Construction Of Sugarcane Yellow Leaf Virus Infectious Clones And Identification Of Sugarcane ScPetC Interacting With Bait Viral Protein P0

Sugarcane yellow leaf disease is a viral disease caused by Sugarcane yellow leaf virus(SCYLV)and has become one of the most serious viral diseases to sugarcane.SCYLV belongs to the Poleovirus of the family Luteoviridae and only is transmitted by aphids.The cultivation of varieties is the most effective method for preventing the prevalence of Sugarcane yellow leaf virus.In this study,the bioinformatics analysis of SCYLV-FJ isolate(Gen Bank accession NO.: GU190159.1)encoding proteins was performed.Then the infectious clone vector of SCYLV was constructed and artificially inoculated on plants(Nicotiana benthamiana,Triticum aestivum,Oryza sativa,and Zea mays).Subsequently,a SCYLV-P0 host interactor,ScPetC,was obtained.And the bioinformatics analysis of ScPetC was also performed,following by analysis of its subcellular location and expression patterns of ScPetC.We hope to help people further understanding the function of SCYLV viral protein and the mechanism of SCYLV infection.The research results are as follows:1、Bioinformatics analysis of SCYLV-encoded viral proteinsBased on the whole genome,the phylogenetic tree of SCYLV was constructed and indicated that SCYLV is a member of the Poleovirus with the closest relationship with Wheat leaf yellow virus.Bioinformatics analysis revealed that there were large differences in the number of amino acid residues(44 aa-676 aa)of SCYLV encoding 7 viral proteins.Among the seven viral proteins,P0,P2,P3,P4 and P5 have no transmembrane domains and are unstable,hydrophobic,nonsecretory proteins.P1 have transmembrane domains and is unstable,hydrophobic,secretory protein.P3 a have transmembrane domains and is stable,hydrophilic,unsecretory protein.The prediction of the secondary and three-dimensional structure indicated that,except P3 a did not contain extended chain,P0,P1,P2,P3,P4 and P5 contain α-helix,random coil structure and extended chain structure.The above results indicated that the physical and chemical properties of the seven virus proteins were different,and they played different biological functions in the process of virus infection The application of viral protein fused GFP type of infectious clone would facilitate to identify the subcellular localization of viral protein under presence of SCYLV condition,by which providing a novel research approach for studying viral protein function and viral infection mechanism.2、Construction of SCYLV infectious clone vector and subcellular localization of viral proteinsUsing yeast homologous recombination and site-specific homologous recombination method,a SCYLV wild-type(p CB301-SCYLV-wt,p CB301-SCYLV-sg),a GFP fused type(p CB301-SCYLV-GFP),and viral protein fused GFP type of infectious clone(p CB301-SCYLV-P0-GFP,p CB301-SCYLV-P1-GFP,p CB301-SCYLV-P2-GFP,p CB301-SCYLV-P3-GFP and p CB301-SCYLV-P4-GFP)were constructed and used for Agrobacterium-mediated transformation of N.benthamiana leaves Comparing the infectious clone based method and the viral protein individual method,we found that the virus proteins,except for P4,were located on cell membranes and nuclei both under the absence or presence of SCYLV.However,comparing to viral proteins under the absence of SCYLV,p CB301-SCYLV-GFP,p CB301-SCYLV-P0-GFP,p CB301-SCYLV-P1-GFP,p CB301-SCYLV-P2-GFP,p CB301-SCYLV-P3-GFP and p CB301-SCYLV-P4-GFP can not only form fluorescent signal on microtubule structure,but form special fluorescent vesicle structure in the cytoplasm under presence of SCYLV(ps:p CB301-SCYLV-P0-GFP 、 p CB301-SCYLV-P3-GFP can not form special fluorescent vesicle structure).In the process of virus infecting the host,the viral proteins do not function individually,but bind to the other viral or host proteins as a complex.It indicate that,viral protein fused GFP type of infectious clone can facilitate the comprehensive understand of the subcellular location of viral proteins.The GFP-fused infectious clones have made it possible to use the model plant system easily,such as N.benthamiana and Arabidopsis thaliana,to study the interaction between host proteins and viral proteins.It also provides a potential novel method for drawing out the process of the SCYLV intruding into host cell and its replication and proliferation in Co-immunoprecipitation(Co-IP).After the leaf-infiltration of plant seedlings in N.benthamiana,T.aestivum,O.sativa,and Z.mays,RT-PCR was used to detect the presence of SCYLV.The RT-PCR test indicated that,SCYLV could be detected in the agroinfection sites of N.benthamiana leaves,but not in its systemic leaves.Similarly,the negative detection of SCYLV was found in the agroinfection leaves of T.aestivum,O.sativa,and Z.mays.These results suggest that the SCYLV complex deriving from artificial infectious clone cannot move cell-to-cell in its non-host plant,N.benthamiana.At the same time,the infectious clone of SCYLV cannot successfully infect Gramineous plants(T.aestivum,O.sativa,and Z.mays.)by agroinfection.3、Cloning and expression analysis of sugarcane Rieske Fe/S protein precursor gene ScPetCP0,a viral silencing suppressor,plays an important role in the host infection process of SCYLV.ScPetC can bind to SCYLV P0 protein in the yeast two hybrid(Y2H)assay.Based on protein-protein interaction,the coding sequence of ScPetC gene was cloned.ScPetC is 824 bp in length,containing a 678 bp open reading frame(ORF)and encoding a226-amino acids protein in length.ScPetC belongs to the PRK13473 superfamily and has a typical Rieske domain at its C-terminus of the amino acid chain.ScPetC is a stable and hydrophilic protein with p I 8.19.Most of the secondary structural elements in ScPetC were random coil.Compared to the Pet C from the other plants,ScPetC contained one more fragment of helix in its third dimensional structure.The transient expression of YFP-fused protein in N.benthamiana leaves showed that ScPetC was located in the chloroplast,cytoplasm and cell membrane.Real-time quantitative PCR analysis showed that ScPetC was expressed constitutively in different tissues of sugarcane,and the highest level of its expression was found in leaves.When sugarcane plant was exposed to abscisic acid stress for 3 h,the expression of ScPetC was significantly up-regulated,but the expression level was then inhibited with longer treatment time.Under the stress of methyl jasmine,salicylic acid,copper chloride,cadmium chloride and sodium chloride,the expression of ScPetC was significantly down-regulated.The study on biological function,expression pattern and interaction of ScPetC with the proteins of sugarcane virus would improve the understanding of the happening of yellow leaf symptom,which caused by SCYLV.