Identification Of Whole Genome Sequence And Gene Silencing Suppressor Of A Novel Mulberry Crinivirus

Mulberry viral disease is one of the most important factors limiting the development of sericultural industry.The genus Crinivirus in the family Closteriviridae includes a variety of viruses causing severe harm to agricultural production.These viruses are efficiently transmitted by whiteflies in a semi-persistent manner.In the natural environment,it causes explosive and epidemic diseases that cause massive agricultural economic losses and attract global attention.In 2019,a novel crinivirus in a mulberry tree with mosaic,mottling and dwarfism symptoms was found through high-throughput sequencing(HTS)and named mulberry crinivirus(MuCV).In this study,we determined the full-length sequence of MuCV and identified the coding post transcriptional gene silencing(PTGS)suppressors and pathogenicity determinants.1.Complete genome characteristics analysis of MuCV and its occurrence in the mulberry fieldDouble-stranded RNA(ds RNA)was extracted from mulberry leaves that originally discovered MuCV and the full-length sequence of MuCV was determined by reverse transcription(RT)-polymerase chain reaction(PCR)and T4 RNA ligase oligonucleotidemediated amplification.MuCV possesses a bipartite genome,RNA1 and RNA2,which are8571 nucleotides(nt)and 8082 nt in length,respectively.RNA1 contains four putative open reading frames(ORFs).ORF1 a encodes a polyprotein.ORF1 b encodes an RNA-dependent RNA polymerase(Rd Rp),which is probably expressed via + 1 ribosomal frameshift.ORF2 and ORF3 encode P5.5 and P6 a at the 3’ end of RNA1,respectively.These two small proteins show no similarity to the amino acid(aa)sequence of any other public protein.RNA2 contains eight ORFs,including the typical "5-gene module" which is consistent with the genomic structure of the genus Crinivirus.The BLASTx search indicated that aa sequences of Rd Rp,major coat protein(CP)and heat shock protein 70 homolog(HSP70h)of MuCV shared the highest identification(71.25%,54.69% and 68.75%,respectively)with those of Jasmine virus A-1.According to the species demarcation criteria for the genus Crinivirus proposed by ICTV,MuCV is a new member of the genus Crinivirus.RT-PCR was performed on forty-three randomly selected mulberry samples in mulberry filed and ten were MuCV positive.The occurrence of MuCV is about 23% in the mulberry field.2.Identification of MuCV post transcriptional gene silencing suppressorsP6a,P60,P9,CP,CPm and P27 of MuCV were selected as candidate genes for identifying PTGS suppressors.The equal Agrobacterium suspension mixtures of the each PVX recombinant expression vector of the six candidate genes and green fluorescent protein(GFP)expression vector were co-infiltrated into the leaves of GFP transgenic N.benthamiana line 16 c.The results showed that the GFP fluorescence intensity of positive control and P6 a remained strong at 5 days post infiltration(dpi),in contrast,the GFP intensity of negative control and the other five had almost totally vanished at 5 dpi.The equal Agrobacterium suspension mixtures of the PVX recombinant expression vector of the the P6 a mutant(m P6a)and GFP expression vector were co-infiltrated into the leaves of N.benthamiana line 16 c.The results showed that the GFP intensity of positive control and P6 a remained strong at 5 dpi,in contrast,the GFP intensity of negative control and m P6 a had almost completely disappeared at 5 dpi.The results of RT-q PCR showed that the GFP m RNA of the co-infiltrated area in the negative control and m P6 a was similar and lowest,and the positive control was highest,while P6 a was lower than the positive control and significantly higher than m P6 a and the negative control.Therefore,P6 a is a PTGS suppressor of MuCV and acts a PTGS suppressor at the protein level but not at the RNA level.3.Identification of MuCV pathogenicity determinantsThe six candidate genes of MuCV were expressed in N.benthamiana by PVX vector.Only P6 a expression resulted in a systematically severe leaf-curling and plant-stunting symptoms in N.benthamiana plants,while the other five showed mild mottle symptoms similar to the negative control.Semi-quantitative RT-PCR analysis revealed that P6 a gene expression is responsible for these symptoms,implying that the P6 a encoded by MuCV acts as a pathogenicity determinant in N.benthamiana.We discovered a new crinivirus in the mulberry tree in this study,and it is also the first crinivirus to infect woody plants.P6 a is both a PTGS suppressor and a pathogenicity determinant.These findings have significant reference and application value for establishing specific molecular detection methods to minimize virus propagation and harm,laying the foundation for further study into the pathogenic mechanism of this virus.