads with sequencing length of 35 bp(target tags plus 3′adaptor).Each read in the library represented a single tag derived from a single transcript.

Sequence annotation and data normalization

“Clean Tags” were obtained by removing 3′adaptor sequences,reads with only adaptor sequences but no tags,tags with unknown sequences “N,”excessively long or short tags,and tags with a copy number of 1(probably a sequencing error).A database containing all possible CATG+17 bases length sequences was created using a large collection of cassava ESTs from TIGR(Manihot_esculenta_release_5,containing 5,189 assemblies,10,214 singletons,released 1 June,2007)(Childs et al.2007),cassava ESTs from NCBI(80,631 ESTs,released 25 August,2010),34,151 predicted cassava transcripts from Phytozome(Prochnik et al.2012),and a 35,400 full-length cDNA RIKEN library(Sakurai et al.2007).All clean tags were mapped to the reference sequences,allowing only 1 bp mismatch.Clean tags mapping to multiple genes were filtered and the remaining clean tags were determined to be unambiguous clean tags.The number of unambiguous clean tags for each gene was calculated and then normalized to TPM(number of transcripts per million clean tags)(’t Hoen et al.2008;Morrissy et al.2009).Mapped genes were locally blasted against the NCBI non-redundant protein database using the blastx program in the blastall package(version 2.2.25),and the top hits were used for gene annotation.

Analysis of differentially expressed genes

A rigorous algorithm was developed to identify genes expressed at different levels in ACMV-infected(INF)and Control(MOCK)samples,according to a method described previously(Audic and Claverie 1997).When the number of total clean tags in the MOCK library is noted as N1,and that in the INF library as N2;gene A holds x unambiguous clean tags in MOCK and y tags in INF.The probability of gene A expression being equivalent in both conditions can be calculated as follows:

P-value corresponds to a differential gene expression test.False Discovery Rate(FDR)was used to determine the threshold of P-value in multiple test and analysis.We used“FDR≤0.001 and the absolute value of Log2(INF/MOCK)≥1”as the threshold to judge the significance of the difference in gene expression(Benjamini and Hochberg 1995;Yoav and Daniel 2001).

Gene ontology functional enrichment analysis

Gene ontology enrichment analysis based on the Gene Ontology database (http://www.geneontology.org/)was used to identify the significantly enriched GO terms in DEGs,calculated as follows:

where N is the number of all genes with GO annotation;n is the number of DEGs in N;M is the number of all genes that are annotated to certain GO terms;m is the number of DEGs in M.Gene ontology terms with Bonferroni-corrected P-value≤0.05 are significantly enriched in DEGs.

Pathway enrichment analysis

Pathway enrichment analysis based on the KEGG(http://www.genome.jp/kegg/)was used to identify significantly enriched pathways in DEGs.The calculating formula is the same as that in GO analysis.Here,N is the number of all reference genes with KEGG annotation,n is the number of DEGs in N,M is the number of all genes annotated to specific pathways,and m is the number of DEGs in M.False Discovery Rate-corrected Q-value was used for determining the threshold of P-value in multiple test and analysis(Benjamini and Hochberg 1995).Pathways with Q-value≤0.05 are significantly enriched in DEGs.

Real-time PCR analysis

To confirm the results of the DGE analysis,the expression levels of 22 selected genes were measured using Real-time PCR.Samples were prepared as described above and total RNA was isolated from the leaf mixture.Experiments were carried out on three independent biological replicates,each containing three technical replicates.First-strand cDNA was synthesized from 2 μg DNase-treated total RNA using“ReverTra Ace reverse transcriptase” (TOYOBO,Osaka,Japan).Specific primer pairs were designed using Primer 3 Plus (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi)to obtain a Tm of 60°C and an amplicon length between 70–200 bp(Table S6).Real-time PCR reactions were performed in a 20-μL volume containing 10 μL 2× SYBR Green Master Mix(TOYOBO),50 ng cDNA,and 400 nM of forward and reverse primers in a Bio-Rad CFX96 thermocycler.The amplification conditions were as follows:95°C for 1 min,followed by 40–50 cycles of 95 °C for 15 s,60 °C for 20 s,and 72°C for 20 s.A melting curve was run after the PCR cycles to test the primers.Cassava beta-actin was used as an internal control to normalize all data(An et al.2012).The fold change in mRNA expression was calculated by the ΔΔCT method(Livak and Schmittgen 2001).

