Emergence of a deletion mutant of GFP-expressing plantago asiatica mosaic virus that has overcome acibenzolar- S -methyl-induced defense response against its long-distance movement

The rapid emergence of new virus mutants that are adapted to host environments increases the threat to crop production. In a previous study, we reported that pretreatment of Nicotiana benthamiana (Nb) with a plant activator, acibenzolar- S -methyl (ASM), inhibited the infection of green fluorescent protein-expressing plantago asiatica mosaic virus (PlAMV-GFP) at two distinct stages: replication in inoculated leaves and the viral loading step for long-distance movement. However, whether the virus might mutate that will overcome host ASM-induced defenses at each of these stages is unknown. To address this question, we performed five serial passages of PlAMV-GFP in either ASM-treated or untreated Nb leaves. A virus lineage passaged in ASM-treated leaves, named the ASM lineage, had no dominant mutations and was unable to overcome ASM-mediated inhibition. The ASM lineage tended to have less genetic diversity than in the control lineage. In contrast, in a petiole of ASM-treated, inoculated leaves where viruses had accumulated after loading into the vascular tissue, we found a mutant with a 693-bp deletion in the GFP-coding region and an extension of 13 amino acids at the C-terminus of the movement protein TGBp3 that had less restricted accumulation in inoculated leaves and moved to the upper leaves faster than the wild-type PlAMV-GFP in ASM-treated plants. The emergence of this defense-evading mutant specifically from the loading stage suggests that the ASM-induced defense could impose selective pressure on viruses at the viral loading stage rather than at replication.