Screening And Identification Of Proteins Interacting With Citrus Yellow Vein Clearing Virus In Eureka Lemon

Citrus yellow vein clearing disease(CYVCD)is a novel viral disease first reported on Pakistani lemons in 1988.CYVCD has been widely distributed in almost all citrus growing provinces in China,and it is considered to be one of the most serious disease in lemon production.CYVCD is caused by Citrus yellow vein clearing virus(CYVCV).Symptoms in affected lemon and other sensitive species consist of strong yellow vein clearing,leaf distortion and water soaking of the veins on the abaxial side of the leaf.CYVCV is a single-stranded positive-sense RNA virus,and it is a member of the genus Mandarivirus.CYVCV genome is around 7.5 kb,contains 6 open reading frames(ORFs),and encodes REP,TGB,CP,and P23 protein.At present,the research on CYVCV is mostly limited to detection and identification,and evolutionary analysis.Few research was forcus on the interaction between CYVCV and host plants,and the pathogenic mechanism is still unknown.In this study,the leaves of Eureka lemon seedlings were used as materials to construct a cDNA library,and bait vectors containing CYVCV TGB,CP and p23genes,respectively were obtained.Eureka lemon proteins which may potentialy interacts with TGB,CP and p23 protein of CYVCV were screened from the cDNA library by using yeast two-hybrid technology.These potential interacting proteins were analyzed by bioinformatics,and corotating validation.This study provided important data to the host response to CYVCV.It is useful for further study the interaction mechanism between CYVCV and citrus.The main results are as follows:1.Gateway technology was used to construct a nuclear cDNA library of Eureka lemon.The titer of the constructed library was 4.0×10~6 CFU/mL,the library capacity was 1.6×10~7CFU,the recombination rate was 100%,and the inserted fragments were all above 1 kb.It shows that the constructed nuclear cDNA library meets the requirements of high-quality libraries and can meet the needs of target protein screening.2.The pGBKT7-TGB,pGBKT7-CP and pGBKT7-P23 were constructed and showed that the three bait vectors had no toxicity and no self-activation,and could be applied to the screening of Eureka lemon cDNA library.3.Using CYVCV TGB as bait,36 potential interaction proteins were screened from Eureka lemon cDNA library.These 36 proteins were divided into 13 categories,such as oxidation-reduction process,carbohydrate metabolic process,hydrogen peroxide catabolic process,cellular oxidant detoxification. These proteins were also grouped 12 categories,such as translation factor activity,protein binding,ligase activity,catalase activity.Among the potential interaction proteins screened by pGBKT7-TGB,15 were selected for co-transformation verification,including 12 interacting proteins,3 non interacting proteins,these potential interacting proteins were new TGB-4, new TGB-5,new TGB-9,new TGB-17,new TGB-14,new TGB-24-2,new TGB-1, new TGB-6,TGB-2,TGB-3,TGB-15,TGB-20.4.Using CYVCV CP as bait,31 potential interaction proteins were screened from Eureka lemon cDNA library.These 31 interacting proteins were divided into 20 categories,such as response to light stimulus,protein-chromophore linkage, photosynthesis,light harvesting in photosystem I.These proteins were also grouped into 11 categories,such as metal ion binding,protein binding, chlorophyll binding,oxidoreductase activity.Among the potential interaction proteins screened by pGBKT7-CP,15 were selected for verification,including 5 interacting proteins,10 non interacting proteins,these potential interacting proteins were new CP-1,new CP-15,new CP-22,new CP-20,new CP-8;5.Using CYVCV P23 as bait,33 potential interaction proteins were screened from Eureka lemon cDNA library.These 33 interacting proteins were divided into 15 categories,such as oxidation-reduction process,protein ubiquitination, cellularoxidantdetoxification,SCF-dependentproteasomal ubiquitin-dependent protein catabolic process.These proteins were also grouped ionto 13 categories,such as ubiquitin protein ligase activity, chlorophyll binding,cullin family protein binding,catalase activity.Among the potential interaction proteins screened by pGBKT7-P23,15 were selected for verification,including 9 interacting proteins,6 non interacting proteins,these potential interacting proteins were new P23-10,new P23-11,new P23-4-1, new P23-17,new P23-22,new P23-20,new P23-24,P23-2,new P23-5.