Pigment content and chlorophyll fluorescence analysis

Total chlorophyll was determined as previously described(Lichtenthaler 1987).Extracts obtained from 100 mg fresh tissue were homogenized in 10 mL of 80%acetone.The homogenate was centrifuged at 18,514 g for 3 min,and the supernatant obtained was used for measurement at 663 and 645 nm.

Chlorophyll fluorescence was measured with a pulse amplitude-modulated fluorometer(PAM-101,H.Walz,Effeltrich,Germany)equipped with a data-acquisition system to record fast changes(Meurer et al.1996).The leaves were darkadapted for at least 30 min before measurement.Chlorophyll fluorescence signals were analyzed,as described by Genty et al.(1989),to estimate the relative quantum efficiency of PSII:Fv/Fm=(Fm-Fo)/Fm,where Fo and Fm are minimum and maximum chlorophyll fluorescence levels of dark-adapted leaves,respectively.The significant difference was evaluated by T-test.

TEM analysis of chloroplasts

Small leaf segments from samples were collected and fixed in 2.5%glutaraldehyde in phosphate buffer(pH 7.2)for 4 h at 4°C.After fixation,the tissue was washed and postfixed with 1%OsO4overnight at 4°C.After washing in phosphate buffer,the samples were dehydrated through a series of ethanol solutions,then infiltrated with a graded series of epoxy resin in epoxy propane,and embedded in Epon 812 resin.Thin sections were stained in 1%uranyl acetate,followed by lead citrate solution and viewed with a transmission electron microscope(HITACHI H-7650,Hitachi,Tokyo,Japan).

ACKNOWLEDGEMENTS

This work was supported by grants from the National Basic Research Program(2010CB126605),the National High Technology Research and Development Program of China(2012AA101204),the National Science Foundation of China(31201254),the Earmarked Fund for China Agriculture Research System(CARS-12-shzp),and Shanghai Municipal Afforestation&City Appearance and Environmental Sanitation Administration(F132427,F122422).Utilization of the data:The data discussed in this publication have been deposited in the Gene Expression Omnibus(GEO)of NCBI(Edgar et al.2002).The accession numbers are:Series,GSE47224;samples,GSM1147066–GSM1147067.

Abbink TE,Peart JR,Mos TN,Baulcombe DC,Bol JF,Linthorst HJ(2002)Silencing of a gene encoding a protein component of the oxygenevolving complex of photosystem II enhances virus replication in plants.Virology 295:307–319

Almási A,Harsányi A,Gáborjányi R(2001)Photosynthetic alterations of virus infected plants.Acta Phytopathol Entomol 36:15–29

Amin I,Patil B,Briddon R,Mansoor S,Fauquet C(2011)Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs.Virol J 8:238

An D,Yang J,Zhang P(2012)Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress.BMC Genomics 13:64

Aubry S,Mani J,Hortensteiner S(2008)Stay-green protein,defective in Mendel’s green cotyledon mutant,acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway.Plant Mol Biol 67:243–256

Audic S,Claverie JM(1997)The significance of digital gene expression profiles.Genome Res 7:986–995

Ayanru DKG,Sharma VC(1982)Effects of cassava mosaic disease on certain leaf parameters of field-grown cassava clones.Phytopathology 72:1057–1059

Balachandran S,Osmond CB,Makino A(1994)Effects of two strains of tobacco mosaic virus on photosynthetic characteristics and nitrogen partitioning in leaves of Nicotiana tabacum cv Xanthi during photoacclimation under two nitrogen nutrition regimes.Plant Physiol 104:1043–1050

Barry CS(2009)The stay-green revolution:Recent progress in deciphering the mechanisms of chlorophyll degradation in higher plants.Plant Sci 176:325–333

