Identification and Characterization of Plant-Interacting Targets of Tomato Spotted Wilt Virus Silencing Suppressor

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) is an economically important plant virus that infects multiple horticultural crops on a global scale. TSWV encodes a non-structural protein NSs that acts as a suppressor of host RNA silencing machinery during infection. Despite extensive structural and functional analyses having been carried out on TSWV NSs, its protein-interacting targets in host plants are still largely unknown. Here, we systemically investigated NSs-interacting proteins in Nicotiana benthamiana via affinity purification and mass spectrometry (AP-MS) analysis. Forty-three TSWV NSs-interacting candidates were identified in N. benthamiana. Gene Ontology (GO) and protein–protein interaction (PPI) network analyses were carried out on their closest homologs in tobacco (Nicotiana tabacum), tomatoes (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). The results showed that NSs preferentially interacts with plant defense-related proteins such as calmodulin (CaM), importin, carbonic anhydrase and two heat shock proteins (HSPs): HSP70 and HSP90. As two major nodes in the PPI network, CaM and importin subunit α were selected for the further verification of their interactions with NSs via yeast two-hybrid (Y2H) screening. Our work suggests that the downstream signaling, transportation and/or metabolic pathways of host-NSs-interacting proteins may play critical roles in NSs-facilitated TSWV infection.