Construcción de un clon sintético de un carlavirus en papaya carica papaya

Abstract

CONDITION FOR PUBLICATION OF PROJECT. Several viruses affecting papaya have been reported, some with high economic impact. Through high-throughput sequencing (HTS) of latex exudate from papaya plants, a viral RNA with high homology to members of the genus Carlavirus was detected, identified as Papaya defective virus 1 (PapDfV1), with 96 % identity at the nucleotide level with Zhejiang betaflexivirus 2 (ZhBV2). Genomic analysis of PapDfV1 revealed an extensive deletion in genes related to viral movement and encapsidation. This study addresses for the first time the construction of a synthetic clone of PapDfV1 to study its infectivity and symptomatology. Synthetic fragments of PapDfV1 and ZhBV2, with and without ribozyme, were designed and assembled using IIS-type restriction enzymes. The constructs were transformed into E. coli Top 10 and subsequently into Agrobacterium tumefaciens (strains GV2260 and AGL1). Host plants (common bean, soybean, Nicotiana benthamiana, N. occidentalis and papaya) were inoculated by two agroinoculation methods. Infectivity was evaluated by reverse-transcription (RT)-PCR and transmission electron microscopy (TEM). Addition of ribozyme enhanced ZhBV2 infection in soybean and bean. PapDfV1 was not infective on any of the inoculated plants under any treatment. The results suggest that PapDfV1 may require helper viruses or host-specific factors for replication and systemic movement. Keywords: Papaya defective virus 1, Zhejiang betaflexivirus 2, RT-PCR, agroinoculation