a939d6e81f89aca97326f187f18daf92 pmc_33925878.pdf 700eacddab3e75ba2b23e2325dfdd24d97aaab76 pmc_33925878.pdf cdca66b7e121911b5ed6fe881e71028fb24433fdf4ec23a38d6b1aca901457ea pmc_33925878.pdf Title: Argonaute 2 Controls Antiviral Activity against Sweet Potato Mild Mottle Virus in Nicotiana benthamiana Subject: RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2-/- mutant Nicotiana benthamiana. Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato. Keywords: RNA silencing; AGO1; AGO2; sweet potato mild mottle virus Author: Erzsébet Kenesi, Juan-Jose Lopez-Moya, László Orosz, József Burgyán and Lóránt Lakatos Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Apr 26 11:57:22 2021 CEST ModDate: Mon Apr 26 12:05:52 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 10 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 14517597 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OPZDHV+VnURWPalladioL Type 1 Custom yes yes no 10 0 TOXNZL+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 JSVCMW+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 TSYUQM+URWPalladioL-BoldItal Type 1 Custom yes yes no 24 0 LOTOZP+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 XMYFHA+CMSY10 Type 1 Builtin yes yes no 32 0 ZZTQUO+VnURWPalladioL-Bold Type 1 Custom yes yes no 36 0 VTLKDP+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 45 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 75 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 79 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 83 0 MDJLZU+EURM10 Type 1 Builtin yes yes no 107 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:19:42 CEST RepresentationInformation: pmc_33925878.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:09:52 CEST Size: 14517597 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 299 FreeObjects: 11 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: The N. benthamiana AGO2-/- Mutant Is More Susceptible to the SPMMV Compared with Wild Type Plants Destination: subsection.2.1 Item: Title: SPMMV Infection Induces AGO2 Destination: subsection.2.2 Item: Title: AGO2 Associates with Viral siRNAs and miRNAs in SPMMV-Infected Plants Destination: subsection.2.3 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Plant Materials Destination: subsection.4.1 Item: Title: Plant Inoculation Destination: subsection.4.2 Item: Title: RNA Analysis Destination: subsection.4.3 Item: Title: Small RNA Analysis Destination: subsection.4.4 Item: Title: Protein Analysis Destination: subsection.4.5 Item: Title: Immunoprecipitation Destination: subsection.4.6 Item: Title: Quantitative Reverse Transcriptase PCR (qRT-PCR) Destination: subsection.4.7 Item: Title: References Destination: subsection.4.7 Info: Title: Argonaute 2 Controls Antiviral Activity against Sweet Potato Mild Mottle Virus in Nicotiana benthamiana Author: Erzsébet Kenesi, Juan-Jose Lopez-Moya, László Orosz, József Burgyán and Lóránt Lakatos Subject: RNA silencing is a sequence specific post-transcriptional mechanism regulating important biological processes including antiviral defense in plants. Argonaute (AGO) proteins, the catalytic subunits of the silencing complexes, are loaded with small RNAs to execute the sequence specific RNA cleavage or translational inhibition. Plants encode several AGO proteins and a few of them, especially AGO1 and AGO2, have been shown to be required for antiviral silencing. Previously, we have shown that the P1 protein of the sweet potato mild mottle virus (SPMMV) suppresses the primary RNA silencing response by inhibiting AGO1. To analyze the role of AGO2 in antiviral defense against the SPMMV, we performed a comparative study using a wild type and ago2-/- mutant Nicotiana benthamiana. Here we show that the AGO2 of N. benthamiana attenuates the symptoms of SPMMV infection. Upon SPMMV infection the levels of AGO2 mRNA and protein are greatly increased. Moreover, we found that AGO2 proteins are loaded with SPMMV derived viral small RNAs as well as with miRNAs. Our results indicate that AGO2 protein takes over the place of AGO1 to confer antiviral silencing. Finally, we provide a plausible explanation for the AGO2 mediated recovery of an SPMMV-infected sweet potato. 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