Bymovirus reverse genetics: requirements for RNA2-encoded proteins in systemic infection

Abstract

Barley yellow mosaic virus (BaYMV), the type species of the genus Bymovirus in the family Potyviridae in the picornavirus-like superfamily, causes a yellow mosaic disease of winter barley with significant yield losses in Europe and East Asia. Until now, infectious in vitro transcripts for the bipartite plus-sense RNA genome of any bymovirus species have not been available, rendering molecular analyses of bymovirus pathogenicity and the host resistance mechanisms difficult. In this study, we constructed the first cDNA clones of BaYMV RNA1 and RNA2, from which infectious RNA can be transcribed in vitro. Using in vitro transcripts, we showed that RNA1, which encodes eight proteins, including a viral proteinase NIa-Pro, the RNA-dependent RNA polymerase NIb, genome-linked viral protein VPg and the capsid protein CP, replicated autonomously in barley mesophyll protoplasts in the absence of RNA2 optimally at 15 degrees C, a temperature similar to the optimum for causing disease in barley fields. For systemic infection of barley plants, RNA1 alone was not sufficient and RNA2 was also required. Of the two proteins encoded on RNA2 (P1 with cysteine proteinase activity and P2 with unknown functions), P1 was essential and P2 was dispensable for systemic infectivity. The expression of both P1 and P2, but not the precursor polyprotein, together with RNA1 increased systemic infection and caused mosaic leaf symptoms. The infectious cDNA clones of BaYMV will be vital for future studies of bymovirus-host-vector interactions at the molecular level.

Figure 1

The genome organizations of Barley yellow mosaic virus (BaYMV) RNA1, RNA2 and RNA2 mutants shown as their full‐length cDNA constructs. (A) pBY1, the full‐length cDNA clone for wild‐type (WT) RNA1. A rectangular box indicates a 271‐kDa polyprotein, which is cleaved into eight proteins by a serine proteinase activity residing in the C‐terminal region of nuclear inclusion protein a (NIa) (indicated by an open star). A thick grey bar above P3 indicates a putative pipo gene expressed by a +2 frame‐shift at GGAAAAAA (nucleotides 676–683) (Chung et al., 2008). Putative cleavage sites are shown by short vertical bars, and the names of the cleaved products are written below the box. A dotted vertical bar in NIa indicates an internal cleavage into the N‐terminal genome‐linked viral protein (VPg) and the C‐terminal nuclear inclusion protein a‐proteinase (NIa‐Pro). A filled square in nuclear inclusion protein b (NIb) indicates the position of the GDD RNA‐dependent RNA‐polymerase motif. (B) pBY2, the full‐length cDNA clone for WT RNA2. A rectangular box indicates a 98‐kDa polyprotein, which is cleaved into P1 and P2 at a glycine–serine bond by a cysteine proteinase activity residing in the C‐terminal region of P1 (indicated by a filled star). (C) Five pBY2 mutants, which were prepared so that P1, P2, P12 or green fluorescent protein (GFP) could be expressed as mentioned in Experimental procedures. Rightward filled pentagons at the 5′ end indicate the T7 promoter.

Figure 2

Viral protein expression in transcript‐transfected barley protoplasts. (A) Protoplasts were transfected with mock, RNA1 transcripts alone, or both RNA1 and RNA2 transcripts and incubated at 15 °C for 48 h. Total proteins were subjected to Western blot analysis. α‐CP, anti‐capsid protein serum; α‐VPg, anti‐genome‐linked viral protein serum; α‐P1, anti‐P1 serum. The sizes of the proteins are indicated in the right margin in kilodaltons. An asterisk indicates the position of the 47‐kDa nuclear inclusion protein a (NIa), which may co‐migrate with the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco). (B) Protoplasts were transfected with both RNA1 and RNA2 transcripts and incubated at 12, 15, 17 or 20 °C for 60 h. N.C. indicates mock‐infected protoplasts which were incubated at 15 °C for 60 h. Total proteins were subjected to Western blot analysis using anti‐CP serum (α‐CP) and anti‐P1 serum (α‐P1). ‘Rubisco’ is the Rubisco large subunit stained by Coomassie Brilliant Blue G‐250.

Figure 5

(A) Upper leaf symptoms after inoculation with wild‐type RNA1 and RNA2 transcripts (WT) and with WT RNA1 and ΔP2 RNA2 transcripts (ΔP2) at 4 weeks and 6 weeks post‐inoculation, respectively. (B) Immunoblot detection of capsid protein (CP) and P1 from leaves infected, from the left, with WT RNA1 and RNA2, WT RNA1 and P1_TAA revertant RNA2, WT RNA1 and ΔP2 RNA2 and uninfected. (C) Genome organization of RNA2 and nucleotide and translated amino acid sequences at the boundary of the P1/P2 cleavage site. WT, bases 905–934 encoding amino acids 221–260 of polyprotein P12. Cleavage occurs between Gly255 and Ser256. P1_TAA, a UAA termination codon was inserted between the glycine and serine codons. P1_TAA Rev, the inserted UAA codon was substituted with a UCA serine codon in this revertant.

Figure 3

Cell‐free translation products from RNA2 mutants in wheat germ extract. (A) Total products after the translation reaction using precharged biotinylated lysine‐tRNA as a label. From the left, water control and RNA2 transcripts from pBY2, pBY2.ΔP1, pBY2.P1_TAA, pBY2.P12V and pBY2.ΔP2. (B) Immunoprecipitated proteins using anti‐P1 serum. From the left, water control and RNA2 transcripts from pBY2, pBY2.P1_TAA, pBY2.P12 and pBY2.ΔP2. The positions of P1, P2 and P12 are shown in the left margin.

Figure 4

Protein expression profiles in protoplasts transfected with wild‐type (WT) RNA1 and RNA2 mutants analysed by Western blotting using anti‐P1 serum (A), anti‐capsid protein (anti‐CP) serum (B, D) and anti‐green fluorescent protein (anti‐GFP) serum (C). (A, B) From the left, water control, RNA1 transcripts only, RNA1 transcripts with WT RNA2 transcripts, ΔP1 RNA2 transcripts, P1_TAA RNA2 transcripts, P12V RNA2 transcripts and ΔP2 RNA2 transcripts. The positions of P1, P12 and CP are indicated in the right margin. (C, D) Left, water control; right, RNA1 transcripts with GFP RNA2 transcripts. The positions of GFP and CP are indicated in the right margin. ‘Rubisco’ is the Rubisco large subunit stained by Coomassie Brilliant Blue G‐250.