Evidence That the Nonstructural Protein ofTomato spotted wilt virusIs the Avirulence Determinant in the Interaction with Resistant Pepper Carrying theTswGene

Abstract

All known pepper cultivars resistant to Tomato spotted wilt virus (TSWV) possess a single dominant resistance gene, Tsw. Recently, naturally occurring resistance-breaking (RB) TSWV strains have been identified, causing major concerns. We used a collection of such strains to identify the specific genetic determinant that allows the virus to overcome the Tsw gene in Capsicum spp. A reverse genetic approach is still not feasible for TSWV; therefore, we analyzed reassortants between wild-type (WT) and RB strains. Our results confirmed that the S RNA, which encodes both the nucleocapsid protein (N) and a nonstructural protein (NSs), carries the genetic determinant responsible for Tsw resistance breakdown. We then used full-length S RNA segments or the proteins they encode to compare the sequences of WT and related RB strains, and obtained indirect evidence that the NSs protein is the avirulence factor in question. Transient expression of NSs protein from WT and RB strains showed that they both can equally suppress post-transcriptional gene silencing (PTGS). Moreover, biological characterization of two RB strains carrying deletions in the NSs protein showed that NSs is important in maintaining TSWV infection in newly emerging leaves over time, preventing recovery. Analysis of another RB strain phenotype allowed us to conclude that local necrotic response is not sufficient for resistance in Capsicum spp. carrying the Tsw gene.

Phylogenetic analysis of Tomato spotted wilt virus (TSWV) NSs protein demonstrates the isolated emergence of resistance-breaking strains in pepper

“…Isolates from the GenBank containing most of these data were chosen to compare variations or mutations occurring in RB pepper strains. Similarly, to the results of Margaria et al [24], we detected several mutations both in NSs and N proteins, but there were not any of them common with all the RB strains. Controversially, with several plant viruses that interfere plant resistance by a 'gene for gene' interaction and the emergence of RB strains can be connected to specific point mutations (at given aa positions), TSWV RB strains evolve by different point mutations/substitutions at various aa positions.…”

“…The rapid adaptation of TSWV to pepper resistance and breakdown of the Tsw resistance gene facilitated the determination of the avr factor (avr) of TSWV, and the study of the evolutionary aspects of emergence of new strains [15,[20][21][22][23][24][25][26][27]. However, contradictory results, lacking information and methodological difficulties hinder or delay advances in these questions.…”

Evolution and structure of Tomato spotted wilt virus populations: evidence of extensive reassortment and insights into emergence processes

“…the exchange of parts of genome segments during replication). Experimental selection of reassortants from plants coinfected by two TSWV isolates with contrasted biological properties allowed mapping the determinant for breakdown of the Tsw resistance to RNA S (Jahn et al, 2000;Margaria et al, 2007). Further studies have suggested contradictorily that the NSs nonstructural protein (Margaria et al, 2007) or alternatively the nucleocapsid protein (NP) (Lovato et al, 2008) coding regions of RNA S were involved in the breakdown.…”

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense

“…A classical test for revealing silencing suppressor activity was initially performed by co-agroinfiltrating 16C N. benthamiana with pBin-GFP (Margaria et al 2007) and each of the pBin61 derivatives of OuMV RdRp, MP, and CP previously described (Crivelli et al 2011). As positive control, we used the well-characterized silencing suppressor p19 of Tomato bushy stunt virus (TBSV), which exhibited suppression, but we failed to detect suppression activity with the OuMV MP, CP, or RdRp (not shown).…”

“…For N. benthamiana agroinfection, each plasmid was transformed into Agrobacterium tumefaciens C58C1 and agroinfiltrated in young leaves, as previously described (Bendahmane et al 1999(Bendahmane et al , 2002Margaria et al 2007). To standardize the experiments, the absorbance (optical density at 600 nm) of the Agrobacterium cultures was measured and each agroclone was adjusted to a final concentration corresponding to an absorbance of 0.25.…”