Fig 1.
Coding potential analyses of barley yellow dwarf luteoviruses and poleroviruses.
A. Map of the ~5.7 kb Barley yellow dwarf virus (BYDV) genome, including the newly identified ORF3a (pink). Subgenomic RNA start sites are from Kelly et al. [30]. ORF1 and (via ribosomal frameshifting) ORF2 are translated from the genomic RNA. ORFs 3a, 3, 4 and 5 are expressed from sgRNA1, with translation of ORF3a predicted to be dependent on non-AUG initiation, translation of ORFs 3 and 4 being dependent on leaky scanning, and ORF5 translated by readthrough of the ORF3 stop codon. ORF6 may be translated from sgRNA2. B. MLOGD analysis, using a 40-codon sliding window, of the coding potential (lines) and positions of stop codons in each aligned sequence (points) in each of the three forward reading frames. The analysis is based on 76 aligned BYDV sequences, including serotypes PAV, PAS, MAV, GAV and Ker-II. Positive MLOGD scores indicate that the sequence is likely to be coding in that reading frame [37]. A conserved absence of stop codons provides independent support for a coding assignment. The pale pink rectangle in the panel corresponding to the +0 frame (blue) indicates the newly discovered ORF3a. To map the analysis onto the coordinates (and reading frames) of a specific sequence, all alignment columns with gaps in a chosen reference sequence, NC_004750.1 (BYDV-PAV), were removed. Remaining alignment gaps in non-reference sequences are indicated with grey rectangles in the stop codon plots. C. Map of the ~5.6 kb Turnip yellows virus (TuYV) genome, including the newly identified ORF3a (pink). ORFs 0, 1 and 2 are translated from the genomic RNA with expression of ORF1 being dependent on leaky scanning and translation of ORF2 via a -1 ribosomal frameshift. ORFs 3a, 3, 4 and 5 are translated from sgRNA1, with expression of ORF3a being dependent on non-AUG initiation, translation of ORFs 3 and 4 via leaky scanning, and of ORF5 via stop codon readthrough. D. MLOGD analysis was performed and displayed as in panel B. The analysis is based on 97 polerovirus sequences aligned separately within the 5' and 3' gene blocks. NC_003743.1 (TuYV) was used as the reference sequence.
Fig 2.
Sequence analysis of ORF3a and flanking regions.
Pseudo-alignment of representative luteovirus and polerovirus NCBI species RefSeqs (GenBank accession numbers indicated at left) for ORF3a and flanking regions. Spaces separate codons in the ORF3a reading frame. The alignment within ORF3a is based on a P3a amino acid alignment (S1 Fig). The 5' and 3' flanking sequences have not been aligned since pan-genus alignments in these regions are ambiguous. Virus name abbreviations: BLRV—Bean leafroll virus; SbDV—Soybean dwarf virus; CYDV-RPV—Cereal yellow dwarf virus-RPV; WYDV-GPV—Wheat yellow dwarf virus-GPV; CYDV-RPS—Cereal yellow dwarf virus-RPS; TuYV—Turnip yellows virus; BrYV—Brassica yellows virus; CpCSV—Chickpea chlorotic stunt virus; MABYV—Melon aphid-borne yellows virus; SABYV—Suakwa aphid-borne yellows virus; CLRDV—Cotton leafroll dwarf virus; ScYLV—Sugarcane yellow leaf virus; TVDV—Tobacco vein distorting virus; PeVYV—Pepper vein yellows virus; BWYV—Beet western yellows virus; BChV—Beet chlorosis virus; BMYV—Beet mild yellowing virus; CABYV—Cucurbit aphid-borne yellows virus; PLRV—Potato leafroll virus; CtRLV—Carrot red leaf virus; RSDaV—Rose spring dwarf-associated virus; MYDV-RMV—Maize yellow dwarf virus-RMV; BYDV-PAS—Barley yellow dwarf virus-PAS; BYDV-MAV—Barley yellow dwarf virus-MAV; BYDV-PAV—Barley yellow dwarf virus-PAV; BYDV-GAV—Barley yellow dwarf virus-GAV; BYDV-KerII—Barley yellow dwarf virus Ker-II.
Fig 3.
Schematic representation of the TuYV-3a mutants and in vitro translation of their corresponding subgenomic RNA1.
A. Mutations introduced into ORF3a: TuYV-3aAUG, TuYV-3aAGC, TuYV-3a2stop and TuYV-3aFLAG. The substitutions or insertions are indicated for each mutant. The translational potential of the different constructs regarding ORF3a is shown by a plain arrow starting at the ACG or modified codon at the same position. The dotted arrow indicates an expected absence of translation from the AGC codon. B. Tricine-SDS-PAGE analysis of proteins translated in wheat germ extracts from the indicated sgRNA1 mutants transcribed in vitro. Mock: no RNA added. After drying the gel, the radioactively labeled proteins were detected by phosphorimager. Positions of CP, P4 and putative P3a are indicated on the left. Sizes of molecular weight markers are indicated on the right.
Fig 4.
Protoplast infection with TuYV-ORF3a mutants.
