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Yeast as a Platform to Dissect Poly(ADP-Ribose) Polymerase Function from Magnaporthe oryzae and Evaluate PARP
Rachel E Kalicharan1, Nalleli Payne1, Jessie Fernandez1
1Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA.
Abstract:
Poly(ADP-ribose) polymerases (PARPs) regulate genome maintenance through NAD+-dependent ADP-ribosylation, yet PARP function in fungi remains poorly defined. Here, we reconstituted the activity of the Magnaporthe oryzae PARP1 homolog (MoPARP1) in Saccharomyces cerevisiae, a genetically tractable organism that lacks endogenous PARP enzymes. Upon galactose induction, expression of MoPARP1 reduced yeast growth, whereas a catalytically inactive mutant showed no defect, indicating that the growth phenotype depends on PARP catalytic activity. Consistent with this requirement, PARylation was detected in MoPARP1-expressing yeast cells but not in the catalytic mutant. In a multidrug transporter-deficient background, the PARP inhibitor 3-aminobenzamide and the clinically used PARP inhibitor olaparib rescued the growth of MoPARP1-expressing strains, establishing a framework for inhibitor testing in vivo. Finally, MoPARP1-GFP localized to the nucleus independent of catalytic activity, supporting correct targeting in this heterologous system. Together, these findings establish yeast as a platform to dissect fungal PARP biology and evaluate chemical inhibition.
Insights
We established yeast as a platform to study fungal Poly(ADP-ribose) polymerases (PARPs). Expressing a fungal PARP in yeast inhibited growth, but this was rescued by PARP inhibitors, enabling drug screening.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Poly(ADP-ribose) polymerases (PARPs) are crucial for genome maintenance via NAD+-dependent ADP-ribosylation.
- The specific functions of PARPs in fungi are not well understood.
Purpose of the Study:
- To reconstitute and characterize the activity of Magnaporthe oryzae PARP1 (MoPARP1) in Saccharomyces cerevisiae.
- To establish a yeast-based system for studying fungal PARP biology and evaluating potential inhibitors.
Main Methods:
- Reconstitution of MoPARP1 activity in a genetically tractable yeast model lacking endogenous PARP enzymes.
- Assessing growth phenotypes and detecting PARylation in yeast expressing MoPARP1.
- Evaluating the efficacy of PARP inhibitors (3-aminobenzamide and olaparib) in rescuing growth defects.
Main Results:
- Expression of active MoPARP1 in yeast reduced cell growth, dependent on catalytic activity.
- PARylation was detected in yeast expressing active MoPARP1, confirming enzyme function.
- PARP inhibitors rescued the growth defect, demonstrating their effectiveness in this heterologous system.
- MoPARP1-GFP localized to the nucleus, indicating proper targeting.
Conclusions:
- Saccharomyces cerevisiae serves as a viable platform for dissecting fungal PARP function.
- This system allows for in vivo evaluation of chemical inhibitors against fungal PARPs.
- Findings pave the way for understanding and targeting fungal-specific PARP pathways.
