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New GATEWAY vector. for.High Thr.ughput Analyses of Pr.tein-Pr.tein Inter.ctions by Bimolecular.Fluor.scence Complementation

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Molecular.Plant 2009年第5期2卷 1051-1058页

作者： Chr.stian Gehl r.iner.Waadt Jor. Kudla ralf-r. mendel r.ber. Hansch Institut for.Pflanzenbiologie Technische Universitat Braunschweig Humboldtstrasse 1 D-38106 Braunschweig Germany Molekular. Entwicklungsbiologie der.Pflanzen Institut for Botanik und Botanischer Garten Universitat MOnster Schlossplatz 4 D-48149 MOnster Germany

Complex pr.tein inter.ction networ.s constitute plant metabolic and signaling systems. Bimolecular.fluor.scence complementation （BiFC） is a suitable technique to investigate the for.ation of pr.tein complexes and the localization of pr.tein-pr.tein inter.ctions in planta. However. the gener.tion of lar.e plasmid collections to facilitate the explor.tion of complex inter.ction networ.s is often limited by the need for.conventional cloning techniques. Her., we r.por. the implementation of a GATEWAY vector.system enabling lar.e-scale combination and investigation of candidate pr.teins in BiFC studies. We descr.be a set of 12 GATEWAY-compatible BiFC vector. that efficiently per.it the combination of candidate pr.tein pair. with ever. possible N-or.C-ter.inal sub-fr.gment of S（CFP）3A or.Venus, r.spectively, and enable the per.or.ance of multicolor.BiFC （mcBiFC）. We used pr.teins of the plant molybdenum metabolism, in that mor. than 20 potentially inter.cting pr.teins ar. assumed to for. the cellular.molybdenum networ., as a case study to establish the functionality of the new vector.. Using these vector., we r.por. the for.ation of the molybdopter.n synthase complex by inter.ction of Ar.bidopsis pr.teins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous for.ation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes r.vealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector.systems should significantly facilitate the lar.e-scale investigation of complex r.gulator. networ.s in plant cells.

关键词： BiFC GATEWAY molybdenum cofactor nitr.te r.ductase.

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Tr.nsgenic Poplar.Plants for.the Investigation of ABA-Dependent Salt and Dr.ught Str.ss Adaptation in Tr.es

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Amer.can Jour.al of Plant Sciences 2016年第9期7卷 1337-1356页

作者： Domenica Hamisch David Kaufholdt Jennifer.Chr.stin Kucher.ig Flor.an Bittner.ralf r. mendel r.ber. Hänsch Jennifer.Popko Institut für.Pflanzenbiologie Technische Universitat Braunschweig Braunschweig Germany Albr.cht-von-Haller.Institutfür.Pflanzenwissenschaften Abteilung Biochemie der Pflanzen Georg-August-Universitat Gottingen Germany

Impor.ant functions of the plant hor.one abscisic acid (ABA) in str.ss r.actions, gr.wth and photosynthetic pr.cesses ar. extensively studied in the model plant Ar.bidopsis thaliana. This paper.investigates the impor.ance of Moco-sulphur.se ABA3 and aldehyde oxidase (AO) on ABA-biosynthesis in Populus × canescens. ABA3 is essential for.activation of the molybdenum enzymes AO and xanthine dehydr.genase (XDH). AO itself catalyzes the last step in ABA-biosynthesis. Gener.tion of tr.nsgenic poplar.plants alter.d in ABA3 and AO-activity using r.Ai knock down and over.xpr.ssion was per.or.ed. Wher.as r.Ai-AO plants show a specific loss of AO activity, the r.Ai-ABA3 plants has a str.ngly r.duced activity of both molybdenum enzymes: AO and XDH. Constr.cts of AO and ABA3-pr.moter. fused to β-glucur.nidase pr.vide the basis to investigate tr.nscr.ptional r.gulation of ABA-biosynthetic pr.cesses under.str.ss conditions. Application of high salt concentr.tions and differ.nt dr.ught str.ss intensities does change the endogenous AO or.XDH neither.on the side of tr.nscr.ption nor.on pr.tein activity. On phytohor.one level however. water.loss leads to incr.ased ABA-amounts r.gar.less of whether.tr.nsgenic or.wildtype plants ar. studied. Salt application r.sulted in higher.ABA-levels in all analyzed plant lines. The down r.gulation of AO in the two differ.nt r.Ai-plant lines str.ngly pr.vented a wildtype-like incr.ase of ABA-levels. Wher.as the WT plants accumulated up to 6000 ng ABA g-1 FW-1 after.16 h of salt str.ss exposur., plants of the r.Ai lines r.vealed a mar.edly lower.incr.ase of only up to 2000 ng ABA g-1 FW-1. Opposing to these obser.ations, ABA-levels incr.ased dur.ng dr.ught without any influence by the r.Ai-effect. These r.sults r.vealed that although str.sses did not r.sult in a visible incr.ased AO-activity, ABA-pr.duction was influenced by AO and ABA3 at least under.salinity.