A. Northern blot analysis of RNA extracted from triplicate samples of C. quinoa protoplasts inoculated with in vitro transcribed genomic RNAs corresponding to the wild type TuYV (WT) or the TuYV-3aAUG (AUG), TuYV-3aAGC (AGC), TuYV-3a2stop (2stop) or TuYV-3aFLAG (FLAG) mutants. Mock, mock-inoculated control; gRNA, genomic RNA; sgRNA1, subgenomic RNA1; LC, loading control, consisting of ethidium bromide-stained ribosomal RNA. Blots indicate results of three separate experiments, each with its own negative (Mock) and positive (WT) control. B. and C. Western blot analyses of proteins extracted from the same infected protoplast samples as in panel A. The blots were incubated with antibody specific to P3a (B), or CP, P4 or RTD protein (C). The molecular weights and mobilities of prestained protein markers are indicated in B. LC, loading control of proteins stained on the membranes by Ponceau red.
Fig 5.
Infection of A. thaliana infiltrated leaves.
A. Northern blot hybridization of RNA extracted from A. thaliana leaves agroinfiltrated with the wild-type TuYV (WT) or the mutants TuYV-3aAUG (AUG), TuYV-3aAGC (AGC), or TuYV-3aFLAG (FLAG). Mock, mock-inoculated control; gRNA, genomic RNA; sgRNA1, subgenomic RNA1, LC, loading control of ethidium bromide stained ribosomal RNA. Each sample corresponds to a mixture of leaves from 3 individual plants. B. and C. Western blot analysis of proteins extracted from the same infected leaf samples. Proteins were blotted on the same membrane and incubated with antibody (@) specific to P3a (B), CP, P4 and RTD protein (C). The blots were then cut in two for the figure. Two different exposures (short and long) are shown for P3a detection. LC, loading control of proteins stained on the membranes by Ponceau red.
Fig 6.
Systemic infection of A. thaliana.
Northern blot analysis of RNA extracted from upper, non-inoculated leaves of A. thaliana plants infiltrated with the wild-type TuYV (WT) or one of the TuYV-3a mutants, TuYV-3aAUG (AUG), TuYV-3aAGC (AGC), TuYV-3a2stop (2stop) or TuYV-3aFLAG (FLAG). All plants were analyzed but only three samples representative for the TuYV-WT and each mutant are shown, with at least one sample of an infected plant, confirmed by RT-PCR. Mock, mock-inoculated control; gRNA, genomic RNA; sgRNA1, subgenomic RNA1, LC, loading control of ethidium bromide stained ribosomal RNA.
Table 1.
Systemic infection by TuYV containing P3a mutants.
Fig 7.
Complementation analysis of ORF3a mutants.
A. Northern blot hybridizations of RNA extracted 21 d.p.i. from N. benthamiana systemic leaves co-infiltrated with the TuYV-3aAGC mutant (AGC) or the TuYV-WT virus (WT), and either the TuYV-3aAUG mutant (AUG) or agrobacterium transiently expressing the P3a, P3a-GFP or GFP protein. Viral mutants are labeled in black; transiently expressed proteins are labeled in green. Controls were conducted with infiltrations of TuYV-3aAGC (AGC), TuYV-3aAUG (AUG), TuYV-WT (WT), the empty vector (M) or agrobacterium expressing either P3a or P3a-GFP or co-infiltrations of TuYV-3aAUG (AUG) and agrobacterium expressing GFP. No additional suppressor of RNA silencing was supplied. Due to the number of samples, the blots are presented in two parts and for each of them a long and a short exposure is shown. B. Quantification of TuYV RNA by qRT-PCR in systemic leaves of N. benthamiana agroinfiltrated or co-agroinfiltrated with the mutants and agrobacterium expressing P3a, P3a-GFP or GFP as shown in panel A. The number of the samples refers to the same plants as those shown in panel A. After normalization with GAPDH reference gene, the values were normalized arbitrarily with sample #22. The insert is a magnification of the values of some samples that were particularly low.
Fig 8.
Viral particles produced by TuYV-3a mutants.
Negatively stained viral particles in A. thaliana inoculated with TuYV-WT, TuYV-3aAUG, TuYV-3aAGC, TuYV-3a2stop and TuYV-3aFLAG. Virus purification was performed from leaves agroinfiltrated with the viral constructs. Viral particles were observed by transmission electron microscopy (TEM). A TuYV polyclonal antiserum was used to capture viral particles on the grids before TEM. Mock: purified sample from leaves agroinfiltrated with an empty pBin plasmid. Scale bar: 50 = nm.
Fig 9.
Subcellular localization of P3a.
GFP- or RFP-fusion proteins were transiently expressed in N. benthamiana leaves and epidermal cells were observed under confocal microscopy. (A-B) Expression of P3a-GFP; (C-D) of P3a-RFP. (E-G) Co-expression of P3a-GFP (E) with Man1-RFP (a Golgi apparatus marker, panel F) and the merged fluorescent signal (G). (H-N) Co-expression of P3a-RFP (I and M) with the plasmodesmata marker PDLP1-GFP (H and L) and the merged fluorescent signal (panels J, K and N). A close-up of the box drawn in J is shown in panel K. Panels L-N show co-localization of the same proteins at a higher magnification. Scale bars, 20 μm for panels A-D and 10 μm for panels E-J and L-N. At least two independent experiments were performed for each condition. Representative images of the observations are presented. Western blot analyses of tissues expressing P3a-GFP and P3a-RFP fusions with GFP- and RFP-specific antibodies showed that the fusion proteins migrated in their monomeric form, with no major breakdown products released (S8 Fig).