Figure 3.

Several RIN4 protein family members are not required for the suppression of MPK4/11 activation. A, Phylogenetic analyses of RIN4/NOI protein family members. Bootstrap test values (%) are depicted next to the branches (for details, see Supplemental Methods S1). Boxes mark members where homozygous T-DNA insertion lines were isolated. B, Disappearance of c-myc-RIN4/NOIs in the presence of AvrRpt2 was monitored in protoplasts (rps2) after successive immunoassays with the indicated antibodies (# indicates remnant c-myc-signals after stripping). C, AvrRpt2-mediated suppression of MPK4/MPK11 action was tested in protoplasts prepared from the indicated genotypes as in Figure 1D (15 min after 100 nm flg22 treatment). Note that only noi4, noi7, noi8, noi9, and noi10 are true knockdown/knockout mutants (see quantitative reverse transcription [qRT]-PCR; Supplemental Fig. S3). Experiments were performed three times with similar results. Amido Black staining after immunoblotting (showing the large subunit of Rubisco) was used to demonstrate equal loading.