1U N I UNIVERSITY OF Plant Celt and Molecular Biology doc. Markéta Šámalové Institut Pasteur jihomoravský kraj I2?3> Oddělení experime biologie rostlin experimentální OUTLINE of the talk Arabidopsis thaliana ► 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 How to make a genetically modified or 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 An ideal inducible system ► High induced expression (e.g. lOOOx or more). ► No uninduced expression (not leaky). ► Rapid uptake and wide distribution of inducer. ► No toxicity, no physiological effects in plants. ► Convenient application by a number of methods. ► Functional in several plant species. Depend on the type of application, the gene being expressed and the plant species! .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 pH-Luc/ LhGR «* > 2 neither DEX nor Lh&k affects endogenous processes in plants ... though ethanol doesl DEX in ethanol DEX in DMSO 0.1% ethanol MS \ ^^^^^^^ Arabidopsis seedlings were grown on plates in the presence or absence of 10 |aM DEX. The pOp6/LhGR system is inducible by various methods 6h 12h 24h pV-TOP/ LhGR pH-TOP/ LhGR Oh 6h 12h 24h pV-TOP/ LhGR pH-TOP/ LhGR Watering plants with DEX bEX distribution through tissues (24h after watering). Painting plants with DEX A leaf painted pV-TOP/LhGR pH-TOP/Lh The pOp6/LhGR system is functional in several Arabidopsis Tobacco (Craft, Samalova et a/., 2005) (Samalova et a/., 2005) (Samalov DEX - DEX 2" ^* i 1 h . pOp6-YFP ► Maize, potato, tomato, Cardamine hirsuta, citrus. [► Detailed step-by-step protocols in Samalova et a/., 2019 CRISPR/Cas9 bacterial system adapted to edit the genome of various species ~ "genetic scissors" The ability of Cas9 (nuclease) to target a specific site of genomic DNA using gRNA ► 2020 Nobel Prize in chemistry awarded to E. Charpentier a J. Doudna ► Genome-edited organism ► Changes in the open reading frame (ORF) generate a stop codon! Creating "knock-out" (KO mutant) CRISPR/Cas9 guide RNA Double-stranded DNA break ax: nun in 1111 X .......1111 I I \/ NHEJ HDR Doriijrejiterflül DNA III Mil! >l_\s X .....I..............l>^N|, .{Jy X. iijiinii..............u^Nf, Expected J NA sequence CCCATGTGCAGGTTGTTAACACAAGAC r T TTATAT CACCG\T G WTGCAGGTT GTTAACrSCAAGACGT 60 70 80 91 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.... ■ 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/., 2024) Arabidopsis leaf G22 ^FP Linkers / \ ER sign ĺ CD BP22 peptide (Transmembranedomain) 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) CW is crucial for plant growth <& developmen ► shapes the plant body ► movement of solutes and nutrients ► protects plants from the environment ► intercellular communication (Wolf eta/.,2012) ► Cellulose is the most abundant biopolymer on Earth! Middle J Lamella I_ Primary Cell Wall Plasma Membrane Pectin Cellulose Microfibril Hemicellulose Soluble Protein 1/7/17 US' ► Load-bearing cellulose microfibrils \ ► embedded into] viscoelastic matrix of hemicellulose and pectin^ How do plant cells grow? ► Plant CW combine strength with extensibility Wall extensibility may be controlled at limited regions, 'biomechanical hotspots' (Cosgrove, 2014; 2018). EXPANSINs are small proteins that disrupt the non-covalent bonds between CW polysaccharides, thus relaxing wall stresses and allowing turgor-driven cell expansion (Cosgrove, 2000). Sites of expansin action! 7 B'omec^an'ca' "hot spot" 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 al., 2024) > Use of mCherry (RFP) instead of pH sensitive GFP Promoter EXPA1 1 nls eGFP eGFP eGFP m EXPANSINS are localized into various root 1 1 :alized into various root 1 ► 3D projection of Z-stack (combined optical slices) taken by a confocal microscope pEXP 10: :EXP 10.mCherry EXPANSINS are localized into various root 1 \,\ '• > 'fjp&P1*:EXP 14:mCherry vVfv-' pEXP15: :EXP 15:mCherry ^ cortex Overexpression of EXPA1 makes the plants smaller by "stiffening" cell walls ► Changes in biomechanical properties of CWs. pRPS5A»EXPA1 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 Plants overexpressing EXP Al are more ^ to bacteria Pseudomonas syringae > P. s. is an agressive bacterial pathogen. > Entres plants thought stomata! resistant EXPA1 localizes in stomata! Bacterial plant infection 14000 12000 g 10000 oi 8000 E ^ 6000 u. U 4000 2000 J 11.... 10-3 13-1 DEX ■ + DEX ■ 25-5 WT \ MI II + DEX Tobacco line 10-3 pEXP 1: :EXP 1 .mCherry Not only plant cells have the ON... 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— ROS toxicity alone is NOT sufficient Magnaporthe oryzae in resistan ito kill y. Pice! Exploring redox state in susceptible & resistant (Samalova 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 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 gj 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