An evaluation of the efficacy of antimicrobial peptides against grapevine pathogens by Marike Visser Thesis presented in partial fulfilment of the requirement for the degree Master of Science at the Department of Genetics, Stellenbosch University. Supervisor: Prof. J.T. Burger Co-supervisor: Dr. D. Stephan March 2011 Stellenbosch University http://scholar.sun.ac.za Declaration By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. March 2011 Copyright © 2011 University of Stellenbosch All rights reserved i Stellenbosch University http://scholar.sun.ac.za Abstract This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem- limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity ii Stellenbosch University http://scholar.sun.ac.za screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development. iii Stellenbosch University http://scholar.sun.ac.za Opsomming Hierdie studie het die gebruik van antimikrobiese peptiede (AMPe) as 'n moontlik bron van weerstand teen 'n reeks van patogene in wingerd ondersoek. Alhoewel die uiteindelike doel sal wees om AMPe uit te druk in wingerd, is transgeniese wingerd ontwikkeling tydrowend en daarom is vooraf evaluering van potensiële AMPe nodig. Hierdie klein molekules, van minder as 50 aminosure in lengte, word uitgedruk deur amper alle organismes as deel van hul nie- spesifieke verdedigingsisteem. In vitro vooraf evaluering van AMP aktiwiteit is van waarde, maar is beperk aangesien die aktiwiteit op kunsmatige media mag verskil van die AMP- aktiwiteit in planta. Hierdie toetse is ook beperk tot patogene wat in vitro gekweek kan word. Hierdie beperkinge kan oorkom word deur gebruik te maak van tydelike uitdrukkingsisteme om die in planta aktiwiteit van AMPe te bepaal teen patogene van belang. In hierdie studie is tydelike uitdrukkingsisteme gebruik om AMPe uit te druk in ontwikkelde plantweefsel om hul effektiwiteite te toets teen wingerdpatogene soos Agrobacterium vitis, Xylophilus ampelinus en aster yellows fitoplasma. Aster yellows fitoplasmas, wat onlangs in plaaslike wingerde ontdek is, is bekend vir die uitgebreide skade wat hul aanrig en hou daarom 'n groot bedreiging in vir die Suid-Afrikaanse wingerd industrie. Om die in planta effek van AMPe teen die bogenoemde patogene te bestudeer is tydelike uitdrukkingsvektore ontwikkel wat die AMPe D4E1 of Vv-AMP1 uitdruk. D4E1 is 'n sinteties-ontwerpte AMP wat aktief is teen bakterieë en fungi, terwyl Vv-AMP1, wat uit druiwekorrels geïsoleer is, alreeds aktiwiteit teen fungi getoon het. In 'n tydelike uitdrukkingsbenadering in wingerd is die uitdrukking van transgene, vanaf virus of nie-virus gebaseerde vektore, bevestig deur die uitdrukking van die merker gene β-glukuronidase en die Groen Fluoresserende Proteïen, terwyl weefsel afdrukkings-immunotoetse virus replisering en sistemiese beweging in Nicotiana benthamiana bevestig het. Die virusvektore was gebaseer op die floëem-beperkte virus, wingerdvirus A. Slegs Agrobacterium-bemiddelde 35S tydelike uitdrukkingsvektore is gebruik om die AMP in planta aktiwiteit te bepaal aangesien die virus- bemiddelde uitdrukking in wingerd onvoldoende was vir evaluering teen A. vitis en X. ampelinus weens die beperking tot die floëem weefsel na infiltrering van die totale blaar. Geen fitoplasma geïnfekteerde materiaal kon gevestig word nie, en daarom is AMP aktiwiteitsevaluering slegs teen A. vitis en X. ampelinus uitgevoer. Kwantifisering van die bakterieë is deur middel van qPCR uitgevoer. Vv-AMP1 het geen aktiwiteit getoon teen enige van die bakterieë in planta nie, terwyl D4E1 aktief was teen beide. Die waargenome in planta aktiwiteit van D4E1 het ooreengestem met die in vitro aktiwiteit soos bepaal deur 'n AMP iv Stellenbosch University http://scholar.sun.ac.za plaat bio-toets. In kontras tot in vitro evaluering kan die in planta AMP-aktiwiteit evaluering 'n meer akkurate voorspelling bied van die potensiële antimikrobiese aktiwiteite van die peptied in 'n transgeniese plant omgewing. Hierdie studie het bewys dat tydelike uitdrukkingsisteme gebruik kan word as 'n voorafgaande evalueringsmetode vir AMP in planta aktiwiteit teen wingerdpatogene, wat die evaluering van 'n verskeidenheid AMPe in 'n relatiewe kort tydperk toelaat voor verbintenis tot die ontwikkeling van transgeniese wingerd. v Stellenbosch University http://scholar.sun.ac.za Abbreviations AMP(s) Antimicrobial peptide(s) ARC Agricultural Research Council ATP Adonine triphosphate BCIP 5-bromo-4-chloro-3-indolyl-phosphate CaMV Cauliflower mosaic virus cDNA Complementary deoxyribonucleic acid cfu Colony forming units CP Coat protein Ct Threshold cycle CTAB N-Cetyl-N,N,N-trimethyl Ammonium Bromide dpi Days post infiltration (inoculation) DTT 1,4-Dithiothreitol E qPCR reaction efficiency EDTA Ethylene Diamine Tetra-Acetic Acid di-sodium salt ELISA Enzyme-linked immunosorbent assay EmGFP Enhanced Green Fluorescent Protein GFP Green Fluorescent Protein GOI Gene of interest GUS(i) β- glucuronidase GVA Grapevine virus A LTP Lipid transfer protein MCS Multiple cloning site MIC Minimum inhibition concentration MLOs Mycoplasma-like organisms MS Murashige and Skoog NA Nutrient agar NBT Nitroblue tetrazolium OD Optical density ORF Open