Progress 10/01/02 to 09/30/05
Outputs
In 2005, we continued our molecular, genetic, and biochemical studies of the BAP1 and BAP2 genes. We identified multiple roles of these two homologous genes in controlling cell death and defense responses in Arabidopsis thaliana. First, BAP1 is a repressor of an R gene SNC1 and the loss of BAP1 function induces enhanced disease resistance to virulent bacterial and oomycete pathogens. Second, BAP1 and BAP2 have overlapping functions in repressing cell death mediated by PAD4 and EDS1 apparently through multiple R genes. Third, BAP1 modulates HR to an avirulent bacterial pathogen. Forth, BAP1 regulates basal defense response and BAP1 overexpression leads to enhanced susceptibility to a virulent oomycete. BAP1 and BAP2 encode small proteins with one calcium-dependent lipid binding C2 domain. They likely function together with an evolutionarily conserved C2 domain-containing BON1/CPN1 family. These proteins thus represent members of a new pathway involving lipids and calcium
regulation in controlling R gene activities, cell death, and basal defense.
Impacts
Functional analysis of BAP1 and BAP2 is revealing essential roles of these two genes in defense responses and growth regulation. Related work from our lab is also revealing intricate links between temperature responses, disease resistance, and growth regulation. These studies will greatly enhance our understanding of the molecular mechanisms in regulating plant disease resistance because BAP1 and BAP2 are novel membrane proteins that negatively regulate R (resistance) genes. They will provide an entry point to dissect the modulation of disease resistance by environmental factors such as temperature. The mechanistic understanding of these processes will give us new tools to modulate disease resistance and growth for plants to best adapt to their environment.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
In the past year, we have made some progress in understanding the functions of BAP1 and BAP2 as summarized below. Using molecular, biochemical and genetic approaches, we found that they have overlapping functions in regulating defense responses and plant growth and they do so by interacting with the BON1 protein localized to the plasma membrane. 1. Interaction between BAP1 and BON1 We purified BAP1 as a MBP-fusion protein and expressed the A domain of BON1by in vitro transcription/translation. The BAP1-MBP fusion protein, but not the MBP alone, can pull down the BON1 A domain in vitro. This confirms our early finding that BAP1 is a BON1 interacting protein from the two-hybrid screen. 2. Subcellular localization of the BAP1 protein We expressed a BAP1::GUS fusion protein under the control of the BAP1 native promoter. Membrane and soluble proteins were prepared from transgenic plants containing the chimeric protein. GUS activity was only detected in membrane proteins,
indicating that BAP1 is associated with membranes. This is consistent with the finding that BON1 is localized to the plasma membrane and BAP1 is a BON1 associated protein. 3. Characterization of the bap1 loss-of-function mutant The bap1 loss-of-function mutant bap1-1 has small curly leaves at 22C, a phenotype similar to but weaker than that of bon1-1. bap1-1 also exhibits a phenotype in defense responses. The expression of PR1, a molecular marker gene for defense responses, is upregulated in bap1-1. bap1-1 is more resistant to pathogen Peronospora parasitica than the wild type. We show that the growth phenotype of bap1 results from an activation of defense responses. The growth defect and the upregulation of PR1 expression in bap1-1 are eliminated by nahG gene, suggesting that a SA mediated defense response induces growth defect. We further show that SNC1 mediates at least partly the defense responses in bap1-1. The SNC1 loss of function mutant snc1-11 suppressed the growth defect
totally and the resistance to P. parasitica largely in bap1-1. 4. Characterization of the BAP1 overexpression phenotype To further analyze the role of BAP1 in disease resistance, we overexpressed BAP1 using the 35S promoter. BAP1 over-expressing lines are more susceptible to P. parasitica than the wild type, confirming an essential role of BAP1 in disease resistance. 5. Investigation of the BAP2 function The loss-of-function mutant bap2-4 does not exhibit abnormal phenotype. Because BAP2 is a close homologue of BAP1, we tested whether it can substitute BAP1 when overexpressed. Expression of BAP2 under the 35S promoter rescued the bap1-1 phenotype, indicating the functional similarity between the two genes. To reveal potential overlapping roles of BAP1 and BAP2, we generated double mutants between bap1-1 and bap2-4. Both bap1-1bap2-4/+ and bap1-1/+bap2-4 exhibit more sever growth defect than bap1-1 single mutant. Homozygous bap1-1bap2-4 is seedling lethal at 22C, dying at two
cotyledons stage. Thus BAP1 and BAP2 have overlapping functions in regulating plant growth and development.
