Samples: 11 Protocols: 6 -- Organism: Arabidopsis thaliana -- Experiment types: RNA-seq of coding RNA, compound treatment design --- Activation of plant immunity is associated with dramatic transcriptome reprogramming to prioritise immune responses over normal cellular functions. Changes in gene expression are coordinated by the immune hormone salicylic acid (SA). Here we investigated the involvement of the HECT-type ligase Ubiquitin Protein Ligase 3 (UPL3) in SA-induced transcriptional reprogramming. We show that UPL3 acts as an amplifier of SA-induced changes in gene expression. Four-week old Arabidopsis thaliana plants of wild-type Col-0 and mutant upl3-4 genotypes were germinated on soil in 100% relative humidity. After 12 days plants were transplanted to larger pots (six plants per pot) and grown for an additional 3 weeks before experimental treatment. Plants were continuously grown in an environmental chamber with 16/8 hour day/night light regime (120 mol m-2 s-1 light intensity), 21/18 degrees celcius day/night cycle and 65% relative humidity. Plants were then sprayed with water or 0.5 mM SA until all leaves were thoroughly covered with fine droplets. After 24 hours leaf tissue was harvested from 6 plants per treatment and pooled together into a single biological repeat. In total two or three independent biological repeats were collected. After harvesting leaf tissue was immediately frozen in liquid nitrogen until further analysis.
Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in UPL1, UPL3 and UPL5 significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of upl3 mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.
Salicylic acid-induced gene expression in wild-type Col-0 and mutant upl3-4 Arabidopsis thaliana plants. James J. Furniss, Heather Grey, Zhishuo Wang, Mika Nomoto, Lorna Jackson, Yasuomi Tada, Steven H. Spoel. ArrayExpress database accession number E-MTAB-7374. https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-7374/