Comparative secretomics and functional analysis of effectors utilized by the Microbotryum genus of anther-smut fungal pathogens, and their role in host-specificity.

Abstract

Understanding how pathogens evolve in response to changes in their host is paramount to combating the spread of emergent strains of disease. This is particularly true for plant pathogens that cause billions of dollars of damages to crops globally, every year. Understanding the molecular interactions between pathogens and their hosts therefore sheds light on the coevolutionary arms race that can result in host-specificity and host-shifts in plant pathogens. This research approaches the question of how fungal pathogens interact with their plant hosts utilizing both unique and shared arsenals of secreted proteins (SPs) during infection, and addresses the question of whether alterations to shared SPs or species-specific SPs play a more important role in host-specificity. To answer these questions, we annotated and compared the secretomes of three species from the Microbotryum genus of anther smuts, two closely related sister species that are able to infect each other’s hosts, albeit to reduced degrees, M. lychnidis-dioicae and M. silenes-dioicae, and one distantly related species that is unable to infect either of the other two species’ host plants and vice versa, M. violaceum var. paradoxa. We then characterized the function of the core SP MVLG_02245, an SP found in all three species with differing levels of conservation at the amino acid sequence level, and tested the importance of two species-specific SPs in host specificity, MvSl_01693 and MvSd_09295, via heterologous expression in each sister species. Finally, for future research into the role of SPs in host pathogenicity, we established a site-specific knockout system in Microbotryum using CRISPR Cas9 technology. Our results demonstrate that while host specificity in the Microbotryum genus is likely the result of alterations to the amino acid sequence of several core SPs, expression of novel SPs can have dramatic effects on pathogenicity. The research is therefore the first to identify key proteins involved in host specificity of the Microbotryum genus, and the first to establish a means of site-specific gene modification and knockout in the Microbotryum system using a CRISPR Cas9.