Plant virus-induced diseases cause significant losses to agricultural crop production worldwide.Reverse genetics systems of plant viruses allow gene manipulation on viral genomes,which greatly facilitates studies of v...Plant virus-induced diseases cause significant losses to agricultural crop production worldwide.Reverse genetics systems of plant viruses allow gene manipulation on viral genomes,which greatly facilitates studies of viral pathogenesis and interactions with host organisms.In addition,viral infectious cDNA clones have been modified as versatile recombinant vectors for virus-mediated protein overexpression,virus-induced gene silencing,and gene editing.Since genome RNAs of plant positive-strand RNA viruses are directly translatable,recovery of these viruses has been achieved more than three decades ago by simply expressing viral genome RNA or viral genome-derived in vitro synthesized transcripts in planta.In contrast,genomes of plant negative-strand RNA(NSR)viruses are complementary to their mRNAs and cannot be translated directly.Therefore,rescue of infectious plant NSR viruses from cDNA clones strictly requires the core replication proteins together with their genome RNAs which can assemble into nucleocapsid(NC)complexes as minimal infectious units.However,it is a major challenge to deliver multiple essential components in single cells and to assemble the NC complexes in vivo.Major breakthroughs in reverse genetics systems of plant non-segmented and segmented NSR viruses were just achieved in recent 5 years through various strategies,such as agroinfiltration,minireplicon systems,insect transmission and airbrush inoculation assays.In this review,we summarized critical steps toward developing reverse genetics systems for recovery of several plant NSR viruses in plants and insects.We also highlighted important applications of these reverse genetics of NSR viruses in viral gene function analyses,investigation of virus-insect-plant interactions,and genomic studies of insect vectors and host plants.展开更多
Strawberry vein banding virus(SVBV)is a double-stranded DNA virus.In our previous studies,we generated an infectious clone of SVBV,pSVBV,which causes light-green vein banding symptoms along the leaf veins in strawberr...Strawberry vein banding virus(SVBV)is a double-stranded DNA virus.In our previous studies,we generated an infectious clone of SVBV,pSVBV,which causes light-green vein banding symptoms along the leaf veins in strawberry plants(Fragaria vesca).In this study,we constructed pSVBV-P1-MCS and pSVBV-P4-MCS,two recombinant virus vectors containing a multiple cloning site(MCS)downstream of the SVBV-encoded movement protein gene(P1)and coat protein gene(P4),respectively.At 35 days post-inoculation,the two SVBV-based vectors could produce light-green vein banding symptoms on the systemic leaves of strawberry plants,indicating that they could successfully cause infection.Furthermore,the infectivity rates of the recombinant virus vectors pSVBV-P1-MCS and pSVBV-P4-MCS were similar to that of the wild-type infectious clone pSVBV,indicating that the insertion of MCS did not affect the infectivity of SVBV-based vectors.Additionally,we engineered SVBV as a transient overexpression vector,which could be used for the overexpression of exogenous green fluorescent protein in strawberry plants.Collectively,these SVBV-based vectors provide a new approach for the analysis of gene functions in strawberry plants.展开更多
Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions.Although bamboo has high economic value and produces much biomass quickly,gene functional rese...Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions.Although bamboo has high economic value and produces much biomass quickly,gene functional research is hindered by the low efficiency of genetic transformation in this species.We therefore explored the potential of a bamboo mosaic virus(BaMV)-mediated expression system to investigate genotype-phenotype associations.We determined that the sites between the triple gene block proteins(TGBps)and the coat protein(CP)of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species.Moreover,we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1,which resulted in the promotion and suppression of intemode elongation,respectively.In particular,this system was able to drive the expression of three 2A-linked betalain biosynthesis genes(more than 4 kb in length)to produce betalain,indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future.Since BaMV can infect multiple bamboo species,we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.展开更多
基金supported by the Natural Science Foundation of China(Grants 31872920 and 31571978 to XBW).
