Evaluating In Vivo Translation Inhibition via Puromycin Labeling in Botrytis cinerea Germlings Treated With Antifungal Peptides.

Antimicrobial peptides are effective agents against various pathogens, often targeting essential processes like protein translation to exert their antimicrobial effects. Traditional methods such as puromycin labeling have been extensively used to measure protein synthesis in mammalian and yeast systems; however, protocols tailored for plant pathogenic filamentous fungi, particularly those investigating translation inhibition by antifungal peptides, are lacking. This protocol adapts puromycin labeling to quantify translation inhibition in Botrytis cinerea germlings treated with antifungal peptides. Optimizing the method specifically for fungal germlings provides a precise tool to investigate peptide effects on fungal protein synthesis, advancing our understanding of translation dynamics during pathogen-host interactions in filamentous fungi. Key features • This protocol is designed for in vivo experiments, enabling the estimation of protein translation inhibition by antifungal peptides in Botrytis cinerea. • It is optimized for antifungal peptides capable of penetrating fungal cells and inhibiting translation, making it applicable to other filamentous fungal pathogens. • The protocol is ideal for laboratories utilizing traditional western blotting techniques, ensuring accessibility and ease of implementation. • This protocol for in vivo application in filamentous fungi is adapted from the SUnSET method [1]. Graphical overview.