RNA4-encoded p31 of beet necrotic yellow vein virus is involved in efficient vector transmission, symptom severity and silencing suppression in roots

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The RNA3-encoded p25 protein of beet necrotic yellow vein virus (BNYVV) is responsible for the production of rhizomania symptoms of sugar beet roots (Beta vulgaris subsp. vulgaris). Here, it was found that the presence of the p25 protein is also associated with the resistance response in rub-inoculated leaves of sugar beet and wild beet (Beta vulgaris subsp. maritima) plants. The resistance phenotype displayed a range of symptoms from no visible lesions to necrotic or greyish lesions at the inoculation site, and only very low levels of virus and viral RNA accumulated. The susceptible phenotype showed large, bright yellow lesions and developed high levels of virus accumulation. In roots after Polymyxa betae vector inoculation, however, no drastic differences in virus and viral RNA accumulation levels were found between plants with susceptible and resistant phenotypes, except at an early stage of infection. There was a genotype-specific interaction between BNYVV strains and two selected wild beet lines (MR1 and MR2) and sugar beet cultivars. Sequence analysis of natural BNYVV isolates and site-directed mutagenesis of the p25 protein revealed that 3 aa residues at positions 68, 70 and 179 are important in determining the resistance phenotype, and that host-genotype specificity is controlled by single amino acid changes at position 68. The mechanism of the occurrence of resistance-breaking BNYVV strains is discussed.

“…Foliar rub-inoculation and root vector inoculation using P. betae were conducted as described previously (Tamada et al, 1989;Tamada, 2007;Rahim et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of amino acids of the beet necrotic yellow vein virus p25 protein required for induction of the resistance response in leaves of Beta vulgaris plants

Chiba

Miyanishi

Andika

et al. 2008

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“…However, lesions on Beta spp. leaves are clearly produced when at least BNYVV RNAs-1, -2 and -3 are present (Tamada et al, 1989;Rahim et al, 2007). Although BSBV and BNYVV both naturally infect sugar beet, BSBV lacks the genetic material necessary to induce clear symptoms on B. macrocarpa leaves whereas BNYVV has this through RNAs-3 and -4.…”

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confidence: 99%

A full-length infectious clone of beet soil-borne virus indicates the dispensability of the RNA-2 for virus survival in planta and symptom expression on Chenopodium quinoa leaves

Crutzen

Mehrvar

Gilmer

et al. 2009

For a better understanding of the functionality and pathogenicity of beet soil-borne virus (BSBV), full-length cDNA clones have been constructed for the three genomic RNAs. With the aim of assessing their effectiveness and relative contribution to the virus housekeeping functions, transcripts were inoculated on Chenopodium quinoa and Beta macrocarpa leaves using five genome combinations. Both RNAs-1 (putative replicase) and -3 (putative movement proteins) proved to be essential for virus replication in planta and symptom production on C. quinoa, whereas RNA-2 (putative coat protein, CP, and a read-through domain, RT) was not. No symptoms were recorded on B. macrocarpa, but viral RNAs were detected. In both host plants, the 19 kDa CP was detected by Western blotting as well as a 115 kDa protein corresponding to the CP-RT.Beet soil-borne virus (BSBV) is a pomovirus transmitted to Chenopodiaceae by the protist Polymyxa betae (Ivanović et al., 1983), which is also the vector of the aetiological agent of the rhizomania syndrome of sugar beet, beet necrotic yellow vein virus (BNYVV) (Tamada & Baba, 1973). Originally reported in Italy (Canova, 1959), rhizomania disease is now widespread in most countries where sugar beet is grown (McGrann et al., 2009) and BSBV is often found in beet infected with BNYVV (Meunier et al., 2003). However, the pathogenicity of BSBV and its contribution to the rhizomania syndrome remain unclear, with opinions still divided on this (Prillwitz & Schlösser, 1992;Kaufmann et al., 1993;Lindsten, 1993;Rush & Heidel, 1995).The BSBV genome consists of three single-stranded RNAs of positive polarity, packaged into rod-shaped particles (Koenig et al., 1996(Koenig et al., , 1997Koenig & Loss, 1997). RNA-1 (5.8 kb) encodes the putative viral replicase. The 19 kDa coat protein (CP) and a putative 85 kDa read-through (RT) domain are encoded by RNA-2 (3.5 kb). RNA-3 (3.0 kb) comprises three open reading frames (ORFs) encoding three putative proteins (48, 13 and 22 kDa) thought to be responsible for the viral cell-to-cell movement, resembling the well-known triple gene block proteins (TGBs) (Fig. 1).In this study, the contribution of each RNA component to virus survival and symptom expression was investigated through the use of full-length cDNA clones on two host plants, Chenopodium quinoa and Beta macrocarpa. This last plant species was preferred to the natural host Beta vulgaris with a view of developing the basis for further molecular analysis of viral systemicity, following the example of previous studies on BNYVV (Lauber et al., 1998).Full-length cDNA sequences were generated from an Iranian BSBV isolate (Nyshabour, Khorasan Razavi Province), which was trapped from infested soil in roots of B. vulgaris. After total RNA extraction from sugar beet roots using the SV total RNA isolation kit (Promega), the three genomic RNAs were reverse transcribed and amplified by PCR (RT-PCR) using expand reverse transcriptase and the expand long template PCR System (Roche). Primers matching the extremities of the t...

“…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.…”

mentioning

confidence: 99%

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

Koenig

Loss

Specht

et al. 2009

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- ...