文摘Plant virus-induced diseases cause significant losses to agricultural crop production worldwide.Reverse genetics systems of plant viruses allow gene manipulation on viral genomes,which greatly facilitates studies of viral pathogenesis and interactions with host organisms.In addition,viral infectious cDNA clones have been modified as versatile recombinant vectors for virus-mediated protein overexpression,virus-induced gene silencing,and gene editing.Since genome RNAs of plant positive-strand RNA viruses are directly translatable,recovery of these viruses has been achieved more than three decades ago by simply expressing viral genome RNA or viral genome-derived in vitro synthesized transcripts in planta.In contrast,genomes of plant negative-strand RNA(NSR)viruses are complementary to their mRNAs and cannot be translated directly.Therefore,rescue of infectious plant NSR viruses from cDNA clones strictly requires the core replication proteins together with their genome RNAs which can assemble into nucleocapsid(NC)complexes as minimal infectious units.However,it is a major challenge to deliver multiple essential components in single cells and to assemble the NC complexes in vivo.Major breakthroughs in reverse genetics systems of plant non-segmented and segmented NSR viruses were just achieved in recent 5 years through various strategies,such as agroinfiltration,minireplicon systems,insect transmission and airbrush inoculation assays.In this review,we summarized critical steps toward developing reverse genetics systems for recovery of several plant NSR viruses in plants and insects.We also highlighted important applications of these reverse genetics of NSR viruses in viral gene function analyses,investigation of virus-insect-plant interactions,and genomic studies of insect vectors and host plants.
基金supported by grants from the National Natural Science Foundation of China(No.32072386 and 31801700)the Key Research and Development Project of Anhui Province(202004a06020013)the Postdoctoral Science Foundation of Anhui Province(No.2019B360).
文摘Strawberry vein banding virus(SVBV)is a double-stranded DNA virus.In our previous studies,we generated an infectious clone of SVBV,pSVBV,which causes light-green vein banding symptoms along the leaf veins in strawberry plants(Fragaria vesca).In this study,we constructed pSVBV-P1-MCS and pSVBV-P4-MCS,two recombinant virus vectors containing a multiple cloning site(MCS)downstream of the SVBV-encoded movement protein gene(P1)and coat protein gene(P4),respectively.At 35 days post-inoculation,the two SVBV-based vectors could produce light-green vein banding symptoms on the systemic leaves of strawberry plants,indicating that they could successfully cause infection.Furthermore,the infectivity rates of the recombinant virus vectors pSVBV-P1-MCS and pSVBV-P4-MCS were similar to that of the wild-type infectious clone pSVBV,indicating that the insertion of MCS did not affect the infectivity of SVBV-based vectors.Additionally,we engineered SVBV as a transient overexpression vector,which could be used for the overexpression of exogenous green fluorescent protein in strawberry plants.Collectively,these SVBV-based vectors provide a new approach for the analysis of gene functions in strawberry plants.
基金funded by the National Key Research and Development Program of China(2021YFD2200505)a grant from the National Natural Science Foundation of China(31971734)+1 种基金the Natural Science Foundation of Fujian Province(2021J02027)the Forestry Peak Discipline Construction Project of Fujian Agriculture and Forestry University(72202200205)。
文摘Bamboo is one of the fastest growing plants among monocotyledonous species and is grown extensively in subtropical regions.Although bamboo has high economic value and produces much biomass quickly,gene functional research is hindered by the low efficiency of genetic transformation in this species.We therefore explored the potential of a bamboo mosaic virus(BaMV)-mediated expression system to investigate genotype-phenotype associations.We determined that the sites between the triple gene block proteins(TGBps)and the coat protein(CP)of BaMV are the most efficient insertion sites for the expression of exogenous genes in both monopodial and sympodial bamboo species.Moreover,we validated this system by individually overexpressing the two endogenous genes ACE1 and DEC1,which resulted in the promotion and suppression of intemode elongation,respectively.In particular,this system was able to drive the expression of three 2A-linked betalain biosynthesis genes(more than 4 kb in length)to produce betalain,indicating that it has high cargo capacity and may provide the prerequisite basis for the development of a DNA-free bamboo genome editing platform in the future.Since BaMV can infect multiple bamboo species,we anticipate that the system described in this study will greatly contribute to gene function research and further promote the molecular breeding of bamboo.
