1U N I UNIVERSITY OF Plant Celt and Molecular Biology doc. Markéta Šámalové Institut Pasteur jihomoravský kraj I2?3> Oddělení experime biologie rostlin experimentální My 1st transgenic tobacco plant that I made (in the last century...:) ► Re-generated in vitro using tissue cultures leaves Ectopic meristems developing into shoot-like structures... Introduced IPTgene encoding isopentenyl transferase which catalysis the first step in cytokinin hormone biosynthesis... roots OUTLINE of the talk ► How to make a genetically modified plant? ► Tobacco, rice ► Arabidopsis thaliana ► How to regulate (trans)gene expression? ► The pOp6/LhGR system ► CRISPR/Cas9 ► Transient gene expression ► Fluorescent proteins ► Plant endomembrane system ► Plant cell wall ► Expansins <& (a)biotic stresses ► Fungal cell wall ► Magnaporthe oryzea - a model organism ► Aspergillus f urn igat us Arabidopsis thaliana How to make a genetically modified/ genome-edited plant? Transformation ► Tissue cultures ► tobacco ► rice DNA Extraction and Isolation Cloning and Destgning Genes ^ Agrobacterium How to regulate (trans)gene expression? 1 Chemically inducible gene expression systems! ► regulate (trans)gene expression at a particular developmental stage and for a specific duration using chemical inducers. ► Expression can be SWITCHED ON or OFF using chemical inducers ► Gene overexpression, knock-down expression by amiRNAs, gene by combining the system with CRISPR/Cas9 (Gehrke ► Essential for expression of gene products that interfere with regeneration, growth or reproduction... Meristem defect + Inducible gene The chemically inducible transcription activation system pOp/LhGR Lh R A TVATO regulatory DNA- transcription domain binding d. activation d. promoter GR LacľHis17> Gal4 i Á i pOp REPORTER 6x lac Operators lac TATA Operators box YOUR GENE ^ J HSP90 complex # + INDUCER DEXAMETHASONE > Developed in the laboratory of Dr Ian MOOR > Use world-wide today... an "ideal" inducible system MMs, UNIVERSITY OF WOXFORD .1 The pOp6/LhGR is highly inducible, fast & v. ► 10,000-fold induction of GUS activity (log scale !) Arabidopsis sensitive 10000 0.01 0.1 T-1-1—I I I I I |-1-1-1—I I I II |-1-1-1—I Mill-1-1-1—I I I I I 10 Time (h) 100 1000 ► Increase of GUS activity in 2h! ► The most sensitive system for tobacco! m The pOp6/LhGR system is tightly regulated ► Basal expression levels tested with ipt gene ► from Agrobacterium (cytokinin biosynthesis) ► physiologically strong transgene + DEX - DEX ■ pOp6-ipt/ LhGR pV-ipt/ LhGR pOp-ipt/ LhGR 4. * t ** > 2 (£4 pH-Luc/ LhGR neither DEX nor Lh&k affects endogenous processes in plants ... though ethanol doesl DEX in ethanol DEX in DMSO 0.1% ethanol MS \ ^^^^^^^ grown on plates ii or absence of The pOp6/LhGR system is inducible by various methods 6h 12h 24h pV-TOP/ LhGR pH-TOP/ LhGR Tobacco pOp6-ipt/ LhGR The pOp6/LhGR system is inducible by various methods Painting plants with DEX Oh 6h 12h 24h pV-TOP/ LhGR pH-TOP/ LhGR A leaf half painted with DEX application on axillary buds pV-TOP/LhGR pH-TOP/LhGR Tobacco >Op6-ipt/Lhl_\s X .....I..............l>^N|, ,{Jy X. iijiinii..............u^Nf, Changes in the open reading frame (ORF) generate a stop codon! Creating "knock-out" (KO mutant) Expected J NA sequence CCCATGTGCAGGTTGTTAACACAAGAC TT TTATAT CACCG\T G WTGCAGGTT GTT.^AC ACAAGACGT 60 70 80 T Nucleotide G insertion! IWWU CRISPR' Clustered Regularly Interspaced Short Palindromic Repeats PAtA- Protospacer Adjacent Motifs https:// www.youtube.com / watch?v=4YKFw2KZA5o&ab_channel=naturevideo Transient gene expression and fluorescent proteins Transient gene expression assay ► AGROINFILTRATION method ► Agrobacterium infiltrated into tobacco plants ► e.g. to study plant + Agrobacterium* endomembrane trafficking * * V 3. TRANSIENT GENE EXPRESSION ASSAY Use of fluorescent proteins (FP) in cell biology Protein localization, protein-protein interactions... GFP ~ green FP from jellyfish Aequorea victoria ^ CLSM „ confocal laser scanning microscope mRFPl - monomeric red FP from biscosoma coral ► Generates optical slices through live specimens. fiffiffffi ► Excitation spectra Emission spectra 500 600 Wavelength (nm) 73^8 18111045 Targeting fluorescent fusion proteins into different Cell Compartments (Samalova et a/., 2006) Location yfp yfp gfp "self-cleaving" 2A peptide mm gfp hdelB mm gfp Cytoplasm Endoplasmic reticulum (ER) Cell wall rfp 2A sp gfp Ts™ rfp 2A sp gfp n rfp j j gfp n rfp gfp Nucleus Cell wall ER ^ . . Cell wa Öolgi app. ER n rfp 2A gfp rfp _ 2A gfp Vacuole Cell wa ER N RFP Jolgi apparatus 5 [im cytoplasm >1 ^ GFP V 5 \im J merge