UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE FARMACIA Departamento de Microbiología y Parasitología TESIS DOCTORAL Efecto de los antibióticos β-lactámicos carbenicilina y penicilina en el desarrollo de Araidopsis thaliana MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Marco Esteban Gudiño Gomezjurado Directores Federico Navarro García Miguel Ángel Blázquez Rodríguez Madrid 2019 © Marco Esteban Gudiño Gomezjurado, 2018 Arabidopsis thaliana AGRADECIMIENTOS ÍNDICE DE CONTENIDOS Arabidopsis thaliana A. thaliana A. thaliana A. thaliana COBL9::GFP A. thaliana Colletotrichum tofieldiae A. thaliana A. thaliana Colletotrichum tofieldiae ÍNDICE DE FIGURAS Arabidopsis thaliana A. thaliana de A. thaliana. CYCB1;1 COBL9 Solanum lycopersicum Oryza sativa. GL2 SKP2B SKP2B tir1-1 axr1-12 CYP81F2 CYP81F3 CYP81F4 y rhd2 Arabidopsis thaliana Colletotrichum tofieldiae Ct ÍNDICE DE TABLAS Arabidopsis thaliana Introducción β in situ β Objetivos β Arabidopsis thaliana Resultados A. thaliana β CYCB1;1 A. thaliana noa1 nia1 nia2 rhd2 DR5 SKP2B SKP2B β CYP71A12, CYP71A13 CYP81F2 CYP81F2 β CYP79F1, CYP79F2, CYP83A1 FMO Gsox4 FMO Gsox2, FMO Gsox3, FMO Gsox5, A. thaliana Colletotrichum tofieldiae Conclusiones A. thaliana A. thaliana Colletotrichum tofieldiae Arabidopsis thaliana Introduction β in situ Objectives Arabidopsis thaliana Results A. thaliana CYCB1;1 A. thaliana noa1 nia1 nia2 rhd2 DR5 SKP2B SKP2B β CYP71A12 CYP71A13 CYP81F2 CYP81F2 CYP79F1, CYP79F2, CYP83A1 FMO Gsox4 FMO Gsox2, FMO Gsox3, FMO Gsox5 A. thaliana Colletotrichum tofieldiae Conclusions A. thaliana 3. A. thaliana Colletotrichum tofieldiae. Arabidopsis thaliana Hordeum vulgare Bouvardia ternifoli Antirrhinum majus Malus Platanus acerifolia Populus tremuloides x P. tremula Citrus sinensis Continúa Lactuca sativa, Medicago sativa, Daucus carota Daucus carota Brassica rapa Pekinensis Triticum aestivum Musa Brassica napus, Triticum aestivum, Apera spica - venti Penicillium chrysogenum Proteus vulgaris Klebsiella pneumoniae Enterobacter Arabidopsis thaliana Solanum lycopersicum Oryza sativa Pisum sativum Arabidopsis thaliana A. thaliana A. thaliana reactive oxygen species 1.3.1.1. Auxinas A. thaliana A. thaliana transport inhibitor response 1 uxin signalling F Box protein auxin/indole–3–acetic acid protein auxin response factor related to ubiquitin 1 auxin resistant 1 – E1 C – terminal related 1 DR5::GUS 1.3.1.2. Especies reactivas de oxígeno y óxido nítrico 1.3.1.2.1 Biosíntesis de las ROS y del NO respiratory burst oxidase homologues Arabidopsis thaliana nitric oxide – associated 1 1.3.1.2.2 Interacción entre las ROS y el NO S S S 1.3.1.2.3. Interacción entre auxinas, ROS y NO A. thaliana 1.3.2.1. El meristemo apical de la raíz de : morfología, crecimiento y diferenciación shoot apical meristem root apical meristem quiescent center Arabidopsis thaliana cyclin dependent kinases 1.3.2.2. Los pelos radiculares A. thaliana A. thaliana A. thaliana GLABRA2 GL2 RHD2 ROOTHAIR DEFECTIVE2, AT5G51060 1.3.2.3. Las raíces laterales GATA23 AT5G26930 MAKR4 AT2G39370 SKP2B AT1G77000 S-Phase Kinase-Associated Protein2 SKP2B Brassicales Brassica Arabidopsis S A. thaliana A. thaliana Actinobacteria Proteobacteria Firmicutes Fusarium Trichoderma