New Tools to Understand Mechanisms of Nutrient Transfer from Plants to Biotrophic Pathogens

The interaction between Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), provides a model for understanding how oomycetes colonize plants. Hpa is a model organism for many highly destructive oomycete pathogens and transcriptomics of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of pathogen-proximal and pathogen-distal plant cells. The most direct interactions between Arabidopsis and Hyaloperonospora arabidopsidis occur in haustoriated cells- where the pathogen can secrete effectors and acquire nutrients needed for successful colonization and reproduction. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to "bulk" transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients.