Screening And Function Study Of Rice Factors Interacted With Rice Black-Streaked Dwarf Virus P5-2 Protein

Rice black streaked dwarf virus(RBSDV)belongs to the reovirus family(Reoviridae)fiji virus(Fijivirus),and it is transmitted by vector,small brown planthoppers(SBPH;Laodelphax sreiate striatellus Fallen),in a persistent and propagative manner.The virus can infect rice,corn and wheat,the rice plants infected by RBSDV show severe dwarf,less tiller and low seed setting rate compared with healthy plants,resulting in serious economic losses.The RBSDV genome is composed of 10 dsRNA fragments,which are called S1-S10 in order of their mobility in PAGE from fast to slow.The S5 genome contains 2 ORF,ORF1 encoding P5-1 protein,and ORF2 encoding P5-2 protein.P5-2 is a non structural protein,and studies have shown that P5-2 protein is located in the chloroplast in Benedict,but its function is not yet clear.In this study,in order to clarify the pathogenic mechanism of RBSDV P5-2,we constructed P5-2 over-expressed Arabidopsis.The results showed that RBSDV P5-2 protein overexpressed in Arabidopsis mutant plants and displayed severe dwarf phenotype,indicating that P5-2 is an important pathogenic factor of virus.Using yeast two hybrid to screen the factor in rice(Nipponbare)cDNA Library which interact with P5-2,the results show that P5-2 protein interacted with several host factor,through the sequence comparison and prediction analysis,The host factors that interacted with P5-2 in yeast include ZFP(TFIIIA zinc finger transcription factor),BBT15(Bowman-Birk trypsin inhibitor),R3H(rice function unknown protein)and so on.For these rice factors,yeast two-hybrid technique was used to verify the interaction between ZFP?BBTI5?R3H and P5-2 protein in yeast.To further verify the interaction of P5-2 with host factors in plants,We constructed a series of fluorescently labeled or Luc(luciferase)plant expression vectors.After infiltrated the Nicotiana benthamiana,The luciferase complementation assay(Split-luciferase)verifies that P5-2 interacts with rice transcription factor ZFP,protease inhibitor BBTI5,and RNA binding protein R3H in plant cells;In a subcellular localization(Co-localization)of the P5-2 protein and its interaction factor,the result show that RBSDV P5-2 interacts with transcription factor ZFP in the nucleus and interacts with the protease inhibitor BBTI5 in the cytoplasm and interacts with R3H in cytoplasm and nucleus.In order to analyze the role of the above rice P5-2 interaction factor in anti-virus,this study uses CRISPR-cas9 gene editing technology to construct rice P5-2 interaction factor mutant rice plants.Transgenic rice T0 plants were obtained by Agrobacterium-mediated transformation methods,according to the results of molecular detection and sequencing of target sequences of rice ZFP,BBTI5,and R3H CRISPR-cas9 knockout mutants,we obtained ZFP,BBTI5,and R3H knockout T0 pure and mutant plants.Inoculation of ZFP and BBTI5 mutant plants with small brown planthoppers with RBSDV,quantitative analysis of virus content by qRT-PCR,the results showed that the virus content and incidence were significantly lower in the ZFP and BBTI5 CRISPR-cas9 mutants compared to wild-type plants.These results indicate that the virus P5-2 rice interaction factors ZFP and BBTI5 play an important role in rice anti-virus.In addition,this study requires the rice that is affected by the rice black-streaked dwarf virus,in order to speed up the progress of the experiment which can detect whether rice plants carry the virus,this study established the recombinant polymerase amplification technology(RPA),and through the exploration of the optimum reaction condition such as temperature,in a certain temperature range,when the reaction temperature is 37 degrees centigrade,the results is obviously,the test results can be observed after 20 min reaction,RPA detection method of RBSDV is simple,fast and efficient,suitable for the detection of RBSDV in rice field and laboratory.The results of this study provide a theoretical basis for the analyzing the pathogenic mechanism of the virus and the interaction mechanism between RBSDV P5-2 and rice host factors ZFP and BBTI5.