A. thaliana Piriformospora indica Colletotrichum tofieldiae Colletotrichum tofieldiae A. thaliana SKP2B::GUS, tir1-1/SKP2B::GUS axr1- /SKP2B::GUS COBL9 CYCB1;1 cyp79b2/3 GL2 nia1nia2 noa1 nia1,nia2,noa1 rhd2 Oryza sativa Solanum lycopersiucum software Analyze, set scale Freehand line Analyze, Measure CYCB1;1::GUS DR5::GUS axr1 SKP2B::GUS tir1 SKP2B::GUS pSKP2B::GUS software software Straight line oftware Straight line, Stacks Dynamic Reslice Straight line Measure Analyze H2DCFDA DAF–FM–DA software Analyze, set scale Freehand line Analyze, Measure rhd2 pCOBL9::GFP el Departament de Ciències Agràries i del Medi Natural Universitat Jaume I rhd2 RNeasy Plant Mini Kit QIAshredder spin column g RNeasy spin g DNA-free kit 10X DNase I rDNase I DNase Inactivation Reagent g PrimeScript Reverse Transcriptase Oligo dT primer dNTP Mixture 5X PrimeScript Buffer RNase Inhibitor PrimeScript Reverse Transcriptase SYBRPremix Ex Taq II (Tli RNAseH Plus) Bulk ROX reference Dye software Fast-Real Time PCR System ACT8. YUCCA 9 TAA1 AMI1/ CYP79B2 CYP79B3 IAMT1 CYP81F2 Continúa CYP81F3 CYP81F4 CYP79F1 CYP79F2/ CYP83A1/ FMO GSox2/ FMO GSox3/ FMO GSox4/ FMO GSox5/ ACT8 RNeasy Plant Mini Kit RNA Integrity Number TruSeq Stranded Total RNA Library Prep Kit FastQC Cutadapt Tophat HTSeq-count principal component analysis PtR mock dgeR p heat maps Matrix2png Interface A. thaliana Colletotrichum tofieldiae A. thaliana cyp79b2/3 Colletotrichum tofieldiae Ct+ , Ct- software A. thaliana A. thaliana mock p CYCB1;1:GUS CYCB1;1 uidA CYCB1;1 de A. thaliana. p p CYCB1;1 CYCB;1::GUS p p p gene ontology p p Heatmap Fold-Change p Continúa Continuación rhd2 rhd2, Moc k COBL9 , COBRA LIKE 9 COBL9::GFP COBL9 Solanum lycopersicum Oryza sativa COBL9 p pCOBL9::GFP Solanum lycopersicum Oryza sativa. S. lycopersicum O. sativa GL2::GUS GLABRA2 GL2 GL2 GL2 A. thaliana GL2::GUS nia1 nia2 noa1 NO–associated protein1 nia1 nia2 noa1 nia1 nia2 noa1 A. thaliana nia1 nia2 noa1 nia1 nia2 noa1 Heatmap Fold-Change p rhd2 Arabidopsis thaliana Arabidopsis thaliana rhd2 p DR5::GUS YUCCA9, TAA1, AMI1, CYP79B2 CYP79B3 IAMT1 CYP79B3 cyc79b2/3 CYP79B3 SKP2B SKP2B SKP2B::GUS, SKP2B SKP2B YUCCA9 TAA1 IAMT1 AMI1 CYP79B2 CYP79B3 A. thaliana p t cyc79b2/3 cyc79b2/3 SKP2B SKP2B::GUS p SKP2B SKP2B SKP2B::GUS tir1-1 axr1-12 SKP2B SKP2B Mock SKP2B tir1- 1 axr1-12 SKP2B::GUS tir1-1 axr1-12 A. thaliana Heatmap Fold-Change p CYP79B3 CYP79B3 CYP79B3 ns p p ns CYP81F2 CYP81F3 CYP81F4 CYP81F2 CYP81F2 rhd2 Arabidopsis thaliana CYP81F2 CYP81F3 CYP81F4 y rhd2 CYP81F2 en rhd2 p t p ns CYP79F1 CYP79F2 CYP83A1 FMO GSox2, FMO GSox3, FMO GSox4 FMO GSox5 FMO GSox2 FMO GSox5 FMO GSox3 FMO GSox4 CYP79F1 CYP79F2 CYP83A1 FMO GSox2 FMO GSox3 FMO GSox4 FMO GSox5 p t A. thaliana Colletotrichum tofieldiae Colletotrichum tofieldiae cyp79b2/3 C. tofieldiae C. tofieldiae Ct C. tofieldiae cyp79b2/3 C. tofieldiae Arabidopsis thaliana Colletotrichum tofieldiae Ct A. thaliana cyp79b2/3 Ct Ct C. tofieldiae A. thaliana cyp79b2/3 Ct p ns p Colletotrichum tofieldiae rhd2 microbe-associated molecular patterns A. thaliana CYP81F CYP81F2 IGMT1 IGMT4 IGMT5 IGMT2 CYP81F2 CYP71F1 CYP71F2 CYP83A1 FMOGS-ox3 FMOGS-ox4 MAM1 MAM3 SUR1 GGP1 UGT74B1 C1 FMOGS-ox mam1 mam3 TGG1 TGG2, TGG3, TGG5, TGG6, BGLU34, PEN2 