Figure 7. Interaction of AtFH2 with actin filaments is required for its function in regulating the permeability of PD.

(A) Protein sequence alignment of the FH2 domains in AtFH2 and three other well-characterized formins. Ile519 (I) and Lys672 (K) in AtFH2 (red triangles) correspond to Ile1431 and Lys1601 in Bni1p. These residues were previously shown to be important for the dimer formation and actin nucleation activity of Bni1p (Xu et al., 2004). S. cerevisiae Bni1p (Bni1), P41832; human diaphanous protein 1 (Dia1), O60610; AtFH1, Q9SE97. (B) SDS-PAGE analysis of recombinant AtFH2-FH2 and AtFH2-FH2M. Lane 1, GST-AtFH2-FH2; lane 2, GST-AtFH2-FH2M. (C) The effect of AtFH2-FH2 and AtFH2-FH2M on spontaneous actin assembly. The conditions for actin assembly are described in Figure 5C. (D) The effect of AtFH2-FH2 and AtFH2-FH2M on seeded actin elongation. The conditions for actin assembly are described in Figure 6A. (E) Elution volume of AtFH2-FH2 (red line) and AtFH2-FH2M (blue line) by size exclusion chromatography. To exclude the potential interference of GST in dimer formation, His-tag fusion proteins were generated. The inset shows SDS-PAGE analysis of 6His-AtFH2-FH2 and 6His-AtFH2-FH2M. The estimated molecular weight is about 100 kDa for AtFH2-FH2 and 50 kDa for AtFH2-FH2M. (F) Subcellular localization of AtFH2M-eGFP and CMV MP-mCherry in epidermal pavement cells of N. benthamiana leaves. Bar = 10 μm. (G) Quantification of eGFP diffusion cell layers in Arabidopsis leaf epidermal cells. AtFH2M represents AtFH2p:AtFH2M;atfh2, and AtFH2-eGFP represents AtFH2p:AtFH2-eGFP;atfh2. The number of diffusion cell layers at 24 hr and 48 hr was plotted. Error bars represent SE, n > 30, **p<0.01 by Mann-Whitney U test. ND, no statistical difference. The experiment was repeated at least three times. (H) Quantification of Fny-CMV infection rates in Arabidopsis plants. The number of symptomatic plants (with curly leaves) was counted at different days and the relative proportion of symptomatic plants was plotted.