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Relevant bibliographies by topics / Yeast two-hybrid

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Academic literature on the topic 'Yeast two-hybrid'

Author: Grafiati

Published: 4 June 2021

Last updated: 6 July 2024

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Journal articles on the topic "Yeast two-hybrid"

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NISHIHARA, Tsutomu, and Jun-ichi NISHIKAWA. "Bioassay for endocrine disruptors by using yeast two-hybrid system." Folia Pharmacologica Japonica 118, no. 3 (2001): 203–10. http://dx.doi.org/10.1254/fpj.118.203.

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Hughes-Davies, L. "The Yeast Two-Hybrid System." Journal of Medical Genetics 35, no. 8 (August 1, 1998): 704. http://dx.doi.org/10.1136/jmg.35.8.704.

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Szeberényi, József. "The yeast two-hybrid system." Biochemistry and Molecular Biology Education 34, no. 4 (July 2006): 306–7. http://dx.doi.org/10.1002/bmb.2006.494034042638.

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NAKAMURA, HIDEMITSU. "Diversification of yeast two-hybrid system." Kagaku To Seibutsu 37, no. 4 (1999): 249–50. http://dx.doi.org/10.1271/kagakutoseibutsu1962.37.249.

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Koegl, M., and P. Uetz. "Improving yeast two-hybrid screening systems." Briefings in Functional Genomics and Proteomics 6, no. 4 (January 22, 2008): 302–12. http://dx.doi.org/10.1093/bfgp/elm035.

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Reece-Hoyes, John S., and Albertha J. M. Walhout. "Generating Yeast Two-Hybrid Bait Strains." Cold Spring Harbor Protocols 2018, no. 7 (July 2018): pdb.prot094979. http://dx.doi.org/10.1101/pdb.prot094979.

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Reece-Hoyes, John S., and Albertha J. M. Walhout. "Gateway-Compatible Yeast One-Hybrid and Two-Hybrid Assays." Cold Spring Harbor Protocols 2018, no. 7 (July 2018): pdb.top094953. http://dx.doi.org/10.1101/pdb.top094953.

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Serebriiskii, Ilya G., Rui Fang, Ekaterina Latypova, Richard Hopkins, Charles Vinson, J. Keith Joung, and Erica A. Golemis. "A Combined Yeast/Bacteria Two-hybrid System." Molecular & Cellular Proteomics 4, no. 6 (March 20, 2005): 819–26. http://dx.doi.org/10.1074/mcp.t500005-mcp200.

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Van Criekinge, Wim, and Rudi Beyaert. "Yeast two-hybrid: State of the art." Biological Procedures Online 2, no. 1 (October 1999): 1–38. http://dx.doi.org/10.1251/bpo16.

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Uetz, Peter. "Editorial for “The Yeast two-hybrid system”." Methods 58, no. 4 (December 2012): 315–16. http://dx.doi.org/10.1016/j.ymeth.2013.01.001.

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Dissertations / Theses on the topic "Yeast two-hybrid"

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Vafiadaki, Elizabeth. "An investigation of the role of dysferlin in skeletal muscle." Thesis, University of Newcastle Upon Tyne, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250105.

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Frei, Eva. "N-C Interaktionen des Ca2+-aktivierten Kaliumkanals, hSK3." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-58883.

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INOUE, MASAHIKO, YOSHIHIRO WAKAYAMA, TAKAHIRO JIMI, SEIJI SHIBUYA, HAJIME HARA, AKIHIKO UNAKI, and KIYOKAZU KENMOCHI. "SKELETAL MUSCLE SYNTROPHIN INTERACTORS REVEALED BY YEAST TWO-HYBRID ASSAY." Nagoya University School of Medicine, 2008. http://hdl.handle.net/2237/10550.

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Guy, Colin Paul. "RadB from archaea : bioinformatics, biochemistry and yeast two-hybrid analyses." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.446393.

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Taylor, Danielle Nicola. "Yeast two-hybrid studies with tobacco mosaic virus replicase proteins." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624336.

