The Hypervariable Amino-Terminus of P1 Protease Modulates Potyviral Replication and Host Defense Responses
Figure 5
RNA silencing suppression is impaired by the lack of separation between P1 and HCPro.
(A) The transient RNA silencing assay was done by co-infiltrating N. benthamiana leaf tissue with an Agrobacterium p35S:GFP culture and cultures of Agrobacterium without plasmid (Ф) or containing pSN.5 P1-S (pS, producing P1 S259A+HCPro), pSN.5 P1-ST2A (pST2A, producing P1 S259A+T2A+HCPro), or pSN.5 PPV (pWT, producing wild-type P1+HCPro). Picture was taken under UV light (6 dpa). Scale bar, 1 cm. (B) N. clevelandii plants were agro-inoculated with wild-type PPV and viral clones S (P1 S259 replaced by alanine) and ST2A (P1 S259A+T2A peptide). Viral accumulation in inoculated leaves was assessed by anti-PPV CP (CP) immunoblot analysis (6 dpi); each lane represents a pool of samples used for signal quantification. Relative CP signal intensities were plotted using average PPV value equal to 100. Histograms show mean ± SD (n = 4 samples/condition, from two independent Agrobacterium cultures); ** p<0.01, Student's t-test. Ponceau red-stained blot is shown as loading control. (C) Symptoms and GFP fluorescence of Arabidopsis dcl2/3/4 plant agro-inoculated with PPV mutant clone S (P1 S259 replaced by alanine). Pictures were taken in an epifluorescence microscope at 23 dpi. Scale bar, 1 cm. (D) Anti-PPV HCPro (HC) western blot assay of agro-inoculated Arabidopsis plants. Samples of upper non-inoculated leaves were collected (23 dpi). H, healthy dcl2/3/4 plant; PPV, pool of 16 Col-0 plants challenged with wild-type PPV; S, plants challenged with viral clone S, where Col-0 samples are pools of four plants and dcl2/3/4 are pools of two/three dcl2/3/4 plants showing GFP fluorescence. Ponceau red-stained blot is shown as loading control. (E) RT-PCR of A. thaliana dcl2/3/4 plants challenged with viral clone S (23 dpi). A PPV cDNA fragment encompassing the mutated regions (nt 1–1197 of the PPV genome) was amplified by RT-PCR from a pool of three plants and analyzed by DNA sequencing. Sanger sequencing results of the RT-PCR fragment and of the inoculated plasmid DNA are shown. The wild-type PPV sequence is shown as reference. Nucleotides that differ from the wild-type sequence are indicated in lower case. The nucleotide that leads to reversion of the amino acid codon is underlined. Encoded amino acids are indicated beneath each nucleotide sequence and between box-drawing characters. The mutated residue is in italic (alanine), reverted residue underlined.