Progress 05/15/05 to 05/14/08
Outputs
OUTPUTS: Due to the restructuring of the original program (NRI 51.8 -Biology of Plant-Microbe Association) from which this project was funded, this two-year (with one-year extension) project does not fit the scope this program any more and thus is now terminated. This project focused on characterization of a protein phosphatase type 2C as a novel regulatory component of plant defense signaling. Specifically, the major results from the project are as follows: 1) A protein phosphates type 2C (At1g22280; also named as DAPP1 for Defense-associated protein phosphatase type 2C 1) was identified as a putative interacting protein of RPW8 in the yeast-two-hybrid system. RPW8 is a resistance (R) protein conferring broad-spectrum resistance to powdery mildew in Arabidopsis. By using the bimolecular fluorescence complementation (BiFC) approach, the RPW8-DAPP1 in vivo interaction has been confirmed. 2) Down-regulation of DAPP1 by RNAi in Col-0 (which lacks RPW8) resulted in constitutive expression of numerous defense-related genes, spontaneous HR-like cell death and enhanced disease resistance to virulent biotrophic fungal (Golovinomyces cichoracearum UCSC1) and oomycete (Hyaloperonospora parasitica Noco2) pathogens and a bacterial pathogen (Pseudomonas syringae pv. tomato DC3000). 3) Genetic analysis indicated that DAPP1 negatively regulates a conserved defense pathway that engages signaling components salicylic acid, EDS1, PAD4 and EDS5, and NPR1, which are also required for function of RPW8 and the classical TIR-NBS-LRR (Toll-interleukin receptor nucleotide-binding-site and leucine-rich-repeat) R genes. 4) Transient expression of HA:DAPP1 in Nicotiana benthamiana and subsequent enzymatic assays demonstrated that DAPP1 is a biologically active phosphatase. 5) Subcellular localization showed that DAPP1 is largely plasma membrane-associated. 6) Plants combining DAPP1-silecing with loss-of-function mutation of EDR1, a MAPKKK gene, develop severe to lethal spontaneous HR-like cell death, suggesting that there is a potential additive or synergistic effect between DAPP1-silecing and loss of EDR1. This result suggests that DAPP1 and EDR1 may function in separate signaling pathways connected to defense signaling. In summary, DAPP1 appears to be an essential negative regulator of plant basal resistance stimulated by RPW8 and therefore may be a promising target for engineering broad-spectrum disease resistance in plants. An international patent application No. PCT/US05/45001 was filed on December 13, 2005 under the Patent Cooperation Treaty, entitled PLANTS WITH REDUCED EXPRESSION OF PHOSPHATASE TYPE 2C GENE FOR ENHANCED PATHOGEN RESISTANCE based on and claiming the priority of the U.S. patent application serial number 60/635,768. PARTICIPANTS: Shunyuan Xiao (PI) supervised the whole project and conducted genetic analysis with signaling mutants; Wenming Wang (postdoctoral research associate; two years) did RNAi silencing, protein expression and enzymatic assays and subcellular localization studies; Robert Berkey (graduate student; one years) helped with disease phenotyping. Besides, this project provided training opportunities for Undral Orgil, Samantha Tangchaiburana (undergraduates; two years), Roy Wang, Kalika Agrawal Francis Dey Jr, Sanam Razeghi (undergraduate students, 1-6 months), Sara Mirghani, Udochi Ohiri, Louie Sun (High school students, 1-12 months). TARGET AUDIENCES: Research findings were reported at meetings to the plant research community. Knowledge gained was incorporated by the PI in the lectures given to graduate students in University of Maryland. This project has also supported training of one postdoc and a graduate student for two years and provided opportunity for training of six undergraduates (two being minority) and 3 high school internship students (two being minority). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Protein phosphorylation/dephosphorylation constitutes an important early signaling step in plant disease resistance. Despite the existence a large number of protein phosphatase type 2C genes in plants genomes (e.g >60 in the Arabidopsis genome), not a single PP2C gene has been definitely shown to be involved in regulation of plant defense signaling. Thus, the identification of the protein phosphatase type 2C (DAPP1) as an essential negative regulator of plant defense is revealing and should help elucidate the signaling mechanisms of plant disease resistance in general. The knowledge gained from this project may also provide new means for engineering broad-spectrum disease resistance in crop species through controlled down-regulation of the corresponding phosphatase genes.
Publications
- Xiao, S. 2006. Current perspectives on molecular mechanisms of plant disease resistance. In:Teixeira da Silva (ed.), "Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues"(1st ed.) Global Science Books, UK. pp 317-333.
- Wang, W., Devoto, A., Turner, J.G. and Xiao, S. 2007. Expression of the membrane-associated resistance protein RPW8 enhances basal defense against biotrophic pathogens. Mol. Plant-Microbe Interact. 20: 966-976.