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Schmidt, Carsten. "Untersuchungen zu Angiogenin-Interakteuren mit Hilfe des Yeast-Two-Hybrid-Systems." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963014544.

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Hung, Kwok Wang. "Identification of the EphA4-interacting proteins by yeast two-hybrid screening /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BICH%202006%20HUNG.

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Kashuba, Elena. "Identification of EBNA binding cellular proteins, using yeast two-hybrid system /." Stockholm, 2002. http://diss.kib.ki.se/2003/91-7349-416-X/.

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Nguyen, Jacqueline Phuong Anh. "Finding Interactions of SCRAMBLED by Using the Yeast Two-Hybrid System." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1453195.

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Thesis (M.S.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed July 1, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 40-42).

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Meddins, Anna Kathryn. "Isolating candidate cyclin-binding proteins using the Yeast Two-Hybrid assay." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627268.

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Books on the topic "Yeast two-hybrid"

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L, Bartel Paul, and Fields Stanley, eds. The yeast two-hybrid system. New York: Oxford University Press, 1997.

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Surtees, Jennifer A. Search for host factors involved in P1 plasmid partition and characterization of ParB-ParB interactions using the yeast two-hybrid system. Ottawa: National Library of Canada, 1996.

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Two-Hybrid Systems: Methods and Protocols. Humana, 2018.

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Two-Hybrid Systems: Methods and Protocols (Methods in Molecular Biology). Humana Press, 2001.

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Two-Hybrid Systems: Methods and Protocols (Methods in Molecular Biology). Humana Press, 2001.

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Two Hybrid Technologies Methods And Protocols. Humana Press, 2012.

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Purintrapiban, Juntipa. Coordination of protease systems on muscle protein degradation and identification of calpain substrates using the yeast two-hybrid system. 1999.

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Newman, Mark. Biological networks. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198805090.003.0005.

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A discussion of a range of different kinds of biological networks. The chapter starts with a discussion of biochemical networks such metabolic and protein interaction networks and methods for determining their structure, particularly focusing on high-throughput methods such as the yeast two-hybrid screen. Next is a discussion of neural networks and other networks in the brain, along with measurement techniques such as slice electron microscopy, optical microscopy, transsynaptic tracing, functional MRI, and diffusion MRI. Finally, there is a discussion of ecological networks, and particularly food webs.

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Book chapters on the topic "Yeast two-hybrid"

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Maple, Jodi, and Simon G. Møller. "Yeast Two-Hybrid Screening." In Methods in Molecular Biology, 207–23. Totowa, NJ: Humana Press, 2007. http://dx.doi.org/10.1007/978-1-59745-257-1_15.

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Smith, M. J., K. Pozo, and F. A. Stephenson. "Yeast Two‐Hybrid Studies." In Handbook of Neurochemistry and Molecular Neurobiology, 409–21. Boston, MA: Springer US, 2007. http://dx.doi.org/10.1007/978-0-387-30401-4_19.

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Donnard, Elisa, Erica M. Queiroz, J. Miguel Ortega, and R. Daniel Gietz. "Yeast Two-Hybrid Liquid Screening." In Methods in Molecular Biology, 97–107. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0799-1_7.

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Roberts, George G., Jodi R. Parrish, Bernardo A. Mangiola, and Russell L. Finley. "High-Throughput Yeast Two-Hybrid Screening." In Methods in Molecular Biology, 39–61. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-455-1_3.

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Lin, Jer-Sheng, and Erh-Min Lai. "Protein–Protein Interactions: Yeast Two Hybrid." In Methods in Molecular Biology, 235–46. New York, NY: Springer US, 2023. http://dx.doi.org/10.1007/978-1-0716-3445-5_15.

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Tikhmyanova, Nadezhda Y., Eugene A. Izumchenko, Ilya G. Serebriiskii, and Erica A. Golemis. "A Bacterial/Yeast Merged Two-Hybrid System." In Gene Function Analysis, 257–90. Totowa, NJ: Humana Press, 2007. http://dx.doi.org/10.1007/978-1-59745-547-3_15.

