Laser microirradiation as a versatile system for probing protein recruitment and protein-protein interactions at DNA lesions in plants

Plant protoplasts are generated by treatment with digestion enzymes, producing plant cells devoid of the cell wall and competent for efficient polyethylene glycol mediated transformation. This way fluorescently tagged proteins can be introduced to the protoplasts creating an excellent system to probe the localization and function of uncharacterized plant proteins in vivo. We implement the method of laser microirradiation to generate DNA lesions in Arabidopsis thaliana, which enables monitoring the recruitment and dynamics of the DNA repair factors as well as bimolecular fluorescence complementation assay to test transient, conditional interactions of proteins directly at sites of DNA damage. We demonstrate that laser microirradiation in protoplasts yields a physiological cellular response to DNA lesions, based on proliferating cell nuclear antigen (PCNA) redistribution in the nucleus and show that factors involved in DNA repair, such as MRE11 or PCNA are recruited to induced DNA lesions. This technique is relatively easy to adopt by other laboratories and extends the current toolkit of methods aimed to understand the details of DNA damage response in plants. The presented method is fast, flexible and facilitates work with different mutant backgrounds or even different species, extending the utility of the system.

Automated summary

This study presents a method for studying the DNA damage response in plants using plant protoplasts. DNA lesions were generated using laser microirradiation, and fluorescently tagged proteins were introduced to study the localization and function of uncharacterized plant proteins in the protoplasts. The results demonstrate that laser microirradiation in protoplasts induces a physiological cellular response and recruits factors involved in DNA repair.