The project combined comparative virology, plant genetics, proteomics and molecular biology through three connected research lines.
First, material was generated to compare healthy plants and plants infected with CaMV, TuMV and CMV. Samples were prepared and submitted to a specialised facility for ubiquitinome profiling using K-ε-GG peptide enrichment, which identifies ubiquitinated proteins and ubiquitination sites. Total proteome analysis was also included to support interpretation. This established the framework for comparative analysis of UPS-related changes across three distinct viral infections. The datasets are currently being generated and analysed and are expected to provide candidate UPS targets.
Second, the project investigated how natural genetic variation in Arabidopsis thaliana influences the relationship between viral infection, proteasome activity and disease outcome. Approximatly 100 accessions were infected with CaMV and analysed for virus accumulation, plant performance, proteasome activity and salicylic acid (SA) quantification. Although the genome-wide association analysis did not reveal a strong genetic hit suitable for immediate mutant follow-up, the dataset provided biological insights. Arabidopsis intraspecific variation showed wide phenotypic ranges for all traits, and our dataset revealed positive correlations between proteasome activity (PA) and both CaMV accumulation and SA levels. In addition, PA, CaMV accumulation and SA positively correlated with biomass loss upon infection. Further analysis supports a model in which SA promotes ubiquitin accumulation, while proteasome activity buffers this accumulation and helps balance defence activation and plant growth. This provides a mechanistic framework to understand the UPS as a rheostat of the growth–defence trade-off.
Third, selected UPS components and potential UPS targets were functionally analysed. Several candidates previously identified through GFP-ubiquitin pull-down experiments were tested in infections with the three viruses, and a subset was investigated in greater detail. UBA1 was particularly informative because it fitted the emerging model linking ubiquitination, proteasome function and plant growth–defence balance. Candidate UPS targets, including members of a small protein family identified in ubiquitin-associated pull-downs, were also analysed. Proteasome-related mutants were used to test how altered proteasome function affects viral infection, ubiquitin accumulation and plant performance.
Overall, the project generated experimental resources, phenotypic datasets and mechanistic insights into the role of the UPS during plant viral infection. The main achievements include a comparative proteomics workflow for three viruses; a large natural-variation dataset linking UPS regulation with infection outcome; the identification of a novel regulatory axis connecting SA-dependent ubiquitin accumulation, proteasome activity and the growth–defence trade-off; and the functional analysis of selected UPS candidates. These results are being consolidated into two scientific manuscripts, while the proteomic datasets are expected to support future publications.
