constructs plx-(pvy p1-6x-hcpro)-ppv  (HCPro Inc)

Journal: Plants

Article Title: Adaptation of a Potyvirus Chimera Increases Its Virulence in a Compatible Host through Changes in HCPro

doi: 10.3390/plants11172262

Figure Lengend Snippet: Schematic representation of the genomic organization of the viruses used in this study. A plum pox virus (PPV)-based construct encoding a GFP-expressing infectious PPV variant, PPV-GFP (PLX-PPV; Pasin et al., 2017), and three constructs expressing chimeric viruses, in which the P1-HCPro bi-cistron of PPV had been replaced by that of a Scottish isolate of Potato virus Y (PVY), shown in red. In two chimeric constructs, the PVY HCPro was expressed either with or without a 6x Histidine tag fused at its N-terminus (chimera with or without 6x His tag). A third construct expressed a viral chimera in which nt 2053 was modified, expressing a 6x His-tagged HCPro with a single substitution at amino acid 352: Ile (A U U) →Thr A C U) (chimera 2053Ile→Thr). All virus variants carry an additional cistron that expresses GFP.

Article Snippet: To do this we compared the virulence in N. benthamiana plants agroinfiltrated with constructs pLX-(PVY P1-6x-HCPro)-PPV vs. pLX-(PVY P1-6x-HCPro 532Ile→Thr )-PPV, expressing the unmodified chimera, or chimera 352 Ile→Thr.

Techniques: Virus, Construct, Expressing, Variant Assay, Modification

Journal: Plants

Article Title: Adaptation of a Potyvirus Chimera Increases Its Virulence in a Compatible Host through Changes in HCPro

doi: 10.3390/plants11172262

Figure Lengend Snippet: The oligonucleotides used to amplify by RT-PCR the three segments of the chimera genome.

Article Snippet: To do this we compared the virulence in N. benthamiana plants agroinfiltrated with constructs pLX-(PVY P1-6x-HCPro)-PPV vs. pLX-(PVY P1-6x-HCPro 532Ile→Thr )-PPV, expressing the unmodified chimera, or chimera 352 Ile→Thr.

Techniques: Sequencing, Construct

Journal: Plants

Article Title: Adaptation of a Potyvirus Chimera Increases Its Virulence in a Compatible Host through Changes in HCPro

doi: 10.3390/plants11172262

Figure Lengend Snippet: Changes in chimera titers after several consecutive mechanical passages through Nicotiana benthamiana plants: ( a ) Schematic representation of the procedure followed: after agroinfiltration of wild type (wt) plants with binary constructs pLX-(PVY P1-6x-HCPro)-PPV or pLX-(PVY P1-HCPro)-PPV, expressing infectious chimeras (generation 1), extracts from these infected plants were used to mechanically inoculate healthy plants (generation 2), which were used in turn to inoculate the next round of plants, creating generations 3→4→5, successively. Two Infected plants A and D that were subsequently used to sequence the genomes of the chimera viruses infecting them are highlighted in green; ( b ) RT-qPCR quantitation of relative systemic titers of genomic viral RNAs of the chimeras found in plants inoculated with extracts from plants A and D (generation 4 and 5 chimeras) vs. those found in generation 1 agroinfiltrated plants. They are all relativized to the levels of PPV-GFP at 7 dai (value 1). After the mechanical passages, chimera titers in systemic plant tissues increased significantly in wt plants. Titers of the passaged chimeras were significantly lower in DCL2/4.5i plants than in wt plants. Different letters indicate significant differences ( p -value < 0.05; Dunett’s T3 test).

Article Snippet: To do this we compared the virulence in N. benthamiana plants agroinfiltrated with constructs pLX-(PVY P1-6x-HCPro)-PPV vs. pLX-(PVY P1-6x-HCPro 532Ile→Thr )-PPV, expressing the unmodified chimera, or chimera 352 Ile→Thr.

Techniques: Construct, Expressing, Infection, Sequencing, Quantitative RT-PCR, Quantitation Assay

Journal: Plants

Article Title: Adaptation of a Potyvirus Chimera Increases Its Virulence in a Compatible Host through Changes in HCPro

doi: 10.3390/plants11172262

Figure Lengend Snippet: Amino acid sequence and substitutions at the C-terminal HCPro/P3 cleavage sites of the parental viruses and the chimeras, as described in . A single Arg to Leu substitution (position P3) in the HCPro expressed by the chimeras differentiates it from the parental PVY HCPro because of the use during the cloning of the binary constructs encoding them maintaining of a SexA I site present in PLX-PPV-GFP ( ACCWGGT . W: A or T) was required. However, RT-PCR showed that in the generation 4 chimera infecting plant D nucleotide substitution 2359 led to HCPro amino acid 454 substitution Leu (C T G)→Arg (C G G), which restored the intact amino acid sequence of PVY HCPro in this chimera, although not its nucleotide sequence. DNA sequences are shown. P′1 and P′2 indicate the 1st and 2nd amino acid positions at the P3 viral protein.

Article Snippet: To do this we compared the virulence in N. benthamiana plants agroinfiltrated with constructs pLX-(PVY P1-6x-HCPro)-PPV vs. pLX-(PVY P1-6x-HCPro 532Ile→Thr )-PPV, expressing the unmodified chimera, or chimera 352 Ile→Thr.

Techniques: Sequencing, Cloning, Construct, Reverse Transcription Polymerase Chain Reaction

Journal: Plants

Article Title: Adaptation of a Potyvirus Chimera Increases Its Virulence in a Compatible Host through Changes in HCPro

doi: 10.3390/plants11172262

Figure Lengend Snippet: Nucleotide variability in agroinfiltrated, generation 1 plants systemically infected with chimeras. ( a ) Detected in four separate deep sequencing libraries of viral small RNA reads: two from plants infected with (PVY P1-HCPro)-PPV-GFP, and two from plant infected with (PVY P1-6x-HCPro)-PPV-GFP. Single nucleotide polymorphisms common to the 4, 3 or 2 libraries, as well as their occurrence along the viral non-coding-regions (NCRs) and cistrons are indicated. ( b ) The four nucleotide polymorphisms common to the 4 libraries were located in the 5′NCR and in the HCPro cistron.

Article Snippet: To do this we compared the virulence in N. benthamiana plants agroinfiltrated with constructs pLX-(PVY P1-6x-HCPro)-PPV vs. pLX-(PVY P1-6x-HCPro 532Ile→Thr )-PPV, expressing the unmodified chimera, or chimera 352 Ile→Thr.

Techniques: Infection, Sequencing