Periodic Reporting for period 1 - Zuppressors (Identifying Zymoseptoria tritici effectors that suppress wheat immune responses)
Okres sprawozdawczy: 2021-04-01 do 2023-03-31
Food security is a major concern to growing world populations and efforts are required to maximise the yield of crops. Plant pathogens result in significant annual losses, and so it is important to better understand these pathogens, and the mechanisms they use to infect plants, in order to develop new strategies for mitigating associated losses. One of the major fungal pathogens of wheat (particularly in Europe) is Zymoseptoria tritici, which is responsible for the necrotic lesion disease known as Septoria tritici blotch (STB). Despite its importance, very little is known about the molecular mechanisms involved in how the pathogen manipulates the host during infection. Before inducing necrotic lesions, Z. tritici undergoes an extended latent growth phase, and is able to evade the host’s immune system. However, we do not know how this fungus actively suppresses the host immune system.
The goal of my Marie Curie Fellowship project was to help develop our understanding of the molecular processes that occur during the latent phase of Z. tritici’s growth in wheat by identifying small-secreted proteins that are involved in host-immune suppression (or targeting plant proteins involved in this process). The three primary objectives of my project were:
• Objective 1: Identify effectors conserved among all Zymoseptoria species that are able to suppress BAK1 dependent immunity.
• Objective 2: Identify effectors unique to Z. tritici, but conserved across all examined isolates of the fungus, and screen for ability to confer host-range specificity
• Objective 3: Express and purify candidate BAK1-suppressing effectors and candidate host-range effectors for infiltration in wheat.
To undertake this project, I used a number of different molecular and bioinformatics techniques and established multiple new collaboration partnerships. From these efforts, I have successfully identified a number of effectors that are involved in plant immune-suppression, and/or that putatively target plant proteins involved in defence signalling.
The goal of my Marie Curie Fellowship project was to help develop our understanding of the molecular processes that occur during the latent phase of Z. tritici’s growth in wheat by identifying small-secreted proteins that are involved in host-immune suppression (or targeting plant proteins involved in this process). The three primary objectives of my project were:
• Objective 1: Identify effectors conserved among all Zymoseptoria species that are able to suppress BAK1 dependent immunity.
• Objective 2: Identify effectors unique to Z. tritici, but conserved across all examined isolates of the fungus, and screen for ability to confer host-range specificity
• Objective 3: Express and purify candidate BAK1-suppressing effectors and candidate host-range effectors for infiltration in wheat.
To undertake this project, I used a number of different molecular and bioinformatics techniques and established multiple new collaboration partnerships. From these efforts, I have successfully identified a number of effectors that are involved in plant immune-suppression, and/or that putatively target plant proteins involved in defence signalling.
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.
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.
Despite its importance as a major wheat pathogen, very little is known about Z. tritici effectors and how they suppress immune responses during infection. Developing our understanding about this these proteins and host systems targeted is important for developing new strategies for controlling the disease (such as finding new sources of resistance or susceptibility genes). The findings of my research have provided new data about these effectors’ activity and potential interacting proteins from wheat.
One of the significant recent advances in the field of effector biology has come from improvements in protein structure predictions. My research used this technological advance to help generate hypotheses about effector function, and, in particular, to successfully predict putative wheat protein interactors. These tools have enabled me to rapidly generate novel data for the Z. tritici-wheat pathosystem, with findings that have potential applications beyond plant-pathogen interactions. I expect to build upon the results of my fellowship to develop my independent research program, and to further advance the community’s understanding about Z. tritici-wheat interactions. Further, I hope that these results generated from my project will be built upon by others researchers, and that I might be fortunate enough establish further collaborations to assist in undertaking future work to study this important disease.
One of the significant recent advances in the field of effector biology has come from improvements in protein structure predictions. My research used this technological advance to help generate hypotheses about effector function, and, in particular, to successfully predict putative wheat protein interactors. These tools have enabled me to rapidly generate novel data for the Z. tritici-wheat pathosystem, with findings that have potential applications beyond plant-pathogen interactions. I expect to build upon the results of my fellowship to develop my independent research program, and to further advance the community’s understanding about Z. tritici-wheat interactions. Further, I hope that these results generated from my project will be built upon by others researchers, and that I might be fortunate enough establish further collaborations to assist in undertaking future work to study this important disease.