Ubiquitylome analysis reveals a central role for the ubiquitin-proteasome system in plant innate immunity

Author:

Ma Xiyu1,Zhang Chao2,Kim Do Young34,Huang Yanyan1,Chatt Elizabeth5,He Ping1ORCID,Vierstra Richard D35ORCID,Shan Libo12ORCID

Affiliation:

1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843

2. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843

3. Department of Genetics, University of Wisconsin–Madison, 425-G Henry Mall, Madison, Wisconsin 53706

4. Advanced Bio Convergence Center, Pohang Technopark, Gyeong-Buk 37668, South Korea

5. Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130

Abstract

Abstract Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

Funder

National Institutes of Health

National Science Foundation

NIH

Robert A. Welch Foundation

China Scholarship Council

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

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