Cucurbit Chlorotic Yellows Virus p22 Protein Interacts with Cucumber SKP1LB1 and Its F-Box-Like Motif Is Crucial for Silencing Suppressor Activity

Siyu Chen,Bingjian Sun,Hangjun Sun,Yan Shi,Ying Wei,Linlin Chen,Yajuan Shi,Honglian Li,Xinyan Sun,Xiaoyu Han

doi:10.3390/v11090818

Abstract

Plants use RNA silencing as a defense against viruses. In response, viruses encode various RNA silencing suppressors to counteract the antiviral silencing. Here, we identified p22 as a silencing suppressor of cucurbit chlorotic yellows crinivirus and showed that p22 interacts with CsSKP1LB1, a Cucumis sativus ortholog of S-phase kinase-associated protein 1 (SKP1). The F-box-like motif of p22 was identified through sequence analysis and found to be necessary for the interaction using a yeast two-hybrid assay. The involvement of the F-box-like motif in p22 silencing suppressor activity was determined. Proteomics analysis of Nicotiana benthamiana leaves expressing p22, and its F-box-like motif deletion mutant showed 228 differentially expressed proteins and five enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways: ABC transporters, sesquiterpenoid and triterpenoid biosynthesis, ubiquitin-mediated proteolysis, riboflavin metabolism, and cysteine and methionine metabolism. Collectively, our results demonstrate the interaction between p22 and CsSKP1LB1 and show that the deletion of F-box-like motif inhibits p22 silencing suppressor activity. The possible pathways regulated by the p22 through the F-box-like motif were identified using proteomics analysis.

Highlights

The members of the genus Crinivirus in the family Closteroviridae cause significant losses of yield and quality in many plant species [1,2,3]
To test for the suppressor activity of p22, N. benthamiana leaves were co-infiltrated with a mixture of Agrobacterium tumefaciens harboring 35S-genomic of the protein (GFP) and p22, or with 35S-GFP and Tomato bushy stunt virus (TBSV) silencing suppressor protein P19 or an empty vector serving as positive and negative controls, respectively (Figure 1B)
We demonstrated the in planta interaction of p22 with CsSKP1LB1 and NbSKP1 using a co-immunoprecipitation (Co-IP) assay

Summary

Introduction

The members of the genus Crinivirus in the family Closteroviridae cause significant losses of yield and quality in many plant species [1,2,3]. CCYV RNA1 contains four ORFs: ORF1a, ORF1b, ORF2, and ORF3 [7]. ORF1a encodes viral methyltransferase and RNA helicase 1. Neither p6 nor p22 shows significant similarity to the corresponding proteins of other criniviruses [7]. The positioned and sized p22 proteins of tomato chlorosis virus (ToCV) and sweet potato chlorotic stunt virus (SPCSV) were identified as effective silencing suppressors [8,9]. ToCV p22 suppressed sense RNA, and dsRNA triggered silencing efficiently but showed no effect on the short or long-distance spread of silencing [9]. SPCSV p22 suppressed RNA silencing more efficiently with the co-expression of RNase 3 [8]

Methods

Results

Conclusion