Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843.
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843.
Plant Physiol. 2021 Apr 23;185(4):1943-1965. doi: 10.1093/plphys/kiab011.
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.
蛋白质泛素化深刻地扩展了蛋白质组的功能，并使细胞信号转导过程多样化，最近的研究充分证明了其对植物免疫的重要性。为了全面了解免疫识别过程中泛素组的动态变化，我们采用了基于 6His 标签泛素（Ub）变体的两步亲和富集方案，结合高灵敏度质谱法，鉴定了拟南芥中在植物感知微生物相关分子模式（MAMP）肽 flg22 后迅速被泛素化的蛋白质。用 flg22 处理 2 周龄幼苗 30 分钟的文库包含 690 个缀合物、64 个 Ub 足迹和所有七种 Ub 连接类型，包括以前未表征的免疫成分缀合物。体内泛素化测定法证实了几种候选物在免疫诱导后的修饰，并揭示了关键免疫成分的不同修饰模式和动力学，包括多泛素化和单泛素化，以及泛素化水平的诱导或降低。该集合的基因本体论和网络分析还揭示了 Ub-蛋白酶体系统本身的快速修饰，这表明存在一个关键的自动调节环，对于有效的 MAMP 触发免疫反应和随后的抗病性是必要的。包括 UBIQUITIN-CONJUGATING ENZYME 13（UBC13）和蛋白酶体成分 REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b（RPN8b），它们的后续生化和遗传分析表明它们在免疫诱导中起负作用。总的来说，我们的蛋白质组学分析进一步加强了泛素化与 flg22 为基础的免疫信号之间的联系，鉴定了调节植物免疫的成分和途径，并增加了植物中泛素化底物的数据库。
