PsCRN108 is a <i>P</i>. <i>sojae</i> secreted effector that acts in the nucleus of plant cells.

<p>(A) Yeast invertase secretion assay of the predicted signal peptide of PsCRN108. The predicted signal peptide sequences and the following two amino acids (1–23) of PsAvr1b was used as the positive control to assay the predicted signal peptide sequences and the following four amino acids (1–21) of PsCRN108. CMD-W (minus Trp) plates were used to select yeast strain YTK12 carrying the pSUC2 vector. YPRAA media was used to indicate invertase secretion. An enzymatic activity test based on reduction of TTC to red-colored formazan was used to confirm invertase secretion. <b>(B)</b> Western blot analysis of proteins in culture supernatants of the <i>P</i>. <i>sojae</i> transgenic lines. PsCRN108:Avr1bCt indicates the full length of PsCRN108 fused to the Avr1b C-terminal regions (Avr1bCt aa 66–138, lacking the predicted signal peptide and RxLR host translocation domain). A <i>P</i>. <i>sojae</i> line expressing Avr1b:mRFP was used as the positive control. The His tag was attached to the C-terminus of all the fusion proteins. The non-transformed <i>P</i>. <i>sojae</i> P6497 line was used as the antibody-specificity control. Proteins extracted from mycelia (M) and culture supernatants (S) were analyzed by Western blotting using anti-His or anti-Actin antibodies. The protein sizes are expressed in kDa. (C) Western blot analysis of proteins isolated from <i>P</i>. <i>sojae</i> transgenic lines during infection. Avr1bCt:mRFP indicates Avr1b C-terminal regions fused to mRFP and is used as a control. The infected hypocotyl tissues (HARO13) including mycelia of <i>P</i>. <i>sojae</i> transgenic lines were collected at 12 hpi for protein extraction, and then Western blotting was performed using anti-His antibodies. The protein sizes are expressed in kDa. PS, Ponceau S staining. (D) Hypocotyl-inoculation assay using two soybean cultivars. Two soybean cultivars, HARO13 (<i>Rps1b</i>) and Williams (<i>rps</i>), were inoculated with the indicated <i>P</i>. <i>sojae</i> transgenic lines. Photographs were taken 2 dpi. Data (e.g. 2/83) indicate the number (2) of dead plants out of the total number (83) of plants tested. (E) Western blot analysis of GFP fusion proteins transiently expressed in <i>N</i>. <i>benthamiana</i>. The indicated proteins were extracted from leaves at 48 h after agoinfiltration and detected using anti-GFP antibodies. GFP:PsCRN108-NLSm and GFP:PsCRN108-HhHm are described in Fig 3. Dotted line indicates lanes not adjacent on the gel. The protein sizes are expressed in kDa. PS, Ponceau S staining (F) Nuclear localization of PsCRN108 in <i>N</i>. <i>benthamiana</i>. Epidermal cells of <i>N</i>. <i>benthamiana</i> leaves transiently expressing GFP alone or GFP:PsCRN108 were observed using confocal microscopy at 2 days post infiltration. DAPI staining was used to visualize the nuclei. Scale bars = 50 μm (upper panel) and 10 μm (lower panel).