Interactions Between Proteins Encoded By Cucurbit Chlorotic Yellows Virus And Functional Identification Host Factors Interacting With P22

Cucumber,watermelon,melon etc.are very important economic crops in China,provide people with abundant sugars,vitamins and other nutrients,are necessities of People's Daily life.Melons caused leaf chlorosis,fruit-set rate drop after the infection of CCYV,seriously affected the quality of melons,cause huge economic losses.This experiment used yeast two hybrid studied coding proteins by CCYV of their interactions,in order to clear understand CCYV encoding protein interactions,and confirm interactions functional domains of the interactions proteins.We ues bimolecular fluorescence complementation identification interactions between CCYV P22 and host factors in vivo.Analysis host proteins function for and subcellular localization,through a point mutation and transient expression mediated agrobacterium verification gene silence suppressors function of P22.The main research contents and results as follows:CCYV coding proteins : P6,P22,P4.9,Hsp70,P6.5,P59,P9,P26,CP,CPm,used Y2 H assays.The P22 fusion was found to be autonomously activated therefore cannot be used for yeast two hybrid experiments.The other 9 of recombinant plasmid respectively two combination,a total of 81 combination,using PEG/LIAC co-transformation Y2 HGold yeast,show only the P9 and P59 have their interactions,other are no interactions between protein combinations.P59 corotating 2 days on board blue,P9 a total of five days turn blue.To determine the interacting regions of P59 and P9,the fulllength sequences of P59 and P9 were truncated in thirds to construct new recombinant AD vectors.The truncated proteins were tested for interactions with full-length P59 and P9 BD fusions.Results indicate that the middle region(nucleotides 517–1032)of P59 was necessary for self-interaction,while the three truncated versions of P9 showed no interaction with full-length P9.P22 was constructed to p SPYCE-35 S BIFC vectors,cucumber M2(Cucumis sativus SKP1-like protein 1A),M39(Cucumis sativus SKP1-like protein 1B),Rpol(Cucumis sativus ribosome-like protein),the Nicotiana benthamiana Nb SKP1(Nicotiana benthamiana SKP1)Were constructed to p SPYNE-35 S BIFC vectors.Recombinant plasmid into agrobacterium respectively,P22 and M2,M39,Nb SKP1,Rpol respectively were co-infiltration the Nicotiana benthamiana of the epidermis cells,cultivate 48-72 h then observe the fluorescence by laser confocal microscope;Results p SPYCE-35Sp22 with p SPYNE-35SM2,p SPYNE-35SM39,p SPYNE-35 S,p SPYNE-35 SNb SKP1,p SPYNE-35 Rpol were all observed fluorescence,P22 and M2,M39,Nb,Rpol have interactions.To M2,Rpol built into GFPII vectors,transformation of cucumber protoplast,laser confocal microscopy subcellular localization,the results showed that the M2,Rpol positioning in the cytoplasm.P22 will build on the p UC-SPYCE BIFC carrier,the M2,M39,Nb SKP1,Rpo built into the p UC-SPYNE BIFC carrier carrier,conversion of agrobacterium,co-infiltration the Nicotiana benthamiana,laser confocal fluorescence microscope observation,the results found in the protoplasm somatoplasm fluorescence,same P22 and M2,M39,Nb SKP1,Rpol interactions.qPCR analysis of cucumber M2,Rpol different organizations expression level,the result shows that organization express specificity.CCYV RNA1 and RNA2 respectively with M2,Rpol co-transformation cucumber protoplast,Northern blot analysis CCYVRNA1 and RNA2,expression level,the results showed CCYV RNA1 and RNA2 increase expression.P22-303 with SKP1 ?SKP1B3 homologous protein(Cucumis sativus SKP1-like protein 1B3),SKP14(Cucumis sativus SKP1-like protein14),SKP21(Cucumis sativus SKP1-like protein 21),M2-C,M2-N,M39(Cucumis sativus SKP1-like protein 1B),respectively,yeast co-transformation,the results P22-303 respectively and Cs SKP1B3 and Cs M39(Cucumis sativus SKP1-like protein 1 b)there are interactions,other all have no interactions.Through point mutations have three mutants of P22: P2253 a,P225354a,P22 Del,at the same time get P22-303 mutants by PCR: P22-303/53 a,P22-303/53a54 a,P22-303/Del.Yeast co-transformation with P22 mutants and M2,Rpol interactions,the results of P22 mutants and P22-303 interaction with Rpol,and have no interactions with M2,explain P22 protein might be have a F-box domain,P22 is similar to the F-box protein,as a SCF(Skp1-Cullin-F-box protein)complex Skpl or Skp1 similar protein bind domain.P22,P2253 a P225354a,P22 Del construct to the PGD vector,35 SGFP co-infiltration the Nicotiana benthamiana,observed under UV lamp silence suppression effect.Results found and P22 have obviously GFP fluorescence,explained P22 is a silence suppressors,and P22 mutant have no fluorescence,mutant lost silence suppressors function,again confirm P22 is likely to be a F –box protein.P22,P2253 a P225354a P22 Del construct to PVX vectors,observe the pathogenic infiltration.The results show PVXP22 pathogenic much more serious than PVX,P22 mutant pathogenic below or agree with PVX,confire P22 is a strong silence suppressors,and mutant loses its pathogenicity.Northern blot results P22 Del with PVX RNA level,again confirm P22 is a silence suppressors.