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INVESTIGATOR: Savory, E. A.; Day, R. B.; Buell, C. R.

PERFORMING INSTITUTION:
MICHIGAN STATE UNIV
EAST LANSING, MICHIGAN 48824

AN INTEGRATED LAB TO FIELD APPROACH TOWARDS UNDERSTANDING AND DEVELOPING RESOURCES FOR MANAGEMENT OF CUCURBIT DOWNY MILDEW (PSEUDOPERONOSPOR

NON-TECHNICAL SUMMARY: Cucumber downy mildew is a major limiting factor to production of cucumber and other cucurbit crops in the United States. This disease is caused by the oomycete pathogen, Pseudoperonospora cubensis, which can destroy an entire crop within 2 weeks under optimum conditions. Despite its economic importance, the basic mechanisms underlying the pathogenicity of Ps. cubensis and its interactions with cucumber are not well studied. This project seeks to establish resources to better understand the interaction between Ps. cubensis and cucumber, investigate determinants of the pathogen's virulence and pathogenicity, and to develop materials for an outreach and education program for stakeholders such as cucumber growers and processors. Genes up- or down-regulated during infection of cucumber with Ps. cubensis will be assessed using RNA-Seq, which enables collection of information from both the pathogen and host simultaneously. Differential regulation of genes will be correlated with the pathogen growth stages and symptom development in the host to determine those important in both pathogenicity and virulence as well as host response. Subsequent characterization of upregulated pathogenicity determinants will be performed to determine those essential for disease development. Finally, data gleaned from the first two objectives will be incorporated into an outreach and education program, coupled with information about basic pathogen biology, epidemiology, and control recommendations to better serve our stakeholders.

OBJECTIVES: Cucurbit downy mildew, caused by the oomycete pathogen Pseudoperonospora cubensis, is the major limiting factor in cucumber (Cucumis sativus) production in the United States. In the US, the cucurbit industry occupies approximately 229,000 hectares, with a market value of ~$1.6 billion. As the #1 producer of cucumbers used for processing, with a farm gate value of $30.6 million, Michigan's 38,000 acres of pickling cucumbers generate a finished product valued at $240.7 million. In recent years however, the re-emergence of cucurbit downy mildew as a major disease problem has placed significant strain on the cucurbit industry, not only in Michigan, but also for growers throughout the country. Thus, understanding the molecular-genetic basis for this interaction and the function of virulence determinants of Ps. cubensis is key to the long-term survival of this industry. This project focuses on 1) understanding the interaction between pathogen and host, 2) elucidating genetic components that contribute to virulence and diversity, and 3) disseminating this knowledge in a timely and accessible manner to the appropriate stakeholders (ie., cucumber growers and producers). Completion of these objectives will result in resources that will contribute to development of an effective disease management system for Ps. cubensis as well as establish an effective outreach and education program for our stakeholders.

APPROACH: This project seeks to develop transcriptomic and genetic resources for the purpose of better understanding the host-pathogen interaction between C. sativus and Ps. cubensis. Profiling transcriptome dynamics in plants in response to pathogen invasion can often provide useful information for not only understanding the basic mechanisms of disease resistance, but also towards the development of strategies to detect and manage the onset and spread of disease in the field. Next-generation sequencing technology assays such as RNA-Seq, allow for the simultaneous collection of expression data not only in the plant, but also in the pathogen. Analysis of these expression patterns in Ps. cubensis throughout the infection process will yield information about the mode of pathogenicity as well as uncover new virulence determinants. Characterization of virulence determinants, such as effector proteins, can tell us about the mechanisms of infection and colonization, and potentially function as targets for breeding resistant cultivars. As more information about the genetic capacity, pathogenicity, and virulence of Ps. cubensis is obtained, it is important that the appropriate stakeholders (i.e., cucumber growers and producers) remain informed. To this end, this project will additionally develop an outreach and education program aimed to educate stakeholders, building on an established collaboration between the Pickle Packers International (PPI) and researchers at Michigan State University.