Benjamini Y,Hochberg Y(1995)Controlling the false discovery rate:A practical and powerful approach to multiple testing.J R Stat Soc B 57:289–300

Briddon RW,Liu S,Pinner MS,Markham PG(1998)Infectivity of African cassava mosaic virus clones to cassava by biolistic inoculation.Arch Virol 143:2487–2492

Ceballos H,Iglesias CA,Pérez JC,Dixon AGO(2004)Cassava breeding:Opportunities and challenges.Plant Mol Biol 56:503–516

Chant SR,Bateman JG,Bates DC(1971)The effect of cassava mosaic virus infection on the metabolism of cassava leaves.Trop Agr 48:263–270

Chant SR,Beck BDA(1959)The effect of cassava mosaic virus on the anatomy of cassava leaves.Trop Agr 36:231–236

Chekounova EC,Voronetskaya VV,Papenbrock JP,Grimm BG,Beck CB(2001)Characterization of Chlamydomonas mutants defective in the H subunit of Mg-chelatase.Mol Genet Genomics 266:363–373

Childs KL,Hamilton JP,Zhu W,Ly E,Cheung F,Wu H,Rabinowicz PD,Town CD,Buell CR,Chan AP(2007)The TIGR plant transcript assemblies database.Nucleic Acids Res 35:D846–D851

Cock J(1982)Cassava:A basic energy source in the tropics.Science 218:755–762

Edgar R,Domrachev M,Lash AE(2002)Gene Expression Omnibus:NCBI gene expression and hybridization array data repository.Nucleic Acids Res 30:207–210

Fargette D,Colon LT,Bouveau R,Fauquet C(1996)Components of resistance of cassava to African cassava mosaic virus.Eur J Plant Pathol 102:645–654

Fradkin LI,Chkanikova RA,Shlyk AA(1981)Coupling of chlorophyll metabolism with submembrane chloroplast particles,isolated with digitonin and gel electrophoresis.Plant Physiol 67:555–559

Fregene M,Matsumura H,Akano A,Dixon A,Terauchi R(2004)Serial analysis of gene expression(SAGE)of host–plant resistance to the cassava mosaic disease(CMD).Plant Mol Biol 56:563–571

Funayama-Noguchi S,Terashima I(2006)Effects of Eupatorium yellow vein virus infection on photosynthetic rate,chlorophyll content and chloroplast structure in leaves of Eupatorium makinoi during leaf development.Funct Plant Biol 33:165–175

Funayama S,Hikosaka K,Yahara T(1997a)Effects of virus infection and growth irradiance on fitness components and photosynthetic properties of Eupatorium makinoi(Compositae).Am J Bot 84:823

Funayama S,Sonoike K,Terashima I(1997b)Photosynthetic properties of leaves of Eupatorium makinoi infected by a geminivirus.Photosynth Res 53:253–261

Funayama S,Terashima I(1999)Effects of geminivirus infection and growth irradiance on the vegetative growth and photosynthetic production of Eupatorium makinoi.New Phytol 142:483–494

Garcia-Cerdan JG,Kovacs L,Toth T,Kereiche S,Aseeva E,Boekema EJ,Mamedov F,Funk C,Schroder WP(2011)The PsbW protein stabilizes the supramolecular organization of photosystem II in higher plants.Plant J 65:368–381

Genty B,Briantais J.-M,Baker NR(1989)The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence.B B A Gen Subjects 990:87–92

Gray JC(2003)Chloroplast-to-nucleus signalling:A role for Mgprotoporphyrin.Trends Genet 19:526–529

Gray JC,Sullivan JA,Wang J.-H,Jerome CA,MacLean D(2003)Coordination of plastid and nuclear gene expression.Philos Trans R Soc London Ser B 358:135–145

Grossman AR,Bhaya D,Apt KE,Kehoe DM(1995)Light-harvesting complexes in oxygenic photosynthesis:Diversity,control,and evolution.Annu Rev Genet 29:231–288

Horie Y,Ito H,Kusaba M,Tanaka R,Tanaka A(2009)Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis.J Biol Chem 284:17449–17456

