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Systemin Involvement in Tomato Defense Priming

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Systemin Involvement in Tomato Defense Priming SYSTEMIN INVOLVEMENT IN TOMATO DEFENSE PRIMING Valentina Madonna Dottorato in Scienze Biotecnologiche – XXVIII ciclo Indirizzo Biotecnologie per le Produzioni Vegetali Università di Napoli Federico II Dottorato in Scienze Biotecnologiche – XXVIII ciclo Indirizzo Biotecnologie per le Produzioni Vegetali Università di Napoli Federico II SYSTEMIN INVOLVEMENT IN TOMATO DEFENSE PRIMING Valentina Madonna Dottoranda: Valentina Madonna Relatore: Prof.ssa Rosa Rao Coordinatore: Prof. Giovanni Sannia A Francesco e alla mia Famiglia INDEX RIASSUNTO ........................................................................................................... 1 SUMMARY ............................................................................................................. 7 1. INTRODUCTION ................................................................................................ 9 1.1 Plant undergo a series of different stresses ................................................... 9 1.2 Plant defenses against biotic stresses: Direct and Indirect .......................... 10 1.3 Time of defenses: Constitutive and Inducible responses ............................. 12 1.4 Plant responses against insects .................................................................. 14 1.5 Priming of defences ..................................................................................... 16 1.6 Solanum lycopersicum defence: Systemin .................................................. 19 1.7 Octadecanoid pathway ................................................................................ 21 1.8 Research Objective ..................................................................................... 23 2. MATERIALS AND METHODS ........................................................................... 25 2.1 Plants Material ............................................................................................ 25 2.2 Molecular characterization of transgenic plants ........................................... 25 2.3 Expression analysis of genes induced by biotic stimuli ................................ 27 2.3.1 Spodoptera littoralis chewing ................................................................ 27 2.3.2 Tomato plants treatment with Systemin peptide .................................... 27 2.4 Exposure of plants to different sources........................................................ 27 2.5 RNA Isolation and Quantification ................................................................. 29 2.6 RNA retrotranscription ................................................................................. 29 2.7 Real Time RT-PCR ..................................................................................... 30 2.8 Bioassays .................................................................................................... 30 2.8.1 Spodoptera littoralis rate increase assay .............................................. 30 2.8.2 Analysis of attractiveness towards the parasitoid Aphidius ervi ............. 31 2.9 Qualitative and quantitative evaluation of volatile blend modifications using GC-MS .............................................................................................................. 31 2.10 RNA-Sequencing and Data Analysis ......................................................... 31 2.10.1 Gene Ontology and enrichment analysis (GOEA) ............................... 32 3. RESULTS ......................................................................................................... 33 3.1 PCR screening of transgenic plants ............................................................ 34 3.2 Expression analysis of defense genes in sources plants ............................. 35 3.2.1 RNA and cDNA quality .......................................................................... 35 3.2.2 Genes induced by leaves chewing from Spodoptera littoralis................ 37 3.2.3 Genes induced by foliar applications of Systemin peptide ..................... 39 3.3 Expression analysis of defense related genes in receiver plants ................. 40 3.3.1 Expression profiles induced by Spodoptera littoralis chewed plants ...... 42 3.3.2 Expression profiles induced by transgenic plants over-expressing the ProSys gene .................................................................................................. 43 3.3.3 Expression profiles induced by plants treated with Systemin ................ 43 3.4 Spodoptera littoralis weight increase and survival rate: Induction of direct defense ............................................................................................................. 