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.

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Kamenice 5 Brno CZ 62500 Czech Republic

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Angelis KJ, Dusinská M, Collins AR. 1999. Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity. Electrophoresis 20: 2133-2138.

Bártová E, Suchánková J, Legartová S, Malyšková B, Hornáček M, Skalníková M, Mašata M, Raška I, Kozubek S. 2017. PCNA is recruited to irradiated chromatin in late S-phase and is most pronounced in G2 phase of the cell cycle. Protoplasma 254: 2035-2043.

Birnbaum K, Shasha DE, Wang JY, Jung JW, Lambert GM, Galbraith DW, Benfey PN. 2003. A gene expression map of the Arabidopsis root. Science 302: 1956-1960.

Charbonnel C, Gallego ME, White CI. 2010. Xrcc1-dependent and Ku-dependent DNA double-strand break repair kinetics in Arabidopsis plants. The Plant Journal 64: 280-290.

Citovsky V, Lee L-Y, Vyas S, Glick E, Chen M-H, Vainstein A, Gafni Y, Gelvin SB, Tzfira T. 2006. Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. Journal of Molecular Biology 362: 1120-1131.

Culligan KM, Robertson CE, Foreman J, Doerner P, Britt AB. 2006. ATR and ATM play both distinct and additive roles in response to ionizing radiation. The Plant Journal 48: 947-961.

De Storme N, Mason A. 2014. Plant speciation through chromosome instability and ploidy change: cellular mechanisms, molecular factors and evolutionary relevance. Current Plant Biology 1: 10-33.

Dinant C, deJager M, Essers J, vanCappellen WA, Kanaar R, Houtsmuller AB, Vermeulen W. 2007. Activation of multiple DNA repair pathways by sub-nuclear damage induction methods. Journal of Cell Science 120: 2731-2740.

Doonan JH, Sablowski R. 2010. Walls around tumours - why plants do not develop cancer. Nature Reviews Cancer 10: 794-802.

Dvorácková M, Rossignol P, Shaw PJ, Koroleva OA, Doonan JH, Fajkus J. 2010. AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin. The Plant Journal 61: 637-649.

Essers J, Theil AF, Baldeyron C, vanCappellen WA, Houtsmuller AB, Kanaar R, Vermeulen W. 2005. Nuclear dynamics of PCNA in DNA replication and repair. Molecular and Cellular Biology 25: 9350-9359.

Exner V, Taranto P, Schönrock N, Gruissem W, Hennig L. 2006. Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development. Development 133: 4163-4172.

Franek M, Kovaříková A, Bártová E, Kozubek S. 2016. Nucleolar reorganization upon site-specific double-strand break induction: DNA repair and epigenetics of ribosomal genes. Journal of Histochemistry and Cytochemistry 64: 669-686.

Fulcher N, Sablowski R. 2009. Hypersensitivity to DNA damage in plant stem cell niches. Proceedings of the National Academy of Sciences, USA 106: 20984-20988.

Ganguly A, Guo L, Sun L, Suo F, Du L-L, Russell P. 2018. Tdp1 processes chromate-induced single-strand DNA breaks that collapse replication forks. PLoS Genetics 14: e1007595.

Gao J, Zhu Y, Zhou W, Molinier J, Dong A, Shen W-H. 2012. NAP1 family histone chaperones are required for somatic homologous recombination in Arabidopsis. Plant Cell 24: 1437-1447.

Gassman NR, Wilson SH. 2015. Micro-irradiation tools to visualize base excision repair and single-strand break repair. DNA Repair 31: 52-63.

González-Arzola K, Díaz-Quintana A, Rivero-Rodríguez F, Velázquez-Campoy A, De la Rosa MA, Díaz-Moreno I. 2017. Histone chaperone activity of Arabidopsis thaliana NRP1 is blocked by cytochrome c. Nucleic Acids Research 45: 2150-2165.

González-Magaña A, Blanco FJ. 2020. Human PCNA structure, function and interactions. Biomolecules 10: E570.

