Plant Experimental 1U N I UNIVERSITY OF WOXFORD Biology Markéta Šámalové Institut Pasteur jihomoravský kraj experimentální K3> Oddělení experime biologie rostlin SUMMARY ► How to make a transgenic plant? ► Tobacco, rice ► Arabidopsis thaliana ► How to regulate (trans)gene expression? ► The pOp6/LhGR system ► CRISPR/Cas9 ► Transient gene expression ► Fluorescent proteins and protein localization ► Plant endomembrane system ► Plant cell wall ► Expansins <& abiotic stresses ► Fungal cell wall ► Magnaporthe oryzea - a model organism ► Aspergillus f urn igat us Arabidopsis thaliana My 1st transgenic tobacco plant that I made during my PhD (in the last century...:) ► Re-generated in vitro using tissue cultures Ectopic meristems developing into shoot-like structures... Introduced IPTgene encoding isopentenyl transferase which catalysis the first step in hormone cytokinin biosynthesis... leaves roots How to make a transgenic plant? Transformation ► Tissue cultures ► tobacco ► rice DNA Extraction and Isolation Cloning and Designing Genes ^* Transformation SBOC-OCX^" ^ Bactena How to regulate (trans)gene expression? 1 Chemically inducible gene expression systems ► used to regulate (trans)gene expression at a particular developmenta stage and for a specific duration using chemical inducers. ► Allow precise temporal and spatial control of a (trans)gene. ► Expression can be SWITCHED ON or OFF using chemical inducers ► Gene overexpression or knock-down expression by amiRNAs. ► Essential for expression of gene products that interfere with regeneration, growth or reproduction... Meristem defect + Inducible gene inducer The chemically inducible transcription activation system pOp/LhGR Lh R A 1VATO pOp REPORTEI 6 Operators m GUS regulatory DNA- transcription domain binding d. activation d. promoter GR LacľHis17> Gal4 i Á i lac TATA operators box HSP90 complex Ö ě + INDUCER DEXAMETHASONE > Developed in the laboratory of Dr Ian MOORE > Use world-wide today... > An "ideal" inducible system? ] GEA/E 14 YOUR GENE 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 pH-TOP 2 pH-TOP 9— pV-TOP 3 pOp-GUS 1—I—Mill-1-1-1—1—I I I I 0.1 10 Time (h) 100 1000 ► Increase of GUS activity in 2h! ► The most sensitive system for tobacco! m 1 The pOp6/LhGR system is tightly regulated ► Basal expression levels tested with ipt gene ► from Agrobacterium (cytokinin biosynthesis) ► physiologically strong transgene + DEX - DEX ■ not toxic! pOp6-ipt/ LhGR pV-ipt/ LhGR pOp-ipt/ LhGR 4. * r ** > 2 (£0 pH-Luc/ LhGR neither DEX nor Lh&k affects endogenous processes in plants ... though ethanol does! DEX in ethanol DEX in DMSO 0.1% ethanol _MS 5$ grown on plates in tj or absence of The pOp6/LhGR system is inducible by varioi 1 methods 6h 12h 24h pV-TOP/ LhGR Watering plants with DEX pH-TOP/ LhGR DEX distribution through tissues (24h after watering). Tobacco 1 The pOp6/LhGR system is inducible by varioi 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/Lh EXPANSINs localised to the CW in vivo for the first time! \ > Use of mCherry (RFP) instead of pH sensitive GF Promoter EXPA 1 n/s eGFP eGFP EXPANSINS are localized into various roo* ► 3D projection of Z-stack (combined optical slices) taken by CLSM with airyscan detector EXPANSINS are localized into various roo Overexpression of