Halterman, Dennis A. 2017. Demonstrating concepts of pathogenesis using effectors of Phytophthora infestans. The Plant Health Instructor. 10.1094/PHI-T-2017-0610-01
Dennis A. Halterman, USDA-ARS Vegetable Crops Research Unit, Madison, WI, Maya C. Hayslett, Victoria Kartanos, and Douglas I. Rouse, Department of Plant Pathology, University of Wisconsin, Madison
The objective of this exercise is to increase student proficiency in pathogenesis-related concepts. Specifically, students should be able to:
Pathogenesis, or how pathogens cause disease, is an important concept in plant pathology. The study of pathogenesis in plant pathology has rapidly expanded and is now a significant portion of plant pathology research conducted at the level of molecular host-pathogen interactions. This laboratory exercise directly addresses some of these pathogenesis concepts, specifically the interaction of host R gene products with pathogen effectors, and the outcome of these interactions (e.g., the hypersensitive response).
Resistance genes (referred to as R genes) are host plant genes that confer resistance to a pathogen. R genes encode a protein (R protein) that detects the pathogen and elicits a resistance response. Effectors are pathogen proteins involved in disease development. The pathogen uses them to infect and cause disease. When a host R protein detects an effector, the cell initiates a resistance response. The response can be the death of the infected host cell, called a hypersensitive response. This localized cell death prevents the pathogen from spreading and colonizing the plant. R proteins may interact directly with effectors to detect the pathogen, or there may be other proteins involved (Gururani et al. 2012).
This activity utilizes Phytophthora infestans effectors and potato resistance genes in a model system developed by Dr. Dennis A. Halterman at the USDA (Halterman et al. 2010). P. infestans is the causal agent of the potato late blight disease (Schumann and D’Arcy 2000). Potato plants with the RB protein can recognize the presence of effector IPI-O1 from P. infestans. When P. infestans infects a plant expressing RB, the effector is detected, which elicits a hypersensitive cell death response (HR) in infected plant cells. In this exercise, students will investigate the interaction of a second effector, the related IPI-O4, with the RB protein and with IPI-O1.
Click here for instructor Note #1: Discussion of learning goals
Transgenic tobacco expressing the RB gene is used for the experiments. Students infiltrate the leaves of RB-expressing transgenic tobacco with Agrobacterium tumefaciens capable of expressing the effectors IPI-O1 and IPI-O4 from P. infestans in the plant. This experiment also provides a platform for discussion of transgenic organisms and their use in agriculture and research.
Click here for instructor note #2: Preparation of Materials
For this exercise, you will look at the interaction between Phytophthora infestans effectors and potato R-genes. You will NOT be using the pathogen P. infestans, instead you will use Agrobacterium tumefaciens that has been transformed with an effector gene from P. infestans (the T region has been removed from the Ti plasmid and the effector gene has been added) (Agrios 2005). Therefore, plants inoculated with these transformed strains of A. tumefaciens will express the effector in the infected cells. You will be using two different strains, one with the effector gene IPI-O1 and one with the effector gene IPI-O4.
The plants you will inoculate are NOT potato plants but transgenic tobacco plants that express a resistance gene (the RB gene) that recognizes the presence of the IPI-O1 effector.
Before starting you may want to visit this YouTube video to see infiltration of a tobacco leaf: https://www.youtube.com/watch?v=GHc7PU_jG2M.
Assuming the interaction is gene-for-gene and that it leads to a hypersensitive response, what do you expect to see next week in each treatment?
Examine your plants for a resistance response. You should get either a hypersensitive response or no response (compare to the negative control). Figure 1 below shows what the HR response should look like. However, it is possible that you may get an intermediate response where the inoculated area has patches of healthy tissue mixed with patches of dead tissue. Record your results in Table 1 below.
Is there is a gene-for-gene interaction as assumed? Explain. Do both effectors have a direct interaction with the RB protein? How do you know that the interaction is or is not gene-for-gene?
What is the advantage to using transgenic organisms in this type of research?
Click here for instructor Note #3: Explanation of the expected results
Agrios, G. 2005. Plant Pathology, 5th edition. Elsevier Academic Press, Burlington, MA.
Gururani, A. M., Venkatesh, J., Upadhyaya, C. P., Nookarajuc, A., Pandey, S. K, Park, S. E. 2012. Plant disease resistance genes: Current status and future directions. Physiological and Molecular Plant Pathology 78:51-65.
Halterman, D. A., Chen, Y., Sopee, J., Berduo-Sandoval, J. and Sánchez-Pérez, A. 2010. Competition between Phytophthora infestans effectors leads to increased aggressiveness on plants containing broad-spectrum late blight resistance. PLoS ONE 5:e10536.
Schumann, G. L., and D’Arcy, C. J. 2000. Late blight of potato and tomato. The Plant Health Instructor. DOI: 10.1094/PHI-I-2000-0724-01