Analysis Of Interaction Mechanism Of The EIf(iso)4E Genes To TuMV VPg In Brassica Rapa L.

The virus disease is one of the three main diseases damaging Chineses cabbage production.Turnip mosaic virus,TuMV,belongs to the genus Potyvirus（type species Potato virus Y）in the family Potyviridae and TuMV is the only potyvirus known to infect brassica crops.To date,there have mapped and cloned about 20 resistance genes to TuMV in brassica crops,and further revealing the resistance mechanism to TuMV would be helpful to improve the germplasm resources in B.rapa.Meanwhile,it may have the scientific guidance and application value for genetic improvement to TuMV in brassica crops.Eukaryotic translation initiation factors were key resistant genes to TuMV in Brassica rapa.In this study,the different copies of eIF4E/eIF（iso）4E genes were cloned from eight B.rapa lines,analyzing the interaction relationship between eIF4E/eIF（iso）4E proteins and TuMV isolates,confirming the key amino acids in the interaction,elucidating the influence of key amino acids on the 3D structure of eIF（iso）4E proteins,then constructing the interaction model between eIF4E/eIF（iso）4E and TuMV isolates.Five results were obtained as follows:1.The different copies of eIF4E/eIF（iso）4E genes were cloned from eight B.rapa highly inbred lines,but only eIF4E.a,eIF4E.c,eIF（iso）4E.a,and eIF（iso）4E.c were obtained.After aligning the eIF4E/eIF（iso）4E protein sequences,it was obvious that there were more variations among eIF4E protein sequences in different Chinese cabbage lines,but there were lesser variations among eIF（iso）4E protein sequences.2.Through yeast two-hybrid system（Y2H）and bimolecular fluorescence complementation（BiFC）analysis,the five TuMV isolates（C4,CDN1,UK1,CHN2,CHN3）all could not interact with the eIF4E proteins,and could interact with eIF（iso）4E proteins.The TuMV C4/CHN2/CHN3 VPgs could interact with B.rapa eIF（iso）4E.a proteins,not with eIF（iso）4E.c proteins,however,the TuMV UK1/CDN1VPgs could interact with B.rapa eIF（iso）4E.c proteins,not with eIF（iso）4E.a proteins.The TuMV UK1isolate belongs to pathotype 1,and TuMV C4/CHN2/CHN3 isolates belong to pathotype 3,and TuMV CDN1 isolate belongs to pathotype 4.The TuMV C4/CHN2/CHN3 isolates belong to the same pathotype and the interaction relationship with B.rapa eIF（iso）4E proteins were no difference,which may indicate that the same pathotype TuMV isolates would interact with the same B.rapa eIF（iso）4E proteins.3.Five site-directed mutagenesis between TuMV C4 VPg and TuMV CDN1 VPg proteins,five site-directed mutagenesis between TuMV UK1 VPg and TuMV CHN2 VPg proteins,and four site-directed mutagenesis between BraA.eIF（iso）4E.c-1 and BraA.eIF（iso）4E.c-2 proteins were obtained by splicing by overlap extension PCR.Using yeast two-hybrid system（Y2H）and bimolecular fluorescence complementation（BiFC）,the I₅₂L and L₁₈₆F amino acids in TuMV VPg and the L₃₆F amino acid in B.rapa BraA.eIF（iso）4E.c protein would play a key role in the interaction process between B.rapa eIF（iso）4E proteins and TuMV VPgs.4.Using the Phyre2 software constructing the three-dimensional（3D）structural model of the BraA.eIF（iso）4E.c-1 protein in B.rapa,the structure of the BraA.eIF（iso）4E.c protein includes eightβ-strands,threeα-helices,and three extended loops.There was a large cavity at its cap-binding site that undergoes conformational changes in the cap-binding loops.There were four different amino acids(F₃₆L,A₅₂V,I₈₀T,P₁₅₀Q)between BraA.eIF（iso）4E.c-1 and BraA.eIF（iso）4E.c-2 proteins in B.rapa,and the 36^th amino acid was located in the cap-free structure of BraA.eIF（iso）4E protein,thereby suggesting that it played an essential role in the allosteric regulation of the BraA.eIF（iso）4E protein.The 36^th amino acid in BraA.eIF（iso）4E.c changed from Phe to Leu,and Phe is an aromatic amino acid that contains a benzene ring.However,Leu is an aliphatic amino acid that is linear and does not contain a benzene ring.Thus,the two amino acid substitutions may influence the biochemical functions and the structure of the protein.Compared with the 36^th amino acid,the 52^th,80^th,and 150^th amino acids were not conserved in the protein,and had little influence on the protein structure.5.Based on the interaction relationship and the key amino acids between different B.rapa eIF（iso）4E proteins and various TuMV isolates,the interaction model was constructed.The eIF（iso）4E proteins of B.rapa could interact with TuMV VPgs,and facilitate TuMV expanding propagation and transportation in the host plants.When some amino acid changed(such as I₅₂L)in TuMV VPg,resulting in the 3D structure of TuMV VPg protein changing,which would influence the interaction with B.rapa eIF（iso）4E.TuMV RNA could not duplicate in plant,so the plant was resistant to the TuMV isolate.When some amino acid changed(such as L₃₆F)in BraA.eIF（iso）4E.c,resulting in the 3D structure of BraA.eIF（iso）4E protein changing,which would influence the interaction with B.rapa eIF（iso）4E,and the plant was resistant to the TuMV isolate.