Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone

Abstract

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Keywords: Prune dwarf virus; fruit tree virus; ilarvirus; infectious clone; next generation sequencing.

Figure 1

Source and genome structure of PDV. (A) A healthy, asymptomatic leaf from a cherry tree showing an even distribution of green colouring, absence of damage or other deformations. (B) A symptomatic leaf from a cherry tree showing symptoms commonly associated with viral infection including chlorosis (yellowing) and uneven distribution of green colouring, vein suturing and leaf curl. (C) A diagram showing the genome structure of PDV. Every genomic ssRNA( + ) fragment is shown with approximate lengths in parentheses. Encoded proteins are shown as gray boxes. All RNA fragments have putative m7G cap structures at the 5′ UTR, and each 3′ UTR is predicted to adopt complex secondary structures. RNA 1 encodes the P1 protein which has Met and Hel domains which are essential for viral replication. RNA 2 encodes the RdRp (P2). RNA 3 directly encodes the MP. The fourth RNA fragment, sgRNA 4 is transcribed from RNA 3 downstream of ORF3a and encodes the viral CP. m7G: 5′-7-methyl-G cap; :3′ UTR secondary structure; aa: amino acids; bp: base pair; P1: replicase protein; P2: RNA dependent RNA polymerase (RdRp); MP: movement protein; CP: coat protein.

Figure 2

Schematic diagram of the infectious clone of PDV for in plant studies. (A) Tripartite infectious clone of PDV is comprised of cDNAs of each PDV genomic RNA separately inserted into the vector pCB-Rz. (B) cDNAs of PDV genomic RNA fragments 1 and 2 were combined into a single vector to increase the infectivity rate of the PDV infectious clone. Single black lines and lined boxes represent noncoding and coding regions of each RNA fragment, respectively. The protein encoded by each coding region is labelled: P1 (replicase), P2 (RdRp), MP and CP. Identified functional domains of proteins are shown as grey boxes. Transcription start sites are shown at the 5′ end of each construct by a bent arrow following the promoter (arrows containing “35 s”) sequence. At the 3′ sequence the uppercase and lowercase letters represent the 3′ sequence of viral RNA and the non-viral sequence of the hammerhead ribozyme (box containing “RZ”), respectively. The bent arrow at the 3′ end indicates the self-cleavage site of the ribozyme. The nucleotide length of RNA1, RNA2 and RNA3 is shown to the right with the number of additional nucleotides after ribozyme self-cleavage provided in parentheses. The single NarI restriction enzyme recognition sequence is shown in pPDV1-301 which was used for the integration of the cDNA corresponding to the viral genomic RNA2 resulting in the construction of pPDV1&2-301.

Figure 3

The PDV infectious clone is infectious on its natural host sweet cherry. Seedlings of cherry were agroinfiltrated with the PDV infectious clone to test the infectivity of this construct on the natural host. (A) Side view of seedlings infiltrated with the PDV infectious clone and empty vector (mock). Seedlings infected by PDV (left) have shorter internodal lengths resulting in a dwarfed or stunted phenotype compared to mock treated plants (right). (B) Aerial view of seedlings infiltrated with the PDV infectious clone or empty vector (mock). PDV infected seedlings (left) produce fewer leaves compared to mock treated plants (right). (C) The presence of PDV in upper non-infiltrated leaves of dwarfed cherry plants (left) was detected by RT-PCR. Lane 1: 1000 bp DNA ladder; 2: non infiltrated distal leaf of PDV infected cherry seedling at 8 wpa; 3: distal leaf of mock treated plant at 8 wpa; 4: a sample of foliar tissue known to be infected with PDV served as a positive control; 5: water was used as a negative control to test PCR reactions. (D) DAS-ELISA confirmed the presence of PDV in the distal, non-infiltrated leaf samples of the seedlings agroinfiltrated with the PDV infectious clone. The absence of PDV was confirmed in mock treated plants. The error bars represent the standard deviation of the means (n = 5). This experiment was performed twice, and each experiment consisted of 5 seedlings receiving each treatment.

