Genetic diversity and silencing suppression effects of Rice yellow mottle virus and the P1 protein

Abstract

Background: PTGS (post-transcriptional gene silencing) is used to counter pathogenic invasions, particularly viruses. In return, many plant viruses produce proteins which suppress silencing directed against their RNA. The diversity of silencing suppression at the species level in natural hosts is unknown.

Sites under positive selection modulate the RNA silencing suppressor activity of rice yellow mottle virus movement protein P1

“…Its first ORF encodes a zinc finger protein with redox-dependent flexibility (Gillet et al, 2013), named P1, which has been described as a multifunctional protein that participates in virus spread and replication (Bonneau et al, 1998). In addition, P1 proteins originating from different RYMV isolates displayed a wide range of ability to suppress RNA silencing, from strong to weak RNA silencing suppression, as in the case of the Tanzanian Tz3 (P1Tz3) and Madagascar Mg1 (P1Mg1) isolates, respectively (Voinnet et al, 1999;Siré et al, 2008). Both were characterized as proteins exhibiting dual functions to suppress local RNA silencing and activate systemic RNA silencing (Lacombe et al, 2010).…”

“…Four plants were used for each mutant (eight leaves per treatment per mutant in total). The time-course of RNA silencing suppression activity on the GUS reporter gene was quantified by fluorimetry, as detailed in Siré et al (2008). Two independent experiments were performed.…”

“…Such a motif has been reported to be essential for silencing suppression activity (van Wezel et al, 2003). Point mutations corresponding to amino acid positions 20 (Ala to Thr), 54 (Lys to Gly), 66 (Glu to Arg) and 110 (Glu to Asp) were introduced according to the protocol described by Siré et al (2008). Each WT amino acid was changed into an amino acid existing in the RYMV natural diversity in at least one WT isolate and having different properties, including charge and hydrophobicity.…”

“…In total, 54 sequences [including 44 new sequences according to the sampling in Siré et al (2008)] representing the RYMV natural diversity were used to detect sites under positive selection. Sequence alignments of P1 are presented in Fig.…”

“…The effect of P1 substitutions A20T, K54G, Q66R and Q110D on the RNA silencing suppression function of P1 was assessed in a Journal of General Virology 95 transient expression assay in Nicotiana benthamiana leaves expressing artificial silencing on a b-glucuronidase-encoding (GUS) reporter gene. Briefly, constructs for the transient expression of GUS, P1Mg1, P1Tz3 WT and P1Tz3 mutants silencing suppressors were inserted into a pCambia 1301 binary vector under the 35S promoter, and all pCambiaderived plasmids were inserted into Agrobacterium tumefaciens strain GV3101 using an electroporation method (Siré et al, 2008). Four plants were used for each mutant (eight leaves per treatment per mutant in total).…”