- Orgil, U., Araki, H., Tangchaiburana, S. and Xiao, S. 2007. Intraspecific genetic variations, fitness cost and benefit of RPW8. a disease resistance locus in Arabidopsis thaliana. Genetics. 176:2317-2333.
- Xiao, S., Wang, W., and Yang, X. 2008. Evolution of resistance genes in plants. In Innate Immunity of Plants, Animals, and Humans, H. Heine, ed (Springer-Verlag Berlin Heidelberg), pp. 1-25.
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Progress 05/15/06 to 05/14/07
Outputs
The RPW8-interacting PP2Ca has been demonstrated to be involved in plant defense and thus has been designated DAPP1 (defense associated protein phosphatase type 2C 1). Our data showed that 1) down-regulation of DAPP1 by RNAi or co-suppression resulted in constitutive expression of PR genes, spontaneous HR-like cell death and enhanced basal resistance against powdery mildew (Erysiphe cichoracearum), downy mildew (Hyaloperonospora parasitica) and a bacterial pathogen (Pseudomonas syringae). 2) Genetic analysis indicated that DAPP1 negatively regulates a conserved defense pathway that engages signaling components salicylic acid, EDS1, PAD4 and EDS5, and NPR1, which are also required for function of RPW8 and the classical TIR-NBS-LRR (Toll-interleukin receptor nucleotide-binding-site and leucine-rich-repeat) R genes. 3) Transient expression of HA:DAPP1 in Nicotiana benthamiana and subsequent enzymatic assay demonstrated that DAPP1 is a biologically active phosphatase. 4)
By using the Bimolecular Fluorescence Complementation (BiFC) method, it has been demonstrated that DAPP1 interacts with RPW8 in vivo. Taken together, DAPP1 appears to be an essential negative regulator of plant basal resistance stimulated by RPW8 and therefore may be a promising target for engineering broad-spectrum disease resistance in plants. A patent application on DAPP1 has been filed (International Patent Application No. PCT/US05/450001; U.S. Patent Application Serial No. 60/635,768).
Impacts
Identification of a protein phosphatase type 2C gene as an essential negative regulator of plant defense will help elucidate the signaling mechanism of plant disease resistance and provide new means for engineering broad-spectrum disease resistance in crop species through down-regulation of the corresponding phosphatase genes.
Publications
- Xiao, S. 2006. Current perspectives on molecular mechanisms of plant disease resistance. In:Teixeira da Silva (ed.), "Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues"(1st ed.) Global Science Books, UK. pp 317-333.
- Wang, W., Devoto, A., Turner, J.G. and Xiao, S. 2007. Expression of the membrane-associated resistance protein RPW8 enhances basal defense against biotrophic pathogens. Mol. Plant-Microbe Interact. (in press).
- Orgil, U., Araki, H., Tangchaiburana, S. and Xiao, S. 2007. Intraspecific genetic variations, fitness cost and benefit of RPW8. a disease resistance locus in Arabidopsis thaliana. Genetics. (in press).
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Progress 05/15/05 to 05/14/06
Outputs
Protein phosphorylation and dephosphorylation processes are implicated in early signaling steps of plant disease resistance. A protein phosphates type 2C (PP2Ca) was identified as a putative interactor of RPW8, a resistance (R) protein conferring resistance to powdery mildew in Arabidopsis. By using bimolecular fluorescence complementation (BiFC) technology, it has been confirmed that RPW8-PP2Ca interaction occurs in vivo. Down-regulation of PP2Ca by RNAi or co-suppression led to constitutive expression of defense marker genes, spontaneous HR-like cell death and enhanced disease resistance to powdery mildew (E. cichoracearum), downy mildew (Peronospora parasitica) and a bacterial pathogen (Pseudomonas syringae). Genetic analysis indicated that PP2Ca negatively regulates a defense pathway that engages signaling components salicylic acid, EDS1, PAD4 and EDS5, which also appear to be recruited by RPW8 and classical TIR-NBS-LRR (Toll-interleukin receptor
nucleotide-binding-site and leucine-rich-repeat) R genes. Close homologues of PP2Ca exist in crop species. There is potential application in engineering disease resistance of crop plants through down regulation of functional homologues of PP2Ca. An international patent application No. PCT/US05/45001 was filed on December 13, 2005 under the Patent Cooperation Treaty, entitled PLANTS WITH REDUCED EXPRESSION OF PHOSPHATASE TYPE 2C GENE FOR ENHANCED PATHOGEN RESISTANCE based on and claiming the priority of the U.S. patent application serial number 60/635,768.
Impacts
Identification of a protein phosphatase type 2C gene as an essential negative regulator of plant defense will help elucidate the signaling mechanism of plant disease resistance and provide new means for engineering broad-spectrum disease resistance in crop species through down-regulation of the corresponding phosphatase genes.
Publications
- No publications reported this period
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