Biomedical subjects

Brad Day

Publications and source records attributed to Brad Day.

7 recordsLinked to original sources

PubMed2006

NDR1 interaction with RIN4 mediates the differential activation of multiple disease resistance pathways in Arabidopsis.

Recognition of pathogens by plants involves the coordinated efforts of molecular chaperones, disease resistance (R) proteins, and components of disease resistance signaling pathways. Characterization of events associated with pathogen perception in Arabidopsis thaliana has advanced understanding of molecular genetic mechanisms associated with disease resistance and protein interactions critical for the activation of resistance signaling. Regulation of R protein-mediated signaling in response to the bacterial pathogen Pseudomonas syringae in Arabidopsis involves the physical association of at least two R proteins with the negative regulator RPM1 INTERACTING PROTEIN4 (RIN4). While the RIN4-RPS2 (for RESISTANCE TO P. SYRINGAE2) and RIN4-RPM1 (for RESISTANCE TO P. SYRINGAE PV MACULICOLA1) signaling pathways exhibit differential mechanisms of activation in terms of effector action, the requirement for NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) is shared. Using a yeast two-hybrid screen, followed by a series of coimmunoprecipitation experiments, we demonstrate that the RIN4-NDR1 interaction occurs on the cytoplasmically localized N-terminal portion of NDR1 and that this interaction is required for the activation of resistance signaling following infection by P. syringae expressing the Cys protease Type III effector protein AvrRpt2. We demonstrate that like RPS2 and RPM1, NDR1 also associates with RIN4 in planta. We suggest that this interaction serves to further regulate activation of disease resistance signaling following recognition of P. syringae DC3000-AvrRpt2 by Arabidopsis.

Arabidopsis↗

PubMed2006

Host-microbe interactions: shaping the evolution of the plant immune response.

The evolution of the plant immune response has culminated in a highly effective defense system that is able to resist potential attack by microbial pathogens. The primary immune response is referred to as PAMP-triggered immunity (PTI) and has evolved to recognize common features of microbial pathogens. In the coevolution of host-microbe interactions, pathogens acquired the ability to deliver effector proteins to the plant cell to suppress PTI, allowing pathogen growth and disease. In response to the delivery of pathogen effector proteins, plants acquired surveillance proteins (R proteins) to either directly or indirectly monitor the presence of the pathogen effector proteins. In this review, taking an evolutionary perspective, we highlight important discoveries over the last decade about the plant immune response.

Bacteria↗

PubMed2006

Domain switching and host recognition.

The collective function of secreted pathogen effector molecules is to enhance the virulence and avirulence activity of the pathogen during the infection of its host. While the activity of a majority of pathogen effectors is unknown, several classes of effector molecules have been well characterized. Among these include proteins which function to modulate host defences either through proteolysis, post-translational modifications, or by directly manipulating the host transcriptional machinery that regulates the induction of defence responses. In recent years, several key advances have been made in the characterization of the latter class of effector molecules. Among these include research characterizing the processes associated with host nuclear import and the targeting of host transcriptional defences. While current research is beginning to reveal the biochemical and genetic mechanisms controlling the induction of host resistance, the signalling events that control host specificity remain largely unknown. In this issue of Molecular Microbiology, work by Nissan et al. sheds light onto the molecular-genetic patterns involved in determining host specificity and pathogen virulence in the Pantoea-gypsophila interaction.

Bacterial Proteins↗

PubMed2005

Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana.

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.

Arabidopsis Proteins↗

PubMed2005

Molecular basis for the RIN4 negative regulation of RPS2 disease resistance.

