Transcriptomic and functional analyses uncover a conserved effector driving genotype-dependent virulence in the...

The introduction of invasive microbes compromises the structure, biodiversity, and function of naïve ecosystems. Sphaerulina musiva, a hemibiotrophic pathogen that causes leaf spot and stem cankers in Populus species, exemplifies an invasive fungal pathogen spread by human activities. However, the genetic mechanisms of pathogenicity and virulence are poorly understood, impeding mitigation strategies. We utilized RNA sequencing to identify fungal effectors linked to stem canker formation, informing the development of future strategies for effective disease management. Our analysis revealed 70 genes differentially expressed at 2 weeks and 110 genes at 3 weeks between inoculated trees and controls. Notably, the gene with the highest expression at 2 weeks and the second highest at 3 weeks was homologous to Extracellular protein 2 (Ecp2). Complementary genome-wide association studies linked sequence polymorphisms in this locus to phenotypic variation in disease severity. Infiltration of S. musiva Ecp2 into Populus trichocarpa leaves induced necrosis in susceptible genotypes. Gene disruption using a CRISPR-Cas9 RNP system resulted in a genotype-dependent reduction of stem canker and disease severity. Tracing the evolutionary history of this effector across the fungal kingdom, we uncovered clade-specific gene-family expansions and orthologs in new species. These findings raise questions about the function and adaptive significance of these gene families in fungal lifestyles. Our study provides the first tractable target for breeding resistant poplar genotypes, addressing the challenges of managing S. musiva and uncovering mechanisms that drive its virulence, and provides deeper insights into the evolutionary dynamics of a conserved small-secreted protein with a diversity of functions.

Populus species, key feedstocks in the bioeconomy, are severely impacted by leaf spot and stem canker caused by Sphaerulina musiva. This disease diminishes biomass, reduces wood quality, and increases tree mortality, jeopardizing industrial sustainability. Invasion of S. musiva into naïve ecosystems exacerbates these challenges by disrupting ecosystem processes. Breeding resistant poplar genotypes has been the primary strategy to combat this pathogen, but it has remained unclear which molecular drivers of infection breeders should target. Our study makes a significant advance by identifying a key necrotrophic effector that increases S. musiva virulence in specific Populus genotypes. We identify clade-specific gene-family expansions of this effector that raise questions about the function of closely related genes. Our research elucidates the ecology and evolution of a small-secreted protein across the fungal kingdom while offering insights that enable host-breeding efforts to reduce the economic impact of S. musiva.