Fig. 2: The ability of TbCSV to express a chloroplast-localized C4 compromises phloem restriction.
From: Defence-mediated phloem restriction of a plant virus facilitates insect transmission
a Viral symptoms induced by TbCSV(Y35), TbCSV(Y41), and TbCSV(Y35-2C4) in Nicotiana benthamiana plants at 14 days post-inoculation (dpi). b Southern blot analysis of TbCSV(Y35), TbCSV(Y41), and TbCSV(Y35-2C4) accumulation in N. benthamiana plants at 14 dpi. Total DNA was hybridized with a TbCSV CP probe. Total nucleic acids (50 μg DNA) were extracted from N. benthamiana plants inoculated with TbCSV(Y35), TbCSV(Y41), or TbCSV(Y35-2C4) infectious clones at 14 dpi. Total genomic DNA visualized by ethidium bromide staining is shown as a loading control. c Immunofluorescence detection of TbCSV in leaves of N. benthamiana plants infected by TbCSV(Y35), TbCSV(Y41), or TbCSV(Y35-2C4). The distribution of TbCSV(Y35), TbCSV(Y41), or TbCSV(Y35-2C4) was visualized in a cross-section using an antibody against the TbCSV coat protein (CP) (green). d TbCSV strains expressing two C4 variants (Y41 and Y35-2C4) overcome phloem restriction and reach parenchyma tissue in N. benthamiana plants. The distribution of TbCSV(Y35), TbCSV(Y41), or TbCSV(Y35-2C4) was visualized in a cross-section using an antibody against TbCSV CP (red). Autofluorescence of highly lignified tissues is shown in blue. PC, parenchyma; P, phloem; X, xylem. White arrowheads indicate TbCSV in parenchyma cells. Scale bar = 50 μm. Statistical analysis showed that TbCSV(Y35) is strictly limited to the phloem in N. benthamiana plants, but TbCSV(Y41) and TbCSV(Y35-2C4) can be observed overcoming phloem restriction and reaching parenchyma cells in over 70% of the images. At least eight individual samples of TbCSV(Y35)-infected N. benthamiana plants, five individual samples of TbCSV(Y41)-infected N. benthamiana plants and six individual samples of TbCSV(Y35-2C4)-infected N. benthamiana plants from three independent biological replicates were used for immunochemistry and statistical analysis. e Immunofluorescence detection of TbCSV in leaf vein sections of AtSWEET11::GFP transgenic N. benthamiana plants. The distribution of TbCSV(Y35), TbCSV(Y41), or TbCSV(Y35-2C4) and phloem cells was visualized using antibodies against TbCSV CP (red) or GFP (green). Autofluorescence of highly lignified tissues is shown in blue. PC, parenchyma; P, phloem; X, xylem. White arrowheads indicate TbCSV in parenchyma cells. Scale bar = 50 μm. Experiments in (b–d, e) were repeated at least three times with similar results.
