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. 2021 Mar 1;118(10):e2021351118. doi: 10.1073/pnas.2021351118

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Copyright © 2021 the Author(s). Published by PNAS.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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Fig. 5. — SUMOylation facilitates WRKY33 interaction with MPK3. (A) WRKY33 interacts with MPK3 in the transient expression system. Total protein extracted from N. benthamiana leaves transiently expressing GFP-WRKY33 with HA-MPK3 was subjected to IP with anti-GFP beads (IP: αGFP) and immunoblotted with anti-HA (IB: αHA) for HA-MPK3 and anti-GFP (IB: αGFP) for GFP/GFP-fusion proteins. Total protein extracts were probed with anti-HA antibody (HA-MPK3 input) for equal HA-fusion protein. (B) SUMOylation is important for facilitating WRKY33-MPK3 interaction. Total protein from N. benthamiana leaves transiently expressing GFP-WRKY33 or GFP-WRKY33 with HA-MPK3 was subjected to IP with anti-GFP beads (IP: αGFP) and immunoblotted with anti-HA (IB: αHA) for HA-MPK3 and anti-GFP (IB: αGFP) for GFP/GFP-fusion proteins. Total protein extracts were probed with anti-HA antibody (HA-MPK3 input) for equal HA-fusion protein. (C) BiFC analysis to show that WRKY33^3K/R interacted weakly with MPK3 in N. benthamiana leaves. Confocal images of N. benthamiana leaves coexpressing the N-terminal domain of enhanced yellow fluorescent protein (nEYFP):WRKY33 or nEYFP:WRKY333K/R with the C-terminal domain of YFP (cEYFP):MPK3. nEYFP with cEYFP and cEYFP-MPK3 and cEYFP with nEYFP-WRKY33 were used as negative controls. An additional control was used by testing the interaction of MPK3 with WRKY4, another group I WRKY member. YFP channel: reconstituted EYFP; DAPI channel: DAPI; Bright field: transmitted; Merge: superimposition of all the three channels; Magnification: 40×. (D) GFP-WRKY33 immunoprecipitated from spf1 spf2 mutant background interacts more strongly with MPK3. GFP-WRKY33 immunoprecipitated from spf1 spf2 mutant was mixed with recombinantly expressed GST-MPK3 protein followed by pulling down GFP-WRKY33. The sample was immunoblotted with anti-GST antibody (IB: αGST) to probe for GST-MPK3 and anti-GFP antibody (IB: αGFP) for probing WRKY33-GFP. Equal amount of GST-MPK3 was used for all samples and is represented as an input lane. Col-0 was used as negative control.