# Characterisation of guided entry of tail-anchored proteins in Magnaporthe oryzae

## TL;DR

This study explores the role of the GET complex in the rice blast fungus, revealing how specific proteins affect growth, pathogenesis, and stress response.

## Contribution

The study identifies and characterizes the GET complex components in Magnaporthe oryzae, revealing their roles in pathogenesis and stress response.

## Key findings

- MoGet1 and MoGet2 are essential for vegetative growth, pathogenesis, and ER stress response in M. oryzae.
- MoGet3 negatively regulates hyphal growth and pathogenesis.
- MoGet1, MoGet2, and MoGet3 physically interact, suggesting coordinated regulation.

## Abstract

Rice (Oryza sativa L.) is one of the most important staple foods for human population worldwide. However, rice production continues to be severely threatened by rice blast disease caused by an ascomycete fungus Magnaporthe oryzae. Tail-anchored (TA) proteins are conserved across diverse organisms and belong to a class of polypeptides that are inserted into the membrane by a hydrophobic sequence located at the C-terminal region. The Guided Entry of Tail-anchored (GET) complex is responsible for the post-translational insertion of nascent TA proteins into the Saccharomyces cerevisiae ER lipid bilayer. In S. cerevisiae, the GET pathway comprises six known associated components Get1, Get2, Get3, Get4, Get5, Sgt2 and Ssa1 that have been identified and extensively studied. However, the role of the GET complex in rice blast fungus has not been elucidated. Here, we identified five proteins of the GET Complex in M. oryzae, namely MoGet1, MoGet2, MoGet3, MoGet4 and MoSgt2 and generated the gene knock-out mutants. Deletion of MoGET1 and MoGET2 revealed that they are required for vegetative growth, asexual reproduction, pathogenesis, and right localization of TA protein, MoYsy6, while MoGet3 negatively regulates hyphal growth, asexual development and pathogenesis of M. oryzae. In contrast, loss of MoGet4 and MoSgt2 had no effect on the normal development of the rice blast fungus. We demonstrated that the MoGet2 is important in osmotic stress response and positively regulates cell wall integrity. The MoGet1 and MoGet2 were ER-localized and indispensable for DTT-induced ER stress response. In vitro and in vivo interaction assay revealed MoGet3 has physical interaction with both MoGet1 and MoGet2, indicating the existence of a possible synergistic function amongst the Get components in rice blast fungus. In summary, this finding provides valuable insight into the biological functions of the GET components in plant fungal pathogens.

Rice blast disease, caused by Magnaporthe oryzae is a devastating disease of rice, causing major economic loss in rice production across the globe. Since its isolation in 1892, M. oryzae has also been implicated in infecting other grasses as wheat, finger millet and barley. M. oryzae initiates pathogenicity by forming a specialized, dome-shaped appressorium when in contact with the hydrophobic surface of its host. The Guided Entry of Tail-anchored (GET) pathway, known to direct tail-anchored proteins to their target organelles in yeast and mammals, remains unexplored in pathogenic fungi. Here, we dissected the role of the GET pathway in M. oryzae pathogenesis using gene knockout strategies. We showed that while MoGet4 and MoSgt2 of the pathway has no effect on the virulence, ER-localized MoGet1 and MoGet2 proved critical for the pathogenesis of M. oryzae. Additionally, we demonstrated that MoGet2 is especially essential for stress tolerance and cell wall integrity, traits vital for host invasion. In contrast, MoGet3 is a negative regulator of vegetative growth and pathogenicity. We also demonstrated through yeast two-hybrid assay and co-immunoprecipitation that MoGet1, MoGet2 and MoGet3 interact with one another in vitro and in vivo, suggesting a coordinated regulatory network.