An aromatic amino acid and associated helix in the C-terminus of the potato leafroll virus minor capsid protein regulate systemic infection and symptom expression

Abstract

The C-terminal region of the minor structural protein of potato leafroll virus (PLRV), known as the readthrough protein (RTP), is involved in efficient virus movement, tissue tropism and symptom development. Analysis of numerous C-terminal deletions identified a five-amino acid motif that is required for RTP function. A PLRV mutant expressing RTP with these five amino acids deleted (Δ5aa-RTP) was compromised in systemic infection and symptom expression. Although the Δ5aa-RTP mutant was able to move long distance, limited infection foci were observed in systemically infected leaves suggesting that these five amino acids regulate virus phloem loading in the inoculated leaves and/or unloading into the systemically infected tissues. The 5aa deletion did not alter the efficiency of RTP translation, nor impair RTP self-interaction or its interaction with P17, the virus movement protein. However, the deletion did alter the subcellular localization of RTP. When co-expressed with a PLRV infectious clone, a GFP tagged wild-type RTP was localized to discontinuous punctate spots along the cell periphery and was associated with plasmodesmata, although localization was dependent upon the developmental stage of the plant tissue. In contrast, the Δ5aa-RTP-GFP aggregated in the cytoplasm. Structural modeling indicated that the 5aa deletion would be expected to perturb an α-helix motif. Two of 30 plants infected with Δ5aa-RTP developed a wild-type virus infection phenotype ten weeks post-inoculation. Analysis of the virus population in these plants by deep sequencing identified a duplication of sequences adjacent to the deletion that were predicted to restore the α-helix motif. The subcellular distribution of the RTP is regulated by the 5-aa motif which is under strong selection pressure and in turn contributes to the efficient long distance movement of the virus and the induction of systemic symptoms.

Fig 1. The effect of C-terminal RTP mutants on the accumulation of virus in different host plants.

(A) Genome organization of PLRV and a description of the readthrough deletion mutants used in this study. (B) Virus titer measured by DAS-ELISA at 5 wpi in three immature leaves collected from each of 15 hairy nightshade plants systemically infected with wild-type (WT) PLRV and the C-terminal mutants shown in panel A and a mutant (Mut-5670-TAA that had TAA stop codon inserted at nucleotide 5670 that would prevent the translation of the five amino acid domain. (C and D) Virus titer measured by DAS-ELISA at 5 wpi in three immature leaves collected from each of 15 N. benthamiana (C) and P. floridana (D) plants systemically infected with WT-PLRV or each of the three C-terminal mutants that defined the 5-aa motif. Letters above the bars in (B), (C), (D) indicate significant differences revealed by Dunn’s multiple comparisons test p<0.05.

Fig 2. The 5-aa motif does not affect virus multiplication or readthrough protein translation.

(A) Virus titer measured 3 dpi by DAS-ELISA on N. benthamiana leaves (n = 3) infiltrated with wild-type (WT) PLRV and the three C-terminal RTD mutants that defined the 5-aa domain. Letters above the bars indicate significant differences revealed by Dunn’s multiple comparisons test p<0.05. (B) Western blot analysis of total protein extracts prepared from N. benthamiana leaves (n = 3) infiltrated three days prior with wild-type (WT) PLRV, three C-terminal mutants, and agrobacterium only (mock). Signal band quantification was measured with ImageJ software (https://imagej.nih.gov/ij/). Levels of RTP (Rel. RTP/CP) detected in tissues infected with the mutants relative to WT-PLRV (set at 100) were calculated as the ratio of RTP/CP. Values represent the means (± standard error) determined from three independent experiments. (C) Northern blot analysis of total RNA extracted 3 dpi from N. benthamiana leaves (n = 3) infiltrated with WT- PLRV, two C-terminal mutants, and agrobacterium only (mock). The positions of the PLRV genomic and subgenomic RNA1 are indicated. (D) Average virus titer measured by DAS-ELISA at 5 wpi in three immature leaves collected from each of 15 hairy nightshade plants systemically infected with WT-PLRV or the NoORF7 mutant.

Fig 3. The 5-aa deletion mutants are inefficient in phloem loading.

(A and B) Representative immunoprints of petiole tissue from hairy nightshade (A) and N. benthamiana (B) plants agroinoculated 5 and 7 days prior, respectively, with wild-type (WT) PLRV or readthrough protein mutants (Mut-Δ5670, Mut-Δ5685, Mut-Δ5670–5685). Tissue prints were developed with antibodies to PLRV and virus was visualized as blue-stained foci of indoxyl-precipitate. The prints were photographed under a microscope at a magnification of 50 X. The graphs to the right of the tissue prints indicate the average number and standard error of virus infection foci counted under the microscope in 10 petioles. Letters indicate significant differences as determined by Dunn’s multiple comparisons test p<0.05.

Fig 4. Sequence duplications within the deletion mutants can restore wild-type PLRV infection phenotypes.

