Dissection of the role of Tox3-PR1 interaction in the Parastagonospora nodorum-wheat interaction.
Creators: name: Sung, Yi-Chang
Publisher: name: The Australian National University
Year: 2021

description: Parastagonospora nodorum is a necrotrophic fungal pathogen causing the wheat disease septoria nodorum blotch which is causal to significant economic losses globally. As a necrotroph, P. nodorum secretes necrotrophic effectors that induce cell death responses in wheat cultivars harbouring dominant susceptibility genes. The Tox3 effector from P. nodorum elicits necrotic responses in wheat cultivars dominant for the Snn3 gene. Independent studies have shown that Tox3 also interacts with several members of wheat pathogenesis-related protein 1 family (TaPR1), although the role of the interaction in disease remains unclear. Therefore, I sought to dissect the interaction of Tox3 and TaPR1 with a focus on understanding the role of these proteins and their interaction in disease. Firstly, I utilized site-directed mutagenesis to identify a Tox3 variant, Tox3P173S, that was unable to interact with TaPR1 but could induce a necrotic response in Snn3 cultivars, suggesting the interaction is not required for necrosis. Additionally, wheat leaves pre-infiltrated with TaPR1 proteins prior to P. nodorum inoculation showed strong disease reduction. Subsequent gene expression profiling of known defence genes indicated TaPR1 induced a host defence response mediated by the conserved C-terminal peptide CAPE1. The priming activity of TaPR1 was compromised by the Tox3-TaPR1 interaction but unaffected by the non-interacting Tox3P173S variant. Furthermore, the incubation of Tox3 and TaPR1-1 in wheat apoplastic washing fluid (TaAWF) abolished the TaPR1 cleavage but the non-interacting Tox3P173S didn't affect the cleavage activity, suggesting Tox3 inhibits PR1-mediated immunity by preventing the cleavage of CAPE1 from PR1. Collectively, these data demonstrate that the Tox3 effector independently functions to induce necrosis and suppress host defence. Understanding that PR1 functions depends on the release of CAPE1, I sought to uncover the mechanisms of PR1 cleavage and the protease involved. A protease inhibition assay revealed that a serine protease was required for cleaving TaPR1. To identify the protease, chromatography-based TaAWF profiling and proteomics approaches were used for TaAWF proteins. Subsequent activity assays and trypsin digestions led to the identification of the wheat subtilase 1.7 (TaSBT1.7) which harboured PR1 cleavage activity. Using similar approaches in the model plant Arabdiopsis thaliana system, AtPR1 was produced and cleaved in A. thaliana apoplastic washing fluid (AtAWF) mediated by a serine protease. Subsequent purification and mass spectrometry approaches identified that the Ara12 serine protease, the homologue of TaSBT1.7, was active against PR1 leading to the release of CAPE1. These data independently confirm the identity of a protease responsible for the cleavage of the CAPE1 defence signalling peptide from PR1 proteins. To demonstrate the role of the serine protease in PR1-mediated immunity in a disease, A. thaliana and Pseudomonas syringae DC3000 (Pst DC3000) system was used for functional characterisation of PR1 and Ara12. Leaves pre-infiltrated with AtPR1 protein and CAPE1 prior to infection showed a significant disease reduction in the wild-type of A. thaliana. However, disease repression activity was significantly attenuated when infiltrating AtPR1 into an Ara12 T-DNA mutagenesis line (ara12-ko), implying that Ara12 is required for PR1-mediated immunity. Importantly, infiltrating AtCAPE1 into the ara12-ko line induced resistance against Pst DC3000 confirming the role of Ara12 as the protease causal to the cleavage of CAPE1 from AtPR1. This study has shown the PR1 mediates host defence signalling through the CAPE1 peptide and the release of CAPE1 depends on a serine protease. Furthermore, PR1-meidated immunity is hijacked by Tox3 via directly targeting to PR1 and preventing CAPE1 release. Collectively, these data have advanced our understanding of the role of PR1 proteins in plant-microbe interactions.