Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments
Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by alteri...
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Veröffentlicht in: | The Plant cell 2017-07, Vol.29 (7), p.1571-1584 |
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creator | Park, Eunsook Lee, Hye-Young Woo, Jongchan Choi, Doil Dinesh-Kumar, Savithramma P. |
description | Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses. |
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These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.</description><identifier>ISSN: 1040-4651</identifier><identifier>EISSN: 1532-298X</identifier><identifier>DOI: 10.1105/tpc.17.00047</identifier><identifier>PMID: 28619883</identifier><language>eng</language><publisher>England: American Society of Plant Biologists</publisher><subject>Animal diseases ; Arabidopsis - genetics ; Arabidopsis - microbiology ; Bacteria ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biological activity ; BREAKTHROUGH REPORT ; Breakthrough Reports ; Compartments ; Effector cells ; Effectors ; Fluorescence ; Gram-negative bacteria ; Green fluorescent protein ; Green Fluorescent Proteins - analysis ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; Host-Pathogen Interactions ; Immune response ; Localization ; Molecular Imaging - methods ; Monitoring systems ; Nicotiana - genetics ; Nicotiana - microbiology ; Pathogens ; Peptide Fragments - analysis ; Peptide Fragments - genetics ; Peptide Fragments - metabolism ; Plant cells ; Plant Diseases - microbiology ; Plant immunity ; Plants, Genetically Modified ; Protein Folding ; Proteins ; Pseudomonas ; Pseudomonas syringae ; Pseudomonas syringae - metabolism ; Pseudomonas syringae - pathogenicity ; Recombinant Proteins - chemistry ; Recombinant Proteins - genetics ; Recombinant Proteins - metabolism ; Transgenic plants</subject><ispartof>The Plant cell, 2017-07, Vol.29 (7), p.1571-1584</ispartof><rights>2017 American Society of Plant Biologists</rights><rights>2017 American Society of Plant Biologists. All rights reserved.</rights><rights>Copyright American Society of Plant Biologists Jul 2017</rights><rights>2017 American Society of Plant Biologists. All rights reserved. 2017</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c500t-59725b19835f427bcef32302e5fe46daf74aea4df3f69df74132b807fcf2417a3</citedby><orcidid>0000-0002-2994-6444 ; 0000-0003-2984-3039 ; 0000-0002-4366-3627 ; 0000-0001-5738-316X ; 0000-0003-3797-889X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/90012748$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/90012748$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,776,780,799,881,27901,27902,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28619883$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Park, Eunsook</creatorcontrib><creatorcontrib>Lee, Hye-Young</creatorcontrib><creatorcontrib>Woo, Jongchan</creatorcontrib><creatorcontrib>Choi, Doil</creatorcontrib><creatorcontrib>Dinesh-Kumar, Savithramma P.</creatorcontrib><title>Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments</title><title>The Plant cell</title><addtitle>Plant Cell</addtitle><description>Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.</description><subject>Animal diseases</subject><subject>Arabidopsis - genetics</subject><subject>Arabidopsis - microbiology</subject><subject>Bacteria</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biological activity</subject><subject>BREAKTHROUGH REPORT</subject><subject>Breakthrough Reports</subject><subject>Compartments</subject><subject>Effector cells</subject><subject>Effectors</subject><subject>Fluorescence</subject><subject>Gram-negative bacteria</subject><subject>Green fluorescent protein</subject><subject>Green Fluorescent Proteins - analysis</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>Host-Pathogen Interactions</subject><subject>Immune response</subject><subject>Localization</subject><subject>Molecular