Functional characterization of the MHD2 motif and the P-loop motif.

<p>(A) Multiple sequence alignment of the MHD motifs from Rp1-D, Rp1-D21 and Rp1-dp2. Positions of the MHD1 and MHD2 motifs are indicated. The numbers indicated the positions of the aspartate (D) corresponding to Rp1-dp2. (B) The MHD1 and MHD2 point mutations in Rp1-dp2 background were generated and transiently expressed in <i>N</i>. <i>benthamiana</i>. (C) Investigating the MHD2 point mutations in V1 and V16, two recombinant constructs that were not autoactive as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004674#ppat.1004674.g006" target="_blank">Fig. 6</a>. (D) Point mutations in P-loop motif of Rp1-D21 and Rp1-dp2(D517V) caused the loss of the ability to induce HR. EV (empty vector) as a negative control. The picture of hypersensitive response (HR) phenotype was taken at 3 days post-infiltration and total protein was detected by anti-HA antibody from agro-infiltrated leaves at 30 hours post infiltration. Equal loading of protein samples was shown by Ponceau-S staining of Rubisco subunit. The experiments were repeated three times with the same results.