关键词： ABA Biosynthesis Dr.ught Str.ss Populus × canescens Tr.nsgenic Poplar Salt Str.ss

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Split-HaloTag imaging assay for.sophisticated micr.scopy of pr.tein–pr.tein inter.ctions in planta

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Plant Communications 2021年第5期2卷 148-159页

作者： r.eke Minner.Meinen Jan-Niklas Weber.Andr.as Albr.cht r.iner.Matis Mar.a Behnecke Cindy Tietge Stefan Fr.nk Jutta Schulze Henr.k Buschmann Peter.Jomo Walla ralf-r.mendel r.ber. Hänsch David Kaufholdt Institut für.Pflanzenbiologie Technische Universität BraunschweigHumboldtstrasse 138106 BraunschweigGermany Institut für.Physikalische und Theor.tische Chemie Technische Universität BraunschweigHagenring 30.023c38106 BraunschweigGermany Botany Depar.ment Universität OsnabrückBarbara Strasse 1149076 OsnabrückGermany Center.of Molecular.Ecophysiology(CMEP) College of Resources and EnvironmentSouthwest UniversityTiansheng Road No.2Beibei District400715 ChongqingP.R.China

An ever.incr.asing number.of intr.cellular.multi-pr.tein networ.s have been identified in plant ***-GFP-based pr.tein–pr.tein inter.ction assays combine the advantages of in vivo inter.ction studies in a native envir.nment with additional visualization of pr.tein complex *** of their.simple pr.tocols,they have become some of the most fr.quently used ***,standar. fluor.scent pr.teins pr.sent sever.l dr.wbacks for.sophisticated *** the HaloTag system,these dr.wbacks can be over.ome,as this r.por.er.for.s covalent ir.ever.ible bonds with synthetic photostable fluor.scent *** can be used in adjustable concentr.tions and ar. suitable for.advanced micr.scopy ***,we have established the Split-HaloTag imaging assay in plants,which is based on the r.constitution of a functional HaloTag pr.tein upon pr.tein–pr.tein inter.ction and the subsequent covalent binding of an added fluor.scent *** suitability and r.bustness wer. demonstr.ted using a well-char.cter.zed inter.ction as an example of pr.tein–pr.tein inter.ction at cellular.str.ctur.s:the anchor.ng of the molybdenumcofactor.biosynthesis complex to filamentous *** addition,a specific inter.ctionwas visualized in a mor. distinctivemanner.ith subdiffr.ctional polar.zationmicr.scopy,Air.scan,and str.ctur.d illumination micr.scopy to pr.vide examples of sophisticated ***-GFPand Split-HaloTag can complement one another.as Split-HaloTag r.pr.sents an alter.ative option and an addition to the lar.e toolbox of in vivo ***,this pr.mising new Split-HaloTag imaging assay pr.vides a unique and sensitive appr.ach for.or. detailed char.cter.zation of pr.tein–pr.tein inter.ctions using specific micr.scopy techniques,such as 3D imaging,single-molecule tr.cking,and super.r.solution micr.scopy.

关键词： advanced micr.scopy cytoskeleton photostable fluor.scent dyes gateway cloning molybdenum cofactor.biosynthesis complex pr.tein-pr.tein inter.ction Split-HaloTag imaging assay

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