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Abstract BackgroundAlternative splicing of precursor mRNAs serves as a crucial mechanism to enhance gene expression plasticity for organismal adaptation. However, the precise regulation and function of alternative splicing in plant immune gene regulation remain elusive. ResultsHere, by deploying in-depth transcriptome profiling with deep genome coverage coupled with differential expression, differential alternative splicing, and differential transcript usage analysis, we reveal profound and dynamic changes in alternative splicing following treatment with microbial pattern flg22 peptides inArabidopsis. Our findings highlight RNA polymerase II C-terminal domain phosphatase-like 3 (CPL3) as a key regulator of alternative splicing, preferentially influencing the splicing patterns of defense genes rather than their expression levels. CPL3 mediates the production of a flg22-induced alternative splicing variant, diacylglycerol kinase 5α (DGK5α), which differs from the canonical DGK5β in its interaction with the upstream kinase BIK1 and subsequent phosphorylation, resulting in reduced flg22-triggered production of phosphatidic acid and reactive oxygen species. Furthermore, our functional analysis suggests that DGK5β, but not DGK5α, contributes to plant resistance against virulent and avirulent bacterial infections. ConclusionsThese findings underscore the role of CPL3 in modulating alternative splicing dynamics of defense genes and DGK5 isoform-mediated phosphatidic acid homeostasis, shedding light on the intricate mechanisms underlying plant immune gene regulation. 

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.