Geneitc Structure Of Rice Black-streaked Dwarf Virus (RBSDV) Populations And Identification Of Maize Protein Interacting With RBSDV P8

Maize is one of the most important food and forage crops and industrial raw materials.Diseases lead to severe losses of maize production in China. Maize rough dwarf disease(MRDD) is devastating and caused significant losses to maize production. As a species of thegenus Fijivirus in the family Reoviridae, Rice black-streaked dwarf virus (RBSDV) can causeMRDD in maize. The biological characteristics and spread of MRDD, genome structure andgene function of RBSDV have been well documented, but its molecular diversity andevolution mechanism of RBSDV remain largely unknown. Because natural hosts in the field,incidence and percentage of viruferous small brown planthopper are closely related tooccurrence of MRDD, quick and efficient detection of RBSDV field hosts and viruferousvector are critical for providing prevention and control measures. Elucidating the factorsaffecting RBSDV evolutio and understanding the interaction between host and viral proteinswill provide theoretical guide for the breeding of resistant maize cultivar to RBSDV and thesustainable control of MRDD.In the study, we established and optimized the detection system for RBSDV, anddetected hosts in the field, sequenced the genome of a RBSDV reassortant, detected SRBSDVfrom Shandong maize, analyzed the genetic structure of RBSDV populations, and identifiedmaize protein interacting with P8. The results were as follows:Firstly, four pairs of primers were designed according to the nucleotide sequences ofRBSDV S10. RT-PCR detection system for RBSDV was optimized after comparing primerpair, concentrations of Mg2+and Taq polymerase and times of RNA dilution. The optimalRT-PCR system was10×PCR buffer2.5μL,25mM MgCl21.5μL, dNTP (each2.5mM)2.0μL, primers F4and R4(10μM) each1.0μL,5U/μL Taq DNA polymerse0.2μL, templateRNA5.0μL, sterilized water11.8μL to make a total volume of25μL. With the methodestablished, RBSDV can be detected from RNA extracted from single Laodelphax striatellusor30ng infected maize leaves. Besides graminceous plants, Sonchus brachyotus andEclipta prostrate from family Asteraceae and Amaranthus retroflexus fromAmaranthaceae are also natural hosts of RBSDV. Secondly, we reported the complete genomic sequences of all the open reading frames(ORFs) in SDZZ10. Comparing with two RBSDV isolates whose complete genomicsequences were available, the most ORFs and corresponding proteins of SDZZ10sharedhigher nucleotide (nt) or amino acid (aa) identities with RBSDV-Hbm; While ORFs3,4,9-2and10of SDZZ10shared higher nt identities with RBSDV-Zjr, P4, P9-1and P9-2sharedhigher aa identities with RBSDV-Zjr. Phylogenetic analyses of ORF8and ORF10showedthat SDZZ10was clustered into different groups, indicating that SDZZ10is a naturalreassortant.Thirdly, we sequencing S7-S10of SRBSDV isolate, JNi4. The S7to S10of JNi4sharenucleotide identities of72.6-73.1%，72.3-73%，73.9-74.5%and77.3-79%, respectively, withcorresponding segments of Rice black-streaked dwarf virus isolates, and identities of99.7%,99.1%-99.7%,98.9%-99.5%, and98.6%-99.2%with those of SRBSDV isolates HN and GD.JNi4forms a separate branch with GD and HN in the phylogenetic trees constructed withgenomic sequences of S7to S10. These results confirm the proposed taxonomic status ofSRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4is an isolate ofSRBSDV. Shandong is so far the norther nmost region where SRBSDV is established.Fourly, we obtained the sequences of101segment8(S8; carries ORF8encoding theminor core capsid protein) and103S10(ORF10encoding the major capsid protein). BothORF8and ORF10are under negative selection. The S8sequences of3isolates and S10sequences of two isolates were ‘clear’ recombinants. The RBSDV population of China can beclassified into three groups according to the S8and two according to the S10sequences,irrespective of their hosts or geographical origins. Among85RBSDV isolates with both S8and S10sequences available,17are between-group reassortants,30are between-subgroupreassortants. The RBSDV subpopulations from different geographical regions and hosts showfrequent gene flow within or between subpopulations. The RBSDV population from maize inChina is in a state of expanding. In this study no new emergent population was detected.Taken together, our results indicate that recombination, reassortment, negative selection andgene flow are important factors that drive evolution of RBSDV in China.Finally, to identify the maize protein interacting with RBSDV P8, a cDNA library wasscreened by yeast two-hybrid system with pGBKT7-S8as bait plasmid which carried theORF8of RBSDV isolate SDZZ10. Totally26maize proteins were obtained as potentialinteracting proteins. We further confirmed that the40S ribosomal protein S13can interactwith RBSDV P8by YTHS and BiFC. To determine the specific interaction region, we dividedthe sequence of the40S ribosomal protein S13to three regions (N, M and C), and constructed five deletion mutants. According to the results of YTHS and BiFC, we concluded that the40Sribosomal protein S13interacted with P8via its N-and C-termini.