Progress 09/01/03 to 08/31/07
Outputs
OUTPUTS: The work was described in multiple invited research seminars and through posters at scientific meetings.
PARTICIPANTS: Andrew Bent, Ruth Genger, Betania Quirino, Ryan Kipp, Kendra Hogan, Mark Howe, Allison Neirode, and James Chandler.
TARGET AUDIENCES: Primary target was the basic science research community, and the plant biotechnology research/development community.
Impacts
The most significant outcome was scientific training of project personnel. Betania Quirino worked as an Assistant Professor at Universidad Catolica Brasilia and is now a scientist at Embrapa (Brazil). Ruth Genger is now researching potato pathogens, diseases and cultivation practices as a researcher at University of Wisconsin - Madison. Undegraduate researchers Ryan Kipp and Kendra Hogan were accepted to and moved on to an excellent medical school and graduate school, respectively.
Publications
- Quirino, B.F., R. Genger, J.H. Ham, G. Zabala and A.F. Bent, 2004. Identification and functional analysis of Arabidopsis proteins that interact with resistance gene product RPS2 in yeast. Physiol. Molec. Plant Pathol. 65:257-267.
- Jurkowski, G.I., R. K. Smith, Jr., I. Yu, J. H. Ham, S. B. Sharma, D. F. Klessig, K. A. Fengler, and A. F. Bent (2004). Arabidopsis DND2, a Second Cyclic Nucleotide-Gated Ion Channel Gene for Which Mutation Causes the "Defense, No Death" Phenotype. Mol. Plant-Microbe Interact. 17:511-520.
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Progress 01/01/06 to 12/31/06
Outputs
The aims of our project are: 1) to examine the in planta role of RPS2-interacting proteins in RPS2-mediated defense activation; and 2) to test specific hypotheses regarding the physical interaction of RPS2 with itself, with the above RPS2-interactors, and with AvrRpt2. To address Aim 1, we continued work on plants carrying epitope-tagged overexpression constructs for RPIs 8 and 11. We saw slight alterations to bacterial growth (compared to wild-type and/or T1 plants transformed with the empty vector) for plants carrying overexpression constructs for RPI11. Western blotting to detect overexpressed proteins has been unsuccessful. We have previously shown that lines carrying T-DNA insertions in RPI 4 and RPI11 show altered bacterial growth compared to wild-type ecotype Columbia. We constructed transgenic lines to allow further characterization of these RPIs. An overexpression construct for RPI4 was introduced into Columbia; the T-DNA insertion line for RPI4 was
transformed with a complementation construct (genomic copy of RPI4). The T-DNA insertion line for RPI11 was transformed with a complementation construct (genomic copy of RPI11). Additionally, a transgenic line expressing HA-tagged RPS2 was transformed with an overexpression construct for RPI11. Experiments with these lines will allow us to determine the effects of RPI overexpression on disease resistance and RPS2 accumulation. We continued work on an Arabidopsis line from the Salk Institute T-DNA collection, carrying a T-DNA insertion in the promoter of an HSP70-like protein that interacts with RPS2 in a yeast-two-hybrid assay. This line was previously shown to have a dwarfed rosette, impaired RPS2-, RPM1- and RPS5-mediated resistance, and a more rapid response to wounding than wild-type ecotype Columbia. Lines homozygous for the T-DNA insertion and a mutated RPS2 allele were generated by crossing. Tests of disease resistance and observations of rosette phenotype demonstrated that the
phenotypes of this mutant are not associated with the insertion in the HSP70-like gene promoter, and presumably result from a T-DNA insertion at another location.
Impacts
Proteins that physically interact with RPS2 may help us to understand this model system even more deeply, and may serve as paradigms for study and use of similar proteins in other plant defense systems.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The aims of our project are: 1) to examine the in planta role of RPS2-interacting proteins in RPS2-mediated defense activation; and 2) to test specific hypotheses regarding the physical interaction of RPS2 with itself, with the above RPS2-interactors, and with AvrRpt2. Dec. 2005 was 2.3 years into the project. To address Aim 1, we continued characterization of an Arabidopsis line from the Salk Institute T-DNA collection, carrying a T-DNA insertion in the promoter of an HSP70-like protein that interacts with RPS2 in a yeast-two-hybrid assay. We determined that this line (previously shown to have impaired RPS2-mediated resistance) is also impaired in RPM1- and RPS5-mediated resistance. Disruption of RPM1- and RPS5-mediated resistance was partial, in that DC3000 expressing the cognate avirulence genes still grew to lower population levels than virulent DC3000. Autofluorescence microscopy demonstrated that the T-DNA insertion line showed a more rapid response to wounding
than wild-type ecotype Columbia. Lines homozygous for the T-DNA insertion and a mutated RPS2 allele were generated by crossing, and will be used to assess wounding and disease resistance phenotypes. Additionally, we constructed lines over-expressing proteins that interact with RPS2 in a yeast assay (RPS2-interacting proteins; RPIs) were constructed for RPIs 2, 3, 5, 7, 8 and 11, and assayed growth of virulent and avirulent (avrRpt2) Pseudomonas syringae pv. tomato on individual T1 plants. Altered bacterial growth (compared to wild-type and/or T1 plants transformed with the empty vector) was seen for plants carrying overexpression constructs for RPIs 8 and 11. Western blotting to detect overexpressed proteins has so far been unsuccessful.
