Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

Nicotiana benthamiana is an important model plant widely employed in plant virology, which involves the nucleotide binding site leucine-rich repeat (NBS-LRR) genes in disease resistance. In this study, firstly, 156 NBS-LRR homologs were identified by HMMsearch and they were divided into three clades in the phylogenetic tree by Clustal W, containing 5 TNL-type, 25 CNL-type, 23 NL-type, 2 TN-type, 41 CN-type, and 60 N-type proteins. Further, conserved motifs and domain composition were detected by MEME, showing 10 conserved motifs dispersed throughout the protein sequences in both typical- and irregular- type NBS-LRRs. Subcellular localization prediction by CELLO v.2.5 and Plant-mPLoc suggested 121 NBS-LRRs were located in cytoplasm, 33 in plasma membrane, and 12 in nucleus. And physicochemical characteristics by EXPASY ProtParam revealed common and unique features among the six types of NBS-LRR protein. Finally, gene structure analysis by TBtools exhibited most NBS-LRR genes were composed of either fewer or two introns. And regulatory cis-elements were assayed by PlantCARE, detecting 29 shared kinds and 4 kinds unique in irregular-type NBS-LRR genes, indicating potential upstream regulation factors. In summary, we provided important information on NBS-LRR genes and potential resources for improving disease resistance in tobacco, and will contribute to breeding programs for virus resistance.