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Serebriiskii, Ilya G., Nadia Milech, and Erica A. Golemis. "A Bacterial/Yeast Merged Two-Hybrid System." In Gene Function Analysis, 291–315. Totowa, NJ: Humana Press, 2007. http://dx.doi.org/10.1007/978-1-59745-547-3_16.

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Huang, Betty C. B., and Ying Luo. "Yeast One and Two Hybrid cDNA Cloning." In Genetic Library Construction and Screening, 167–85. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-56408-6_9.

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Maier, Richard H., Christina J. Maier, and Kamil Önder. "Construction of Improved Yeast Two-Hybrid Libraries." In Methods in Molecular Biology, 71–84. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-065-2_5.

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Mallick, Jaideep, Gregor Jansen, Cunle Wu, and Malcolm Whiteway. "SRYTH: A New Yeast Two-Hybrid Method." In Methods in Molecular Biology, 31–41. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3052-4_3.

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Conference papers on the topic "Yeast two-hybrid"

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Liu, Jiao, Shaoping Fu, Yuqiang Guo, Shuo Wang, Ruijun Duan, Ruimei Li, Yuan Yao, and Jianchun Guo. "Construction of a High-Quality Yeast One/two-hybrid cDNA Library from Cassava (Manihot Esculenta Crantz)." In International Conference on Biomedical and Biological Engineering. Paris, France: Atlantis Press, 2016. http://dx.doi.org/10.2991/bbe-16.2016.70.

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Polterauer, Stephan, Dharmarao Thapi, Nikolaus Schultz, Ouathek Ouerfelli, Nancy Chen, Nestor Rosales, Xiu Yan, and David R. Spriggs. "Abstract 3033: Identification of the MUC16/CA125 interaction network: Results of a high-throughput yeast two-hybrid screening." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-3033.

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Polterauer, Stephan, Ryan Parlett, Dharmarao Thapi, Nestor Rosales, David R. Spriggs, and Xiu J. Yan. "Abstract 1946: Detecting protein-protein interactions between MUC16 CDΔ102 (80AA) and a SKOV-3 cDNA library using yeast two-hybrid screening." In Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL. American Association for Cancer Research, 2011. http://dx.doi.org/10.1158/1538-7445.am2011-1946.

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Reports on the topic "Yeast two-hybrid"

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Avni, Adi, and Kirankumar S. Mysore. Functional Genomics Approach to Identify Signaling Components Involved in Defense Responses Induced by the Ethylene Inducing Xyalanase Elicitor. United States Department of Agriculture, December 2009. http://dx.doi.org/10.32747/2009.7697100.bard.

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Plant-microbe interactions involve a large number of global regulatory systems, which are essential for plants to protect themselves against pathogen attack. An ethylene-inducing xylanase (EIX) of Trichoderma viride is a potent elicitor of plant defense responses, like hypersensitive response (HR), in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum). The central goal of this proposal was to investigate the molecular mechanisms that allow plants to specifically activate defense responses after EIX treatment. We proposed to identify cellular signaling components involved in the induction of HR by the EIX elicitor. The molecular genetic analysis of the signal transduction pathway that modulates hypersensitive responses is an important step in understanding the induction of plant defense responses. The genes that mediate LeEIX2-EIX dependent activation of resistance mechanisms remain to be identified. We used two approaches to identify the cellular signaling components that induce HR mediated by the EIX elicitor. In the first approach, we performed a yeast two-hybrid screening using LeEix2 as bait to identify plant proteins that interact with it. In the second approach, we used virus-induced gene silencing (VIGS) for a high-throughput screen to identify genes that are required for the induction of LeEIX2-EIX mediated HR. VIGS will also be used for functional characterization of genes that will be identified during the yeast two-hybrid screen. This investigation will shed light on cellular processes and signaling components involved in induction of general plant defense against pathogens and will provide the basis for future biotechnological approaches to improve plant resistance to pathogens. Several genes were indentified by the two approaches. We used the VIGS and yeast two hybrid approaches to confirm that activity of the genes initially identified by different procedure. Two genes inhibit the induction of HR by the fungal elicitor in the different systems; Tobacco-Harpin binding protein 1 and cyclopropyl isomerase.

