Studies On The Influence Of ZmCYP450 On The Infection Of Maize By Sugarcane Mosaic Virus

Sugarcan mosaic virus(SCMV)is a member of genus Potyvirus,family Potyviridae,which is the main pathogen affecting maize(Zea mays L.)production in China.The pipo(Pretty Interesting Potyviridae ORF)embedded within the P3 cistron of members of Potyviridae encode a transframe protein P3N-PIPO which is involved in virus cell-to-cell movement.In this research,a maize cDNA library in the yeast GAL4 activation domain expression vector was constructed using RNAs extracted from the leaves and stems of maize seedlings by SMART(Switching mechanism at 5' end of the RNA transcript)techinques.The cDNA sequence encoding SCMV PIPO was inserted into the vector pGBKT7 to generate the recombinant plasmid pGBK-PIPO,which had no self-activating effect in yeast strains AH 109 and Y187.The interaction was retested in yeast and a-galactosidase activity analysis,and 51 candidate clones displayed interaction with PIPO in yeast.The sequences of 23 positive clones were obtained and analyzed.These cDNAs can potentially encode twenty-three proteins and their possible functions were analyzed by BLAST.Among the 23 positive clones selected,four clones share the identical sequence of 723 bp containing a single open reading frame(ORF),encoding a predicted protein of 212 amino acids,and 5' terminal and 3'-untranslated regions.Sequence analyses showed that the predicted protein was identical to a CYP450 protein of unknown function in deduced amino acid sequence(GenBank accession number:BT034973.1)and only three nucleotides difference exitsted in the ORF,thus this protein was refered to as ZmCYP450 and selected for further studies.Bimolecular fluorescence complementation(BiFC)assay showed that ZmCYP450 could interact with SCMV PIPO in planta.In order to identify the specific region of ZmCYP450 necessary for the interaction,the cDNAs encoding the N-terminal half(1-100aa)and the C-terminal half(101-212aa)of CYP450 were inserted in frame into the GAL4 activation domain vector pGADT7,respectively.The results showed that the region of ZmCYP450 interacting with the SCMV PIPO located within the C-terminal half.To investigate whether ZmCYP450 interacted with SCMV P3 and P3N-PIPO,the cDNA fragments containing the full-length ORF encoding either P3 or P3N-PIPO from SCMV-BJ were inserted in frame into the vector pGBKT7 to generate pGBK-P3 and pGBK-P3N-PIPO,respectively.The results showed that ZmCYP450 and P3N-PIPO could interact in both yeast and the leaf epidermis of Nicotiana benthamiana,but SCMV P3 could not interact with ZmCYP450 in yeast.To analyze the expression level of the ZmCYP450 gene,leaf tissues from one-week-old plants of maize inbred line(Zheng 58)(mock-and SCMV-inoculated)were harvested at 2d,6d,and 12d postinoculation(dpi),respectively.Real-time reverse transcription-ploymerase chain reaction(RT-PCR)analysis showed that the ZmCYP450 mRNA levels in the second upper un-inoculated leaves of SCMV-infected leaves was increased significantly,and Western blot showed that SCMV infection up-regulated ZmCYP450 expression of similar level.A Brome mosaic virus(BMV)infectious clone,pC-BMVA/G,which was used as a vector for virus-induced gene silencing(VIGS)in monocot plants,was employed to transiently silence ZmCYP450 in maize(cv.Va35).The second upper non-inoculated leaf of the ZmCYP450-silenced plants and control maize plants were challenge-inoculated with SCMV,and the results showed that the second upper un-inoculated leaves of the ZmCYP450-silenced plants developed more severe systemic mosaic symptoms than those of the control plants at 10dpi.Real-time RT-PCR and Western blot showed that the levels of SCMV RNA and SCMV CP in the second upper un-inoculated leaves of ZmCYP450-silenced plants were higher than in the ZmCYP450-unsilenced control plants.These results suggest that silencing of the ZmCYP450 increased the accumulation of SCMV in the plants.Taken together,ZmCYP450 is likely involved in the defense of maize against infection by SCMV.