EXPA1 reduces the roo' ACTIVATOR i* DEX REPORTER GUS pH5.8 pH4 |H4 Line 8-4 + DEX \ \ \ - DEX / + DEX WT % 1 t - DEX ) ) I, + DEX AtEXPAl ^ WT WT + 8-4 8-4 + 5-4 5-4 + DEX DEX DEX Significant reduction of root size of EXPA1 overexpressing plants (7day-old Arabidopsis seedlings) 1 Overexpression of EXPA1 changes biomechani properties "stiffens" CW> Momic Force Microscopy (AFM) (br Alexis Peaucelle, INRAE, Versailles) EA (MPa) 3e+06 2e+06 pH 4 1e+06 Oe+00 WT WT+DEX 8-4 8-4+DEX MOC pH 4 > Brillouin Light Scattering (BLS) \ (br Kareem Elsayad, Biocentre, Vienna) WT WT+DEX 8-4 8-4+DEX [► Samalova et al., 2020 BioRxiv Overexpression of EXPA1 leads to smaller, compact plants *^L* 5-4 8-4 31 WT * ^ ^5 4i ^§sc ^te 35 38 45dpg EXPANSINs can improve stress tolerance of > Explore the role EXPANSINs under stress: > ABIOTIC > drought > salt > heat > cold > H202 > Cd ... > BIOTIC - viruses, bacteria, fungi, brown planthopper ] r Knocking-out multiple EXPANSINs genes ► CRISPR/Cas9 - multicomplex mutagenesis (Richter et a/., 201 ► To create expalexpal Oexpal4expal 5 multiple knock-out CRISPR/Cas9 Cas9 I lift1 Double-stranded DNA break ax / X '.......'.....X"11 u^^s*L Donor ejcteifldl UNA III Mill ifnTTTTTfl ITTTTTTTTTT Ho« DNA ■'■'"III......I..... Expected sequence m ii/4 CCCATGTGCAGGTTGTTAACACAAGAC n TT ATAT D\CCC^T Plant no.8 A T G GCAG GT T GTTAACAC -\ A G AC GT 70 SO 91 > Insertion changes the reading frame! > Createsxf stop codon after 47 aa (ip-ffie first exon of EXPA10). 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 £ Chitin synthase Glycoprotein ^^Chitin Q^J P-(l,3)-glucan synthase |3-(l(6)-glucan ^•Mannan ^^j)Cell wall enzymes £ |3-(l(3)-glucan ► 6PI (6lycosylPhosphatidylInositol) Anchored Proteins = GAP ► CW modifying enzymes ► E.g. Glucan Elongation (Gel) proteins elongating 6-1,3-glucan chain 1 of rice! Magnaporthe oryzae the most devastating pathoger ► 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 pGEL3: :GEL3:mCherry ~ Germling with extracellular matrix c _ I r> I Conidium Spore tip mucilage Autophagy 24hpi Samalaova et a/., 2017 Exploring redox state in susceptible & res is tan ROS toxicity alone is NOT sufficient to kill Magnaporthe oryzae in resistant rice! (Samalaova 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 £ Chitin synthase Glycoprotein ^^Chitin Q^J P-(l,3)-glucan synthase |3-(l(6)-glucan ^•Mannan ^^j)Cell wall enzymes £ |3-(l(3)-glucan ► 6PI (GlycosylPhosphatidyllnositoI) Anchored Proteins = GAP ► CW modifying enzymes ► E.g. Glucan Elongation (Gel) proteins elongating 6-1,3-glucan chain Aspergillus fumigatus is a fungal saprotroph BUT opportunistic human pathogen! ► Causes aspergillosis in immunocompromised patients.... deadly How to knock-out 132 genes in one summei CM MM 1 Ku80 ~"75 7*8* Ku80 75 78 U* 94 122 123 0" " Ku80 18 33 34 55 34 55 59 61 ^v£0^ 58 61 ^70^ MM+CR MM + CF W Single KOs of all GAP proteins!!! > Growth defects /phenotype on different type of media: > Complete <& minimal medium > MM+CR or CFW ~ CW stress > MM+ SDS - PM stress > MM+H202 ~ oxidative stress > Spore phenotype GPI51 ~ chains of conidia > A candidate gene is in a medical trial to test for reduced immune response....(Samalova et a/., 2020) Acknowledgement Ian Moore, Sarah Gurr, Oxford Jean-Paul Latge, Paris Jan Hejatko, Brno