Boscalid - a new selectable marker for Ascochyta lentis and Ascochyta rabiei

Ascochyta lentis and Ascochyta rabiei are fungal pathogens affecting lentil and chickpea crops, respectively. They employ effector proteins to facilitate infection, and understanding the role of effector genes is crucial for unravelling host-pathogen interactions and developing disease-resistant crops. Traditional methods for studying effectors in lentil and chickpea face challenges, such as the ability to perform gene overexpression or knockout studies, due to the difficulty of effector protein infiltration and the limitations of using non-host plants for expression studies. Here, we introduce an alternative tool to enhance the genetic modification toolkit for A. lentis and A. rabiei by developing boscalid-resistant mutants using targeted mutations in the succinate dehydrogenase subunit B (SdhB). This allows for the generation of multiple gene knockouts and gene complementation in A. lentis, where previously only one selectable marker was available. By using the SdhB H277L mutation, we transformed both pathogens and successfully selected transformants using the fungicide boscalid as the selective agent. The method was validated through gene complementation studies of AlScd1 in A. lentis and ArPks1 in A. rabiei, restoring wild type melanin production phenotypes and demonstrating the utility of the new marker system. Additionally, we generated double knockouts in both pathogens, highlighting the potential for more sophisticated genetic studies. The boscalid resistance marker system described here represents a significant advancement in the functional genomics of Ascochyta species, providing a new tool for dissecting the molecular mechanisms underlying pathogenicity and host-pathogen interactions. This approach opens new avenues for research on disease management strategies for lentil and chickpea.