Fig. 2: P7 promotes ToCV infection by activating the MAPK pathway and is associated with membrane localization of P7.

a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of all differentially expressed genes in OE-P7 N. benthamiana plants. b The key genes in the MAPK signalling pathway verified by qRT-PCR. Nbactin was used as the internal reference gene to normalize relative expression. Each treatment included three biological replicates. Values are means ± SDs of three biological replicates (one-sided t-test, P = 0.0095, 0.0308, 0.0494, 0.0326, respectively). *P < 0.05, **P < 0.01. c The phosphorylation level of the MAPK pathway was detected by western blot using an anti-pTEpY antibody. Expression of P7 was detected by western blot analysis with an anti-P7 antibody. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. The molecular weight of P7 is approximately 10 kDa. d Symptoms of P7-enhanced PVX-infected N. benthamiana plants (scale bar = 1 cm). e Accumulation of PVX CP in the assayed N. benthamiana samples was determined through western blot analysis using a PVX CP-specific antibody. Coomassie blue (CBB) staining was used to assess protein loading. The molecular weight of the PVX CP is 30 kDa. f The CP expression level was quantified by qRT-PCR using PVX CP gene-specific primers. Data were presented as means ± SDs and were analysed using Student’s t-test (one-sided, P = 0.0037, 0.0096, 0.0010, respectively). Each treatment had three biological replicates. **P < 0.01. g The subcellular location of P7 tagged with GFP in mesophyll cells was determined by laser scanning confocal microscopy (scale bar = 50 μm). The first and second columns show the subcellular localization of P7 and AtPIP2A determined by the GFP and mCherry channels, respectively. The third column shows the subcellular localization of P7 and AtPIP2A determined by merging the GFP and mCherry channels, with a magnified view inserted. The fourth column shows overlapping fluorescence spectra analysis of P7-GFP and AtPIP2A-mCherry signals marked with white arrows. h Analysis of the subcellular location of P7 tagged with GFP by western blot using proteins extracted from cell fractions. T, total protein; M, membrane protein; C, cytoplasmic protein; N, nuclear protein. H⁺-ATP served as a plasma membrane marker. The molecular weights of P7-GFP and H⁺-ATP are 37 and 100 kDa, respectively. i The subcellular location of mP7 tagged with GFP in mesophyll cells (scale bar = 50 μm). mP7-GFP and AtPIP2A-mCherry were transiently coexpressed in leaves of N. benthamiana. The first and second columns show the subcellular localization of mP7 and AtPIP2A determined by the GFP and mCherry channels, respectively. The third column shows the subcellular localization of mP7 and AtPIP2A determined by merging the GFP and mCherry channels, with a magnified view inserted. The fourth column shows overlapping fluorescence spectra analysis of mP7-GFP and AtPIP2A-mCherry signals marked with white arrows. j Subcellular location analysis of mP7 tagged with GFP by western blot using proteins extracted from cell fractions. T total protein; M membrane protein; C cytoplasmic protein; N nuclear protein. Actin served as a cytoplasmic marker. H3 served as a nuclear marker. The molecular weights of mP7-GFP and H3 are 37 and 17 kDa, respectively. k Symptoms of N. benthamiana plants infiltrated with ToCV and ToCV^mP7 at 30 dpi (scale bar = 3 cm); a magnified view is shown. N. benthamiana plants were infiltrated with Agrobacterium containing empty vector (EV) as a mock control. l The ToCV CP mRNA level quantified by qRT-PCR. Data are presented as means ± SDs and were analysed using Student’s t-test (one-sided, P = 0.0008). Each treatment had three biological replicates. ***P < 0.001. m Accumulated levels of ToCV or MAPK phosphorylation were detected by western blot analysis with an anti-CP or anti-pTEpY antibody. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. For (g–j) the experiments were repeated three times with similar results. Source data are provided as a Source Data file.