Fungal plant pathogen “mutagenomics” reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor

Blyth, Hannah R.; Smith, Dan; King, Robert; Bayon, Carlos; Ashfield, Tom; Walpole, Hannah; Venter, Eudri; Ray, Rumiana V.; Kanyuka, Kostya; Rudd, Jason J.

doi:10.3389/fpls.2023.1140824

Fungal plant pathogen “mutagenomics” reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor

Blyth, Hannah R.; Smith, Dan; King, Robert; Bayon, Carlos; Ashfield, Tom; Walpole, Hannah; Venter, Eudri; Ray, Rumiana V.; Kanyuka, Kostya; Rudd, Jason J.

Authors

Hannah R. Blyth

Dan Smith

Robert King

Carlos Bayon

Tom Ashfield

Hannah Walpole

Eudri Venter

Professor RUMIANA RAY RUMIANA.RAY@NOTTINGHAM.AC.UK
PROFESSOR OF PLANT PATHOLOGY

Kostya Kanyuka

Jason J. Rudd

Abstract

“Mutagenomics” is the combination of random mutagenesis, phenotypic screening, and whole-genome re-sequencing to uncover all tagged and untagged mutations linked with phenotypic changes in an organism. In this study, we performed a mutagenomics screen on the wheat pathogenic fungus Zymoseptoria tritici for altered morphogenetic switching and stress sensitivity phenotypes using Agrobacterium-mediated “random” T-DNA mutagenesis (ATMT). Biological screening identified four mutants which were strongly reduced in virulence on wheat. Whole genome re-sequencing defined the positions of the T-DNA insertion events and revealed several unlinked mutations potentially affecting gene functions. Remarkably, two independent reduced virulence mutant strains, with similarly altered stress sensitivities and aberrant hyphal growth phenotypes, were found to have a distinct loss of function mutations in the ZtSSK2 MAPKKK gene. One mutant strain had a direct T-DNA insertion affecting the predicted protein’s N-terminus, while the other possessed an unlinked frameshift mutation towards the C-terminus. We used genetic complementation to restore both strains’ wild-type (WT) function (virulence, morphogenesis, and stress response). We demonstrated that ZtSSK2 has a non-redundant function with ZtSTE11 in virulence through the biochemical activation of the stress-activated HOG1 MAPK pathway. Moreover, we present data suggesting that SSK2 has a unique role in activating this pathway in response to specific stresses. Finally, dual RNAseq-based transcriptome profiling of WT and SSK2 mutant strains revealed many HOG1-dependent transcriptional changes in the fungus during early infection and suggested that the host response does not discriminate between WT and mutant strains during this early phase. Together these data define new genes implicated in the virulence of the pathogen and emphasise the importance of a whole genome sequencing step in mutagenomic discovery pipelines.

Citation

Blyth, H. R., Smith, D., King, R., Bayon, C., Ashfield, T., Walpole, H., Venter, E., Ray, R. V., Kanyuka, K., & Rudd, J. J. (2023). Fungal plant pathogen “mutagenomics” reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor. Frontiers in Plant Science, 14, Article 1140824. https://doi.org/10.3389/fpls.2023.1140824

Journal Article Type	Article
Acceptance Date	Mar 29, 2023
Online Publication Date	May 3, 2023
Publication Date	May 3, 2023
Deposit Date	Jan 26, 2024
Publicly Available Date	Jan 30, 2024
Journal	Frontiers in Plant Science
Electronic ISSN	1664-462X
Publisher	Frontiers Media
Peer Reviewed	Peer Reviewed
Volume	14
Article Number	1140824
DOI	https://doi.org/10.3389/fpls.2023.1140824
Keywords	Dothideomycete, genome instability, Triticum aestivum, oxidative stress, Septoria Tritici Blotch, mitogen activated protein kinase
Public URL	https://nottingham-repository.worktribe.com/output/21098587
Publisher URL	https://www.frontiersin.org/articles/10.3389/fpls.2023.1140824/full