PROGRESS: 2011/08 TO 2013/08
OUTPUTS: Cucurbit downy mildew, caused by the oomycete pathogen Pseudoperonospora cubensis, is the major limiting factor in cucumber (Cucumis sativus) production in the United States, and despite its economic importance, has been relatively understudied. The overall goal of goals of this project were to establish transcriptomic and genomic resources for the Ps. cubensis-C. sativus interaction, to utilize these resources for better understanding of the pathogenicity of Ps. cubensis through characterization of effector proteins, and to share our findings with members of the research community and our stakeholders. In this year of funding, we were able to make good progress in accomplishing these goals. Utilization of next-generation sequencing techniques allowed us to sequence the genome of Ps. cubensis as well as to simultaneously capture gene expression during an infection time course of both Ps. cubensis as well as a susceptible C. sativus host. We generated a draft sequence of the 88.2 Mb Ps. cubensis genome and predicted 271 candidate effector proteins with potential roles in virulence and pathogenicity. Using a combination of molecular biology, cell biology, and genetic approaches, we characterized the roles of these effector proteins. Additionally, using RNA-Seq, we generated transcriptome data for 6 time points of Ps. cubensis infection on a susceptible C. sativus host. The results generated through the research supported by this proposal were disseminated to other members of our research community as well as our stakeholders and the community via a combination of research presentations, posters, and outreach activities. The findings from this project were presented to current and potential collaborators at the ICuGI/Sol Meeting (2011; Kobe, Japan) the Oomycete Genetics Meeting (2012; Nanjing, China), the XV Congress on Molecular-Plant Microbes Interactions (2012; Kyoto, Japan), as well as via seminars at both Bowling Green State University (2012; Bowling Green, OH), and Oregon State University (2012; Corvallis, OR). To reach our stakeholders, results were reported at the annual Pickle Packers International, Inc. Meeting (2011; Las Vegas, NV) as well as at the Great Lakes Fruit, Vegetable and Farm Market EXPO (2011; Grand Rapids, MI). In addition, an interactive educational program utilizing the Ps. cubensis-C. sativus interaction was developed for the Michigan State University Girls Math and Science Day to introduce 6th grade girls to the concepts of plant pathology, biology, and microscopy. Overall, this project made a significant contribution to the understanding of the Ps. cubensis-C. sativus interaction by sequencing and assembly the genome, identifying a suite of candidate effector proteins, and generating the first large-scale gene expression data elucidating the transcriptional changes that occur during infection of a susceptible host. In addition, we identified the first occurrence of an effector protein generated via alternative splicing of a non-effector gene, leading to additional collaborative research looking at alternative splicing throughout infection. PARTICIPANTS: PI Elizabeth Savory conducted the research described in this proposal under the guidance of project mentors R.B. Day and C. R. Buell. This research and the subsequent publications were the basis for her PhD dissertation. TARGET AUDIENCES: The research undertaken during this funding year was presented to peers and collaborators at several national and international meetings as well as to project stakeholders including growers and processors. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2011/08 TO 2013/08
Establishing genomic and transcriptomic resources of economically important plant pathogens not only provides valuable resources for the research community, but also yields insight into the infection mechanisms and lifestyles of these pathogens, providing information that can be used to develop better control methods. Through this project we were able to increase our coverage of the 88.2 Mb Ps. cubensis genome as well as to utilize our assembly and annotation to predict a candidate list of effector proteins. Through analyses of one of these candidate effector proteins, PscRXLR1, we demonstrated that it arises as a product of alternative splicing. This is the first example of an alternative splicing event in plant pathogenic oomycetes transforming a non-effector gene to a functional effector protein. Through this proposal we also developed the first large scale gene expression data set for Ps. cubensis infection of cucumber. We assembled transcripts generated from RNA-Seq of a sporangia library as a preliminary analysis of the gene expression of Ps. cubensis. This yielded nearly 30,000 transcripts, of while nearly all (98%) mapped to our assembled Ps. cubensis genome, providing expression data support for 61% of the predicted genes. Through analysis of our time course we captured differential expression of 2383 Ps. cubensis genes and identified similarities in expression to another important oomycete phytopathogen, Phytophthora infestans. We also identified differentially expressed C. sativus genes in response to infection. Overall, these findings provide the first look at gene expressing of Ps. cubensis during infection and have advanced our understanding of key molecular and genetic events in the virulence of Ps. cubensis providing a foundation for identifying mechanisms that could be used for future work developing more effective control measures.

PUBLICATIONS (not previously reported): 2011/08 TO 2013/08
1. Savory EA, Zou C, Adhikari BN, Hamilton JP, Buell CR, Shiu S-H, Day B. 2012. Alternative splicing of a multi-drug transporter from Pseudoperonospora cubensis generates an RXLR effector protein that elicits a rapid cell death. PLoS ONE 7(4): e34701.
2. Savory EA, Adhikari BN, Hamilton JP, Vaillancourt B, Buell CR, and Day B. 2012. RNA-Seq Analysis of the Pseudoperonospora cubensis transcriptome during cucumber (Cucumis sativus L.) infection. PLoS ONE 7(4): e35796.
3. Adhikari BN, Savory EA, Vaillancourt B, Childs KL, Hamilton JP, Day B, and Buell CR. 2012. Expression profiling of Cucumis sativus in response to infection by Pseudoperonospora cubensis. PLoS ONE 7(4): e34954.
4. Runge F, Telle S, Ploch S, Savory E, Day B, Sharma R and Thines M. 2011. The inclusion of downy mildews in a multi-locus-dataset and its reanalysis reveals a high degree of paraphyly in Phytophthora. IMA Fungus 2: 163.