Researches Of Small RNA Deep Sequencing On The Identification Of New Citrus Viruses And Novel Antiviral Silencing Pathway Triggered By TuMV Infection

Citrus is the most important fruit throughout the world,till now there have been almost twenty kinds of viruses on citrus,some of which would cause great losses to the citrus production,so both viral identification and prevention would play key roles for the citrus industry.Since it is much harder to purify the virion from fruit trees than from the herbaceous plants,traditional methods are of much limitation on fruit virus identification.Small RNA(sRNA)deep sequencing technology,based on the RNA interfering mechanism,has been widely used in researches on plant virus identification and virus-host interactions.In this research,sRNA deep sequencing method was deployed to identify two viruses from lemon trees,and both viral and host endogenous small RNA profiles were analyzed,as to explore more information for the antiviral research on fruit trees.To identity the potential pathogen associated with the yellow vein clearing symptom on lemon,the profiles of virus-derived small interfering RNAs(vsiRNAs)from citrus samples were obtained and analyzed by deep sequencing method in this study.Twenty-seven contigs obtained by the sRNA deep sequencing technology almost cover the full-length genome of Citrus yellow vein clearing virus(CYVCV)isolate YN.Analysis showed that this isolate CQ shared the highest nucleotide identity with isolate Y1(JX040635)and YN(KP313242),both of which belong to the family Alphaflexiviridae genus Mandarivirus.Mapping analysis of vsiRNAs profiles revealed an uneven distribution pattern of their generations along both the positive and negative genome strands,22-and 21-nt vsiRNAs ranked the majority.In addition,5'-terminal nucleotides preference of the vsiRNAs was also analyzed.BLAST against viroids and other viral databases confirmed that the sample was of single-infection by CYVCV,which indicated that CYVCV was the exact causal agent for the yellow clearing symptom occurring on lemon.CCDD(Citrus chlorotic dwarf disease,CCDD),was firstly discovered in Turkey in 1995,it could infect many citrus varieties.In this research,sRNA deep sequencing technology was deployed to assemble the entire genome of a circular ssDNA virus,citrus chlorotic dwarf-associated virus(CCDaV).Results revealed the complete genome of this isolate CN001 of 3,642-nts,BLASTn analysis showed an overall nucleotide identity of 99.3% with isolate CCDaV TK4(JQ920490),which was a novel member of the family Geminiviridae.Open reading frames were also determined within both the sense and anti-sense strands.Phylogenetic tree further supported the evidence that the CCDaV isolate belonged to the family Geminiviridae.The vsiRNAs analysis showed an unequivalent distribution pattern of their biogenesis,5'-terminal nucleotides preferred A on both sense and anti-sense strands.Combined with the vsiRNAs profiles,an intron region was predicted and verified within its anti-sense strand.Analysis also confirmed that the sample was infected only by CCDaV,without any other viruses,viroids or associated satellite molecules,these evidence indicated that CCDaV would be the causal agent for the chlorotic dwarf symptom occurring on lemon in China,which further supported that the technology could be a powerful tool for fruit virus identification.RNA silencing is an evolutionary conserved sequence-specific gene inactivation mechanism that contributes to the control of development,maintains heterochromatin,acts in stress responses and defends against invading nucleic acids like transposons and viruses.In plants RNA silencing functions as one of the main immune systems.Antiviral immunity controlled by RNA interference(RNAi)in plants and animals is thought to specifically target only viral RNAs by the vsiRNAs.Here we showed that activation of antiviral RNAi in Arabidopsis plants was accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and ribosomal RNAs(rRNAs).These virus-activated siRNAs(vasiRNAs)are predominantly 21-nucleotides long with an approximately equal ratio of sense and antisense strands.Genetically,vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4(DCL4)and RNA-dependent RNA polymerase 1(RDR1)for biogenesis.However,loss of EXORIBONUCLEASE4/THYLENE-INSENSITIVE5(ein5)enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs.We showed that vasiRNAs were active in directing widespread silencing of the targe host genes.Production of vasi RNAs was readily detectable in Arabidopsis after infection by Turnip mosaic virus(TuMV),as the similar result with the previous mutant Cucumber mosaic virus(CMV-?2b).Gene ontology(GO)analysis revealed that both CMV-?2b and TuMV-GFP could cause 369 genes to be the target of RDR1,which were relevant to the basic cellular activities for the Arabidopsis and would be silenced by vasiRNAs.These findings revealed RDR1 production of Arabidopsis were endogenous siRNAs,and identified production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses.In addition,sRNAs profiles from both the mock and CYVCV-infected samples were also analyzed,results revealed that there was a distinct phenomenon for the vasiRNAs pathways between fruit trees and Arabidopsis,sRNA profiles of host rRNAs and coding genes from the CYVCV-infected sample were almost invariable as the mock control,which indicated that vasiRNAs pathway in Arabidopsis may be not work for the lemon trees.This is the first attempt to apply the vasiRNAs pathway from Arabidopsis to fruit trees.