Metaviromics-based Studies On Diversity And Molecular Evolution Of Citrus Viruses

Citrus is the top fruit crop in China,and the world,while virus diseases are important factors that restrict development of citrus industries.More than 20 viral species have been reported to infect citrus,yet species of citrus viruses may be far more than this number.Identification of viruses lays important basis for prevention and control of their occurrence and dispersal,and it is necessary to strengthen investigation of virus species diversity in citrus,so that management and control system of citrus viral diseases can be further improved,which can contribute to sustainable development of citrus industries.High-throughput sequencing technique has been used in identification of some citrus viruses,but it has some disadvantages: 1)for metaviromes in large-scale citrus samples,there is lack of a set of standard analysis method that can refer to,and 2)the virus annotation method(BLASTX)generally used is insufficient for some viruses with split genomes.For these problems this research constructed systematically a citrus metavirome analysis platform that is applicable to both monopartite and segmented/multipartite viruses.Using this platform,plant virome in sample B2-R20 of single plant was analyzed,and one new multipartite-like virus named CJVV1(citrus jivi-related virus 1)identified,and this virus was further studied in molecular characteristics and evolutionary status.Using this platform,a new monopartite virus named citrus-associated rhabdovirus(CiaRV)was found during analyses of plant virus species diversity in one mixed sample CV1 with 50 plants,and on this basis,molecular and biological characteristics of coevolution of CiaRV and plants were further studied.Below are detailed results.1.Construction of citrus metavirome analysis platformIt is firstly to construct citrus metavirome analysis platform systematically.This platform is based on ribosomal RNA-depleted paired-end RNA sequencing(PE RNA-seq),and it adopts BLASTX and untranslated region-based iterative BLAST(UTR-i BLASTN)to annotate contigs of monopartite and segmented/multipartite viruses,respectively.RNA-seq and small RNA sequencing(sRNA-seq)are two virus analysis strategies widely used in citrus.However,using UN8 as an example,nearly complete genomes of known viruses can be recovered from RNA-seq but not sRNA-seq.With BLASTX and UTR-i BLASTN,monopartite and multipartite viruses in UN8 can be fully annotated,respectively.Analysis with statistics showed that UTR-i BLASTN is widely applicable to segmented/multipartite viruses,which embrace at least 17 families involving hosts of invertebrates,plants,fungi,bacteria,and protists.These results indicate practicability and application value of this platform.2.Molecular characterization of new citrus multipartite-like virus CJVV1Application of the platform allows identification in a field citrus sample of a new multipartitelike virus CJVV1,based on which seven homologous viruses newly identified in NCBI databases,and then sequence and phylogeny of CJVV1 and related viruses were systemically studied.Analyzing CJVV1: 1)RNA1-3 of CJVV1 are monocistronic,among them RNA1 encodes a putative protein of about 147.4 k Da,which contains conserved domains for methyltransferase(Mtr),and helicase(Hel);2)putative protein encoded by RNA2 is approximately 115.9 k Da in size,and characterized by Rd RP(RNA-dependent RNA polymerase)domain;3)73.9-k Da putative protein of RNA3 shows a domain typical of another kind of helicase(DEAD-like＿helicase＿N Superfamily);4)RNA4-5(a,b)are bicistronic,while their putative proteins are functionally unknown,of which RNA5 a encodes P18a(18.2 k Da),and P46(46.8 k Da),and RNA5 b encodes P18b(18.98 k Da),and P50(50.8 k Da).P46 and P50 are likely to have the same origin according to the BLASTP＿e-value of 6.96E-10,but only P46 possesses identifiable domains Zn F＿U1 and zf-3Cxx C of the zinc finger,and this protein is related to proteins from fungi according to the PSI-BLASTP＿e-value of E-10.Then,analyzing CJVV1 and related viruses by sequence comparisons and phylogenetics: 1)CJVV1 and the viruses shared similar conserved sequences in their 5′-UTRs;2)all viral proteins are homologous with BLASTP＿evalues<6.16E-6;3)putative proteins encoded by RNA1s(or RNA2s)and RNA3 s are most phylogenetically related to the families Togaviridae(phylum Kitrinoviricota)and Potyviridae(phylum Pisuviricota),respectively,and this indicates that CJVV1 and related viruses are possibly generated by recombination of the two types of viruses,which may represent a new taxon in the order Martellivirales.These results further support practicability of the platform.3.Research on coevolution of new citrus monopartite virus CiaRV and three plant speciesIt is firstly to conduct a metavirome study in citrus with the platform,and four known and two new viruses were identified in a collection of mixed samples in fields,among them a new monopartite virus CiaRV was selected for further systemic studies in molecular and biological characteristics of its coevolution with three plant species.Three main aspects of CiaRV were further studied: 1)on the basis of seven quasispecies and reads mapping,box-plot,heatmap,and Pearson correlation analyses were used to estimate variation levels in the G gene;2)using RT-PCR to investigate its distribution in different plants and whitefly populations;3)using movement protein complementary and grating experiments to assess its movement ability between cells and plants.Here are results and conclusions:1)variation of G gene is high in seven quasispecies,and populations,and is correlated between them,correlation is confident(P-value<0.05),and strong(|r|>0.8),and these indicate G gene is an evolutionary hotspot in CiaRV genomes;2)with the same geographic origins,detection rate of CiaRV in passion fruits and paper bushes is much higher than that in citrus,and CiaRV can be detected in whiteflies(Bemisia tabaci)from passion fruits,and paper bushes but not in whiteflies(Dialeurodes citri)from citrus,thus suggesting CiaRV is possibly vectored by B.tabaci,and CiaRV from citrus may lose the vector transmissions due to defects in G gene;3)CiaRV-C1＿P3 can recovery intercellular trafficking of two kinds of movement-deficient plant viruses,and CiaRV-C1 was detected in systemic leaves from two kinds of rootstocks grafted with skins from the infected plant,suggesting its movement abilities between cells and plants.Based on these data,a hypothesis for CiaRV–three plant species coevolution was proposed: higher variation levels,lack of selection pressures from vectors,and changes of transmission route in citrus are key factors for defects in CiaRV-G gene.This hypothesis provides an important and practical reference to explain origin,emergence,and prevalence of citrus viruses.