Whole-genome Expression Analysis Of Rice Black-streaked Dwarf Virus In Different Hosts And Screening Of Host Factors Interacted With P7-1 Encoded By RBSDV

Rice black-streaked dwarf virus(RBSDV)is transmitted by the small brown planthopper(Laodelphax striatellus,SBPH)in a persistent circulative manner,and is the pathogen of the rice black streaked dwarf disease and maize rough dwarf disease.The RBSDV genome contains ten segments of double-stranded RNA(dsRNA)which included 13 open reading frames(Open reading frame,ORF).The virus can infect plants such as rice,maize and wheat and cause symptoms such as stunting,darkening of leaves.The expression of RBSDV in different plant hosts and the differences between the expressions in plant and insect are unclear.In this study,the relative RNA expression levels of the thirteen RBSDV genes in rice,maize,wheat and SBPH were analyzed by real-time quantitative PCR.The results showed that P7-1 and P10 genes were predominantly expressed whereas P8 and P7-2 genes were expressed at relatively low levels in plant hosts.Similar to the expression in rice,P7-1 was the most abundantly expressed gene and P8 was expressed at the lowest level in SBPH,indicating that RBSDV adopts the same strategy to infect distinct hosts.The high expression levels of the P7-1 gene in both plants and insect suggest that it can be used as the target gene for disease diagnostics.P5-1,P6 and P9-1,the components of the RBSDV viroplasm,are differentially expressed in different hosts,and virus may adjust viral gene expression to replicate in different hosts.RBSDV infection affected the increase of ABA content,and analysis of real-time gene expression revealed that P7-1 stands out as the only gene highly expressed at the earliest time point and its expression precedes all others throughout infection from 8 to 24 days post inoculation.The high expression levels of the P7-1 gene suggest that it plays a significant role in RBSDV-host interaction.Those viral genes that were differentially expressed in distinct hosts may be involved in the interactions between the virus and specific hosts.Previous studies showed that RBSDV P7-1 was localized in the plasmodesmata and could form tubular structure in plant and insect,which suggested that it may be involved in cell movement.P7-1 is considered to be a pathogenicity determinant as over-expression of P7-1 in Arabidopsis resulted in male sterility.In addition,our gene expression analysis results show that P7-1 was highly expressed in both the plants and insect.To reveal the interaction between P7-1 and insect host,P7-1 was used as the bait for screening the cDNA library of SBPH.P7-1 gene was amplified by RT-PCR,and yeast two hybrid vector pGBKT7-P7-1 was constructed.33 positive clones were selected by sequential transformation.The size of the inserted fragments of the positive clones were analyzed by PCR.The plasmid of the positive clones with the PCR product size larger than 500 bp were transformed into E.coli and then sequenced.The sequences of the positive clones were analyzed by BLAST in GenBank database.Sequence analysis showed that these 33 positive clones encode 15 kinds of proteins,including ATPase B subunit,40 S ribosomal protein,enolase,serpin,carboxylesterase,and protein phosphatase.The results lay a foundation for further analysis ofthe interaction between RBSDV and insect host.In order to analyze the interaction between RBSDV P7-1 and plant,we constructed a rice cDNA library,and screened the library using the P7-1 as a bait to investigate the interaction factors in rice.To construct the rice cDNA library,total RNA from Nipponbare seedlings were extracted and the three-frame cDNA entry library was prepared by homologous recombination.The entry library then transferred to the hybrid library vector pGADT7-DEST through gateway technology.The yeast two hybrid cDNA library contained 1.3×107 independent clones and the titer of the amplified library was 4.34×109 cfu/mL.Its recombination rate was above 95%and its average size of inserts was more than 1 kb.The results showed that the library database met the requirements of the standard cDNA library.P7-1 was used as a bait to screen the rice cDNA library and 28 positive clones were obtained.Sequence analysis showed that these positive clones encodes 11 kinds of proteins,including cysteine protease synthesis,superoxide dismutase,proteinase inhibitor,Myb transcription factor,general transcription factor IIH subunit,catalase and thiamine.The results lay a basis on further study of the interaction between RBSDV and plant.