44 3.4.1 Effect of the exposure to chewed plants source on direct defense against S. littoralis larvae ........................................................................................... 44 3.4.2 Effect of the exposure to transgenic plants overexpressing Prosys on survival and weight increase of S. littoralis larvae .......................................... 45 3.4.3 Effect of the exposure to plants with foliar applications of Systemin on survival and weight increase of S. littoralis larvae .......................................... 46 3.5 Induction of indirect defenses in receiver plants .......................................... 47 3.6 Qualitative and quantitative evaluation of VOCs from source and receiver plants ................................................................................................................ 48 3.7 Transcriptomic reprogramming of plants exposed to source plants treated with systemin peptide ........................................................................................ 52 3.7.1 EdgeR analysis ..................................................................................... 54 3.7.2 Functional annotation of differentially expressed genes: Gene Ontology and enrichment analysis (GOEA)................................................................... 55 4.DISCUSSIONS AND CONCLUSIONS ............................................................... 71 5. REFERENCES ................................................................................................. 77 STAGE .............................................................................................................. 91 PUBLICATIONS ................................................................................................ 93 APPENDIX ........................................................................................................ 97 RIASSUNTO Le piante sono organismi sessili incapaci di sfuggire alle variazioni sfavorevoli dell’ambiente. Per contrastare tale condizione, questi organismi hanno evoluto meccanismi elaborati per percepire lo stress e adattarsi ad esso attraverso alterazioni rapide, dinamiche e complesse. Le difese delle piante possono essere distinte in base al tempo di attivazione, in difese costitutive, sempre espresse, e difese inducibili se espresse dopo attacco dei parassiti. Sulla base del meccanismo d'azione, le difese della pianta possono essere distinte in dirette e indirette: le prime interferiscono direttamente con la crescita e lo sviluppo degli insetti, le altre sono attive nel reclutamento dei nemici naturali degli insetti. Sono incluse nelle difese dirette le barriere fisiche quali spine, silice, tricomi, che rendono difficoltosa la colonizzazione da parte dei parassiti, metaboliti primari o secondari come inibitori di proteasi e polifenolo ossidasi che determinano una diminuzione dei livelli di digestione e dell’assimilazione dei nutrienti, compromettendo la crescita degli insetti, ma anche composti tossici come alcaloidi, terpenoidi e fenoli che interferiscono con il metabolismo dell’insetto (Bennet e Wallsgrove, 1994; Kessler e Baldwin, 2002; Campbell et al., 2013). Le difese indirette includono la produzione di composti organici volatili (VOCs), coinvolti nell’attrazione di nemici naturali dei degli insetti, come i predatori e i parassitoidi (Dudareva et al. 2006). I VOCs includono inoltre semiochimici coinvolti nella segnalazione aerea inter-pianta e pianta-pianta. E' noto, infatti che i VOCs possono segnalare 'pericolo di attacco' da parte di erbivori o altri parassiti. e possono segnalare pericolo di attacco da parte di erbivori. Le piante allertate sono in uno "stato innescato", ossia più rapide ed efficienti nell’induzione delle difese a seguito di attacco di erbivori (Hilker e Meiners, 2009). Quindi, l'esposizione ai composti organici volatili prodotti da piante attaccate da erbivori può innescare nella pianta esposta non ancora danneggiata meccanismi molecolari che la preparano ad una risposta difensiva, incrementando così la probabilità di sopravvivenza ad un attacco. Tale fenomeno è denominato “Priming”, di cui sono ancora ancora poco noti i meccanismi molecolari che lo sottendono. Nelle Solanaceae, le sistemine sono una famiglia di peptidi coinvolti nell’attivazione di geni di difesa in risposta a ferite e agli attacchi di erbivori masticatori (Ryan e Pearce, 2003). La Sistemina (Sys) è un ormone peptidico di 18 amminoacidi, rilasciato inizialmente dai siti di danno e rappresenta un segnale primario di ferita in pomodoro. La Sistemina è rilasciata da una proteina precursore, citosolica, di 200 aminoacidi chiamata ProSistemina (ProSys) mediante un meccanismo ancora non noto. L'attivazione di geni di difesa mediata da Sistemina avviene attraverso la via degli octadecanoidi, i cui prodotti sono acido jasmonico (JA) e suoi derivati. Dopo l’attacco da parte di erbivori,
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