Abstract

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that interfere with the cellular silencing machinery through various mechanisms not always well understood1.Mungbean yellow mosaic virus (MYMV) is a plant bipartite geminivirus responsible for a devastating plant disease in some areas of tropics and sub-tropics where its natural host, Vigna mungo, is one of the major sources of food. In some bipartite Begomoviruses both AC2 and AC4 viral proteins act as VSRs, albeit with different efficiency in the same viral species2. Previous studies demonstrated that the MYMV-encoded AC2, which transactivates transcription of late viral genes, functions as a strong VSR3. We examined the role of MYMV AC4 and showed by quantitative real-time PCR that it is essential for infectivity but not for virus replication. MYMV AC4 acts as a determinant of pathogenicity and when expressed from the heterologous Potato virus X (PVX) increases the severity of viral-induced symptom in Nicotiana benthamiana. Importantly, we showed that AC4 counteracts virus induced gene silencing (VIGS) in PVX-GFP-infected N. benthamiana 16c lines, and strongly suppresses the systemic phase of silencing but is an inefficient suppressor of local gene silencing in Agrobacterium co-infiltration experiments. These results demonstrate that MYMV hosts two silencing suppressors, AC2 and AC4, that, probably target distinct steps of the silencing machinery. Interestingly, while most of the known VSRs have cytoplasmic localization we observed that MYMV AC4 specifically accumulates to the plasma membrane (PM) and secondarily to the nucleus. Despite its hydrophilic nature, MYMV AC4 is not predicted to be a globular protein and, probably because of its short length (only 100 aminoacids), it is very poorly structured. AC4 has no trans-membrane domain but hosts a typical nuclear localization signal consensus sequence (KRR) and a cysteine (C11), flanked by two nonpolar phenylalanines, that is predicted to be palmitoylated. Palmitoylation is a post-translational modification that regulates membrane-protein interactions and serves to increase relative membrane affinity. It consists of the reversible linkage of palmitate to proteins at cysteine residues belonging to a variety of sequence motifs. Site-specific mutagenesis experiment revealed that these two domains are functional and responsible for the subcellular localizations of AC4 to the PM (C11) and for nuclear targeting (KRR). Furthermore, the evidence that the N-terminal 12 aminoacids of AC4 expressed in fusion with GFP specifically redirect the cytosolic protein to the PM, supports the indication that C11 mediates protein binding to PM via a process of S-palmitoylation previously unreported for any plant virus protein. We obtained biochemical confirmation of AC4 palmitoylation by means of biotin switch assay4.Remarkably, replacement of C11 with

Autore Pugliese

• Stavolone Livia

Tutti gli autori

• Carluccio A.V.; Stavolone L.

Titolo volume/Rivista

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Anno di pubblicazione

2014

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ISBN

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Numero di citazioni Wos

Nessuna citazione

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Settori ERC

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Codici ASJC

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