Preliminary Analysis On Symptom Development Mechanism Of Wheat Dwarf Disease

In recent years,wheat dwarf disease is becoming one of the severe geminivirus disease on triticeae crops in the northwest of China,which is caused by Wheat dwarf virus(WDV)and transmitted by leafhopper(Psammotettix alienus L)in a persistent,non-propagative manner.Infected plants show typical symptoms such as severe dwarfing,leaf yellowing and effective tiller reduction,resulting in heavy yield losses in cereal crop worldwide.However,the molecular mechanism of WDV pathogenicity has not been studied in depth.In this study,RNA-Seq was used to perform analysis of gene expression and small RNA derived from WDV,contributing to unveiling the mechanism of WDV pathogenesis from the molecular level.Firstly,RNA-Seq was used to perform gene expression analysis in wheat plants in response to WDV infection.The results showed that hormone biosynthesis-related and chloroplast photosynthesis-related genes might play active roles in symptom development during WDV infection.According to the transcriptome data,hormone related genes including ent-kaurene synthase like-3 gene,cytokinin-N-glucosyltransferase 1 gene and brassinosteroid LRR receptor kinase 1 gene,designated as TaKSL3,TaUGT76C1 and TaBRI1,were cloned from wheat plants(cv.Yangmai 12)by homologous cloning technology for the first time.The full length of their coding regions were 1827 bp,1344 bp and3663 bp,respectively.All the coding regions of these genes were analyzed by bioinformatics method in detail,contributing to further study their interaction between wheat and WDV.Among the above genes,ent-kaurene synthase gene is one of the key enzymes in the biosynthesis of gibberellin,which is usually considered to be related to the development of host dwarfing symptom.In order to clarify the relationship between the change of gibberellin content and the formation of dwarfing symptom in WDV-infected wheat,the transcription level of TaKSL3 was analyzed by RT-qPCR,GA3 content was analyzed by indirect ELISA and spraying of exogenous GA3 was applied to observe the change of biological phenotype.The results showed that the infection of WDV could lead to the decrease of gibberellin content in the plant,and the decrease of relative expression of TaKSL3.To some extent,the dwarfing symptom can be relived after spraying of exogenous GA3.It is speculated that WDV infection suppressed the expression of gibberellin biosynthesis-related genes,resulting in the decrease of gibberellin content and then induce dwarfing symptom in infected plants.In addition,increasing studies indicated that virus-derived siRNA(vsiRNA)can be directly involved in the regulation of host gene expression,and then mediate the virus causing yellowing symptom.In order to study the relationship between vsiRNA and symptom development,we firstly analyzed the virus-derived siRNA in wheat by RNA-Seq.A total of 15,236,231 reads were obtained,of which 14,481,641 of the length of 19-26 nt were recorded,accounting for 95.05% and 1,726,212 reads,accounting for 11.92%,could be blasted to the reported sequences.The size of 21 nt,22 nt and 24 nt were dominant and the polarity proportion of complementary strand was slightly larger than that of the viral chain.The size of 18-26 nt were analyzed and found that their 5’ base preference were various,especially 25 nt and 26 nt siRNA with base A content much larger than the other three bases(U,G and C).The vsiRNA was centrally distributed in the 5’ end of RNA genomic,the transcriptional regionencoding coat protein(CP).The results of small RNA analysis derived from WDV,combined with the transcriptome data,may provide clues to the discovery of the key genes related to symptom development,which will promote to elucidate the molecular mechanism of WDV pathogenesis in the future.