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

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.

Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro

“…Although a non-specific in vitro RNA binding activity alone cannot be used to predict the in vivo function of a protein, the finding that CCYV p22 binds in vitro ss-and ds siRNAs firstly suggests a connection with its RNA silencing suppression activity, and secondly, that it may contribute to the reduced GFP-specific 24 nt-long siRNAs accumulation reported by Chen et al, (2019). Interestingly, our experiments also show increased binding affinity for the 24 nt-long ss siRNAs.…”

“…In recent years, it has become apparent that a number of proteins encoded by 3'-proximal ORFs of crinivirus RNA 1 are able to suppress RNA silencing (Kreuze et al, 2005;Cañizares et al, 2008;Cuellar et al, 2009;Kataya et al, 2009;Kubota & Ng, 2016;Mashiko et al, 2019;Orfanidou et al, 2019;Chen et al, 2019) or/and to bind RNA (Wang et al, 2010;Weinheimer et al, 2014;Landeo-Ríos et al, 2016). For these proteins, typically expressed from the earliest stages of infection (Klaassen et al, 1996;Salem et al, 2009;Orilio et al, 2014), there is increasing evidence that significant divergence in their manner(s) of action exists.…”

“…6C). These results suggest that the F-box (Chen et al, 2019), which is entirely included in DEL 2, is not essential for ssRNA binding but that within the terminal amino acid residues 76-188 (DEL 3 and DEL 4) there are elements essential for dsRNA binding.…”

“…1) includes 0-3 similarly-sized ORFs encoding proteins that share low levels (25-40%) of amino acid homology. A number of these proteins, including CCYV p22, have been reported to be viral suppressors of RNA silencing (VSRs): Lettuce chlorosis virus p23, Sweet potato chlorotic stunt virus (SPCSV) RNase3 and p22, Tomato chlorosis virus (ToCV) p22, CYSDV p25 and Tomato infectious chlorosis virus p27 (Kreuze et al, 2005;Cañizares et al, 2008;Cuellar et al, 2009;Kataya et al, 2009;Kubota & Ng, 2016;Mashiko et al, 2019;Orfanidou et al, 2019;Chen et al, 2019).…”

“…Taking into consideration these findings and the reported reduced accumulation of GFP-specific siRNA in GFP/ CCYV p22 co-infiltrated leaf patches (Chen et al, 2019), we examined the ability of CCYV p22 to bind RNA in vitro. Our data show that CCYV p22 is able to bind ss, dsRNA and siRNAs and suggests a higher degree of diversity in the modes of action of crinivirus-encoded VSRs.…”