Hortensteiner S(2006)Chlorophyll degradation during senescence.Annu Rev Plant Biol 57:55–77

Huang L,Hoffman NE(1990)Evidence that isolated developing chloroplasts are capable of synthesizing chlorophyll b from 5-aminolevulinic acid.Plant Physiol 94:375–379

Johanningmeier U,Howell SH(1984)Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi.Possible involvement of chlorophyll synthesis precursors.J Biol Chem 259:13541–13549

Krause GH,Weis E(1991)Chlorophyll fluorescence and photosynthesis:The basics.Annu Rev Plant Physiol Plant Mol Biol 42:313–349

Kusaba M,Ito H,Morita R,Iida S,Sato Y,Fujimoto M,Kawasaki S,Tanaka R,Hirochika H,Nishimura M,Tanaka A(2007)Rice NONYELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence.Plant Cell 19:1362–1375

Leal N,Lastra R(1984)Altered metabolism of tomato plants infected with tomato yellow mosaic virus.Physiol Plant Pathol 24:1–7

Legg JP,Owor B,Sseruwagi P,Ndunguru J(2006)Cassava mosaic virus disease in East and Central Africa:Epidemiology and management of a regional pandemic.Adv Virus Res 67:355–418

Legg JP,Thresh JM(2000)Cassava mosaic virus disease in East Africa:A dynamic disease in a changing environment.Virus Res 71:135–149

Lehto K,Tikkanen M,Hiriart JB,Paakkarinen V,Aro EM(2003)Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus.Mol Plant-Microbe Interact 16:1135–1144

Lichtenthaler HK(1987)Chlorophylls and carotenoids:Pigments of photosynthetic biomembranes.In:Lester Packer RD,ed.Methods in Enzymology.Academic Press,Waltham.pp.350–382

Livak KJ,Schmittgen TD(2001)Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod.Methods 25:402–408

Maloney MA,Hoober JK,Marks DB(1989)Kinetics of chlorophyll accumulation and formation of chlorophyll–protein complexes during greening of Chlamydomonas reinhardtii y-1 at 38°C.Plant Physiol 91:1100–1106

Matthews REF(1991)Plant Virology.Academic Press,Waltham.

Mecey C,Hauck P,Trapp M,Pumplin N,Plovanich A,Yao J,He SY(2011)A critical role of STAYGREEN/Mendel’s I Locus in controlling disease symptom development during Pseudomonas syringae pv tomato infection of Arabidopsis.Plant Physiol 157:1965–1974

Meguro M,Ito H,Takabayashi A,Tanaka R,Tanaka A(2011)Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis.Plant Cell 23:3442–3453

Meurer J,Meierhoff K,Westhoff P(1996)Isolation of high-chlorophyllfluorescence mutants of Arabidopsis thaliana and their characterisation by spectroscopy,immunoblotting and Northern hybridisation.Planta 198:385–396

Mochizuki N,Brusslan JA,Larkin R,Nagatani A,Chory J(2001)Arabidopsis genomes uncoupled 5(GUN5)mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction.Proc Natl Acad Sci USA 98:2053–2058

Morrissy AS,Morin RD,Delaney A,Zeng T,McDonald H,Jones S,Zhao Y,Hirst M,Marra MA(2009)Next-generation tag sequencing for cancer gene expression profiling.Genome Res 19:1825–1835

Oster U,Brunner H,Rüdiger W(1996)The greening process in cress seedlings.V.Possible interference of chlorophyll precursors,accumulated after thujaplicin treatment,with light-regulated expression of Lhc genes.J Photoch Photobio B Biol 36:255–261

Oster U,Tanaka R,Tanaka A,Rudiger W(2000)Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis(CAO)from Arabidopsis thaliana.Plant J 21:305–310

Park SY,Yu JW,Park JS,Li J,Yoo SC,Lee NY,Lee SK,Jeong SW,Seo HS,Koh HJ,Jeon JS,Park YI,Paek NC(2007)The senescence-induced STAYGREEN protein regulates chlorophyll degradation.Plant Cell 19:1649–1664