Imaging - methods</subject><subject>Monitoring systems</subject><subject>Nicotiana - genetics</subject><subject>Nicotiana - microbiology</subject><subject>Pathogens</subject><subject>Peptide Fragments - analysis</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - metabolism</subject><subject>Plant cells</subject><subject>Plant Diseases - microbiology</subject><subject>Plant immunity</subject><subject>Plants, Genetically Modified</subject><subject>Protein Folding</subject><subject>Proteins</subject><subject>Pseudomonas</subject><subject>Pseudomonas syringae</subject><subject>Pseudomonas syringae - metabolism</subject><subject>Pseudomonas syringae - pathogenicity</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - metabolism</subject><subject>Transgenic plants</subject><issn>1040-4651</issn><issn>1532-298X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkc2LFDEQxYMo7rp686oEvHiwx3x2OhdBlh0dWHFhdsFbyHQnY4bupE26Fwb8461x1kE9Jan65VU9HkIvKVlQSuT7aWwXVC0IIUI9QudUclYx3Xx7DHciSCVqSc_Qs1J2gFBF9VN0xpqa6qbh5-jnerRTSJMbxpRtj7-kGKaUQ9zi5PFNcXOXhhRtwWV_qFqHr7x3LTAF3weLb_ejw6vVCq9dmx1IRXxXDt_XYx8mvOznlF1pXZzwTYY5IeJlttsBCuU5euJtX9yLh_MC3S2vbi8_V9dfP60uP15XrSRkqqRWTG5gYS69YGrTOs8ZJ8xJ70TdWa-EdVZ0nvtad_CinG0aonzrmaDK8gv04ag7zpvBdYdlwKsZcxhs3ptkg_m3E8N3s033RkqYLSQIvH0QyOnH7MpkhgCe-t5Gl-ZiqKZEaQ4HoG_-Q3dpzhHsGUZIramCnIB6d6TanErJzp-WocQcYjUQq6HK_I4V8Nd_GzjBf3IE4NUR2BVI5tTXkDhTouG_AAsXqp8</recordid><startdate>20170701</startdate><enddate>20170701</enddate><creator>Park, Eunsook</creator><creator>Lee, Hye-Young</creator><creator>Woo, Jongchan</creator><creator>Choi, Doil</creator><creator>Dinesh-Kumar, Savithramma P.</creator><general>American Society of Plant Biologists</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>4T-</scope><scope>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>K9.</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-2994-6444</orcidid><orcidid>https://orcid.org/0000-0003-2984-3039</orcidid><orcidid>https://orcid.org/0000-0002-4366-3627</orcidid><orcidid>https://orcid.org/0000-0001-5738-316X</orcidid><orcidid>https://orcid.org/0000-0003-3797-889X</orcidid></search><sort><creationdate>20170701</creationdate><title>Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments</title><author>Park, Eunsook ; Lee, Hye-Young ; Woo, Jongchan ; Choi, Doil ; Dinesh-Kumar, Savithramma P.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c500t-59725b19835f427bcef32302e5fe46daf74aea4df3f69df74132b807fcf2417a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Animal diseases</topic><topic>Arabidopsis - genetics</topic><topic>Arabidopsis - microbiology</topic><topic>Bacteria</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Biological activity</topic><topic>BREAKTHROUGH REPORT</topic><topic>Breakthrough Reports</topic><topic>Compartments</topic><topic>Effector cells</topic><topic>Effectors</topic><topic>Fluorescence</topic><topic>Gram-negative bacteria</topic><topic>Green fluorescent protein</topic><topic>Green Fluorescent Proteins - analysis</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>Host-Pathogen Interactions</topic><topic>Immune response</topic><topic>Localization</topic><topic>Molecular Imaging - methods</topic><topic>Monitoring systems</topic><topic>Nicotiana - genetics</topic><topic>Nicotiana - microbiology</topic><topic>Pathogens</topic><topic>Peptide Fragments - analysis</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - metabolism</topic><topic>Plant cells</topic><topic>Plant Diseases - microbiology</topic><topic>Plant immunity</topic><topic>Plants, Genetically Modified</topic><topic>Protein Folding</topic><topic>Proteins</topic><topic>Pseudomonas</topic><topic>Pseudomonas syringae</topic><topic>Pseudomonas syringae - metabolism</topic><topic>Pseudomonas syringae - pathogenicity</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - metabolism</topic><topic>Transgenic plants</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Park, Eunsook</creatorcontrib><creatorcontrib>Lee, Hye-Young</creatorcontrib><creatorcontrib>Woo, Jongchan</creatorcontrib><creatorcontrib>Choi, Doil</creatorcontrib><creatorcontrib>Dinesh-Kumar, Savithramma P.