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Hodges, Thomas K., and David Gidoni. Regulated Expression of Yeast FLP Recombinase in Plant Cells. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7574341.bard.

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Research activities in both our laboratories were directed toward development of control of the FLP/frt recombination system for plants. As described in the text of the research proposal, the US lab has been engaged in developing regulatory strategies such as tissue-specific promoters and the steroid-inducible activation of the FLP enzyme while the main research activities in Israel have been directed toward the development and testing of a copper-regulated expression of flp recombinase in tobacco (this is an example of a promoter activation by metal ions). The Israeli lab hat additionally completed experiments of previous studies regarding factors affecting the efficiency of recombinase activity using both a gain-of-function assay (excisional-activation of a gusA marker) and loss of function assay (excision of a rolC marker) in tobacco. Site-specific recombinase systems, in particular the FLP/frt and R/RS systems of yeast and the Cre/lox system of bacteriophage P1, have become an essential component of targeted genetic transformation procedures both in animal and plant organisms. To provide more flexibility in transgene excisions by the recombinase systems as well as gene targeting, and to widen possible applications, the development of controlled or regulated recombination systems is highly desirable and was therefore the subject of this research proposal. There are a few possible mechanisms to regulate expression of a recombinase system. They include: 1) control of the recombination system by having the target sites (e.g. frt) in one plant and the flp recombinase gene in another, and bringing the two together by cross fertilization. 2) regulation of promoter activities by external stimuli such as temperature, chemicals, metal ions, etc. 3) regulation of promoter activities by internal signals, i.e. cell- or tissue-specific, or developmental regulation. 4) regulation of enzyme activity by providing cofactors essential for biochemical reactions to take place such as steroid molecules in conjunction with a steroid ligand-binding protein (domains). During the course of this research our major emphasis have been focused toward studying the feasibility of hybrid seed production in Arabidopsis, using FLP/frt. Male-sterility was induced using the antisence of a pollen- and tapetum-specific gene, bcp1, isolated from Arabidopsis. The sterility inducing gene was flanked by frt sites. Upon cross pollination of flowers of male-sterile plants with pollen from FLP-containing plants, viable seeds were produced, and the progeny hybrid plants developed normally. The major achievement from this work is the first demonstration of using a site-specific recombinase to restore fertility in male-sterile plants (see attached paper, Luo et al., Plant J 2000; 23:423-430). The implication from this finding is that site-specific recombination systems can be applied in crop plants as a useful alternative method for hybrid seed production.

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Sadot, Einat, Christopher Staiger, and Mohamad Abu-Abied. Studies of Novel Cytoskeletal Regulatory Proteins that are Involved in Abiotic Stress Signaling. United States Department of Agriculture, September 2011. http://dx.doi.org/10.32747/2011.7592652.bard.