PEN3 CYP71A12 CYP71A13 Sclerotinia sclerotiorum Penicillium simplicissimum induced systemic resistance Pseudomonas syringae tomato Pst Pst Penicillium P. chrysoge- num Pst MYB72 cyp79B2 cyp79B3 Solanum lycopersicum Oryza sativa in situ Penicillium CYP71F1 F2 A. thaliana Colletotrichum tofieldiae Pseudomonas pyocyanea Nature 215 Arabidopsis Proceedings of the National Academy of Sciences 114 Bioinformatics 31 Nature Reviews Microbiology 12 The Arabidopsis Book 9 Piriformospora indica Frontiers in Plant Science 6 The Plant Cell Online 21 Journal of Environmental Quality 11 Science 323 PLoS ONE 3 Arabidopsis thaliana Plant Physiology 116 Plant Physiology 129 Cell 115 Arabidopsis thaliana Developmental Biology 194 Arabidopsis thaliana Development (Cambridge, England) 125 PLoS Genetics 10 Molecular Plant-Microbe Interactions 22 Physiologia Plantarum 97 Phytochemistry 67 COBRA Plant Physiology 143 Trends in Plant Science 9 The Plant Cell Online 13 Nature 543 Journal of Environmental Quality 38 Plant Physiology 154 Plant Journal 20 Plant Physiology 151 Nature Reviews Molecular Cell Biology 8 The Plant Cell 14 Science 280 Plant Mol Biol 49 Acacia tortilis raddiana Balanites aegyptiaca Environmental Microbiology 6 Trends in Molecular Medicine 18 Nature 411 Arabidopsis thaliana Development 119 Nucleic Acids Research 38 Proceedings of the National Academy of Sciences USA 107 Microbial Ecology 57 Antonie van Leeuwenhoek 106 New Phytologist, 174 Bacteria in Agrobiology: Crop Productivity New Phytologist 213 Proceedings of the Natural Academy of Sciences USA 108 Rhizobium World Journal of Microbiology and Biotechnology 28 Staphylococcus aureus IUBMB Life 66 Plant & Soil 232 Nature 422 Plant Physiology 155 Arabidopsis thaliana PLoS ONE 6 Auxin and Its Role in Plant Development Plant Physiology 141 Arabidopsis thaliana Fungal Diversity 60 Sulphur in Plants Arabidopsis thaliana Plant Journal 18 Science of the Total Environment 565 Nature Biotechnology 29 Nature 414 The Arabidopsis Book 12 Science 302 Trends in Plant Science 16 Cell Host and Microbe 17 Glucosinolates (80) Annual Review of Plant Biology 57 Plant Journal 50 Plant Journal 74 Colletotrichum tofieldiae Cell 165 Journal of Experimental Botany 66 Penicillium simplicissimum Arabidopsis thaliana Plant and Cell Physiology, 8 Molecular Plant 7 Trends in Microbiology 22 The EMBO Journal 6 Sinorhizobium Medicago Nature Reviews Microbiology 5 FEBS Letters 579 Science Translational Medicine 5 Applied Microbiology and Biotechnology 92 Arabidopsis thaliana Microthlaspi perfoliatum Fungal Diversity 66 Journal of Microscopy 231 Arabidopsis thaliana Genetics 159 Nature, 286, Arabidopsis thaliana Phytochemistry Reviews, 8 Plant and Cell Physiology 56 Journal of Experimental Botany 64 Bacterial Metabolites in Sustainable Agroecosystem Pharmaceuticals in the environment: Sources, fate, effects and risks Plant Molecular Biology 90 Development (Cambridge, England) 121 Molecular Pharmacology 50 Plant Physiology 148 Plant Physiology 167 Plant Physiology 152 Development 140 The Plant Cell 17 Plant Physiology 152 Arabidopsis thaliana Nature 488 Development (Cambridge, England) 124 Proceedings of the National Academy of Sciences 114 Plant Physiology 171 Plant