Fig. 3: NbPEN3-dependent callose deposition determines phloem restriction of TbCSV.
From: Defence-mediated phloem restriction of a plant virus facilitates insect transmission
a Cellular distribution of NbPEN3-GFP in epidermal cells of H2B-RFP transgenic N. benthamiana plants. Scale bar = 50 μm. b Immunofluorescence detection of NbPEN3 in leaf vein sections of wild-type (WT) and nbpen3 knock-out N. benthamiana plants. The distribution of NbPEN3 was visualized in a cross-section using an antibody against PEN3 (green). Autofluorescence of highly lignified tissues is shown in blue. PC, parenchyma; P, phloem; X, xylem. Scale bar = 50 μm. c Viral symptoms induced by TbCSV(Y35), TbCSV(Y41), and TbCSV(Y35-2C4) in WT and nbpen3 knock-out N. benthamiana plants at 14 days post-inoculation (dpi). d Immunoblot analysis of TbCSV(Y35), TbCSV(Y41), and TbCSV(Y35-2C4) coat protein (CP) accumulation in WT and nbpen3 knock-out N. benthamiana plants at 14 dpi. TbCSV CP was detected using a monoclonal antibody. Actin was used as a loading control. e Immunofluorescence detection of TbCSV in leaf vein sections of WT and nbpen3 knock-out N. benthamiana plants. The distribution of TbCSV(Y35), TbCSV(Y41), or TbCSV(Y35-2C4) was visualized in a cross-section using an antibody against the TbCSV CP (red). Autofluorescence of highly lignified tissues is shown in blue. White arrowheads indicate TbCSV in parenchyma cells. Scale bar = 50 μm. In TbCSV(Y35)-infected pen3-ko N. benthamiana plants, over 50% of images show virus signal in parenchyma cells. At least eleven individual samples of TbCSV(Y35)-infected pen3-ko N. benthamiana plants, ten individual samples of TbCSV(Y41)-infected pen3-ko N. benthamiana plants and twelve individual samples of TbCSV(Y35-2C4)-infected pen3-ko N. benthamiana plants from three independent biological replicates were used for immunochemistry and statistical analysis. f Callose deposition in leaf vein sections of WT and nbpen3 knock-out N. benthamiana plants infected by TbCSV(Y35), TbCSV(Y41), or TbCSV(Y35-2C4) at 14 dpi. Callose was stained by using aniline blue. White arrowheads indicate virus-induced callose deposition around the vasculature. Scale bar = 50 μm. Experiments in (a, c–f) were repeated at least three times with similar results.
