Construction of full-length infectious cDNA clones of Apple stem grooving virus using Gibson Assembly method.

Apple stem grooving virus (ASGV) belongs to the genus Capillovirus within the family Betaflexiviridae. In this work, we described the construction of full-length infectious cDNA clones of ASGV isolate jilin-shaguo (JL-SG) using the Gibson Assembly approach (New England BioLabs). The isolate was previously detected in a Chinese pear-leaf crab apple (Malus asiatica Nakai.) in Baicheng, Jilin province, China. Two full-length cDNA clones of ASGV JL-SG were obtained, and they are identical to each other in sequence. The full-length cDNA clone was infectious on Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis 37B via agroinfiltration. Through sap inoculation, the infection was additionally spread to C. amaranticolor. N. benthamiana could not be infected, neither through agroinfiltration nor sap inoculation. In infected herbaceous plants, typical ASGV particles with morphology of flexuous filaments were observed by transmission electron microscope (TEM). Moreover, seeds of infected N. glutinosa and N. occidentalis 37B were collected and germinated, the seedlings were ASGV-free in RT-PCR test, suggesting ASGV JL-SG is not seed-transmissible in the tested Nicotiana species. In addition, the cDNA clone was agroinfiltrated into seedlings of Malus pumila cv. Fuji. The infection was symptomless, and can be spread to C. quinoa via sap inoculation, causing typical symptoms. ASGV JL-SG was also detected by RT-PCR in inoculated the infected Fuji plants, however, no virion was observed by TEM.