The Genomic Sequence Analysis And Primary Study On Virulence-attenuation Mechanism Of An Attenuated ANRSV Isolate(DAT)

Potyviridae is the largest plant RNA virus family that infects a variety of crops worldwide,causing huge economic losses.To date,Potyviridae is divided into 12 genera by the International Committee on Taxonomy of Viruses(ICTV),containing 288 species.Areca palm is the second largest economic crop in Hainan,China.The industry of areca nut of areca palm produces over 10 billion yuan annually.However,Areca palm is severely damaged by various diseases as the tropical climate in Hainnan is suitable for pathogen propagation.Areca palm necrotic ringspot disease(ANRSD)is a wide spread virus disease that threaten the areca nut industry in Hainan.The diseased plant shows severe necrotic ring spots on the infected leaves and eventually leads to the decline of tree vigor with decreased or even no fruit production.Previously,our group identified that ANRSD is closely-related with areca palm necrotic ringspot virus(ANRSV),completed the ANRSV genome sequencing and annotation,and classified ANRSV as a new member of Potyviridae.In this study,an attenuated isolate of ANRSV(DAT)was discovered and identified in areca palm planting area in Ding’an,Hainan,and its complete genomic sequence was determined.In comparison with the virulent strain ANRSV-XC1,it was found that the ANRSV-DAT genome is missing 95 nucleotides(nts)in its 5’-untranslated region(5’-UTR).Furthermore,we analyzed the genome characteristics of the attenuated strain and preliminarily explored the mechanism underlying the attenuation phenotype.Those works could lay the foundation for the prevention and control of ANRSV through cross protection.Eventually,this study attempts to explore the areca plum transfection system of ANRSV,which could help the successful development of areca plum transfection system.1.Genome sequencing and annotation of the attenuated ANRSV isolate DATFrom June,2018 to June,2020,the research team continuously observed the infected symptom of areca palm plant-DAT,and found that DAT plant showed consistent mild and scattered chlorotic ring spots in different growing seasons.First of all,this study used ANRSV specific primers to perform RT-PCR analysis on the suspected plant.The test results showed that DAT plant was indeed infected by ANRSV.Therefore,combined with the mild symptoms,this study speculates that ANRSV-DAT is a potential attenuated ANRSV isolate.Furthermore,we have finished the whole genomic sequencing and annotation of ANRSV-DAT.The genomic sequence length is 9339 nts,including a9060-nt open reading frame(ORF)which encodes a 3019-aa polyprotein,as well as 110-nt5’-UTR and 169-nt 3’-UTR.2.ANRSV-DAT molecular characteristics analysisUsing Lasergene 7.0 sequence analysis package and clusterw2 online tool to compare the nt and amino acid sequence of DAT and XC1,respectively.It was found that ANRSV-DAT was missing 95 nts from in the N-terminal end of the 5’-UTR in comparison with ANRSV-XC1 genome.There are 63 aa differences between two isolates,which are distributed in 10 cistronic regions to varying degrees: HCPro1(6),HCPro2(7),P3(16),7K(2),CI(2),9K(3),NIa-Vpg(1),NIa-Pro(8),NIb(15)and CP(3).3.Research on the method of ANRSV transfection of areca palmUsing fresh leaf homogenates(sap)from ANRSV-XC1-infected N.benthamiana plants and ANRSV-XC1 infectious clone,we attempted the following three sap-inoculation methods in areca palm;1)sap inoculation of areca palm leaves by rubbing;2)sap inoculation of areca palm leaves by injection with syringe;3)sap inoculation of areca palm stem by injection with syringe.In addition,two additional agrobacterium-mediated approaches were employed for the inoculation of ANRSV in areca palm;1)Agrobacterium(harboring infectious clone)inoculation of areca palm leaves by injection;and 2)Agrobacterium(harboring infectious clone)inoculation of areca palm stem by injection with syringe.The results showed none of the methods mentioned above could successfully infect areca palm.4.A preliminary study on the attenuation mechanism of ANRSV-DATPreviously,it was demonstrated that sap from ANRSV-XC1-infected areca palm can successfully infect N.benthamiana plants by rub inoculation.In this study,we used the same inoculation method but found ANRSV-DAT is not able to infect N.benthamiana plants.In view of the lack of 95 nts from the N-terminal of the 5’-UTR of the ANRSV-DAT genome(compared to the virulent isolate ANRSV-XC1),we speculated that the 95-nt sequence deletion may be related to the weak infectivity of ANRSV-DAT.Therefore,this study used the GFP-labeled ANRSV-XC1 infectious clone(ANRSV-XC1-GFP)to construct a mutant with 95-nt deletion in the 5’-UTR of ANRSV-XC1,and investigated the infectivity of the deletion mutant.The results showed that the deletion of 95-nt sequence in the 5’-UTR caused the virus to lose the ability to infect N.benthamiana plants.Moreover,in order to explore whether the deletion of 95-nt affects virus translation,this study used ANRSV-XC1-GFP to construct deletion mutant from the 5’-UTR to the NIb region(termed TM3)and the P1 to NIb region Deletion mutant(termed as TM3);using ANRSV-XC15’-UTR region 95 nt deletion clone mutant to construct P1 to NIb region deletion mutant(termed TM1)to explore the impact of 5’-UTR 95-nt deletion on virus translation.The results showed that 95-nt deletion affects virus translation,and the relatedness between this effect and the loss of virus infection activity needs to be further studied.