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In the original proposal we planned to focus on two proteins related to the actin cytoskeleton: TCH2, a touch-induced calmodulin-like protein which was found by us to interact with the IQ domain of myosin VIII, ATM1; and ERD10, a dehydrin which was found to associate with actin filaments. As reported previously, no other dehydrins were found to interact with actin filaments. In addition so far we were unsuccessful in confirming the interaction of TCH2 with myosin VIII using other methods. In addition, no other myosin light chain candidates were found in a yeast two hybrid survey. Nevertheless we have made a significant progress in our studies of the role of myosins in plant cells. Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (1, 2), plasmodesmata function (3-5), organelle movement (6-10), cytokinesis (4, 11, 12), endocytosis (4, 5, 13-15) and targeted RNA transport (16). Plant myosins belong to two main groups of unconventional myosins: myosin XI and myosin VIII, both closely related to myosin V (17-19). The Arabidopsis myosin family contains 17 members: 13 myosin XI and four myosin VIII (19, 20). The data obtained from our research of myosins was published in two papers acknowledging BARD funding. To address whether specific myosins are involved with the motility of specific organelles, we cloned the cDNAs from neck to tail of all 17 Arabidopsis myosins. These were fused to GFP and used as dominant negative mutants that interact with their cargo but are unable to walk along actin filaments. Therefore arrested organelle movement in the presence of such a construct shows that a particular myosin is involved with the movement of that particular organelle. While no mutually exclusive connections between specific myosins and organelles were found, based on overexpression of dominant negative tail constructs, a group of six myosins (XIC, XIE, XIK, XI-I, MYA1 and MYA2) were found to be more important for the motility of Golgi bodies and mitochondria in Nicotiana benthamiana and Nicotiana tabacum (8). Further deep and thorough analysis of myosin XIK revealed a potential regulation by head and tail interaction (Avisar et al., 2011). A similar regulatory mechanism has been reported for animal myosin V and VIIa (21, 22). In was shown that myosin V in the inhibited state is in a folded conformation such that the tail domain interacts with the head domain, inhibiting its ATPase and actinbinding activities. Cargo binding, high Ca2+, and/or phosphorylation may reduce the interaction between the head and tail domains, thus restoring its activity (23). Our collaborative work focuses on the characterization of the head tail interaction of myosin XIK. For this purpose the Israeli group built yeast expression vectors encoding the myosin XIK head. In addition, GST fusions of the wild-type tail as well as a tail mutated in the amino acids that mediate head to tail interaction. These were sent to the US group who is working on the isolation of recombinant proteins and performing the in vitro assays. While stress signals involve changes in Ca2+ levels in plants cells, the cytoplasmic streaming is sensitive to Ca2+. Therefore plant myosin activity is possibly regulated by stress. This finding is directly related to the goal of the original proposal.

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Avni, Adi, and Gitta L. Coaker. Proteomic investigation of a tomato receptor like protein recognizing fungal pathogens. United States Department of Agriculture, January 2015. http://dx.doi.org/10.32747/2015.7600030.bard.

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Maximizing food production with minimal negative effects on the environment remains a long-term challenge for sustainable food production. Microbial pathogens cause devastating diseases, minimizing crop losses by controlling plant diseases can contribute significantly to this goal. All plants possess an innate immune system that is activated after recognition of microbial-derived molecules. The fungal protein Eix induces defense responses in tomato and tobacco. Plants recognize Eix through a leucine-rich-repeat receptor- like-protein (LRR-RLP) termed LeEix. Despite the knowledge obtained from studies on tomato, relatively little is known about signaling initiated by RLP-type immune receptors. The focus of this grant proposal is to generate a foundational understanding of how the tomato xylanase receptor LeEix2 signals to confer defense responses. LeEix2 recognition results in pattern triggered immunity (PTI). The grant has two main aims: (1) Isolate the LeEix2 protein complex in an active and resting state; (2) Examine the biological function of the identified proteins in relation to LeEix2 signaling upon perception of the xylanase elicitor Eix. We used two separate approaches to isolate receptor interacting proteins. Transgenic tomato plants expressing LeEix2 fused to the GFP tag were used to identify complex components at a resting and activated state. LeEix2 complexes were purified by mass spectrometry and associated proteins identified by mass spectrometry. We identified novel proteins that interact with LeEix receptor by proteomics analysis. We identified two dynamin related proteins (DRPs), a coiled coil – nucleotide binding site leucine rich repeat (SlNRC4a) protein. In the second approach we used the split ubiquitin yeast two hybrid (Y2H) screen system to identified receptor-like protein kinase At5g24010-like (SlRLK-like) (Solyc01g094920.2.1) as an interactor of LeEIX2. We examined the role of SlNRC4a in plant immunity. Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defense responses while silencing SlNRC4 reduces plant immunity. We propose that SlNRC4a acts as a non-canonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perception. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 inVHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune . The interaction of LeEIX2 with SlRLK-like was verified using co- immunoprecipitation and a bimolecular fluorescence complementation assay. The defence responses induced by EIX were markedly reduced when SlRLK-like was over-expressed, and mutation of slrlk-likeusing CRISPR/Cas9 increased EIX- induced ethylene production and SlACSgene expression in tomato. Co-expression of SlRLK-like with different RLPs and RLKs led to their degradation, apparently through an endoplasmic reticulum-associated degradation process. We provided new knowledge and expertise relevant to expression of specific be exploited to enhance immunity in crops enabling the development of novel environmentally friendly disease control strategies.