Physiology 160 EMBnet.Journal 17 GLABRA2 Arabidopsis thaliana Development 122 Nature Education Knowledge 4 Pseudomonas putida Gluconacetobacter azotocaptans Azospirillum lipoferum Microbial Ecology 51 The Plant Cell Online 18 Panicum miliaceum Pisum sativum Zea mays Agriculture, Ecosystems and Environment 52 Chemosphere 37 AoB PLANTS 9 Current Opinion in Plant Biology 35 Proceedings of the National Academy of Sciences 104 Arabidopsis thaliana Journal of Biological Chemistry 283 Microbiologia e Bioquímica do Solo Annual Review of Cell and Developmental Biology 13 Plant Physiology 134 Physiologia Plantarum 15 Trends in Plant Science 18 EMBO Journal 28 Plant Physiology 143 Enterobacteriaceae Trends in Molecular Medicine 18 Frontiers in Plant Science 7 Glucosinolates (80) Journal of Environmental Quality 9 Bioinformatics 19 Annals of Botany 87 Plant Physiology 166 Frontiers in Plant Science 6 O Plant Physiology, 172 Nicotiana benthamiana Arabidopsis The Plant Cell 23 Annual Review of Cell and Developmental Biology 28 Plant Journal 34 Arabidopsis thaliana Scientific Reports 5 Arabidopsis thaliana Nature Immunology 16 MAM1 MAM3 Arabidopsis thaliana Physiology and Biochemistry, 53, Plant Journal 53 Journal of Biological Chemistry 279 Frontiers in Plant Science 8 Bioinformatics 26 Biotechnology Advances 17 Fármacos antimicrobianos. Mecanismos de acción y resistencia Development 143 The Plant Cell Online 27 Plant Physiology 166 Arabidopsis thaliana The Plant Cell 2 Nature Methods 9 Arabidopsis thaliana Proceedings of the National Academy of Sciences USA 111 Arabidopsis thaliana Proceedings of the National Academy of Sciences USA 105 Trends in Plant Science 15 Sclerotinia sclerotiorum The Plant Journal 67 Applied Soil Ecology 15 Environmental Science and Technology, 49 Applied and Environmental Microbiology 77 PLoS ONE 6 Science 319 Plant Signaling & Behavior 8 Frontiers in Plant Science 6 Science 319 Science of the Total Environment 563–564 Plant Journal 70 Plant Physiology, 144 Journal of Plant Nutrition and Soil Science 166 Secondary Metabolites in Soil Ecology Penicillium chrysogenum Arabidopsis thaliana Physiological and Molecular Plant Pathology 67 Trends in Plant Science 19 The ISME Journal 9 Plant, Cell & Environment, (40) Phytochemistry Reviews 8 Nature Protocols 7 Plant Signaling & Behavior 7 Plant Cell 20 Plant Science 197 Current Biology 19 The Plant Cell 9 Nature 378 Arabidopsis thaliana Plant Journal 39 Developmental Cell 35 Arabidopsis thaliana Plant Signaling & Behavior 1 Plant Physiology 147 Plant Physiology 135 BioEssays 37 Frontiers in Plant Science 4 Arabidopsis thaliana Molecular & General Genetics 239 The Penicillins Mayo Clinic Proceedings, 62 Current Biology 25 FEBS Letters 468 S Plant Physiology 167 Plant Signaling & Behaviour 8 Nature 478 Journal of Agricultural and Food Chemistry 60 Molecules 17 Developmental Cell 20 Arabidopsis snc2-1D The Plant Cell 22 The Plant Journal 81 Plant Signaling & Behavior 2 Genes & Development 16 Tesis Marco Esteban Gudiño Gomezjurado PORTADA AGRADECIMIENTOS ÍNDICE DE CONTENIDOS ÍNDICE DE FIGURAS ÍNDICE DE TABLAS RESUMEN SUMMARY 1. INTRODUCCIÓN 2. OBJETIVOS 3. MATERIALES Y MÉTODOS 4. RESULTADOS 5. DISCUSIÓN 6. CONCLUSIONES 7. REFERENCIAS