This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
This summer I conducted a research project in the Orosa Lab, investigating the role of the ubiquitin pathway as a modulator of the immune response in barley during Puccinia hordei infection. Puccinia hordei is an emerging biotrophic pathogen of barley that can reduce crop yields by up to 30%. Its proliferation is favoured by mild weather conditions, which are becoming more frequent due to climate change. Additionally, this pathogen is increasingly developing resistance to fungicides, posing a significant threat to barley production. To address this challenge, new and sustainable strategies to manage P. hordei are urgently needed. Developing such approaches requires a thorough understanding of how barley mounts its defence against this pathogen, and my research focused on elucidating the role of ubiquitin-mediated immune regulation in this context.
Upon infection, plants sense and react to pathogens, triggering the plant’s immune response. This involves changes in protein ubiquitination followed by transcriptional reprogramming. Much research has been done on the transcriptional changes in the plant immune response, however the role of protein ubiquitination is not yet understood. Ubiquitin is a post-translational modification which can become reversibly attached to proteins. In Orosa lab found that immune activation of the ubiquitin pathway is much faster than gene transcription, enabling a fast response needed in pathogen defence.
Previous studies carried out in the Orosa lab by Karolina Brzezinska, identified ubiquitinated proteins and transcribed genes in non-infected and inoculated barley with P. hordei. She extracted protein and identified ubiquitinated proteins using mass spectrometry and measured gene transcription using RNA sequencing. During my summer internship, I analysed proteomics datasets to identify proteins ubiquitinated in response to P. hordei infection. I processed the mass spectrometry data, classifying proteins as either positively or negatively ubiquitinated upon infection. Subsequently, I mapped these proteins to their corresponding genes using RNA sequencing data to integrate transcriptomic and proteomic insights.
From the proteins that were positively ubiquitinated without corresponding changes in gene transcription upon infection, I identified two candidates for further investigation. Ankyrin Repeat Domain-Containing Protein 2A (Akr2A) is monoubiquitinated upon infection, suggesting a potential alteration in its function. Akr2A is a chaperone protein that binds newly synthesised transmembrane proteins, protecting them from degradation. Similarly, Heat Shock Cognate Protein 70 (Hsc70-1) is also monoubiquitinated. Hsc70-1 acts as a molecular linker and is primarily involved in protein folding but has diverse functions, including roles in pathogen defence and facilitating protein ubiquitination. The monoubiquitination of Hsc70-1 may influence its function in immune responses by promoting protein ubiquitination pathways.
Due to the limited genetic resources currently available for barley, I conducted further investigations of these genes in Arabidopsis thaliana. I induced an immune response in wild-type (Col-0) Arabidopsis plants and in hsc70-1 and akr2a knockout mutants using salicylic acid, a plant hormone that activates the plant immune system. RNA was extracted, and the expression of immune marker genes was measured. Specifically, I quantified the expression of PR1, NHL1, and WRK33, which are key indicators of immune response intensity. In Col-0, the expression of PR1, NHL1, and WRK33 increased significantly following immune activation. However, in akr2a knockouts, this response was significantly reduced (PR1: p=0.031, NHL1: p<0.001, WRK33: p=0.049). Similarly, in hsc70-1 knockouts, the expression of PR1 and WRK33 was diminished (p=0.030, p=0.001, respectively). The results for NHL1 in hsc70-1 mutants were inconclusive (p=0.099).
Additionally, I assessed the impact of these knockouts on protein ubiquitination. Following immune activation, I extracted protein, performed western blotting, and probed for ubiquitin. In Col-0, ubiquitination levels increased significantly after immune induction. However, this increase was notably reduced in both akr2a and hsc70-1 knockouts. These findings suggest that upon infection, AKR2A and HSC70-1 get ubiquitinated, leading to altered protein function. This modification enhances the transcription of defence-related genes and promotes protein ubiquitination, both of which are critical components of the immune response.
From the bioinformatics analysis, I also identified Low Sulphur Upregulated 2 (LSU2) as a candidate of interest. LSU2 exhibited increased ubiquitination and transcription following P. hordei infection, suggesting it plays a role in both rapid and sustained aspects of the plant immune response. LSU2 is known to regulate oxidative stress, chloroplast signalling, and transcriptional regulation. Notably, lsu2 knockouts display susceptibility to bacterial pathogens, though the precise mechanism by which LSU2 modulates plant immunity remains unclear. To investigate the role of LSU2 in plant immunity, I developed an expression vector to transiently overexpress the gene. Barley RNA was reverse transcribed into cDNA, and the LSU2 fragment was amplified via PCR. This fragment was then inserted into a donor vector and propagated in Escherichia coli. Using vector amplification in E. coli offers greater sequence fidelity compared to traditional PCR, which is more prone to introducing mutations. The next steps involve creating an additional expression vector containing a modified LSU2 sequence. By substituting the ubiquitinated lysine residue with arginine, ubiquitination will be prevented, allowing a direct comparison between the ubiquitinated and non-ubiquitinated forms of LSU2. This approach will enable us to further investigate the role of LSU2 in regulating the plant immune response.
I would like to express my gratitude to Dr Beatriz Orosa, Karolina Brzezinska, and the members of the Orosa and Spoel labs for their invaluable teaching and supervision. I am also deeply thankful to the BSPP for funding this fantastic opportunity. This experience has been incredibly enriching, and I have gained significant knowledge and skills. Having thoroughly enjoyed this project, I am inspired to pursue a career in plant research.
Patricia O’Riordan
The University of Edinburgh
Cover image:
- A) Summary of the overlap between the mass spectrometry and RNA-seq datasets, highlighting key candidate proteins identified in each category.
- B) Schematic representation illustrating the role of selected candidates in the accumulation of ubiquitinated proteins and their impact on gene expression of immune marker genes.
- C) Gene expression data showing the regulation of immune marker genes following validation of candidate contributions.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
