Transcriptomic and functional analyses uncover a conserved effector driving genotype-dependent virulence in the Sphaerulina musiva-Populus trichocarpa interaction

Authors: SØNDRELI, KELSEY - Oregon State University; RUSH, TOMAS - Oak Ridge National Laboratory; Drott, Milton; MCTAGGART, ALISTAIR - University Of Queensland; ALEXANDER, WILLIAM - Oak Ridge National Laboratory; SAWYER, COLE - Oak Ridge National Laboratory; CHHERTI, HARI - Oak Ridge National Laboratory; HYATT, DOUG - Oak Ridge National Laboratory; BRUEGGEMAN, ROBERT - Washington State University; RICHARDS, JONATHAN - Louisana State University; Friesen, Timothy; JACOBSON, DANIEL - Oak Ridge National Laboratory; ABRAHAM, PAUL - Oak Ridge National Laboratory; TANNOUS, JOANNA - Oak Ridge National Laboratory; LEBOLDUS, JARED - Oregon State University

Submitted to: mBio
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2026
Publication Date: 4/16/2026
Citation: Søndreli, K.L., Rush, T., Drott, M.T., Mctaggart, A., Alexander, W., Sawyer, C., Chherti, H.B., Hyatt, D., Brueggeman, R., Richards, J.K., Friesen, T.L., Jacobson, D., Abraham, P., Tannous, J., Leboldus, J.M. 2026. Transcriptomic and functional analyses uncover a conserved effector driving genotype-dependent virulence in the Sphaerulina musiva-Populus trichocarpa interaction. mBio. 17(5). Article 03120-25. https://doi.org/10.1128/mbio.03120-25.
DOI: https://doi.org/10.1128/mbio.03120-25

Interpretive Summary: Invasion of non-indigenous fungal species is acknowledged as one of the significant external drivers altering ecosystems' structure, biodiversity, and functions. Understanding the genetic mechanisms of pathogenicity and the interaction of these invaders with their hosts is a crucial step toward implementing mitigation strategies to sustain normal ecosystem functions as well as agricultural sustainability. Sphaerulina musiva is a well-characterized example of an invasive fungal species spread by human activities. Originally native to Eastern North America, S. musiva was only recently introduced and established in the Pacific Northwest of North America, resulting in deleterious effects on susceptible Populus genotypes, a foundational bioenergy crop. We sought to identify factors contributing to the virulence of this pathogen by comparing what genes were being used during infection as compared to normal growth. This analysis identified a subset of candidate genes. Among candidates we identified a gene that strongly resembles a virulence-associated protein from other organisms, ECP2. We identify that ECP2 is produced by S. musiva and contributes to virulence, but only on specific host cultivars. We also map the evolution of this protein and identify three different ECP2 variants across the fungal kingdom, including in common and notorious plant pathogens like Fusarium graminearum. Our results identify a new source of virulence that is challenging diverse agricultural efforts from biofuel to food production. In identifying ECP2, we offer new opportunity to protect agriculture from emerging threats through targeted mediation of this virulence factor.

Technical Abstract: Introduction of microbes into naive ecosystems alters their structure, biodiversity, and function. Understanding the genetic mechanisms of pathogenicity and the interaction of these invaders with their hosts is crucial. This knowledge facilitates the implementation of mitigation strategies to sustain native biodiversity and normal ecosystem function. Sphaerulina musiva is a well-characterized example of an invasive fungal species spread by human activities. Originally endemic to Eastern North America, S. musiva was recently introduced to the western United States and Canada. RNA-sequencing was used to identify fungal effectors associated with stem canker formation. Analysis of fungal reads identified 70 genes at two weeks post-inoculation and 110 genes at three weeks post-inoculation that were differentially expressed between the inoculated trees and the control. The gene with the highest expression at two weeks and the second highest expression at three weeks had homology to Extracellular protein 2 (Ecp2), a broadly conserved gene in the Dothidiomycetes. The infiltration of S. musiva Ecp2 (SmEcp2) protein into P. trichocarpa leaves induced necrosis on susceptible genotypes. A disruption of this gene using a CRISPR-Cas9 RNP system resulted in a genotype-dependent reduction of stem canker and leaf spot severity. Lastly, we used a large-scale comparative genomic approach to improve our current understanding of this effector's evolution and distribution throughout the fungal kingdom. These analyses have enabled the establishment of a putative connection between the ECP2 class information and the ecological functions of fungi.