Genetic, Biochemical, and Structural Studies of the Hyaloperonospora arabidopsidis Effector ATR13 and its Cognate Arabidopsis thaliana R-gene, RPP13
- Author(s): Leonelli, Lauriebeth
- Advisor(s): Staskawicz, Brian
- et al.
The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana. Since oomycetes are a particularly important agricultural pest, this pathosystem is an ideal setting for exploration of interactions between host and microbe, as genome sequences for both are available and possess a high level of genetic diversity between naturally occurring populations. Plant recognition of Hpa infection occurs when resistance proteins (R-genes) in the plant host recognize pathogen-derived effectors, which are proteins delivered to the host. One such protein, the Hpa effector ATR13 Emco5, is examined in this study. Herein, we use NMR to solve the backbone structure of a highly disordered protein, ATR13 Emco5, revealing a loosely packed protein possessing a great deal of flexibility. In addition to solving this structure, we use site-directed and random mutagenesis to expose several amino acid residues involved in the recognition response conferred by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches composed of residues that are under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with weak structural similarities to RAN-GTP that contains two surface-exposed patches, only one of which is involved in RPP13 recognition specificity. Furthermore, we have identified two potential protein targets of ATR13 (the RCC1 and PRP39 families). Finally, and we have performed several different EMS mutagenesis screens leading to the isolation of three lines of A. thaliana plants disrupted in RPP13 signaling.