Molecular Identification And Transmission Study On Two Novel Geminiviruses Associated With Paper Mulberry (Broussonetia Papyrifera) Leaf Curl Disease

Paper mulberry(Broussonetia papyrifera)is a broad-leaf,deciduous perennial woody plant,historically known as the source material of Cai Lun paper-making,which is also used in traditional Chinese medicine,livestock feed,barkcloth making and afforestation among other uses.Due to its shorter growth period and own higher protein content,the plant paper mulberry is used as a local poverty alleviation material cultivated in poverty-stricken and rocky-desert areas of China,which has helped 200,000 people out of poverty in 35 counties of 10 provinces,including Shanxi,Guizhou and Henan.However,during the fast development of paper mulberry industry,the research of the diseases and insect pests on paper mulberry is relatively insufficient,which has laid a hidden danger for the healthy development of the industry.To identify the pathogen in paper mulberry plants affected by a disease with leaf curl symptoms and formulate the further targeted prevention and control programs,the high-throughput sequencing(HTS)based on viral metagenomics was performed.Three kinds of viral contigs were gotten from the databases: geminivirus 1,geminivirus2 and cytorhabdovirus.Field detection testified the two geminiviruses are closely correlated to symptoms.Here the genome structure,phylogenetic relationship,biological features of the two viruses were researched,which make taxonomic status and transmission model of the two geminiviruses clear.The main results are summarized as follows:1.Three kinds of viral fragments were found in paper mulberry with the leaf-curl symptom,and two geminiviruses are associated with symptoms.The three transcriptome databases(GS-HY,GS-SWU,GS-TL)from leaf curl paper mulberry was analyzed: one cytorhabavirus and two kinds of geminiviruses(geminivirus 1 and geminivirus 2)fragments were existed in symptomatic samples,while the GS-TL sample only contained geminivirus 2.Reverse transcription-polymerase chain reaction(RT-PCR)and polymerase chain reaction(PCR)were performed to confirm the existence of three pathogens in the symptomatic samples.Of the collected91 samples from field,81 of them show obviously leaf curl and chlorotic symptoms,and 10 samples are non-symptomatic.The results of PCR and RT-PCR showed the incidences of cytorhabdovirus,geminivirus 1,and geminivirus 2 were 24.69%,90.12% and 93.82%,respectively.Therefore,these results confirm the close association between the presence of geminirirus 1,geminivirus 2 and paper mulberry leaf curl disease.Besides,we found that two geminiviruses almost co-infected the paper mulberry and caused more severe leaf curl symptom on leaves.2.The full genomes of the two geminiviruses were obtained,and the molecular biological characteristics such as genomic structure,protein function and genetic evolutionary relationship were analyzed.Rolling-circle amplification(RCA)and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants,with the names paper mulberry leaf curl virus 1 and paper mulberry leaf curl virus 2(PMLCV-1 and PMLCV-2)proposed.The genomes of PMLCV-1(3056 nt)and PMLCV-2(3757-3763 nt)encode six open reading frames(ORFs),with four ORFs in the virion-sense strand(V1,V2,V3,and V4)and two in the complementary-sense strand(C1 and C1:C2).The ORFV1 of both PMLCV-1 and PMLCV-2 encode a protein which was identified as a geminivirus coat protein(CP).The V2ORFs potentially encode proteins of which are related to the MP-like V2 proteins of MMDaV and CaCDaV based on BLASTp analysis.Although no significant similarity was found for related V3 genes when searching against databases,a transmembrane domain that may play a role in geminivirus trafficking was identified as a common characteristic.The deduced V4 protein of PMLCV-2(302 aa)is much larger than that of PMLCV-1(100 aa).The PMLCV-1 V4 as well as the MMDa VV4 and V5 proteins have no similarity with any protein in the databases,while the secondary structures of PMLCV-2V4 and those of related viruses(PCMoV,CCDaV,Ca CDaV)shows a typical 30 K movement protein pattern with a series of unfolded α-helices and β-sheets(αA,β1,β2,αB,β3-β7)from the N to the C terminus.The two coding regions in the complementarysense strand,C1 and C1:C2,potentially encode replication-associated proteins RepA and Rep,respectively.The aa sequence identities among PMLCV-1,PMLCV-2 and the four related viruses(MMDaV,PCMoV,CCDaV,CaCDaV)in C1 and C1:C2 are 34.8–48.9% and 43.7–55.8%,respectively.In addition,the aa motifs reported in other geminivirus Reps were also found in the amino-terminal sequences of the Rep proteins of PMLCV-1 and PMLCV-2.Alternative splicing of an intron,akin to that of mastre-,becurto-,capula-and grabloviruses,was identified by small RNA(sRNA)-seq and rRNA-depleted RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts.Phylogenetic analysis based on nucleotide sequences of the full-length viral genome and amino acid sequences of CP and Rep found that the CP and Rep cladograms showed a similar topology,with PMLCV-1,PMLCV-2,CCDaV,MMDaV,CaCDaV,and PCMoV clustering together.In the full genome tree,PMLCV-1 and PMLCV-2 were placed in two different monophyletic clusters,with PMLCV-1 sharing a closer evolutionary link to MMDaV,while PMLCV-2 is closer to CCDaV,CaCDaV,and PCMoV.Pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to but distinct from two unassigned geminiviruses,CCDaV and MMDaV,thus we suggest ICTV set a new genus which contain two subgenera,one including PMLCV-1 and MMDaV,and the other including PMLCV-2 and CCDaV.3.The transmission mode of PMLCV-1 and PMLCV-2 was preliminarily defined :PMLCV-1 could not be transmitted by seeds,while PMLCV-2 could be transmitted by seeds and virulent branches.Through PCR and RT-PCR detection assay from the insect vectors on the paper mulberry plant,we preliminarily verified the candidate vectors of PMLCV-1 and PMLCV-2as leafhopper Empoascanara and leafhopper Jacobiasca boninensis.Seeds on the symptomatic paper mulberry were collected and cultivated to three-month-old seedlings.Virus detection on seedlings found that cytorhabdovirus and PMLCV-1 can’t be detected in all seedlings,while PMCLV-2 can be detected in some seedlings(12.6%).Using PMLCV-2 primers amplifying viral whole genome,the genome-sized fragments were obtained from 11 samples and verified by sangersequencing.Through transcriptome sequencing of seedling Z28,we found that PMLCV-2 can be transcribed in plant.Grafed scions of seedlings Z28 on healthy paper mulberry,the existence of PMLCV-2 can be verified after six months,which proved the PMLCV-2 can replicate in the plant.