Hyaloperonosprora arabidopsidis/Arabidopsis Interactions

Hyaloperonospora arabidopsidis on Arabidopsis thaliana

National Science Foundation 2010 Oomycete Effector Proteins as Molecular Probes to Elucidate Arabidopsis Disease Resistance Signaling Networks

We have been awarded a National Science Foundation Arabidopsis 2010 grant to study effector proteins from the oomycete pathogen, Hyaloperonosprora arabidopsidis.

Our understanding of the molecular basis of plant disease resistance has greatly benefited from studies that have featured different classes of pathogens interacting with Arabidopsis thaliana. In fact, current theories about plant innate immunity have heavily relied on knowledge generated from these studies.

It has been hypothesized that early land plants originally contained basal immune receptors that were capable of recognizing pathogen associated molecular patterns (PAMPs) leading to the activation of basal host defense responses (8). The ability of plants to actively recognize various PAMPs has now been referred to as PTI (PAMP-Triggered Immunity) and most likely represents an ancient form of plant defense that allowed plants to survive microbial invasions and colonize land. These events put a selection on microbial populations to acquire the ability to overcome basal resistance leading to the acquisition of virulence effector proteins that have now been demonstrated to either suppress or modulate basal defense. Furthermore, the ability to deliver virulence effector proteins directly into the plant cell to reprogram host defense has become a unifying theme among successful phytopathogens.

In an evolutionary struggle for survival, plants evolved the capability to actively recognize pathogen effector proteins, resulting in a robust defense response leading to the inhibition of pathogen proliferation in the infected plant. This form of resistance is often referred to R-protein mediated resistance or ETI (Effector-Triggered Immunity). 

Phytopathogenic oomycetes cause some of the most devastating diseases affecting agricultural crops. Hyaloperonospora arabidopsidis is a native oomycete pathogen of Arabidopsis and is related to other oomycete phytopathogens that include several species of Phytophthora, including the causal agent of potato late blight. 

Recently, four oomycete effector genes have been isolated, and several oomycete genomes have been sequenced. We have developed an efficient and genetically amenable system to test putative effector genes using the bacterial pathogen Pseudomonas syringae pv.tomato DC3000. The Hpa effector protein ATR13 was delivered via P. syringae by fusing the ATR13 gene with the avrRpm1 type three secretion signal peptide, a bacterial sequence that allows transfer of proteins into the host cell through the bacterial type III secretion system. We have also inserted ATR13 into the genome of the turnip mosaic virus, a single-stranded RNA virus.

Arabidopsis thaliana

Our results show that delivery of ATR13 via the bacterial or viral pathogen triggers defense responses in plants containing the cognate resistance protein RPP13Nd, which restricts proliferation of both pathogens. Hence, recognition of ATR13 by RPP13 initiates defense responses that are effective against oomycete, bacterial and viral pathogens, pointing to a common defense mechanism.

Finally we have characterized regions of the RPP13Nd resistance protein that are essential for effector recognition and/or downstream signaling, using transient co-expression in Nicotiana benthamiana.More recently, we have made significant discoveries in the area of NLR immune receptor activation. Specifically, we have developed a surrogate host pathogen system that has allowed us to recapitulate the molecular basis of ATR1/RPP1 specificity. We have shown that when the Arabidopsis RPP1 disease resistance gene is co-delivered with the cognate recognized ATR1 allele that we can observe a hypersensitive cell death response that recapitulates the recognition that occurs between isolates of Hyaloperonospora arabidopsidis that are specifically recognized by resistant lines of Arabidopsis thaliana. 

Furthermore, we have shown that only the recognized alleles of ATR1 co-immunoprecipate with the cognate RPP1 alleles in planta. These experiments are extremely significant as this suggests that these proteins directly interact in the plant. Since the primary amino sequence of the ATR1 effector protein does not share homology with any know proteins in the database, we have focused our attention on solving the 3-D structure of these proteins. The solving of the 3-D crystal structure of the ATR1 effector protein has revealed an unprecedented, two-domain, dimeric fold in this protein. We have identified conserved hydrophobic surface residues that can drive the design of targeted experiments to discover the functional regions of the protein. These studies exemplify the power of combining structural and biological approaches to reveal critical domains involved in pathogen effector function.