Plants use receptors to monitor the extracellular space for pathogen-associated molecular patterns (PAMPs), which are specific conserved molecules, such as bacterial flagellin protein or fungal cell-wall chitin. These receptors can also monitor for specific pathogen effectors. My project was to identify Z. tritici effectors that could suppress the response of such immune receptor-related pathways. Below I highlight a few key examples identified.
I identified a secreted leucine rich repeat (LRR) (Zt-sLRR) effector that is highly expressed during the latent phase of Z. tritici infection. Based on structural predictions, Zt-sLRR closely resembles a plant LRR protein (structural predictions provided by the Krasileva Lab, UC Berkeley) (Fig. 1). In collaboration the Kettles Lab (University of Birmingham), we found that Zt-sLRR could suppress PAMP-triggered immunity when expressed in the model plant, Nicotiana benthamiana. This was confirmed by collaborators in the Saur Lab (University of Cologne), who expressed Zt-sLRR in wheat protoplasts and were able to confirm our initial findings in the Z. tritici host system (Fig. 2).
I performed a Yeast 2-hybrid (Y2H) assays and an immunoprecipitation assay of the Zt-sLRR (expressed in N. benthamiana) that was used in a protein mass-spectrometry analysis, performed by the collaborating Tholey Lab, in Kiel. From this analysis, we identified interacting proteins that have direct roles in plant immune signalling (Fig. 3).
I screened additional effector candidates for immune-suppressing activity and identified four additional effectors (ZtNIS1, Zt1278, Zt132, and Zt190) that are each able to suppress BAK1-dependent ROS burst and cell-death. Two of the three candidate effectors, Zt1278 and Zt132, are homologues of each other and belong to a structural family described as killer protein-like 6 (KP6) effectors. One other effector from this screen, Zt1276 also belonged to this family, but did not suppress host immune responses (Fig. 4).
I identified effector candidates that were putatively unique to Z. tritici. After examining the predicted structures of these effectors, three, Zt156, Zt35, and Zt_1_4, stood out as strong candidates for studying Z. tritici virulence. These effectors’ predicted structures resemble SnTox3, a host-specific effector from a wheat pathogen, Parastagonospora nodorum (Fig. 5); however, they share no sequence similarity. SnTox3 interacts with pathogenesis-related protein 1 (PR-1).
Together with a bachelor thesis student, Fiene Knuth, we screened these effectors, and SnTox3 for their ability to interact with wheat PR-1; however, we observed no interaction between PR-1 and the Z. tritici effectors. I reached out to my PhD Lab, the Solomon Lab (ANU), who had identified the SnTox3-PR-1 interaction. In their initial wheat cDNA Y2H library screen, performed by Dr Susan Breen, they also identified that SnTox3 might interact with SnRK1, a regulatory kinase of sugar metabolism and defence-related cell-death. We performed an additional Y2H assay and observed interaction between the Z. tritici effectors and SnRK1 (Fig. 6).
I have also identified additional candidate effector homologues of interest, and using structural predictions, was able to match these with an interaction partner involved in wheat immune responses. This data is currently confidential as it may have additional applications, but I intend to release the data as soon as possible.
The project was successful, and I identified multiple effectors that interact with and/or suppress components of the plant immune system. I have presented this data at conferences, and I am currently preparing the first two manuscripts (the Zt-sLRR data and the additional immune suppressing effector data). Subsequently, I will perform the final assays to finalise the SnTox3-like effector project, and aim to publish this as an additional manuscript. All of the manuscripts will be made available as open-access pre-prints before publication.
