Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2024 Jun 13;15:5059. doi: 10.1038/s41467-024-49504-6

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 4 — a Yeast two-hybrid analysis of the interaction between BnaA07.MKK9^KR and BnaC03.MPK3/BnaC03.MPK6. Yeast cells were grown on synthetic defined (SD)-Leu-Trp or SD-Leu-Trp-His-Ade/X-α-Gal medium. BD DNA-binding domain, AD activation domain. BD-53/AD-RecT was the positive control. BD-Lam/AD-RecT was the negative control. b Luciferase complementation imaging in N. benthamiana cells confirmed the interaction between BnaC03.MPK3/BnaC03.MPK6 and BnaA07.MKK9^KR, with luciferase signals recorded at 40 h post-infiltration (hpf), scale bar: 1 cm. c, d Co-immunoprecipitation assays using anti-flag beads on protein extracts from N. benthamiana leaves showed the association of BnaA07.MKK9^KR with BnaC03.MPK3/6. Western blotting with anti-flag and anti-myc antibodies was conducted on the input and immunoprecipitated proteins, respectively. e, f In vitro pull-down assays further revealed direct interactions between GST-tagged BnaA07.MKK9^DD and His-tagged BnaC03.MPK3/BnaC03.MPK6, which were pulled down with anti-GST antibodies and analyzed by immunoblotting (IB) with anti-GST and anti-His antibodies, respectively. g, h Kinase assays conducted in vitro indicated that BnaA07.MKK9^DD phosphorylates BnaC03.MPK3/BnaC03.MPK6, with phosphorylation detected by IB using the anti-pTEpY antibody and the input proteins detected with anti-GST and anti-His antibodies. i In Bnamkk9 mutants, phosphorylation of BnaMPK3/6 was reduced compared to J9712 controls after S. sclerotiorum inoculation, with phosphorylation detected by IB with the anti-pTEpY antibody and protein levels detected with anti-BnaMPK6 and anti-BnaMPK3 antibodies. Actin served as the loading control, with Atmpk3 and Atmpk6 mutants providing a reference for the phosphorylation bands of MPK6 and MPK3. Hr+S.s represents the time, in hours, at which inoculation with S. sclerotiorum was conducted. In (c–i), the molecular mass markers in kilodaltons are shown on the left. All experiments were repeated three times, yielding consistent results. Source data are provided as a Source Data file.