MULTIPLE TOSPOVIRUS RESISTANCE BY iRNA IN PLANTS

DEBAT, H. J.; GRABIELE, M.; LOPEZ LAMBERTINI P.; DUCASSE, D. A

Tucumán

Congreso; 45th Annual Meeting Argentine Society for Biochemistry and Molecular Biology (SAIB); 2009

SAIB

Tospoviruses are among the ten most detrimental plant viruses in the world causing severe economic losses in a wide range of vegetables and ornamental crops. In Argentina, the three main species of this genus are TSWV, GRSV and TCSV, affecting mainly lettuce, tomato, potato, pepper and several ornamental plants. Here we present a RNA interference resistance strategy based in the generation of double stranded RNA with sequence identity to a highly conserved region of the nucleocapsid gene of TSWV, GRSV and TCSV. In order to induce the host iRNA pathway to target this viral gene a 173bp region was obtained by multiple alignment of the species, amplified, cloned and inserted as an inverted repeat flanking an intron in a plant expression vector. Nicotiana benthamiana plants were agroinoculated with this construction and challenged against each of the Tospovirus species in transient expression experiments. At 35 days post inoculation 100% of TSWV, 70% of GRSV and 60% of TCSV plants displayed a symptomless phenotype and no virus presence was detected by DAS-ELISA. The approach presented here, based in a hairpin RNA construct targeting a short and highly conserved viral sequence, combines high specificity, a reduced off-target effect, does not relay in viral protein expression, and results in a high frequency broad range virus immunity to three tospoviruses.plants were agroinoculated with this construction and challenged against each of the Tospovirus species in transient expression experiments. At 35 days post inoculation 100% of TSWV, 70% of GRSV and 60% of TCSV plants displayed a symptomless phenotype and no virus presence was detected by DAS-ELISA. The approach presented here, based in a hairpin RNA construct targeting a short and highly conserved viral sequence, combines high specificity, a reduced off-target effect, does not relay in viral protein expression, and results in a high frequency broad range virus immunity to three tospoviruses.