A single U/C nucleotide substitution changing alanine to valine in the beet necrotic yellow vein virus P25 protein promotes increased virus accumulation in roots of mechanically inoculated, partially resistant sugar beet seedlings

Abstract

Beet necrotic yellow vein virus (BNYVV) A type isolates E12 and S8, originating from areas where resistance-breaking had or had not been observed, respectively, served as starting material for studying the influence of sequence variations in BNYVV RNA 3 on virus accumulation in partially resistant sugar beet varieties. Sub-isolates containing only RNAs 1 and 2 were obtained by serial local lesion passages; biologically active cDNA clones were prepared for RNAs 3 which differed in their coding sequences for P25 aa 67, 68 and 129. Sugar beet seedlings were mechanically inoculated with RNA 1+2/RNA 3 pseudorecombinants. The origin of RNAs 1+2 had little influence on virus accumulation in rootlets. E12 RNA 3 coding for V 67 C 68 Y 129 P25, however, enabled a much higher virus accumulation than S8 RNA 3 coding for A 67 H 68 H 129 P25. Mutants revealed that this was due only to the V 67 'GUU' codon as opposed to the A 67 'GCU' codon.The Polymyxa betae-transmitted beet necrotic yellow vein virus (BNYVV) is the causal agent of the devastating rhizomania disease. Its genome consists of four, and in some geographical areas five, different RNA species. RNA 1 and 2 contain the genetic information necessary to enable replication, encapsidation, cell-to-cell movement and suppression of RNA silencing in local lesion hosts (Koenig, 2008; Dunoyer et al., 2002). The additional presence of RNA 3, which codes for a 25 kDa protein (P25), is necessary for virus movement in infected sugar beet roots and production of typical rhizomania symptoms. RNA 4 enables efficient vector transmission and suppression of gene silencing in Nicotiana benthamiana roots (Rahim et al., 2007). The severity of symptoms in sugar beet may be enhanced by the additional presence of RNA 5. The three major strain groups of BNYVV, i.e. the A, B and P types, have highly conserved genomes. Nucleotides 199-210 of the P25-coding region, however, show considerable sequence variation, especially in A type BNYVV (Tamada et al., 2003;Meunier et al., 2003;Schirmer et al., 2005;Ward et al., 2007;Kutluk Yilmaz et al., 2007;Li et al., 2008;Koenig et al., 2008). This stretch of nucleotides codes for the P25 aa 67-70, which in the following will be referred to as the 'tetrad'. Also for the sake of brevity we will refer to the variability of the tetrad rather than its coding sequence, although we do not know whether the effects described below are due to changes at the protein or the nucleic acid level. More than 15 variants of the tetrad have been identified so far, raising the question whether these variations influence the pathogenicity of the virus and may be involved in resistance-breaking phenomena. Klein et al. (2007) studied the influence of P25 sequence variation on its oligomerization and pathogenicity for Tetragonia expansa leaves. Chiba et al. (2008) investigated the influence of mutations in the P25 aa 68, 69 and 70 on the local lesion response in mechanically inoculated leaves of lines of Beta maritima and of the sugar beet varieties Rizor and Monomidori. Acosta- ...

Efficiency of Rz1-based rhizomania resistance and molecular studies on BNYVV isolates from sugar beet cultivation in Greece

“…Earlier studies on pathogenicity and ability of BNYVV isolates to overcome Rz1-based resistance, point to a possible involvement of certain amino acid residues of p25 and particularly those in positions 67-70, 129, 135 and 179 (Tamada et al 2002;Schirmer et al 2005;Acosta-Leal et al 2008;Chiba et al 2008;Koenig et al 2009). Although a significantly higher variation occurs at amino acid residues 67-70 of p25 -aa 67-70 , referred to as "tetrad"-, especially within BNYVV A-type , evidence for the importance of a drift resulting from positive selection to overcome resistance has so far been obtained only for valine at position 67 (Koenig et al 2009).…”

“…Although a significantly higher variation occurs at amino acid residues 67-70 of p25 -aa 67-70 , referred to as "tetrad"-, especially within BNYVV A-type , evidence for the importance of a drift resulting from positive selection to overcome resistance has so far been obtained only for valine at position 67 (Koenig et al 2009). Such finding could explain the previously observed Rz1-resistance breaking ability of A-type isolates from Spain and the USA Pferdmenges et al 2009).…”

“…High sequence variability of A-type BNYVV RNA 3-encoded p25, specifically at amino acids 67-70 (aa 67-70 ), has been associated with varying pathogenicity (Tamada et al 2002;Schirmer et al 2005;Chiba et al 2008). In addition to aa 67-70 , aa residues at positions 129, 135 and 179 have also been proposed as possible factors in variable pathogenicity and resistance breaking incidence (Acosta-Leal et al 2008;Chiba et al 2008;Koenig et al 2009). …”

“…Additionally, resistant varieties used throughout the country seem to effectively protect the crop against rhizomania. However, the long time elapsed since the identification of pathotype A in only one of all growing regions and, particularly in view of the findings on "resistance-breaking" in fields sown to varieties endowed with the Rz1 gene (Liu et al 2005;Schirmer et al 2005;Rush et al 2006;Acosta-Leal et al 2008;Chiba et al 2008;Koenig et al 2009), necessitated a thorough investigation of the situation currently evolved in the country. The objectives of this study were a) a detailed field evaluation of the efficiency of Rz1 gene-based rhizomania resistance b) the molecular characterization of BNYVV isolates from representative fields in all five major sugar beet growing regions, in order to determine the type and genetic diversity of the virus and c) the determination of the amino acid composition of RNA3-encoded p25 protein of isolates collected from the above areas.…”

Generation and dispersion of resistance breaking variants ofBeet necrotic yellow vein virus in the field.

“…At the biochemical level, resistance is associated with differential expression of genes involved in pathogenesis and hormone-mediated plant development (Burketová et al, 2003;Larson et al, 2008). Reverse genetics experiments indicate that a viral determinant for resistance breakdown is the presence of valine at position 67 of p25 (Koenig et al, 2009). This amino acid change was previously associated with rhizomania in field samples from California Imperial Valley (CIV, Acosta-Leal and Rush, 2007).…”