Impacts
Proteins that physically interact with RPS2 may help us to understand this model system even more deeply, and may serve as paradigms for study and use of similar proteins in other plant defense systems.
Publications
- Quirino, B.F., R. Genger, J.H. Ham, G. Zabala and A.F. Bent, 2004. Identification and functional analysis of Arabidopsis proteins that interact with resistance gene product RPS2 in yeast. Physiol. Molec. Plant Pathol. 65:257-267. (Note: final version submitted, accepted and published in Feb. 2005, but the journal (curiously) assigned it to a 2004 issue of the print journal)
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Progress 01/01/04 to 12/31/04
Outputs
The aims of our project are: 1) to examine the in planta role of RPS2-interacting proteins in RPS2-mediated defense activation; and 2) to test specific hypotheses regarding the physical interaction of RPS2 with itself, with the above RPS2-interactors, and with AvrRpt2. Dec. 2004 was 1.3 years into the project. To address Aim 1, we have continued characterizing the disease reaction of Arabidopsis lines from the Salk Institute T-DNA collection that carry T-DNA insertions in genes encoding proteins that interact with RPS2 in a yeast assay (RPS2-interacting proteins; RPIs). Out of 11 lines screened (representing 10 RPIs), we have identified three lines that exhibit reproducibly altered growth of bacterial pathogen, relative to wild-type Arabidopsis (Col-0). The disrupted genes encode, respectively, a protein with ankyrin and tetratricopeptide repeats, an HSP70-like protein, and a protein of unknown function. In each case, growth of avirulent Pseudomonas syringae pv.
tomato strain DC3000 expressing avrRpt2 is greater in the T-DNA insertion line than in Columbia. Growth of virulent DC3000 was unaltered. For each line, disruption of RPS2-mediated resistance was partial, in that DC3000 expressing avrRpt2 still grew to lower population levels than virulent DC3000. This work was recently published. We have also made epitope-tagged over-expression constructs for six RPIs. These constructs will be transformed into wild-type Columbia in order to assess the impact of over-expression on resistance mediated by RPS2. To address Aim 2, we have continued work towards testing physical interactions of RPS2 with RPIs. Five FLAG epitope-tagged RPIs have been transformed into an rps2 null line containing a functional RPS2 transgene with an HA epitope tag. For three of these constructs, transformants have been identified, and co-immunoprecipitation experiments to assess interactions between HA-tagged RPS2 and the FLAG-tagged RPI have been initiated.
Impacts
Proteins that physically interact with RPS2 may help us to understand this model system even more deeply, and may serve as paradigms for study and use of similar proteins in other plant defense systems.
Publications
- Quirino, B.F., R. Genger, J.H. Ham, G. Zabala and A.F. Bent, 2005. Identification and functional analysis of Arabidopsis proteins that interact with resistance gene product RPS2 in yeast. Physiol. Molec. Plant Pathol. in press (available online 24 March 2005).
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Progress 01/01/03 to 12/31/03
Outputs
Our double mutant analyses involving Arabidopsis dnd mutants and the npr1, ndr1, ein2, and eds16/sid2 mutations, and also the nahG gene, have progressed to near-completion. Grace Jurkowski, the Ph.D. student who carried out much of this work, successfully completed her degree in Jan. 2004, and only a few experiments remain prior to submission of a revised thesis chapter as a manuscript for publication. This work reveals the instances where constitutive defense activation and cell death suppression are or are not impacted by the other mutations. We also made significant progress in studying other aspects of dnd1 and dnd2 mutants, including publication of a paper on the identity of the DND2 gene and the behavior of dnd2 mutants. Collaborative work on the role of DND cyclic nucleotide-gated ion channels in plant responses was carried out with Dr. Petra Dietrich, and a manuscript is under review on that work. The news is less promising in projects involving candidate
genetic suppressors of dnd2. We had eight candidate suppressor lines from a very large mutant screen, but with the cloning of dnd2 and identification of an allele-specific PCR marker, we were able to genotype the lines this year and discovered that they are either DND2/dnd2 heterozygotes or else wild-type at the DND2 locus. For dnd1 suppressors, two lines were identified that exhibit reproducible phenotypic suppression (or enhancement), and that were genotypically confirmed to be homozygous for the dnd1-1 allele. One of these lines exhibits partial size increase, and a second exhibits accentuated lesion mimicry (which is often associated with the elevated-defense phenotype of certain Arabidopsis mutants). However, significant difficulty was encountered in making crosses with the suppressor lines, with one line in particular showing reproducible fertility defects. Hence our primary contributions from this grant regard molecular description of DND2, illumination of genetic interactions
between DND1 and DND2 (mutations of which knock out functionally non-redundant CNG channels), and information on interactions between the dnd mutations and the set of Arabidopsis defense loci mentioned above.
Impacts
Characterization of genes that alter plant defense and HR cell death can help us understand the molecular basis of these processes. Double-mutant characterization of the functional interactions between known genes can enhance what is known, and reveal unexpected aspects of the processes previously studied in single-gene mutants.
Publications
- Jurkowski, G.I., R. K. Smith, Jr., I. Yu, J. H. Ham, S. B. Sharma, D. F. Klessig, K. A. Fengler, and A. F. Bent (2004). Arabidopsis DND2, a Second Cyclic Nucleotide-Gated Ion Channel Gene for Which Mutation Causes the "Defense, No Death" Phenotype. Mol. Plant-Microbe Interact. 17:511-520.
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