(A) Sequence of an 85 nucleotide insertion found between nt 5670 and 5740 in virus recovered from two of 30 plants infected with the Mut-Δ5670 that developed a WT-PLRV infection phenotype 10–12 wpi (Rev1, Rev2). The blue box on the schematic of the WT-PLRV genome indicates the origin of the 85 nucleotide duplicated sequence that was inserted after nt 5670 of the revertant viruses. The black line above the nucleotide sequence indicates a stop codon. The amino acids encoded by the insertion in the revertants (rev) and the WT virus are provided. (B) Percentage of the revertant virus population as determined by Amplicon-seq that contained either the 85 nt insertion, the original Mut-Δ5670 sequence, or other various insertions. (C) Number of virus infection foci counted 7 dpi in tissue prints from petioles of 10 leaves infected with WT-PLRV, Mut-Δ5670, a mutant with the 85 nucleotide insertion (Rev-85nt) and a mutant with a 24 nucleotide insertion (Rev-8aa) encoding the 8 amino acids defined in (A). (D) Average virus titer measured 5 wpi by DAS-ELISA in hairy nightshade leaves (n = 3) from 15 plants infiltrated with the mutants used in (C). Letters above the bars indicate significant differences as determined by Dunn’s multiple comparisons test p<0.05.

Fig 5. The role of aromatic residue and associated helix structure in PLRV infection of HNS.

(A) Alignment of the 5-aa motifs in 32 PLRV RTD sequences available in Genbank. Each logo consists of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino at that position. The red rectangular box delineates the 5-aa motif, and the arrows indicate the position of the first two amino acids. (B) Description of helix and the aromatic residue mutants and their ability to infect and induce symptom in hairy nightshade plants (n: no interveinal chlorosis symptom). (C) Virus titer of each of the mutants was measured by DAS-ELISA at 5 wpi in immature systemically infected leaves (n = 3) from each of 15 hairy nightshade plants. Letters above the bars indicate significant differences revealed by Dunn’s multiple comparisons test p<0.05.

Fig 6. Cellular localization of GFP fluorescence infiltrated with GFP-RTP or GFP-RTP (Δ5AA) plus the infectious PLRV clone.

(A) Full length WT-RTP sequence and the RTP sequence from mutant Mut-Δ5670–5685 (RTP-Δ5AA) were fused with GFP (C terminal fusion) and 35S promoter sequence and agroinfiltrated into N. benthamiana in source leaves either alone (a-b) or co-infiltrated with a full-length WT-PLRV infectious clone (d-e). GFP fluorescence was visualized by confocal microscopy 3 dpi. Arrows the nucleus (a-b) or aggregates near the cell periplasm (d-e), respectively. Bars = 10 μm. The last column shows the images of GFP-RTP+PLRV (c) and GFP-RTP(Δ5AA) +PLRV (f) magnified from white box regions in d and e, respectively. (B) The graph indicates the average number of inclusion-like bodies counted under the microscope from 30 single sections from an average of 20 cells. Membrane targeting was strictly controlled on single sections. Significance was determined by a t-test.

Fig 7. Association of RTP inclusion-like bodies (ILBs) with plasmodesmata.

The first to third columns show GFP, RFP, and GFP and RFP merged image fluorescence, respectively observed in the same cells. (A-C) GFP-RTP was co-infiltrated with P17-mCherry into mature N. benthamiana leaves. A, GFP-RTP; B, P17-mCherry; C, Overlay of A and B. Arrow in B indicates P17-mCherry labeled plasmodesmata. (D-F) GFP-RTP and P17-mCherry co-infiltrated with the PLRV infectious clone into mature N. benthamiana leaves. Arrows indicate the RTP ILBs that co-localize with P17. (G-I) GFP-RTP was co-infiltrated with the PLRV infectious clone and the plasmodesmata marker mCherry-PDLP1. Yellow arrows represent the ILBs that co-localized with PDLP1-labeled plasmodesmata; white arrows represent the ILBs that are independent of PDLP1-labeled plasmodesmata. Bar = 20 μm. (J to L) Co-localization of mCherry-PDLP1 with GFP-RTP plus PLRV infectious virus in plasmolyzed cells of N. benthamiana. J, GFP-RTP; K, mCherry-PDLP1; L, Overlay of J and K. N. benthamiana cells were plasmolyzed by infiltration of 30% glycerol. Yellow arrows represent the RTP ILBs that co-localized with PDLP1-mCherry. Bar = 5 μm.

Fig 8. The effect of the aromatic residue and the C-terminal helix in RTP localization when agroinfiltrated with infectious PLRV.

(A) Cellular localization of GFP fluorescence in mature N. benthamiana leaves agroinfiltrated with a full-length infectious PLRV clone and GFP-RTP fusions using RTP sequences from WT-PLRV (a), Rev-8AA (b), Mut-LYE (c), Mut-PFG (d), Mut-AAA (e), and Mut-PGE (f). GFP fluorescence was visualized by confocal microscopy at 3 dpi. Arrows indicate inclusion like bodies (ILBs) along the cell periplasm or in the cytoplasm. Bar = 10 μm. (B) The average number of ILBs were determined in 12 cells and counted in each of 30 single sections representing each cell. ILBs were categorized as being located along the periplasm or in the cytoplasm and the ratio of ILBs in the periplasm/cytoplasm, with that for wild-type GFP-RTP set as 1 is shown. Error bars indicate the standard deviation for 12 individual cells.