Recent studies have demonstrated that RPS2, a plasma membrane-localized nucleotide binding site/leucine-rich repeat protein from Arabidopsis thaliana, associates with RPM1 Interacting Protein 4 (RIN4) and that this association functions to modulate the RPS2-mediated defense pathway in response to the bacterial effector protein AvrRpt2. In addition to negatively regulating RPS2 activity, RIN4 is also a target of AvrRpt2, a Cys protease and cognate bacterial effector protein of RPS2. Nicotiana benthamiana has been employed as a heterologous expression system to characterize the RPS2-RIN4 association, defining the domains in RIN4 required for the negative regulation of RPS2 activity. Upon inoculation of N. benthamiana leaves with Agrobacterium tumefaciens expressing RPS2, a rapid hypersensitive response (HR) is detected with 22 h of infiltration. The HR can be blocked by infiltrating the leaf with A. tumefaciens expressing RPS2 in the presence of RIN4, recapitulating the ability of RIN4 to interfere with RPS2-mediated resistance in Arabidopsis. Moreover, in the presence of RIN4, the RPS2-mediated HR can be restored by the delivery of AvrRpt2 via A. tumefaciens. This assay has been developed as a phenotypic marker for (1) the HR-inducing phenotype associated with RPS2, (2) negative regulation of RPS2 by RIN4, and (3) the AvrRpt2-mediated disappearance of RIN4. Here, we present a series of deletion and site-directed mutation analyses to identify amino acids in RIN4 required for the RPS2-RIN4 association and to distinguish these from residues in RIN4 that serve as a target sequence for AvrRpt2. In addition to characterizing the RPS2-RIN4 association in N. benthamiana, we have moved forward to show that the biological relevance of these amino acid changes is applicable in Arabidopsis as well. To this end, we have identified specific amino acids within the C-terminal half of RIN4 that are required for RPS2 regulation and association.

Agrobacterium tumefaciens↗

PubMed2005

Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2.

During infection of Arabidopsis thaliana, the bacterium Pseudomonas syringae pv tomato delivers the effector protein AvrRpt2 into the plant cell cytosol. Within the plant cell, AvrRpt2 undergoes N-terminal processing and causes elimination of Arabidopsis RIN4. Previous work established that AvrRpt2 is a putative cysteine protease, and AvrRpt2 processing and RIN4 elimination require an intact predicted catalytic triad in that AvrRpt2. In this work, proteolytic events that depend on AvrRpt2 activity were characterized. The amino acid sequence surrounding the processing site of AvrRpt2 and two related sequences from RIN4 triggered Avr-Rpt2-dependent proteolytic cleavage of a synthetic substrate, demonstrating that these sequences are cleavage recognition sites for AvrRpt2 activity. Processing-deficient AvrRpt2 mutants were identified and shown to retain their ability to eliminate wild-type RIN4. Single amino acid substitutions were made in each of the two RIN4 cleavage sites, and mutation of both sites resulted in cleavage-resistant RIN4. Growth of Pseudomonas expressing AvrRpt2 was significantly higher than catalytically inactive mutants on Arabidopsis rin4/rps2 mutant plants, suggesting there are additional protein targets of AvrRpt2 that account for the virulence activity of this effector. Bioinformatics analysis identified putative Arabidopsis proteins containing sequences similar to the proteolytic cleavage sites conserved in AvrRpt2 and RIN4. Several of these proteins were eliminated in an AvrRpt2-dependent manner in a transient in planta expression system. These results identify amino acids important for AvrRpt2 substrate recognition and cleavage as well as demonstrate AvrRpt2 protease activity eliminates multiple Arabidopsis proteins in a transient expression system.

Amino Acid Sequence↗

PubMed2004

Overexpression of the plasma membrane-localized NDR1 protein results in enhanced bacterial disease resistance in Arabidopsis thaliana.

Previous studies have established that mutations in the NDR1 gene in Arabidopsis thaliana suppress the resistance response of three resistance proteins, RPS2, RPM1, and RPS5, to Pseudomonas syringae pv. tomato (Pst) strain DC3000 containing the cognate effector genes, avrRpt2, avrRpm1, and avrpPhB, respectively. NDR1 is a plasma membrane (PM)-localized protein, and undergoes several post-translational modifications including carboxy-terminal processing and N-linked glycosylation. Expression of NDR1 under the NDR1 native promoter complements the ndr1-1 mutation, while overexpression of NDR1 results in enhanced resistance to virulent Pst. Sequence analysis and mass spectrometry suggest that NDR1 is localized to the PM via a C-terminal glycosylphosphatidyl-inositol (GPI) anchor. GPI modification would potentially place NDR1 on the outer surface of the PM, perhaps allowing NDR1 to act as a transducer of pathogen signals and/or interact directly with the pathogen.

Arabidopsis↗