Plumley GF,Schmidt GW(1995)Light-harvesting chlorophyll a/b complexes:Interdependent pigment synthesis and protein assembly.Plant Cell 7:689–704

Pogson BJ,Albrecht V(2011)Genetic dissection of chloroplast biogenesis and development:An overview.Plant Physiol 155:1545–1551

Prochnik S,Marri P,Desany B,Rabinowicz P,Kodira C,Mohiuddin M,Rodriguez F,Fauquet C,Tohme J,Harkins T,Rokhsar D,Rounsley S(2012)The cassava genome:Current progress,future directions.Trop Plant Biol 5:88–94

Pruzinska A,Tanner G,Anders I,Roca M,Hortensteiner S(2003)Chlorophyll breakdown:Pheophorbide a oxygenase is a Riesketype iron-sulfur protein,encoded by the accelerated cell death 1 gene.Proc Natl Acad Sci USA 100:15259–15264

Reiter RS,Coomber SA,Bourett TM,Bartley GE,Scolnik PA(1994)Control of leaf and chloroplast development by the Arabidopsis gene pale cress.Plant Cell 6:1253–1264

Rojas MR,Hagen C,Lucas WJ,Gilbertson RL(2005)Exploiting chinks in the plant’s armor:Evolution and emergence of geminiviruses.Annu Rev Phytopathol 43:361–394

Sakurai T,Plata G,Rodriguez-Zapata F,Seki M,Salcedo A,Toyoda A,Ishiwata A,Tohme J,Sakaki Y,Shinozaki K,Ishitani M(2007)Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response.BMC Plant Biol 7:66

Sato Y,Morita R,Katsuma S,Nishimura M,Tanaka A,Kusaba M(2009)Two short-chain dehydrogenase/reductases,NON-YELLOW COLORING 1 and NYC1-LIKE,are required for chlorophyll b and lightharvesting complex II degradation during senescence in rice.Plant J 57:120–131

Sayre R,Beeching JR,Cahoon EB,Egesi C,Fauquet C,Fellman J,Fregene M,Gruissem W,Mallowa S,Manary M,Maziya-Dixon B,Mbanaso A,Schachtman DP,Siritunga D,Taylor N,Vanderschuren H,Zhang P(2011)The BioCassava Plus Program:Biofortification of cassava for Sub-Saharan Africa.Annu Rev Plant Biol 62:251–272

Schelbert S,Aubry S,Burla B,Agne B,Kessler F,Krupinska K,Hortensteiner S(2009)Pheophytin pheophorbide hydrolase(pheophytinase)is involved in chlorophyll breakdown during leaf senescence in Arabidopsis.Plant Cell 21:767–785

Schmid VH(2008)Light-harvesting complexes of vascular plants.Cell Mol Life Sci 65:3619–3639

Schuchalter-Eicke G,Jeske H(1983)Seasonal changes in the chloroplast ultrastructure in Abutilon mosaic virus(AbMV)infected Abutilon spec.(Malvaceae).J Phytopathol 108:172–184

Shimada Y,Tanaka A,Tanaka Y,Takabe T,Takabe T,Tsuji H(1990)Formation of chlorophyll-protein complexes during greening.1.Distribution of newly synthesized chlorophyll among apoproteins.Plant Cell Physiol 31:639–647

Sui C,Fan Z,Wong SM,Li H(2006)Cloning of cDNAs encoding the three subunits of oxygen evolving complex in Nicotiana benthamiana and gene expression changes in tobacco leaves infected with tobacco mosaic virus.Physiol Mol Plant Pathol 68:61–68

Sundberg E,Slagter JG,Fridborg I,Cleary SP,Robinson C,Coupland G(1997)ALBIN03,an Arabidopsis nuclear gene essential for chloroplast differentiation,encodes a chloroplast protein that shows homology to proteins present in bacterial membranes and yeast mitochondria.Plant Cell 9:717–730

Takahashi H,Ehara Y,Hirano H(1991)A protein in the oxygen-evolving complex in the chloroplast is associated with symptom expression on tobacco leaves infected with cucumber mosaic virus strain Y.Plant Mol Biol 16:689–698

