Name: identification of growth inhibition phenotypes induced by expression of bacterial type iii effectors in yeast
Uploaded: 2010-03-30T16:30:00
Description: Watch this Scientific Journal Video about Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast at JoVE.com

यह काम इसराइल विज्ञान फाउंडेशन द्वारा समर्थित किया गया था.

I. प्रकार III effectors के लिए एक खमीर अभिव्यक्ति सिस्टम डिजाइनिंग एक खमीर प्रकार effector III (ओं) के ब्याज की अभिव्यक्ति के लिए उपयुक्त प्रणाली calibrating एक महत्वपूर्ण कार्य है और कुछ परीक्षण और त्रुटि की आवश्यक?...

<ol>
	<li>Siggers, K. A., Lesser, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+Saccharomyces+cerevisiae:+a+versatile+model+system+for+the+identification+and+characterization+of+bacterial+virulence+proteins.">The yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.</a> Cell Host Microbe. 4, 8-15 (2008).</li><li>Parsons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat. Biotechnol. 22, 62-69 (2004).</li><li>Munkvold, K. R., Martin, M. E., Bronstein, P. A., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+Pseudomonas+syringae+pv.+tomato+DC3000+type+III+secretion+system+effector+repertoire+reveals+several+effectors+that+are+deleterious+when+expressed+in+Saccharomyces+cerevisiae.">A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae.</a> Mol. Plant-Microbe Interact. 21, 490-502 (2008).</li><li>Curak, J., Rohde, J., Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+as+a+tool+to+study+bacterial+effectors.">Yeast as a tool to study bacterial effectors.</a> Curr. Opin. Microbiol. 12, 18-23 (2009).</li><li>Slagowski, N. L., Kramer, R. W., Morrison, M. F., LaBaer, J., Lesser, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+yeast+screen+to+identify+pathogenic+bacterial+proteins.">A functional genomic yeast screen to identify pathogenic bacterial proteins.</a> PLoS Pathog. 4, e9-e9 (2008).</li><li>Huang, J., Lesser, C. F., Lory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+role+of+the+CopN+protein+in+Chlamydia+pneumoniae+intracellular+growth.">The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth.</a> Nature. 456, 112-115 (2008).</li></ol>

इस प्रस्तुति में, हम कैसे प्रकार III बैक्टीरियल effector प्रोटीन और effector प्रेरित विकास निषेध phenotypes की पहचान कैसे की अभिव्यक्ति के लिए एक heterologous प्रणाली के रूप में नवोदित खमीर Saccharomyces cerevisiae का उपयोग करने के लिए सचित?...

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identification of growth inhibition phenotypes induced by expression of bacterial type iii effectors in yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

Watch this Scientific Journal Video about Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast at JoVE.com

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

इस वीडियो में, हम खमीर और effector प्रेरित विकास निषेध phenotypes की पहचान में बैक्टीरिया प्रकार III effectors की अभिव्यक्ति के लिए एक प्रक्रिया का वर्णन. ऐसे phenotypes बाद effector कार्यों और लक्ष्यों को स्पष्ट करने के लिए शोषण कर सकते हैं.

खमीर में बैक्टीरियल प्रकार III effectors की अभिव्यक्ति से प्रेरित विकास निषेध phenotypes की पहचान

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

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