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Coplin, David L., Shulamit Manulis, and Isaac Barash. roles Hrp-dependent effector proteins and hrp gene regulation as determinants of virulence and host-specificity in Erwinia stewartii and E. herbicola pvs. gypsophilae and betae. United States Department of Agriculture, June 2005. http://dx.doi.org/10.32747/2005.7587216.bard.

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Gram-negative plant pathogenic bacteria employ specialized type-III secretion systems (TTSS) to deliver an arsenal of pathogenicity proteins directly into host cells. These secretion systems are encoded by hrp genes (for hypersensitive response and pathogenicity) and the effector proteins by so-called dsp or avr genes. The functions of effectors are to enable bacterial multiplication by damaging host cells and/or by blocking host defenses. We characterized essential hrp gene clusters in the Stewart's Wilt of maize pathogen, Pantoea stewartii subsp. stewartii (Pnss; formerly Erwinia stewartii) and the gall-forming bacterium, Pantoea agglomerans (formerly Erwinia herbicola) pvs. gypsophilae (Pag) and betae (Pab). We proposed that the virulence and host specificity of these pathogens is a function of a) the perception of specific host signals resulting in bacterial hrp gene expression and b) the action of specialized signal proteins (i.e. Hrp effectors) delivered into the plant cell. The specific objectives of the proposal were: 1) How is the expression of the hrp and effector genes regulated in response to host cell contact and the apoplastic environment? 2) What additional effector proteins are involved in pathogenicity? 3) Do the presently known Pantoea effector proteins enter host cells? 4) What host proteins interact with these effectors? We characterized the components of the hrp regulatory cascade (HrpXY ->7 HrpS ->7 HrpL ->7 hrp promoters), showed that they are conserved in both Pnss and Fag, and discovered that the regulation of the hrpS promoter (hrpSp) may be a key point in integrating apoplastic signals. We also analyzed the promoters recognized by HrpL and demonstrated the relationship between their composition and efficiency. Moreover, we showed that promoter strength can influence disease expression. In Pnss, we found that the HrpXY two-component signal system may sense the metabolic status of the bacterium and is required for full hrp gene expression in planta. In both species, acyl-homoserine lactone-mediated quorum sensing may also regulate epiphytic fitness and/or pathogenicity. A common Hrp effector protein, DspE/WtsE, is conserved and required for virulence of both species. When introduced into corn cells, Pnss WtsE protein caused water-soaked lesions. In other plants, it either caused cell death or acted as an Avr determinant. Using a yeast- two-hybrid system, WtsE was shown to interact with a number of maize signal transduction proteins that are likely to have roles in either programmed cell death or disease resistance. In Pag and Pab, we have characterized the effector proteins HsvG, HsvB and PthG. HsvG and HsvB are homologous proteins that determine host specificity of Pag and Pab on gypsophila and beet, respectively. Both possess a transcriptional activation domain that functions in yeast. PthG was found to act as an Avr determinant on multiple beet species, but was required for virulence on gypsophila. In addition, we demonstrated that PthG acts within the host cell. Additional effector genes have been characterized on the pathogenicity plasmid, pPATHₚₐg, in Pag. A screen for HrpL- regulated genes in Pnsspointed up 18 candidate effector proteins and four of these were required for full virulence. It is now well established that the virulence of Gram-negative plant pathogenic bacteria is governed by Hrp-dependent effector proteins. However; the mode of action of many effectors is still unresolved. This BARD supported research will significantly contribute to the understanding of how Hrp effectors operate in Pantoea spp. and how they control host specificity and affect symptom production. This may lead to novel approaches for genetically engineering plants resistant to a wide range of bacterial pathogens by inactivating the Hrp effectors with "plantabodies" or modifying their receptors, thereby blocking the induction of the susceptible response. Alternatively, innovative technologies could be used to interfere with the Hrp regulatory cascade by blocking a critical step or mimicking plant or quorum sensing signals.