Gottschalk AJ, Timinszky G, Kong SE, Jin J, Cai Y, Swanson SK, Washburn MP, Florens L, Ladurner AG, Conaway JWet al. 2009. Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler. Proceedings of the National Academy of Sciences, USA 106: 13770-13774.

Green CM, Almouzni G. 2003. Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo. EMBO Journal 22: 5163-5174.

Haince J-F, McDonald D, Rodrigue A, Déry U, Masson J-Y, Hendzel MJ, Poirier GG. 2008. PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites. Journal of Biological Chemistry 283: 1197-1208.

Harding SM, Boiarsky JA, Greenberg RA. 2015. ATM dependent silencing links nucleolar chromatin reorganization to DNA damage recognition. Cell Reports 13: 251-259.

Holton NW, Andrews JF, Gassman NR. 2017. Application of laser micro-irradiation for examination of single and double strand break repair in mammalian cells. Journal of Visualized Experiments: JoVE 2017: 56265.

Hu C-D, Chinenov Y, Kerppola TK. 2002. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Molecular Cell 9: 789-798.

Hurst V, Challa K, Shimada K, Gasser SM. 2021. Cytoskeleton integrity influences XRCC1 and PCNA dynamics at DNA damage. Molecular Biology of the Cell 32: br6.

Jean-Baptiste K, McFaline-Figueroa JL, Alexandre CM, Dorrity MW, Saunders L, Bubb KL, Trapnell C, Fields S, Queitsch C, Cuperus JT. 2019. Dynamics of gene expression in single root cells of Arabidopsis thaliana. Plant Cell 31: 993-1011.

Kaya H, Shibahara KI, Taoka KI, Iwabuchi M, Stillman B, Araki T. 2001. FASCIATA genes for chromatin assembly factor-1 in Arabidopsis maintain the cellular organization of apical meristems. Cell 104: 131-142.

Kerppola TK. 2008. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annual Review of Biophysics 37: 465-487.

Kirik A, Pecinka A, Wendeler E, Reiss B. 2006. The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants. Plant Cell 18: 2431-2442.

Kolářová K, Nešpor Dadejová M, Loja T, Lochmanová G, Sýkorová E, Dvořáčková M. 2021. Disruption of NAP1 genes in Arabidopsis thaliana suppresses the fas1 mutant phenotype, enhances genome stability and changes chromatin compaction. The Plant Journal 106: 56-73.

Kozak J, West CE, White C, daCosta-Nunes JA, Angelis KJ. 2009. Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance. DNA Repair 8: 413-419.

Lan L, Nakajima S, Komatsu K, Nussenzweig A, Shimamoto A, Oshima J, Yasui A. 2005. Accumulation of Werner protein at DNA double-strand breaks in human cells. Journal of Cell Science 118: 4153-4162.

Lan L, Nakajima S, Oohata Y, Takao M, Okano S, Masutani M, Wilson SH, Yasui A. 2004. In situ analysis of repair processes for oxidative DNA damage in mammalian cells. Proceedings of the National Academy of Sciences, USA 101: 13738-13743.

Lee J-H, Paull TT. 2007. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene 26: 7741-7748.

Lengert L, Lengert N, Drossel B, Cardoso MC, Muster B, Nowak D, Rapp A. 2015. Discrimination of kinetic models by a combination of microirradiation and fluorescence photobleaching. Biophysical Journal 109: 1551-1564.

Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J. 2013. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnology 31: 688-691.

Liu Z, Hong SW, Escobar M, Vierling E, Mitchell DL, Mount DW, Hall JD. 2003. Arabidopsis UVH6, a homolog of human XPD and yeast RAD3 DNA repair genes, functions in DNA repair and is essential for plant growth. Plant Physiology 132: 1405-1414.

Lopez FB, Fort A, Tadini L, Probst AV, McHale M, Friel J, Ryder P, Pontvianne F, Pesaresi P, Sulpice Ret al. 2021. Gene dosage compensation of rRNA transcript levels in Arabidopsis thaliana lines with reduced ribosomal gene copy number. Plant Cell 33: 1135-1150.

Mah L-J, El-Osta A, Karagiannis TC. 2010. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24: 679-686.