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Docstoc</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Plant cell</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Park, Eunsook</au><au>Lee, Hye-Young</au><au>Woo, Jongchan</au><au>Choi, Doil</au><au>Dinesh-Kumar, Savithramma P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments</atitle><jtitle>The Plant cell</jtitle><addtitle>Plant Cell</addtitle><date>2017-07-01</date><risdate>2017</risdate><volume>29</volume><issue>7</issue><spage>1571</spage><epage>1584</epage><pages>1571-1584</pages><issn>1040-4651</issn><eissn>1532-298X</eissn><abstract>Pathogenic gram-negative bacteria cause serious diseases in animals and plants. These bacterial pathogens use the type III secretion system (T3SS) to deliver effector proteins into host cells; these effectors then localize to different subcellular compartments to attenuate immune responses by altering biological processes of the host cells. The fluorescent protein (FP)-based approach to monitor effectors secreted from bacteria into the host cells is not possible because the folded FP prevents effector delivery through the T3SS. Therefore, we optimized an improved variant of self-assembling split super-folder green fluorescent protein (sfGFPOPT) system to investigate the spatiotemporal dynamics of effectors delivered through bacterial T3SS into plant cells. In this system, effectors are fused to 11th β-strand of super-folder GFP (sfGFP11), and when delivered into plant cells expressing sfGFP1-10 β-strand (sfGFP1-10OPT), the two proteins reconstitute GFP fluorescence. We generated a number of Arabidopsis thaliana transgenic lines expressing sfGFP1-10OPT targeted to various subcellular compartments to facilitate localization of sfGFP11-tagged effectors delivered from bacteria. We demonstrate the efficacy of this system using Pseudomonas syringae effectors AvrB and AvrRps4 in Nicotiana benthamiana and transgenic Arabidopsis plants. The versatile split sfGFPOPT system described here will facilitate a better understanding of bacterial invasion strategies used to evade plant immune responses.</abstract><cop>England</cop><pub>American Society of Plant Biologists</pub><pmid>28619883</pmid><doi>10.1105/tpc.17.00047</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0002-2994-6444</orcidid><orcidid>https://orcid.org/0000-0003-2984-3039</orcidid><orcidid>https://orcid.org/0000-0002-4366-3627</orcidid><orcidid>https://orcid.org/0000-0001-5738-316X</orcidid><orcidid>https://orcid.org/0000-0003-3797-889X</orcidid><oa>free_for_read</oa></addata></record> |
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source | Jstor Complete Legacy; Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals |
subjects | Animal diseases Arabidopsis - genetics Arabidopsis - microbiology Bacteria Bacterial Proteins - genetics Bacterial Proteins - metabolism Biological activity BREAKTHROUGH REPORT Breakthrough Reports Compartments Effector cells Effectors Fluorescence Gram-negative bacteria Green fluorescent protein Green Fluorescent Proteins - analysis Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Host-Pathogen Interactions Immune response Localization Molecular Imaging - methods Monitoring systems Nicotiana - genetics Nicotiana - microbiology Pathogens Peptide Fragments - analysis Peptide Fragments - genetics Peptide Fragments - metabolism Plant cells Plant Diseases - microbiology Plant immunity Plants, Genetically Modified Protein Folding Proteins Pseudomonas Pseudomonas syringae Pseudomonas syringae - metabolism Pseudomonas syringae - pathogenicity Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Transgenic plants |
title | Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments |
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