Oomycete plant pathogens cause extremely destructive diseases on crops and in natural ecosystems. Little is known about the molecular weapons that enable oomycetes to be such successful pathogens. Analyses of four recently completed oomycete genomes suggest that oomycetes export dozens or hundreds of their own 'effector' proteins to the interior of plant cells. These pathogen effectors are expected to sabotage plant immune responses and trigger other effects that benefit the pathogen. However, almost nothing is known about the molecular functions of oomycete effectors. This project will investigate the functions of six pairs of effectors that are conserved between two distantly related oomycetes: the Arabidopsis downy mildew pathogen Hyaloperonospora parasitica and the soybean root rot pathogen Phytophthora sojae. It is expected that a focus on conserved effectors will reveal insights that are applicable to the majority of oomycete pathogens. The investigators will perform a series of molecular genetic experiments to determine whether these effectors are capable of suppressing plant immunity or altering plant cell structure or physiology, and whether the pathogen's ability to cause disease is affected when these genes are silenced or overexpressed. Finally, one pair of conserved proteins will be selected for experiments to identify their exact targets inside plant cells. These experiments are expected to reveal important, broadly conserved molecular strategies that underpin oomycetes' ability to cause disease, which will in turn open new avenues toward more sophisticated disease control strategies. The project will provide multidisciplinary training for graduate students and undergraduates. Some aspects of the project will be collaboratively integrated with an outreach program at Virginia Tech that uses Arabidopsis reverse genetics in high school science classrooms.
Intellectual Merit Very little is known about the molecular mechanisms through which oomycete pathogens cause diseases in plants. It is increasingly clear that a major strategy for oomycetes, and other pathogens, is to secrete effector proteins to the interior of plant cells. Once inside plant cells, effectors can target specific plant regulatory proteins, to disable or reprogram plant regulatory networks and thereby increase the plant's susceptibility to disease. This project was designed to better understand how effectors from oomycete pathogens function, evolve, and contribute to infection of host plants. This project contributed to the identification of a large family of effector proteins from Hyaloperonospora arabidopsidis, which is a pathogen used in studies of the model plant Arabidopsis thaliana. We used comparative genomic approaches to identify effector genes that are evolutionarily conserved in distantly related pathogens (i.e., the destructive Phytophthora genus). We prioritized these genes for functional analysis, under the rationale that these genes have been conserved during evolution because they make important contributions to pathogenicity. Our working hypothesis is that these effectors target the same/similar proteins in diverse plants. We then established defense-suppressive roles for several previously uncharacterized effectors and we showed that these effectors can suppress or provoke immune responses in distantly related plant species. This finding counters the viewpoint that effectors are "optimized" to work most efficiently in narrow range of plant hosts. Additionally, we provided the most conclusive evidence to date that oomycete effectors can promote immunity from within the plant nucleus. We identified probable targets of one pair of conserved effector proteins that open a connection to understand the mechanisms through which proteins and other compounds are secreted to specific subcellular locations during the plant immune response, and how these processes are subverted by pathogens. We also used a bioinformatic approach to identify an oomycete effector that is structurally and functionally similar to an effector that is conserved in bacterial plant pathogens, paving the way towards identifying the plant target of these effectors. Finally, we started a project that potentially reveals a mechanism by which H. arabidopsidis manipulates a plant hormone response pathway to suppress immunity. These studies added to the foundation of knowledge about how oomycetes deploy effectors to facilitate infection. We are in a strong position to build on these studies towards a clear understanding of the molecular basis of oomycete diseases. Additionally, the effectors will serve as valuable functional probes to explore the functions of the plant proteins that they target. Broader Impacts Relevance to agriculture: Annual losses to Phytophthora diseases are estimated at 10’s of billions of dollars in losses on a global scale, and downy mildews account for ~25% of the $5 billion dollar global fungicide market. Although the proposed research is focused on fundamental aspects of oomycete-plant interactions, our long-term vision is that foundational knowledge on molecular aspects of these interactions will inspire new control strategies, perhaps based on suppression of effector function. Training: This project provided training in bioinformatics, molecular biology, and functional genomics for three Ph.D. students and 12 undergraduates. Outreach and Public Engagement: Some aspects of the research were integrated with an outreach program at Virginia Tech that uses Arabidopsis reverse genetics in high school science classrooms. In this collaboration, the students conduct original research (e.g., developmental phenotypes and abiotic stress resistance) on transgenic plants expressing effector genes. McDowell lab researchers interacted with the students and teachers to provide the biological context of the research, advice on experimental design, and interpretation of the results. This collaboration integrated current plant biology research into high school education, while developing the researchers’ teaching and communication skills. During the granting period, the PI and students interacted with 1007 students and 26 teachers at 14 schools. Community resources: This project partially supported development of high quality, annotated assembly of the H. arabidopsidis genome, along with web browsers, a training conference, and an annotation jamboree to facilitate full utilization of the genome. The project also supported publications on methodology for experiments with H. arabidopsidis; PI McDowell organized and edited a book on research methods for plant immunity. PI McDowell also authored invited perspective articles on recent advances in pathogen genomics and immune signaling, and a review of recent advances in oomycete effector biology.
