Tomato spotted wilt virus (TSWV) and related thrips-borne orthotospoviruses are a threat to food and ornamental crops. Orthotospoviruses have the capacity for rapid genetic change by genome segment reassortment and mutation. Genetic resistance is one of the most effective strategies for managing orthotospoviruses, but there are multiple examples of resistance gene breakdown. Our goal was to develop effective multigenic, broad-spectrum resistance to TSWV and other orthotospoviruses. The most conserved sequences for each open reading frame (ORF) of the TSWV genome were identified, and comparison with other orthotospoviruses revealed sequence conservation within virus clades; some overlapped with domains with well-documented biological functions. We made six hairpin constructs, each of which incorporated sequences matching portions of all five ORFs. Tomato plants expressing the hairpin transgene were challenged with TSWV by thrips and leaf-rub inoculation, and four constructs provided strong protection against TSWV in foliage and fruit. To determine if the hairpin constructs provided protection against other emerging orthotospoviruses, we challenged the plants with tomato chlorotic spot virus and resistance-breaking TSWV and found that the same constructs also provided resistance to these related viruses. Antiviral hairpin constructs are an effective way to protect plants from multiple orthotospoviruses and are an important strategy in the fight against resistance-breaking TSWV and emerging viruses. Targeting of all five viral ORFs is expected to increase the durability of resistance, and combining them with other resistance genes could further extend the utility of this disease control strategy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Keywords: Sw-5 resistance-breaking; hairpin constructs; orthotospovirus; tomato chlorotic spot virus; tomato spotted wilt virus; transgenic resistance.