Construction Of Cucumber,Mosaic Virus-induced Gene Silencing Vector For Maize Functional Genomics Research

Maize(Zea mays L.)is widely used to feed humans and livestock,as an industrial crop producing alcohol,sucrose,and starch,and as a model species for genetic research.The completion of maize inbred lines "B73" and "Mo17" genome sequences and the utilization of transcriptome and proteomic techniques provide important sequences sources for characterizing maize gene function and identified a wealth of differentially expressed genes/proteins associated with tissue development,metabolism,and response to biotic and abiotic stresses.Currently,major technologies for maize functional genomic research are mainly based on mutation or gene engineering,both are time-consuming,expensive,and not suitable for a large number of maize genes.Virus-induced gene silencing(VIGS)provides a powerful tool,which is rapid,efficient,and suitable for high throughput screening to enable plant genes functional analysis.In this study,a naturally maize-infecting Cucumber mosaic virus(CMV)strain ZMBJ-CMV was identified.The infectious clone of ZMBJ-CMV and ZMBJ-CMV VIGS vectors were constructed.Then,an efficient inoculation method for maize based on vascular puncture inoculation(VPI)was improved.The silencing efficiency of ZMBJ-CMV VIGS vector was evaluated.This ZMBJ-CMV VIGS vector induces mild symptoms,efficient and lone-time gene silencing in maize.ZMBJ-CMV VIGS vector is suitable for most maize inbred lines,including B73,Mo17,Zheng58 and other important cultivars.In a survey of maize virus disease,a plant showing mild irregular mosaic stripe symptoms on upper leaves was found.Through biological assay,RT-PCR detection,and performing Koch’s postulate,the pathogen was identified to be CMV and was named CMV Zea mays Beijing(ZMBJ-CMV).Sequence alignment of the 5’ untranslational region and phylogenetic analysis of CP nucleotide sequences with other 18 CMV strains revealed that ZMBJ-CMV belongs to CMV subgroup IB.The infectious clone of ZMBJ-CMV under double 35S promoters was constructed and it could systemically infect maize inbred lines B73 and Zheng58 in a short time(12～16 days)and induced mild mosaic symptoms in maize leaves.A serial ZMBJ-CMV VIGS vectors with whole 2b or truncated 2b(2bN94,2bN81,and 2bN61)were made.Comparison of silencing efficiency of different ZMBJ-CMV VIGS vectors showed the vector containing 2bN81,(ZMBJ-CMV-2bN81,)induced the best silencing(silenced 83%).A technical system for ZMBJ-CMV VIGS vector to efficiently silence maize genes was established.Compared with mechanical inoculation,Vascular Puncture Inoculation could produce higher infection of ZMBJ-CMV in maize.Inserts with length of 100-300 bp produced the most complete transcript and visual silencing phenotypes.The optimal growth condition of maize for ZMBJ-CMV induced silencing was 16 h light at 20℃/8 h dark at 18℃ cycles.With ZMBJ-CMV VIGS vector,an IPP and DMAPP synthase gene,ZmIspH,was silenced efficiently in maize.The chlorophyll and carotenoid content was reduced significantly in ZmIspH-silenced leaves.Chloroplast ultrastructure was modified with less stacked thylakoids in the cells from the albino issue in ZmIspH-silenced leaf.The albino phenotype was maintained in leaves originally silenced for IspH expression and appeared in upper new emerging leaves for at least 60 days after inoculation.In this study,it was firstly revealed that autophagy was involved in starch degradation in monocotyledon.Autophagy-related genes,ZmATG3 and ZmATG8a were silenced with ZMBJ-CMV-2bN81 vector in maize,respectively.In ZmATG3 or ZmATG8a-silenced maize plants,excessive starch accumulation was observed after 4 h darkness treatment.This result indicated that ZmATG3 and ZmATG8a contributed to leaf starch degradation in maize.Assay of silencing of ZmIspH by ZMBJ-CMV-2bN81 on 25 important inbred lines showed that ZMBJ-CMV-2bN81 VIGS vector was suitable for 17 maize lines,including Oh43,PH207,w22,8112,Mo 17,LH196,K22,178,F42,Q319,Zheng58,Shen5005,5003,Lv28,HuangC,Huangzao4,and Q381.In summary,an efficient VIGS vector using a Cucumber mosaic virus strain that naturally infects maize,and which can therefore be used for functional genomics studies in most maize inbred lines,including B73,the reference sequenced genome was developed.The vector will promote gene mining and identification that involved in maize development,resistance to disease/insect and abio-stress.