Expression Of P4-ATPases Genes AtALA1,AtALA7 And GhALA4 Enhances Plant Resistance To Verticillium Dahliae

Verticillium wilt seriously harms the growth of plants,causing irreparable economic losses to many crops.It is one of the most devastating diseases in the world.Verticillium dahliae is the main pathogen that causes wilt.V.dahliae is a soil-borne pathogenic fungus that has strong microsclerotin viability and strong spread ability.It can spread with the roots,stems,leaves and seeds of plants,and can reproduce rapidly under appropriate conditions,causing a new round of infection.For the lack of effective resistance genes and germplasm resources,Verticillium wilt is difficult to be controlled.The previous study of our group showed that ALA7 and ALA1 of Arabidopsis,which belong to the P4-ATPases subfamily,had the ability to transport toxins into vacuoles;overexpression of AtALA7 promoted the transport of cinnamyl acetate(CIA),a lipophilic toxin produced by V.dahliae,to vacuoles and thus increased the tolerance of Arabidopsis to both CIA and V.dahliae;overexpression of AtALA1 promoted the transport of hydrophilic DON(Deoxynivalenol)produced by Fusarium graminearum/culmorum to the vacuoles and increases the tolerance of Arabidopsis to the toxin.The expression of AtALA7 and AtALA1 could reduce the damage to Arabidopsis thaliana by promoting the transport of toxins to vacuoles.The toxins secreted by V.dahliae include both hydrophobic and hydrophilic toxins.Whether the combination expression of AtALA7 and AtALA1 can further increase the resistance to the disease?Besides,the function of AtALIS1,a CDC50 humongous of Arabidopsis in the toxin resistance and the endogenous AtALA7 gene of cotton in the wilt disease control are to be investigated.Accordingly,in this study,transgenic tobacco and cotton expressing these genes were generated,and the individual or stacking role of the genes in the resistance was analyzed.The main results are as follows:1.Expression of AtALA7 or AtALA1 increases the tolerance of tobacco to CIAThere was no significant difference in growth between wild type and transgenic tobacco.Treated with CIA(450?g/m L),the leaf damage rate of AtALA7 transgenic tobacco was reduced by 66.3%compared with that of the wild type;Treated with CIA(300?g/m L),the leaf damage rate of AtALA1 transgenic tobacco decreased by 63.0%compared with wild type.AtALA7 transgenic tobacco showed higher tolerace to CIA than that of AtALA1 transgenic tobacco.2.Expression of AtALA7 or AtALA1 increases the resistance of tobacco and cotton to V.dahliae,but stacking with AtALIS1 is unable to further increaseCompared with the control(empty-vector),transient expression of AtALA7 and AtALA1 significantly increased cotton resistance to V.dahliae.The disease index of AtALA7 and AtALA1 transgenic tobacco decreased by 30.4%and 25.0%relative to the control,respectively.In contrast,the co-expression of P4-ATPase chaperone gene AtALIS1 in tobacco did not further enhance the resistance to V.dahliae.3.Expression of Cotton GhALA4 increases the tolerance of tobacco to CIA and V.dahliaeThe homologous gene,GhALA4,was obtained in Gossypium hirsutum by BLAST alignment using AtALA7 amino acid sequence.The homology of GhALA4 was 74%with AtALA7.Sequence alignment indicates that GhALA4 contains 10 transmembrane domains and the protein belongs to the P4-ATPases subfamily.Expressing the GhALA4gene in the AtALA7 mutant ala7-24,(GhALA4/ala7-24)could restore the transport of CIA to vacuoles in Arabidopsis and the tolerance to CIA,indicating that GhALA4 has similar function to AtALA7.Treated with CIA(450?g/m L),the leaf damage rate of GhALA4 transgenic tobacco was reduced by 55.8%,compared with that of the wild type.Transient expression of the gene in cotton cotyledons and inoculated the cotyledons with with V.dahliae strain V991,the disease index of the cotyledons expressing GhALA4 was reduced by 50.9%compared to that of the control.Inoculated with V.dahliae V991(5×10~8 Conidia/m L,50 m L per plant),the disease index of GhALA4 transgenic tobacco was 27.5%lower than that of the wild type.Conversely,silenced GhALA4 gene in Gossypium hirsutum,the disease index was increased by40.0%.The results indicate that GhALA4 plays an important role in CIA tolerance and V.dahliae resistance in Gossypium hirsutum.4.Gene stacking of AtALA7 and AtALA1 further increase resistance of tobacco to CIA and V.dahliaeAtALA7 and AtALA1 were stacked together through cross.Treated with CIA(550?g/m L),the leaf damage rate of two hybrid offspring were 46.9%and 51.1%lower than that of wild type and parents,respectively,indicating that stacking of AtALA7 and AtALA1 could further increase the tolerance of tobacco to CIA.Incubated with V.dahliae V991(5×10~8 Conidia/m L,50 m L per plant)three weeks later,the disease index of the hybrid was 8.8%and 3.3%lower than their parent AtALA1 and AtALA7,respectively.