Figure 4

PDV does not infect Arabidopsis and infects N. benthamiana latently. The cDNA clone of PDV was used to infiltrate commonly used experimental host plants. (A) Arabidopsis seedlings agroinfiltrated with the full-length cDNA clone of PDV (right) did not develop any visible symptoms and could not be differentiated from mock inoculated plants (left) at 21 dpa. (B) N. benthamiana plants agroinfiltrated with the full-length cDNA clone of PDV did not develop any visible symptoms and could not be differentiated from mock treated plants at 21 dpa. (C) RT-PCR was used to detect PDV in both plants. The coding sequence of the CP was amplified as 900 bp in size. Lane 1: 1000 bp DNA ladder; 2: locally infiltrated Arabidopsis rosette leaf at 7 dpa; 3: distal leaf of PDV infiltrated Arabidopsis at 21 dpa; 4: mock infiltrated rosette leaf of Arabidopsis; 5 distal leaf of mock infiltrated plant; 6: upper leaf of PDV infiltrated N. benthamiana plant at 21 dpa; 7: upper leaf of mock infiltrated N. benthamiana plant at 21 dpa; 8: A sample of foliar tissue known to be infected with PDV served as a positive control; 9: water was used as a negative control to test PCR reactions. (D) Relative levels of PDV accumulation in locally infiltrated leaves of Arabidopsis at 7 dpa and distal leaf tissues of Arabidopsis and N. benthamiana at 21 dpa were determined by DAS-ELISA. Error bars represent the standard error of the means. Arabidopsis plants were maintained for a total of 5 weeks after infiltration, studies on Arabidopsis were performed as 3 separate experiments consisting of 12 Arabidopsis seedlings receiving each treatment per experiment. N. benthamiana plants were maintained for a total of 5 weeks after infiltration and studies on N. benthamiana were performed as 3 separate experiments. During each experiment, 12 N. benthamiana seedlings received each treatment. Mock inoculated Arabidopsis and N. benthamiana plants did not generate PDV specific amplicons by RT-PCR nor were positive results obtained by DAS-ELISA from these plants.

Figure 5

Cucumber serves as a symptomatic host of PDV. To evaluate the use of cucumber as an experimental host for PDV infection, cotyledons of cucumber cv. ‘Wisconsin’ were agroinfiltrated with the full-length cDNA clone of PDV. Left, mock-inoculated; middle, agroinfiltrated with the PDV infectious clone; right, enlarged true leaf of the middle plant. (A) At 7 dpa, PDV symptoms are visible on the newly emerging first true leaves as small chlorotic spots and some vein clearing (red arrows). (B) At 10 dpa chlorotic leaf spotting is present on the fully expanded first true leaf (red arrows). (C) At 12 dpa the second true leaf of PDV infected cucumber exhibits stronger symptoms including chlorosis and leaf deformation (red arrows). (D) Isometric shaped virions isolated from PDV infected cucumber leaves were visualized by transmission electron microscopy. (E) The presence of PDV in upper non-inoculated leaves of symptomatic cucumber plants was detected by RT-PCR. Lane 1: 1000 bp DNA ladder; 2: non infiltrated first true leaf of PDV infected plant at 12 dpa; 3: distal leaf of mock treated plant at 12 dpa; 4: A sample of foliar tissue known to be infected with PDV served as a positive control; 5: water was used as a negative control to test PCR reactions. (F) Relative levels of PDV accumulation in upper, non-infiltrated leaves were determined by DAS-ELISA. Error bars represent the standard deviation of the mean of 9 seedlings for each treatment. This study was performed in 3 independent experiments consisting of 9 cucumber seedlings receiving each treatment during each experiment. For all studies, mock inoculated plants did not generate PDV specific amplicons nor positive results by DAS-ELISA.

Figure 6

PDV derived from the cDNA clone is mechanically transmissible. The cotyledons of squash and cucumber seedlings were mechanically inoculated with leaf tissues from healthy control plants or with the symptomatic leaves of PDV infected cucumber plants. (A) PDV symptoms are clearly seen as chlorotic leaf spots at 9 dpi (red arrows). (B) When squash seedlings were mechanically inoculated at 9 dpi chlorotic spots were seen at the margins of the first true leaves (red arrows). (C) DAS-ELISA was used to confirm PDV infection and was used to compare relative abundance of PDV at nine dpi. Error bars represent the standard deviation of the mean. Studies on mechanical transmission were performed as three independent experiments each consisting of 5 seedlings of each species for each treatment during each experiment. In all experiments, no positive results were obtained by DAS-ELISA from mock inoculated plants.