Tanaka A,Ito H,Tanaka R,Tanaka NK,Yoshida K,Okada K(1998)Chlorophyll a oxygenase(CAO)is involved in chlorophyll b formation from chlorophyll a.Proc Natl Acad Sci USA 95:12719–12723

Terao T,Katoh S(1989)Synthesis and breakdown of the apoproteins of light-harvesting chlorophyll a/b proteins in chlorophyll b-deficient mutants of rice.Plant Cell Physiol 30:571–580

’t Hoen PAC,Ariyurek Y,Thygesen HH,Vreugdenhil E,Vossen RHAM,de Menezes RX,Boer JM,van Ommen G-JB,den Dunnen JT(2008)Deep sequencing-based expression analysis shows major advances in robustness,resolution and inter-lab portability over five microarray platforms.Nucleic Acids Res 36:e141

Thresh JM,Fargette D,Otim-Nape GW(1994)Effects of African cassava mosaic geminivirus on the yield of cassava.Trop Sci 34:26–42

Vanderschuren H,Akbergenov R,Pooggin MM,Hohn T,Gruissem W,Zhang P(2007)Transgenic cassava resistance to African cassava mosaic virus is enhanced by viral DNA-A bidirectional promoterderived siRNAs.Plant Mol Biol 64:549–557

Vanderschuren H,Alder A,Zhang P,Gruissem W(2009)Dosedependent RNAi-mediated geminivirus resistance in the tropical root crop cassava.Plant Mol Biol 70:265–272

Varma A,Malathi VG(2003)Emerging geminivirus problems:A serious threat to crop production.Ann Appl Biol 142:145–164

Yang J,An D,Zhang P(2011)Expression profiling of cassava storage roots reveals an active process of glycolysis/gluconeogenesis.J Integr Plant Biol 53:193–211

Zhang P,Futterer J,Frey P,Potrykus I,Puonti-Kaerlas J,Gruissem W(2003)Engineering Virus-Induced African Cassava Mosaic Virus Resistance by Mimicking a Hypersensitive Reaction in Transgenic Cassava.Plant Biotechnology 2002 and Beyond.Kluwer Academic Publisher,Dordrecht.pp.143–145

Zhang P,Vanderschuren H,Futterer J,Gruissem W(2005)Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes.Plant Biotechnol J 3:385–397

SUPPORTING INFORMATION

Additional supporting information can be found in the online version of this article:

Figure S1.Symptoms development on plants and leaves of cassava after agro-inoculation with ACMV-NOg.The first unfolded leaf from the shoot apex of systemic infectious cassava(B)shows the difference with the one from MOCK(A);Panel C and D show the whole plants from MOCK and ACMV-infected cassava

Figure S2.Saturation analysis of sequencing.Here,y-axis means the percentage of mapped genes,and x-axis means sequencing amount(total tag number)

Table S1.Significantly differentially expressed genes(DEGs)in the INF and MOCK libraries and gene annotation.Excel file contains the 2,272 significantly upregulated genes and 938 significantly downregulated genes in INF.The parameters“FDR＜0.001”and“Log2 Ratio≥1”were used as the threshold to judge the significance of gene expression difference

Table S2.Gene ontology(GO)terms analysis for cellular component of significantly differentially expressed genes(DEGs).Excel file contains significantly DEGs with mapped 110 terms from the component ontology and their corresponding GO entries

Table S3.Gene ontology(GO)terms analysis for biological process of significantly differentially expressed genes(DEGs).Excel file contains significantly DEGs with mapped 403 terms from the process ontology and their corresponding GO entries

Table S4.Gene ontology(GO)terms analysis for molecular function of significantly differentially expressed genes(DEGs).Excel file contains significantly DEGs with mapped 231 terms from the function ontology and their corresponding GO entries

Table S5.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of significantly differentially expressed genes(DEGs).Excel file contains significantly DEGs with mapped 120 KEGG pathways and their corresponding pathway entries

Table S6.Primers used for real-time reverse transcriptionpolymerase chain reaction(RT-PCR)verification.All the forward and reverse primer sequences were included