Research Accomplishment Reports 2010

Localization of Secreted Proteins During Penetration and Iinvasive Growth of the Rice Blast Fungus Magnaporthe oryzae

M. Farman, M. M. Goodin, B. Valent, C. Soderlund
Department of Plant Pathology

 

Non-Technical Summary

Many fungi that cause diseases of plants are biotrophs that develop specialized infection hyphae within living plant cells. Interestingly, biotrophic fungi do not breach the host cell's plasma membrane. Instead, the membrane expands to accommodate the hyphae as they grow within the cell lumen. How the fungus is able to promote the required proliferation of plant membranes without triggering a defensive response is unknown. There is increasing evidence that proteins secreted by the fungus play critical roles in the establishment and maintenance of biotrophy. Some secreted proteins appear to be translocated into the plant cell cytoplasm, where they function to suppress defense responses and modify the host cell's metabolism. Others are deposited in the interface between the fungal cell wall and the plant membrane, where they may serve structural/protective roles, or function in the transfer of nutrients from the plant to the fungus.

To gain a comprehensive insight into the functions of secreted proteins in plant pathogenesis, we plan to use Magnaporthe oryzae infection of rice as a model pathosystem to study the many (>900) secreted proteins that are predicted to be encoded in the M. oryzae genome. To do this, we will fuse each secreted protein gene to another gene that codes for a fluorescent protein. The resulting fusion proteins will fluoresence under UV light. This will allow us to see when each proteins is expressed, and where it is distributed as the fungus grows inside rice cells. In this manner, we will classify secreted proteins based on their in planta localization patterns.

We expect to identify secreted proteins that are involved in the following processes: penetration into host cells, establishment of biotrophic hyphae inside the cells, formation of the interface between pathogen and host, passage through host cell walls (via plasmodemata), and, eventually, destruction of host tissues. Successful completion of this study will yield unique insights into the roles of secreted proteins in the development of the biotrophic interface. The formation of this interface is a crucial step in the pathogenic process and, therefore, a better understanding of this unique structure could lead to the development of improved strategies for controlling fungal diseases of plants.

2010 Project Description

Activities:

The Farman lab:

1) developed new vectors for protein localization. These included a Gateway version of a vector carrying a nuclear-targeted mCHERRY protein, a GUS reporter construct, as well as CFY, GFP, RFP and YFP fusion vectors that confer geneticin resistance;

2) Tested the localization for more than 200 proteins;

3) Developed protocols for immunodetection of fungal secreted proteins in planta; 4) Developed a system for automated uploading of images into the Magnaporthe Secreted Protein Localization database (with the Soderlund lab); 5) Uploaded localization images to the database.

The Valent lab:

1) performed cloning and localization experiments for 75 proteins previously determined to be specifically expressed during early biotrophic invasion;

2) performed gene disruption experiments for selected proteins;

3) Initiated confocal microscopy studies;

4) attempted to enhance sensitivity in fluorescence localization, including use of a stronger biotrophy-specific promoter and addition of a C-terminal nuclear localization signal to fusion proteins.

The Soderlund lab made improvements to the database that is used to house the images generated in the project. A pipeline for automated uploading of images into the database was developed. Goodin consulted on immunodetection protocols, provided confocal microscope training and advice on imaging.

Events: 5th International Rice Blast Conference, Little Rock, AR. Aug, 2010. Plant and Animal Genome Conference, San Diego, CA. Jan, 2010 (2010) & Jan 2011 (Valent). Department of Plant Pathology Seminar, Ohio State University, May, 2010 (Farman); & Jan 2011 (Valent) Department of Plant Pathology Seminar, Kansas State University, Oct, 2010. 5th KY Innovation & Entrepreneurship Conference, Apr 2010. Mycological Society of America-International Symposium on Fungal Endophytes of Grasses Meeting, Lexington, KY. Aug, 2010. USDA-NRI Microbial Functional Genomics Program Awardee Workshop Washington DC, Aug, 2010. Seminar, AgResearch Limited, Grasslands Research Centre, Palmerston North, New Zealand, December, 2010. New Zealand Microbiological Society & New Zealand Society for Biochemistry and Molecular Biology, Auckland, New Zealand, December, 2010. Symposium on Fungal and Yeast Cell Biology, Massey University, Auckland, New Zealand, November, 2010. 9th International Mycological Congress, Edinburgh, Scotland, August, 2010. Richard R. Nelson Memorial Lecture, Pennsylvania State University, April, 2010.

Services: The Farman lab provided Gateway localization vectors to the following individuals: Richard O' Connell, ETH Zurich, Switzerland; Lisa Vaillancourt, Univ. of KY; Benjamin Matthews, BARC-USDA; Kathy Dobinson, Agriculture and Agri-Food, Canada; Carolyn Young, Noble Foundation; Ane Sesma, Sainsbury Lab, UK; Richard Wilson, Univ. of NE. Goodin provided confocal microscope training to all new users of the Plant Sciences Confocal Imaging facility.

Products:

1) pFPL plasmid vectors for fungal protein localization.

2) Magnaporthe oryzae Secreted Protein Localization database and web page.

2010 Impact

Farman: Several new localization patterns were identified among the more than 200 new proteins that were studied in 2010. In particular, a novel subcellular structure was identified in spores and appressoria and one fungal secreted protein was found to accumulate in the nuclei of infected plant cells. Two fungal proteins were successfully detected using immunodetection. Based on these findings, it is likely that the constructs and protocols developed in this project will be of broad utility for studying secretion processes in other fungi. The Farman lab has already received numerous requests for the vectors that were developed in this project.

Valent: For the biotrophy-associated secreted proteins studied in the Valent laboratory, 93 out of 132 RFP-fusion proteins (70%) displayed detectable fluorescent signals in one or more infection stages. Among these 93 proteins, 80 showed accumulation in biotrophic interfacial complexes (BICs). Twenty-five of the BIC-associated proteins were translocated across the plant-derived extrainvasive-hyphal membrane surrounding biotrophic invasive hyphae in rice cells, and all but one of these translocated proteins moved into uninvaded neighboring rice cells, probably to prepare them before fungal invasion. One of these translocated proteins was specifically localized in the rice nucleus, and 3 were localized at the points where invasive hyphae had crossed into neighboring cells.

We are pursuing the hypothesis that the pathogen proteins that accumulate where hyphae had crossed the host cell walls play roles in the movement of the hyphae through plasmodesmata. We hypothesized that the 55 BIC-associated proteins that did not appear to be translocated to the rice cytoplasm might be translocated at levels below our detection limit. Therefore, we tested if addition of a nuclear localization signal to the fusion proteins would increase sensitivity by targeting translocated fluorescence to the rice nucleus. However, initial results did not support enhanced sensitivity in detecting rice translocation in these experiments.