Impacts
Functional analysis of BAP1 and BAP2 is revealing essential roles of these two genes in defense responses and growth regulation. Related work from our lab is also revealing intricate links between temperature responses, disease resistance, and growth regulation. These studies will greatly enhance our understanding of the molecular mechanisms in regulating plant disease resistance because BAP1 and BAP2 are novel membrane proteins that negatively regulate R (resistance) genes. They will provide an entry point to dissect the modulation of disease resistance by environmental factors such as temperature. The mechanistic understanding of these processes will give us new tools to modulate disease resistance and growth for plants to best adapt to their environment.
Publications
- Yang, S., and Hua, J. 2004. A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growth control in Arabidopsis. Plant Cell 16, 1060-1071.
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Progress 01/01/03 to 12/31/03
Outputs
1. Regulation of expression of BAP1 and BAP2. We found that more RNA expression for both genes in bon1 than in wild type, suggesting a feedback regulation between BON1 and BAP1/2. In addition, BAP1expression is regulated by temperature. There is more BAP1 RNA transcript in plants grown at 22C than those grown at 28C. We further analyzed the response of BAP1 transcription to temperature changes by shifting plants from 28C to 22C and collect samples at 3 hr, 6 hr, 12 hr and 24 hr after the shift. BAP1 transcript level increased by four-fold 3 hours after the shift and stays induced at 22C. This induction by lower temperature is observed in both the wild type and in bon1. To analyze the expression of BAP1 and BAP2 during development, we have constructed GUS reporter gene under the control of the promoters of the two genes respectively. Promoter fragments were fused to the GUS reporter gene and the fusions were transformed into Arabidopsis. Transgenic plants for each
promoter fusion were obtained and homozygous lines are now being selected. The representative lines will be grown to different developmental stages (germinating, two leaf stage, before bolting, after bolting, mature) and stained for GUS activity. 2. Protein localization of BAP1 and BAP2. BON1 is shown to localize to the plasma membrane. Because BAP1 and BAP2 interact BON1 in the yeast two-hybrid assay and they also contain C2 domains, we would like to know whether they also localize to membranes and if yes which membrane(s) BAP1 and BAP2 bind to. BAP1 and BAP2 were tagged with green fluorescent protein (GFP) to facilitate the protein localization. Full-length cDNAs of BAP1 and BAP2 were fused to the N-terminal of GFP and the chimeric proteins were expressed under a strong promoter (CaMV 35S) and transiently expressed in Arabidopsis protoplasts mediated by PEG. Preliminary data suggests that BAP1 and BAP2 when overexpressed in protoplasts are present both on the plasma membrane and in
cytoplasm. In the meantime, we have generated stable transgenic plants with BAP1-GFP fusion under the control of its native promoter. Preliminary data suggests that the transgene can complement the bap1 mutant phenotype (see below) indicating the fusion protein is functional. The GFP signals will be analyzed once homozygous plants are obtained. 3. Characterization of bap1 and bap2 mutants. To elucidate the function of BAP1 and BAP2, we have isolated their loss-of-function mutants using a reverse genetics approach. Collections of T-DNA insertion lines publicly available were screened and two mutant alleles were identified for BAP1 and one mutant allele was identified for BAP2. Homozygous mutants were isolated and their phenotypes are now being characterized. Preliminary data shows that bap1 mutant has a weaker but similar phenotype to bon1. It is slightly dwarf with curly leaves and this phenotype is present at 22C but not at 28C. These data indicate that BAP1 has similar function to
BON1 and likely is a functional partner of BON1. The bap2 mutant does not exhibit any obvious phenotype, and we are now constructing bap1 bap2 double mutant and analyzing the double phenotype.
Impacts
Identification of genes essential in growth homeostasis regulation will give us tools to modulate growth responses to temperature variations in crops.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
During the first three months of the project, we are starting to analyze the expression patterns of BAP1 and BAL (BAP2). We are now constructing the promoter fusions of these two genes with the reporter gene GUS as well as the full-lenght protein fusions with GFP. These constructs will be transformed into plant cells to visualize the expression of these two genes during development and their protein subceullalar localization. We have started the search for bap1 and bap2 mutants in order to study their biological functions. By searching through the T-DNA insertion collections, we have found putative mutants in both genes. The seeds of these mutants have been obtained and the phenotypes of these mutants will be analyzed.
Impacts
Since we are just starting the project, we have not made much discoveries for any impact yet.
Publications
- No publications reported this period
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