Research On Pathogenic Mechanism Of Apple Chlorotic Leaf Spot Virus Movement Protein

Apple chlorotic leaf spot virus（ACLSV）belongs to the genus Trichovirus of the family Betaflexivirdae,and is an important latent virus that harms the apple industry in China and occurs widely around the world.At present,studies on ACLSV mainly focus on biology,molecular characteristics,genetic evolution,and detection methods,etc.There are fewer studies on pathogenesis,and the mechanism of interactions with hosts after ACLSV infestation of plants is still unclear.The ACLSV genome encodes a total of three open reading frames（ORFs）,of which ORF2 is expressed by the subgenome and encodes the movement protein（MP）.In addition to being involved in viral movement,the movement protein of ACLSV has been shown to be a viral suppressor of RNA silencing（VSR）.Based on previous work,this study further demonstrated that the movement protein of ACLSV is a viral symptom determinant and is involved in the formation of pathogenic symptoms.To further clarify the pathogenic mechanism of MP,a c DNA yeast library of the ACLSV laboratory host Nicotiana occidentalis was constructed.Using yeast two-hybrid technology,13 interaction proteins were screened in the c DNA library of Nicotiana occidentalis and retransformation experiments were performed to verify the interaction.The main findings were as follows.1.ACLSV MP and coat protein（CP）genes were cloned,and they were inserted into the multiple cloning sites of expression vector PVX by seamless cloning technology.The PVX-MP and PVX-CP recombinant vectors were successfully constructed and they were transformed into Agrobacterium tumefaciens strain AGL1.Agrobacterium tumefaciens carrying the plasmid was infiltrated at an OD₆₀₀ of 0.9 on leaves of laboratory host Nicotiana occidentalis.It was observed that the symptoms of PVX-MP recombinant vector infiltrated plants appeared earlier and more severe than PVX.The GAPDH gene of Nicotiana occidentalis was used as reference gene,the expression level of PVX CP gene was detected by real-time quantitative reverse transcription PCR.The results showed that the expression level of PVX CP in PVX-MP recombinant vector infiltrated plants was the highest,indicating that MP was a symptom-related protein.2.Using the library vectors p DONR222 and p GADT7,the yeast c DNA library of Nicotiana occidentalis was successfully constructed.Primary library titer and amplification library titer were higher,the result showed c DNA library of Nicotiana occidentalis has good quality that enough for subsequent experiments.3.The bait vector p GBKT7-MP was successfully constructed,and it was verified that it has no self-activation and toxicity.A total of 39 positive clones were obtained by cotransformation screening of Nicotiana occidentalis c DNA library.Using yeast two-hybrid technique,the 39 candidate positive clones were verified by retransformation experiment,and 16 positive genes were obtained.After bioinformatics analysis,13 protein sequences that could be annotated to function were obtained.