reading frame PLRV Potato leafroll virus PTGS Post-transcriptional gene silencing PVDF Polyvinylidene fluoride PVX Potato virus X vi Stellenbosch University http://scholar.sun.ac.za PVY Potato virus Y qPCR Quantitative real-time PCR R2 Correlation coefficient RCA Rolling circle amplification rDNA Ribosomal DNA REST Relative Expression Software Tool RNA Ribonucleic Acid rpm Revolutions per minute rRNA Ribosomal RNA RT-PCR Reverse transcription - polymerase chain reaction SAP Shrimp alkaline phosphatase SDS Sodium dodecyl sulphate SDS-PAGE SDS-Poli-acrylamid gel electrophoresis S.E. Standard error sgRNA Subgenomic RNA SOC Super optimal broth with catabolite repression T-DNA Transfer DNA Ti-plasmid Tumour inducing plasmid TMV Tobacco mosaic virus TPIA(s) Tissue-print immuno-assay(s) VIGS Virus-induced gene silencing vii Stellenbosch University http://scholar.sun.ac.za Acknowledgments I would like to express my sincere gratitude to the following people and institutes: o My supervisor Prof. J.T. Burger, for giving me the opportunity to perform this research and be part of this exceptional group, and for continuous support throughout the study. o Dirk, for guidance, support, encouragement and teaching me everything from cloning to fixing a centrifuge. o All the other members of the Vitis-lab, for advise, encouragement and lots of fun moments, for being the best team to work with. o The staff of the IWBT, for the use of their facilities and for friendly assistance. o The staff of the IPB, for the use of their facilities and for help with the western blots. o The NRF, THRIP, Winetech and Stellenbosch University for financial support. o My friends and family, for support throughout the project. o Bernard, for endless patience, encouragement and support. o To my Heavenly Father, to whom I owe everything. viii Stellenbosch University http://scholar.sun.ac.za Contents Declaration ................................................................................................................................. i Abstract ..................................................................................................................................... ii Opsomming .............................................................................................................................. iv Abbreviations ........................................................................................................................... vi Acknowledgments .................................................................................................................. viii Contents .................................................................................................................................... ix List of Figures ........................................................................................................................ xiii List of Tables ........................................................................................................................ xviii 1. Introduction .......................................................................................................................... 1 1.1 Research background and motivation......................................................................... 1 1.2 Project proposal .......................................................................................................... 2 2. Literature review .................................................................................................................. 4 2.1 Introduction ................................................................................................................ 4 2.2 Antimicrobial peptides ............................................................................................... 4 2.2.1 AMPs originating from plants ................................................................................ 5 2.2.2 Mechanisms of action ............................................................................................. 6 2.2.3 Factors influencing AMP activity .......................................................................... 8 2.2.4 Target specificity .................................................................................................... 8 2.2.5 Pathogen resistance against AMPs ......................................................................... 8 2.2.6 Synthetic AMPs .................................................................................................... 10 2.2.7 Application in science .......................................................................................... 11 2.3 Transient gene expression and viral expression vectors ........................................... 11 2.3.1 Agrobacterium-mediated transient expression systems ....................................... 12 2.3.2 Viral transient expression systems ....................................................................... 13 2.4 Quantification of plant pathogens............................................................................. 15 2.5 Grapevine infecting pathogens ................................................................................. 17 ix