Identification Of Proteins That Interact With The RNAi Suppressor HC-pro In Tobacco(Nicotiana Tabacum L.)

Potyviral helper-component proteinase (HC-pro)of PVY is a potent RNAi suppressor. To investigate the molecular mechanism in which the HC-pro suppresses RNAi, we used the HC-pro as bait to screen for interacting proteins from a tobacco cDNA library in a yeast two- hybrid system. The bait plasmid was constructed with the full length PVY HC-pro fused with the pGBKT7 DNA-binding domain (pGBKT7-HC-pro, Trp+) ,and then was transferred into the yeast strains Y187. The tobacco leaf cDNA library fused to the GAL4 activation domain ( pGADT7-Rec, Leu+) was in the yeast strain AH109. Let the library host strain AH109 mating with the bait strain Y187, the mating mixture was spread directly on the SD/-Ade/-His/-Leu/-Trp selection medium. In the screen, there were 136 positive clones for the a-X- Galactosidase activity. 120 cDNA plasmids from the 136 clones were sequenced. Twenty-one proteins were enconded by the 120 cDNA plasmids. The 21 purified cDNA plasmids were retransformed into the yeast strain AH109 together with the bait plasmid pBGKT7-HC-pro to test the a-X- Galactosidase activity and interaction specificity. Twenty cDNA plasmids displayed specific and significant interaction with PVY HC-pro. Two of the 20 cDNA plasmid show stronger interaction and were designated SZ1 and SZ2 respectively.The full length cDNA of SZ1 and SZ2 were cloned with 5'RACE and RT-PCR. SZ1 encods a multi zinc-finger protein with 8 CCHC zing-finger motifs, a RS motif at N end and a RGG box between the seventh and eighth finger. A isomer of SZ1 was found with six amino acids lost (16-21) and two amino acids changed (7: S to G; 35: D to H) near the N end- A beta subunit of 20S proteasome was encoded by SZ2.SZ1 and SZ2 were cloned on the pESC-Leu vector with HC-pro respectively for co-immunoprecipitation in yeast cells. HC-pro could be pulled down by SZ1 and SZ2 respectively.The results showed that SZ1 and SZ2 can interact with HC-pro respectively.To obtain proteins of SZ1 and SZ2, SZ1 was cloned on the pMAL-c2x vecror and SZ2 was cloned on the pET30a vector.The recombinant plasmids were transformed into Exoli expression strain TB1 and BL21 respectively. The fusion proteins of SZ1 with MBP and SZ2 with 6xHis can be expressed after induced with 1PTG The native fusion proteins of SZ1 and SZ2 have been purified.The relation between the SZ1/SZ2 and RNAi is under studied.