The Mechanism Of The Eukaryotic Translation Initiation Factor 4E Family Genes-Mediated Recessive Resistance In Watermelon

The genus Potyvirus is the largest genus of plant-infecting RNA viruses.Viruses in this genus can infect a wide range of grain crops and cash crops,severely affecting crop yield and quality and causing significant economic losses.The plant genes that are essential for virus infection called susceptibility genes,and mutations in susceptibility genes can confer recessive resistance to viruses.The selective pressure of recessive resistance on virus populations can also lead to the co-evolution of mutants that can break host resistance.In this study,we identified a mutation site in the eukaryotic translation initiation factor 4E（eIF4E）family genes that is resistant to potyviruses,elucidated the molecular mechanism of naturally-occurring viral mutants breaking resistance to the eIF4E family resistance alleles,and further identified a new target site for potyvirus resistance.The main results of the study are as follows:1.The naturally occurring amino acid substitution D71G in eIF4E of resistant watermelon accesions confers resistance to multiple potyviruses in watermelon.The eIF4E family proteins of watermelon include eIF4E,eIF（iso）4E and n CBP.The VPg of papaya ring spot virus watermelon strain（PRSV-W）,zucchini yellow mosaic virus（ZYMV）or watermelon mosaic virus（WMV）individually interacts with watermelon eIF4E.Multiple amino acid sequence analysis of eIF4E from multiple potyvirus-resistant watermelon accesions showed that many of the potyvirus-resistant watermelon accesions had the amino acid mutation D71G in eIF4E,which break the interactions between watermelon eIF4E and PRSV VPg,ZYMV VPg,or WMV VPg.This suggests that the resistance of most potyvirus-resistant watermelon accesions is highly correlated with amino acid mutations in eIF4E.Overexpression of watermelon eIF4E wild type(CleIF4E^WT)and watermelon eIF4E mutant(CleIF4E^D71G)in eIF4E^D71G-carrying potyvirus-resistant watermelon accesion PI244019 and PI 485583,respectively,demonstrated that overexpression of CleIF4E^WTcould break the resistance of PI 244019 and PI 485583 to PRSV and ZYMV,while overexpression of CleIF4E^D71Gcould not,suggesting that the amino acid mutation D71G in watermelon eIF4E could confer resistance to PRSV and WMV in watermelon.2.Mutation of D to G in the conserved WFDN motif of the cap-binding pocket of eIF4E family genes confers resistance to multiple potyviruses.The naturally occurring amino acid substitution D71G in resistant watermelon accesions is located in the WFDN motif of the eIF4E cap-binding pocket.Multiple sequence alignment of amino acid sequences of the eIF4E family genes from multiple plants showed that the WFDN motif in the cap-binding pocket of eIF4E and eIF（iso）4E are conserved in plants.Amino acid mutation D55G in the WFDN motif of tobacco eIF4E cap-binding pocket could confer resistance to potato virus Y（PVY）in tobacco by disruptting the interaction with PVY VPg.The mutations of amino acid D to G in WFDN motif of eIF（iso）4E cap-binding pocket in arabidopsis,tobacco,and maize respectively break the interactions between eIF（iso）4E and turnip mosaic virus（Tu MV）VPg,tobacco vein banding mosaic virus（TVBMV）VPg,or sugarcane mosaic virus（SCMV）VPg.Overexpression of arabidopsis eIF（iso）4E wild type(AteIF（iso）4E^WT)and arabidopsis eIF（iso）4E mutant(AteIF（iso）4E^D37G)in eIF（iso）4E knockout mutants,respectively,and the results showed that overexpression of AteIF（iso）4E^D37Gconferred resistance to Tu MV which suggests that the amino acid mutation D37G in eIF（iso）4E confers resistance to Tu MV in arabidopsis.3.PRSV VPg carrying the naturally occurring amino acid substitution K105Q or E108G in theα1-α2 domain can break the resistance to PRSV in watermelon accession that carrying the eIF4E resistance allele by interacting with eIF（iso）4E.Theα1-α2domain of PRSV VPg is the key domain for binding eIF4E and also the most variable domain of PRSV VPg.Analysis of the VPg mutation sites of potyviruses that break eIF4E family gene-mediated recessive resistance showed that most of the mutation sites were located in theα1-α2 domain of VPg.Multiple sequence analysis of VPg amino acid sequences of multiple PRSV-W isolates showed that mutants of PRSV VPg with the naturally occurring amino acid mutations K105Q and E108G in theα1-α2 domain could break the resistance to PRSV in watermelon accesion PI 244019 carrying the eIF4E resistance allele by interacting with eIF（iso）4E.4.Mutations of the DNQS motif to GAAA in the watermelon eIF（iso）4E cap-binding pocket break the interaction between eIF（iso）4E and VPg ^K105Qor VPg ^E108G.The amino acid analysis of eIF（iso）4E from multiple watermelon accesions showed that the two naturally occurring amino acid substitutions A48T and L58P in the cap-binding pocket could not break the interactions between eIF（iso）4E and VPg ^K105Gor VPg ^E108G.In contrast,mutation of the DNQS motif to GAAA in the eIF（iso）4E cap-binding pocket of watermelon could break the interactions of eIF（iso）4E with VPg ^K105Qor VPg ^E108G.Thus,DNQS motif can be used as a target for breeding watermelon plants resistant to resistance-breaking PRSV isolates.In conclusion,this study identified that the mutation of D to G in the conserved WFDN motif of cap-binding pocket of eIF4E or eIF（iso）4E breaks the interaction with VPg and thereby confers resistance to multiple potyviruses.The naturally-occurring mutations in theα1-α2 domain of PRSV VPg break the resistance of eIF4E resistance allele by interacting with eIF（iso）4E,and further identified a new resistant target in eIF（iso）4E.This study proposes a work model for molecular mechanism of the arms race of coevolution of the eIF4E family genes in watermelon and PRSV,and provides basis and targets for improving the persistence of crop resistance to potyviruses.