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Wagner, D. Ry, Eliezer Lifschitz, and Steve A. Kay. Molecular Genetic Analysis of Flowering in Arabidopsis and Tomato. United States Department of Agriculture, May 2002. http://dx.doi.org/10.32747/2002.7585198.bard.

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The primary objectives for the US lab included: the characterization of ELF3 transcription and translation; the creation and characterization of various transgenic lines that misexpress ELF3; defining genetic pathways related to ELF3 function regulating floral initiation in Arabidopsis; and the identification of genes that either interact with or are regulated by ELF3. Light quality, photoperiod, and temperature often act as important and, for some species, essential environmental cues for the initiation of flowering. However, there is relatively little information on the molecular mechanisms that directly regulate the developmental pathway from the reception of the inductive light signals to the onset of flowering and the initiation of floral meristems. The ELF3 gene was identified as possibly having a role in light-mediated floral regulation since elj3 mutants not only flower early, but exhibit light-dependent circadian defects. We began investigating ELF3's role in light signalling and flowering by cloning the ELF3 gene. ELF3 is a novel gene only present in plant species; however, there is an ELF3 homolog within Arabidopsis. The Arabidopsis elj3 mutation causes arrhythmic circadian output in continuous light; however, we show conclusively normal circadian function with no alteration of period length in elj3 mutants in dark conditions and that the light-dependent arrhythmia observed in elj3 mutants is pleiotropic on multiple outputs regardless of phase. Plants overexpressing ELF3 have an increased period length in constant light and flower late in long-days; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. Indeed, the ELF3 protein interacts with the photoreceptor PHYB in the yeast two-hybrid assay and in vitro. The phase ofELF3 function correlates with its peak expression levels of transcript and protein in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting. Furthermore, flowering time is dependent upon proper expression ofELF3. Scientifically, we've made a big leap in the understanding of the circadian system and how it is coupled so tightly with light reception in terms of period length and clock resetting. Agriculturally, understanding more about the way in which the clock perceives and relays temporal information to pathways such as those involved in the floral transition can lead to increased crop yields by enabling plants to be grown in suboptimal conditions.

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Levy, Maggie, Raymond Zielinski, and Anireddy S. Reddy. IQD1 Function in Defense Responses. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7699842.bard.

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The main objective of the proposed research was to study IQD1's mechanism of action and elucidate its role in plant protection. Preliminary experiments suggest that IQD1 binds CaM in a Ca²⁺-dependent manner and functions in general defense responses. We propose to identify proteins and genes that interact with IQD1, which may provide some clues to its mechanism of action. We also plan to dissect IQD1's integration in defense pathways and to study and modulate its binding affinity to CaM in order to enhance crop resistance. Our specific objectives were: (1) Analysis of IQD1's CaM-binding properties; (2) Identification of IQD1 targets;(3) Dissection of IQD1 integration into defense signaling pathways. Analysis of IQD1's CaM-binding properties defined four potential classes of sequences that should affect CaM binding: one is predicted to raise the affinity for Ca²⁺-dependent interaction but have no effect on Ca²⁺-independent binding; a second is predicted to act like the first mutation but eliminate Ca²⁺-independent binding; a third has no predicted effect on Ca²⁺-dependent binding but eliminates Ca²⁺-independent binding; and the fourth is predicted to eliminate or greatly reduce both Ca²⁺-dependent and Ca²⁺-independent binding. Following yeast two hybrid analysis we found that IQD1 interact with AtSR1 (Arabidopsis thalianaSIGNALRESPONSIVE1), a calcium/calmodulin-binding transcription factor, which has been shown to play an important role in biotic and abiotic stresses. We tested IQD1 interaction with both N-terminal or C-terminal half of SR1. These studies have uncovered that only the N-terminal half of the SR1 interacts with the IQD1. Since IQD1 has an important role in herbivory, its interaction with SR1 suggests that it might also be involved in plant responses to insect herbivory. Since AtSR1, like IQD1, is a calmodulin-binding protein and the mutant showed increased sensitivity to a herbivore, we analyzed WT, Atsr1 and the complemented line for the levels of GS to determine if the increased susceptibility of Atsr1 plants to T. ni feeding is associated with altered GS content. In general, Atsr1 showed a significant reduction in both aliphatic and aromatic GS levels as compared to WT. In order to study IQD1's molecular basis integration into hormone-signaling pathways we tested the epistatic relationships between IQD1 and hormone-signaling mutants. For that purpose we construct double mutants between IQD1ᴼXᴾ and mutants defective in plant-hormone signaling and GS accumulation. Epitasis with SA mutant NahG and npr1-1 and JA mutant jar1-1 suggested IQD1 function is dependent on both JA and SA as indicated by B. cinerea infection assays. We also verified the glucosinolate content in the crosses siblings and found that aliphatic GSL content is reduced in the double transgenic plants NahG:IQD1ᴼXᴾ as compare to parental lines while the aliphatic GSL content in the npr1-1:IQD1ᴼXᴾ and jar1-1: IQD1ᴼXᴾ double mutants was intimidated to the parental lines. This suggests that GSL content dependency on SA is downstream to IQD1. As a whole, this project should contribute to the development of new defense strategies that will improve crop protection and reduce yield losses and the amount of pesticides required; these will genuinely benefit farmers, consumers and the environment.

