Comparative Analysis Of Gene Expression Profiling And MiRNA Of Wheat Response To Heterodera Avenae Infection

Cereal cyst nematode,Heterodera avenae,is an important soil-borne pathogenic nematode of cereal crops and causes significant impact upon quality and quantity of wheat.It is widely distributed in major wheat producing areas around the world and showed a trend of increase year by year.However,the molecular mechanism of interaction between H.avenae and wheat is largely un-known.Gene expression profiling of a susceptible wheat cultivar ’Bainong Aikang 58’ response to H.avenae infection was performed using the GeneChip（?）Wheat Genome Array.Compared to mock-inoculated control plant,4,915,5,556 and 4,715 diffrerentially expressed genes were obtained at 4,8 and 16 dpi（days post inoculation）respectively with P value≤0.05 and |log2-ratio|≥1.Sixty-nine genes from the three time points were validated using quantitative real-time PCR（qPCR）and the consistency between qPCR data and microarray data indicates the reliability of microarray.GO enrichment analyses showed that the differentially expressed genes from 4 and 8 dpi were mainly enriched in response to stress,transmembrane transport and metabolic process,and those from 16 dpi were enriched in oxidation reduction,biosynthetic process,sucrose metabolic process and glycogen biosynthetic process.Genes involved in response to stress and defense were up-regulated at 4 and 8 dpi,the phases of formation and development of syncytium,such as heat shock protein,pathogenesis-related protein,chitinase,xylanase inhibitor,late embryogenesis abundant protein and germin-like protein,and genes associated with cell wall modifcition were also up or down-regulated,which included expansin and glucanase.At 16 dpi,the maintenance phase of syncytium,genes involved in energy supply were up-regulated,such as sucrose synthase and starch synthase.In addition,expression of genes associated with jasmonic acid signaling,phenylpropanoid and lignin biosynthetic pathway were also changed following the H.avenae infection.Small RNA library constructed from H.avenae-inoculated and mock-inoculated wheat roots at 4,8 and 16 dpi were sequenced by high-throughput sequencing.One hundred and eighty-four known miRNAs and 393 novel miRNAs were identified and predicted respectively.Then,40,29 and 58 differentially expressed known miRNAs and 21,20 and 24 differentially expressed novel miRNAs were obtained at the three time points with P value≤0.01 and |log2-ratio|≥1.Twenty miRNAs were validated by qPCR and the expression pattern of 17 miRNAs were consistent with sequencing data,and the results indicate the reliability of sequencing.Expression of miRNAs associated with plant defense were altered following the H.avenae infection,in which miR950,miR1157-3p,miR1069-5p,miR1850.2,miR5485 and miR6170 were up-regulated at three time points.The miRNAs involed in auxin signaling including miR156c-3p,miR396c-3p,miR167e-3p,miR171k-5p,miR393b-3p and miR393h were up or down-regulated follow infection with H.avenae.These miRNAs may negatively regulate their targets to influence the plant defense and the information and development of syncytium.The targets of differentially expressed miRNAs were then predicted which includ some important transcription factors,such as NAC,bHLH,WRKY,AP2/EREBP.A comparative analysis between predicted targets and differentially expressed genes obtained from gene chip was conducted.Forteen differentially expressed genes were matched to the predicted targets,and NAC69-1 and DFR may be regulated by novel_mir₅4 at 16 dpi.We also investigated the occurrence regularity of Beijing population of H.avenae in field.The results showed that there were two hatching peaks for second stage juveniles（J2s）during a wheat growing season and the main peak occured from late March to early April in spring.The investigation also discovered the autumn infection and the overwintering of juveniles in wheat roots.Further studies confirmed that a period of low temperature for new season’s cysts is essential for the hatching and infection of J2s,and the nematodes that penetrated in roots in autumn may come from the residual cysts that have experienced one or more winters.Analysis of the differentially expressed genes and miRNAs response to H.avenae infection will definitely shad light on the understanding the pathogenesis between nematode and host interaction and provide new idea for prevention strategies and measures.Also,the investigation of occurrence regularity of H.avenae in field will improve our understanding on their biology,in turn help us to discover effective control strategies for reducing the damage and losses of wheat.