A Novel Approach to Engineering Tomato Spotted Wilt Virus Infectious Clones by Disarming Key Nodes in Anti-Viral Defenses.

Abstract

Tomato spotted wilt virus (TSWV) is an economically devastating pathogen that rapidly overcomes genetic resistance in major crops. Reverse genetic systems are crucial for investigating plant-virus interactions and resistance breaking mechanisms, and developing these tools for segmented ambisense RNA viruses remains a crucial challenge. Current TSWV-clones rely on extensively modified Asian isolates requiring co-delivery of multiple replication helpers and viral silencing suppressors. Streamlining these systems for regionally significant strains with minimal genetic alterations is essential. Here we developed the first infectious clone of a USA TSWV isolate (PA01). Three binary plasmids contain cDNAs for the antigenomic L and S segments, and the genomic M segment, with enhanced GFP replacing NSs on the S segment. Co-delivery of the cucumovirus 2b alone or in combination with tombusvirus P19 or begomovirus AL2, achieved a high proportion of systemically infected Nicotiana benthamiana and Capsicum annuum plants. In N. tabacum, co-delivering the Caenorhabditis elegans cell death suppressor CED-9, or using NahG transgenic plants produced 20 to 62% systemically infected plants. These data indicate that in addition to the anti-viral RNA silencing machinery, additional host defense pathways influence TSWV rescue and systemic infection from cDNA.