Two Novel Motifs of Watermelon Silver Mottle Virus NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

Abstract

The NSs protein of Watermelon silver mottle virus (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in Nicotiana benthamiana plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (109KFTMHNQ117), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal β-sheet motif (397IYFL400) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated Zucchini yellow mosaic virus vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.

The conserved aromatic residue W122 is a determinant of potyviral coat protein stability, replication, and cell‐to‐cell movement in plants

“…The aromatic residues W, F, and Y are known to be important for protein stability and function. For example, the aromatic ring of Y 398 in the WSMoV NSs protein has been shown to affect NSs stability and its RNA silencing suppression activity (Huang et al, 2015). Deletion of five residues, including an aromatic residue, from the readthrough protein of potato leafroll virus affects viral systemic infection and disease symptom induction (Xu et al, 2018).…”

Identification and localization of Tospovirus genus-wide conserved residues in 3D models of the nucleocapsid and the silencing suppressor proteins

“…In case of Watermelon silver mottle virus (WSMV), Huang et al [20] showed that mutations at H 113 in the common epitope (CE) ( 109 KFTMHNQ 117 ) and Y 398 at the C-terminal β-sheet motif ( 397 IYFL 400 ) affect NSs mRNA stability, and protein stability, respectively, and concluded that both are critical for silencing suppressor activity of NSs. The H 113 of WSMV corresponds to H 115 in TSWV sequence and is also conserved in all species of the genus.…”

Characterization of the genome of a phylogenetically distinct tospovirus and its interactions with the local lesion-induced host Chenopodium quinoa by whole-transcriptome analyses

“…The crosstalk of RNA silencing and SA signaling was also demonstrated [ 59 ]. Since the RSS NSs of a tospovirus can restore a non-local lesion HC-Pro mutant of ZYMV [ 61 ], it is inferred that the GCFSV NSs might also be involved in HR formation on C . quinoa plants.…”