Construção e caracterização de clones infecciosos dos begomovírus Bean golden mosaic virus e Macroptilium yellow spot virus

Abstract

Bean plants (Phaseolus spp.) are of great socioeconomic importance in tropical and subtropical regions and are among the most traditional Brazilian culinary items, presenting remarkable nutritional value. The genus Begomovirus (family Geminiviridae) is among the group of most harmful pathogens that infect these crops, being considered a limiting factor in producing areas. Begomoviruses have single-stranded circular DNA and are transmitted by a complex of cryptic species of Bemisia tabaci, with noncultivated hosts acting as reservoirs of these viruses. Bean golden mosaic virus (BGMV) and Macroptilium yellow spot virus (MaYSV) are the two most economically important species infecting beans in Brazil. In this context, the present study aimed to obtain infectious clones from BGMVe MaYSV isolates using the Gibson Assembly (GA) technique, and to determine the host range of MaYSV. Clones of the DNA-A and B-DNA components of BGMV (173AL) and MaYSV (100AL) were used to apply the methodology for constructing infectious clones by GA. Aliquots of the GA reactions were used to transform Escherichia coli DH10B electrocompetent cells, binding of the viral fragments into pJL-89 binary vector was confirmed by enzymatic digestion and sequencing. Plasmid DNA from the confirmed constructs was used for transformation of Agrobacterium tumefaciens (strain GV3101), and the infectivity of the clones of each viral species was tested individually via agroinoculation in common bean 'cv. Pérola ', which were monitored for 30 days to observe the appearance of symptoms. Total DNA was extracted from leaf samples (systemically infected) collected at 15- and 30-days post agroinfiltration (dpa), and used as template for viral detection using species-specific primers. Inoculated plants with infectious clones showed symptoms of severe yellow mosaic 15 daa, whereas control plants (agroinoculated with pJL-89 without viral inserts) were asymptomatic. Both genomic DNA-A and B-DNA components of BGMV and MaYSV were detected in systemically infected leaves. GA has been shown to be a rapid and useful technique for the construction of infectious clones of plant viruses with circular DNA. For the host range experiments, among the analyzed species, viral infection was verified in the species P. vulgaris, N. benthamiana and S. lycopersicum. Symptoms of mosaic and stunting were observed only in common bean.