Moldovan G-L, Pfander B, Jentsch S. 2007. PCNA, the maestro of the replication fork. Cell 129: 665-679.

Mortusewicz O, Leonhardt H. 2007. XRCC1 and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions. BMC Molecular Biology 8: 81.

Mortusewicz O, Schermelleh L, Walter J, Cardoso MC, Leonhardt H. 2005. Recruitment of DNA methyltransferase I to DNA repair sites. Proceedings of the National Academy of Sciences, USA 102: 8905-8909.

Nakajima S, Sugiyama M, Iwai S, Hitomi K, Otoshi E, Kim ST, Jiang SZ, Todo T, Britt AB, Yamamoto K. 1998. Cloning and characterization of a gene (UVR3) required for photorepair of 6-4 photoproducts in Arabidopsis thaliana. Nucleic Acids Research 26: 638-644.

Nakamura AJ, Rao VA, Pommier Y, Bonner WM. 2010. The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks. Cell Cycle 9: 389-397.

Nisa M-U, Huang Y, Benhamed M, Raynaud C. 2019. The plant DNA damage response: signaling pathways leading to growth inhibition and putative role in response to stress conditions. Frontiers in Plant Science 10: 653.

Olive PL, Banáth JP. 2006. The comet assay: a method to measure DNA damage in individual cells. Nature Protocols 1: 23-29.

Sanchez A, Gadaleta MC, Limbo O, Russell P. 2017. Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast. PLoS Genetics 13: e1007013.

Schep R, Brinkman EK, Leemans C, Vergara X, van derWeide RH, Morris B, vanSchaik T, Manzo SG, Peric-Hupkes D, van denBerg Jet al. 2021. Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance. Molecular Cell 81: 2216-2230.

Schořová Š, Fajkus J, Záveská Drábková L, Honys D, Schrumpfová PP. 2019. The plant Pontin and Reptin homologues, RuvBL1 and RuvBL2a, colocalize with TERT and TRB proteins in vivo, and participate in telomerase biogenesis. The Plant Journal 98: 195-212.

Shibahara K, Stillman B. 1999. Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96: 575-585.

van Sluis M, McStay B. 2015. A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage. Genes & Development 29: 1151-1163.

Tashiro S, Walter J, Shinohara A, Kamada N, Cremer T. 2000. Rad51 accumulation at sites of DNA damage and in postreplicative chromatin. Journal of Cell Biology 150: 283-291.

Tharkar-Promod S, Johnson DP, Bennett SE, Dennis EM, Banowsky BG, Jones SS, Shearstone JR, Quayle SN, Min C, Jarpe Met al. 2018. HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia. Leukemia 32: 49-60.

Uziel T, Lerenthal Y, Moyal L, Andegeko Y, Mittelman L, Shiloh Y. 2003. Requirement of the MRN complex for ATM activation by DNA damage. EMBO Journal 22: 5612-5621.

Varga D, Majoros H, Ujfaludi Z, Erdélyi M, Pankotai T. 2019. Quantification of DNA damage induced repair focus formation via super-resolution dSTORM localization microscopy. Nanoscale 11: 14226-14236.

Wang Y, Zhong Z, Zhang Y, Xu L, Feng S, Rayatpisheh S, Wohlschlegel JA, Wang Z, Jacobsen SE, Ausin I. 2020. NAP1-RELATED PROTEIN1 and 2 negatively regulate H2A.Z abundance in chromatin in Arabidopsis. Nature Communications 11: 2887.

Whelan DR, Rothenberg E. 2021. Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination. Proceedings of the National Academy of Sciences, USA 118: e2021963118.

Xing S, Wallmeroth N, Berendzen KW, Grefen C. 2016. Techniques for the analysis of protein-protein interactions in vivo. Plant Physiology 171: 727-758.

Yokoyama R, Hirakawa T, Hayashi S, Sakamoto T, Matsunaga S. 2016. Dynamics of plant DNA replication based on PCNA visualization. Scientific Reports 6: 29657.

Histone Chaperone Deficiency in Arabidopsis Plants Triggers Adaptive Epigenetic Changes in Histone Variants and Modifications