M vacuole merge * YFP 2A sp GFP The Golgi apparatus moving along the ER network in living tobacco cells.... PLANTS A ARE MOVING! Create your own compartment :) A tool for plant synthetic biology > substantial expansion of the endomembrane system in each cell of the plant (Sandor, Samalova et a/., 2023) Arabidopsis leaf G22 Linkers / \ ER sign ĺ CD BP22 peptide (Trans me m b rane d o ma in) OSER Samalosome 10 nM G22Y + RFP-HDEL A Organised Smooth Endoplasmic Reticulum Potential applications of the synthetic compartment for the metabolic engineering of plants, e.g. recombinant or toxic proteins. No detrimental effects in plants! Plant cell wall (CW) ON is crucial for plant growth and develop ► shapes the plant body ► movement of solutes and nutrients ► protects plants from the environment ► intercellular communication (Wolf et a/., 2012) ► Cellulose is the most abundant biopolymer on Earth! Middle J Lamella \_ Primary Cell Wall Plasma Membrane Pectin Cellulose Microfibril •-^ Hemicellulose Soluble Protein ► Load-bearing cellulose microfibrils ► embedded into viscoelastic matrix of hemicellulose and pectins Plant CWs combine strength with extensibi ► Wall extensibility may be controlled at limited regions, 'biomechanical KotSpOtS* (Cosgrove, 2014; 2018). ► EXPANSINs discovered as the most pH-responsive substance in the CW (McQueen-Mason et a/., 1992). ► They do not have a hydro lytic activity ► but disrupt the non-covalent bonds between CW polysaccharides, thus relaxing wall stresses and allowing turgor-driven cell expansion (Cosgrove, 2000). EXPANSINS are localized in the cell wall Promoter EXPA1 ^ AtEXPAl ~mCheiry~^J > EXPANSINs localised to the ON in vivo for the first time! (Samalova et a/., 2023) > Use of mCherry (RFP) instead of pH sensitive 6FP Promoter EXPA1 1 nls eGFP eGFP eGFP m EXPANSINS are localized into various roo tissues EXPANSINS are localized into various rooi ► 3D projection of Z-stack (combined optical slices) taken by a confocal microscope EXPANSINS are localized into various rooi tiss""* Overexpression of EXPA1 leads to smaller, compact plants that are more resistant to (a)biotic stresses 5-4 8-4 WT * ^ ^5 4i ^§sc ^te 31 35 38 45dpg Not only plant cells have the ON... Unique composition of the fungal cell wall ► makes it an ideal target for the development of fungicides! Glycoprotein-rich outer layer Chitin/p-glucar\ matrix ] Cell membrane J| Chitin synthase Glycoprotein ^•Chitin Q^J P-(l,3)-glucan synthase ^ |3-(l(6)-glucan ^»Mannan ^^y*Cell wall enzymes ^ |3-(l(3)-glucan ► 6PI (6lycosylPhosphatidylInositol) Anchored Proteins = GAP ► Cell wall modifying enzymes ► e.g. Glucan Elongation (Gel) proteins elongating 6-1,3-glucan chain of rice! Magnaporthe oryzae the most devastating path ► Model organism for plant pathogens: 1st sequenced (Deanetal., 2005) Hemibiotrophic filamentous Ascomycete fungus causing rice blast! Haploid, short (asexual) life cycle, gene deletions by homologous recombination. > Food security & climate change Magnaporthe oryzae asexual life-cycle r> P&EL3: :mCherry:6EL3 ~ Germling with extracellular matrix | Conidium labs— Exploring redox state in susceptible & res is tan rice IR68 rice resistant cultivar ROS toxicity alone is NOT sufficient to kill Magnaporthe oryzae in resistant rice! (Samalova et a/., 2013; 2014) 1 growth, TripleAgellAgel3Agel4 KO has reduced my< hyper branching phenotype and is non-pathogenic!!! igel\ Age\3Ai Unique composition of the fungal cell wall ► makes it an ideal target for the development of fungicides! Glycoprotein-rich outer layer Chitin/p-glucar\ matrix ] Cell membrane J| Chitin synthase Glycoprotein ^•Chitin Q^J P-(l,3)-glucan synthase ^ |3-(l(6)-glucan ^»Mannan ^^y*Cell wall enzymes ^ |3-(l(3)-glucan ► 6PI (6lycosylPhosphatidylInositol) Anchored Proteins = GAP ► Cell wall modifying enzymes ► e.g. Glucan Elongation (Gel) proteins elongating 6-1,3-glucan chain pGEL4::eGFP:GEL * • * * * # \ * mt * it V pGEL4::GEL4:eGFP Aspergillus fumigatus is a fungal saprotroph BUT opportunistic human pathogen! ► Causes aspergillosis in immunocompromised patients.... deadly How to knock-out 132 genes in one summer . . . ► Single KOs of all GAP proteins!!! > Growth defects /phenotype > Spore phenotype GPI51 ~ chains of conidia 100- MA/L+ CR MM + CFW 01 GO re 4-> C Ol u 5 50 H re > 3 (/I 0J u ► sivg/4 gene (SWollen and Germinated conidia) is essential for the nitrogen metabolism - AkuSO - AswgA AswgA::swgA T 5 "T~ 10 15 Days > Samalova et a/., 2020, 2023 Thank you for your attention! Acknowledgement Ian Moore, Sarah Gurr, Oxford Jean-Paul Latge, Paris Jan Hejatko, Brno