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Chamovitz, Daniel, and Albrecht Von Arnim. Translational regulation and light signal transduction in plants: the link between eIF3 and the COP9 signalosome. United States Department of Agriculture, November 2006. http://dx.doi.org/10.32747/2006.7696515.bard.

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The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.

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Ohad, Nir, and Robert Fischer. Regulation of Fertilization-Independent Endosperm Development by Polycomb Proteins. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695869.bard.

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Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to identify members of the complex, analyze their interactions, and understand their function. What is the Temporal and Spatial Pattern of Polycomb Proteins Accumulation? The use of transgenic plants expressing tagged FIE and MEA polypeptides as well as specific antibodies were proposed to localize the endogenous polycomb complex. How is Polycomb Protein Activity Controlled? To understand the molecular mechanism controlling the accumulation of FIE protein, transgenic plants as well as molecular approaches were proposed to determine whether FIE is regulated at the translational or posttranslational levels. The objectives of our research program have been accomplished and the results obtained exceeded our expectation. Our results reveal that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms (Publication 1). Moreover our data show that FIE has additional functions besides controlling the development of the female gametophyte. Using transgenic lines in which FIE was not expressed or the protein level was reduced during different developmental stages enabled us for the first time to explore FIE function during sporophyte development (Publication 2 and 3). Our results are consistent with the hypothesis that FIE, a single copy gene in the Arabidopsis genome, represses multiple developmental pathways (i.e., endosperm, embryogenesis, shot formation and flowering). Furthermore, we identified FIE target genes, including key transcription factors known to promote flowering (AG and LFY) as well as shoot and leaf formation (KNAT1) (Publication 2 and 3), thus demonstrating that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Using the Yeast two hybrid system and pull-down assays we demonstrated that FIE protein interact with MEA via the N-terminal region (Publication 1). Moreover, CURLY LEAF protein, an additional member of the SET domain family interacts with FIE as well. The overlapping expression patterns of FIE, with ether MEA or CLF and their common mutant phenotypes, demonstrate the versatility of FIE function. FIE association with different SET domain polycomb proteins, results in differential regulation of gene expression throughout the plant life cycle (Publication 3). In vitro interaction assays we have recently performed demonstrated that FIE interacts with the cell cycle regulatory component Retinobalsoma protein (pRb) (Publication 4). These results illuminate the potential mechanism by which FIE may restrain embryo sac central cell division, at least partly, through interaction with, and suppression of pRb-regulated genes. The results of this program generated new information about the initiation of reproductive development and expanded our understanding of how PcG proteins regulate developmental programs along the plant life cycle. The tools and information obtained in this program will lead to novel strategies which will allow to mange crop plants and to increase crop production.

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