Research Collection Doctoral Thesis Functionality of the FB_MR5 fire blight resistance gene of Malus x robusta 5 Author(s): Kost, Thomas D. Publication Date: 2016 Permanent Link: https://doi.org/10.3929/ethz-a-010656323 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 23355 FUNCTIONALITY OF THE FB_MR5 FIRE BLIGHT RESISTANCE GENE OF Malus ×robusta 5 A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by THOMAS DOMINIQUE KOST MSc in Biology, University of Zurich (UZH) born on 31.03.1987 citizen of Wädenswil (ZH) accepted on the recommendation of Prof. Dr. Bruce A. McDonald, examiner Dr. Giovanni A. L. Broggini, co-examiner Dr. ir. Henk J. Schouten, co-examiner 2016 The Rose Family The rose is a rose, And was always a rose. But now the theory goes That the apple's a rose, And the pear is, and so's The plum, I suppose. The dear only knows What will next prove a rose. You, of course, are a rose-- But were always a rose. Robert Frost (1874-1963) Abstract Abstract Fire blight is a disease triggered by the bacterium Ewinia amylovora that threatens global apple and pear production. The number of effective approaches to manage fire blight is limited, and useful strategies against this disease consist mainly of eradication of the complete orchard (or heavy pruning), treatment with biocontrol agents, application of copper or aluminium sulphate containing chemicals or the use of antibiotics. A more environmentally friendly approach is the use of fire blight resistant cultivars, but albeit such cultivars exist, the consumers and producers prefer popular fire blight susceptible cultivars that meet their expectations in terms of fruit quality and agronomic properties. The breeding for fire blight resistant cultivars is one of the major apple breeding goals in Switzerland, but crossing always results in novel varieties that must succeed in outcompeting well-established susceptible cultivars. Biotechnology could circumvent this problem, amending susceptible established cultivars with resistance genes without affecting other cultivar properties. Such an approach, where only apple genes controlled by their respective native regulatory sequences are present in the final product, is defined as the cisgenic approach. Recently, the identification of a candidate resistance gene (FB_MR5) responsible for the fire blight resistance of the wild apple accession Malus ×robusta 5 was reported, but no confirmation of the functionality of this gene had previously been performed, whereas transgenic lines carrying FB_MR5 had been generated from the fire blight susceptible cultivar 'Gala Galaxy'. In a first experiment the previously developed transgenic lines containing FB_MR5 were tested for their level of fire blight resistance in the greenhouse (chapter 1). It was shown that integration of FB_MR5 leads to fire blight resistance in the usually highly fire blight susceptible cultivar 'Gala Galaxy'. Moreover the functionality of FB_MR5 was confirmed under a strong promotor and terminator as well as under its native regulatory elements (as present in the original wild apple), indicating that it is a promising resistance gene (a so-called “cisgene”) and could therefore be used to develop a cisgenic apple line. Further inoculations performed by our partners at the Julius-Kühn Institute in Germany revealed that a mutant strain of E. amylovora, that carries a deletion of a sequence coding for an effector protein, bypasses the recognition by FB_MR5. FB_MR5 is the first cloned fire blight resistance gene and the investigated transgenic lines represent an ideal tool to investigate the resistance response triggered by FB_MR5. This first finding formed the fundament for two further aims of this thesis: i) the development of fire blight resistant apple lines that could match the definition of “cisgenic” (chapter 2) and ii) to get insights into the FB_MR5-mediated transcriptional defense response after contact with E. amylovora (chapter 3). Four cisgenic lines were generated by transforming the fire blight susceptible cultivar 'Gala Galaxy' with FB_MR5 controlled by its native regulatory sequences using Agrobacterium tumefaciens. Two different vectors were used in order to obtain genotypes free of any apple foreign genes, whose use as selectable marker is unavoidable to achieve efficient transformation in apple. After transformation, these vectors allowed the removal of the unwanted apple foreign genes. The four cisgenic lines resulted as being significantly more fire blight resistant than the untransformed genotype 'Gala Galaxy', and symptoms were reduced in average from 85.3 % percentage lesion length (in 'Gala Galaxy') to 13.4 % (in the four cisgenic lines) percentage lesion length. One of those lines, line C44.4.146, was further characterized and showed a single gene insertion of the FB_MR5 resistance gene in chromosome 16. In C44.4.146 a similar FB_MR5 transcription level as in conventionally bred accessions i Abstract and no significant deviation from conventional cultivars were found considering several investigated morphological traits (annex to chapter 2). Line C44.4.146 will possibly be investigated in a field trial. A cisgenic line with enhanced durability was attempted to be developed by pyramiding FB_MR5 into the fire blight robust variety 'Ladina', although no cisgenic lines could be regenerated so far. Eventually the lines used to confirm the functionality of FB_MR5 were used to investigate the transcriptional defense response triggered by the FB_MR5 specific recognition of the pathogen. Normally the comparison of fire blight resistant (FB_MR5) genotypes with susceptible cultivars is hampered by the heterozygosity of apple. In this thesis the transcriptome of a transgenic, fire blight resistant 'Gala Galaxy' line (amended with FB_MR5) and a line of the susceptible untransformed 'Gala Galaxy' wild-type were compared. To understand if and how FB_MR5 modulates the 'Gala Galaxy' transcriptome after contact with E. amylovora, an RNASeq-based approach on three biological replicates, 24 hours post fire blight inoculation by scissors was performed and differentially expressed transcripts were identified. In this comparison 206 transcripts were differentially expressed. More transcripts related to photosynthesis and secondary metabolites were observed in the susceptible plants than in the resistant ones. In resistant plants transcripts involved in protein modification, transport, cell-wall synthesis and biotic stress were more abundant. Despite application of three biological replicates and a very similar genetic background, no clear transcriptional response related to known defense response was observed. It is probable that the FB_MR5-dependent reaction is not only of transcriptional nature, and further investigations are needed to understand whether the reaction involves post-translational modification of already existing proteins. ii Zusammenfassung Zusammenfassung Feuerbrand ist eine bakterielle Krankheit welche durch das Bakterium Erwinia amylovora ausgelöst wird und weltweit die Apfel und Birnen Produktion gefährdet. Die Anzahl an effektiven Bekämpfungsstrategien um Feuerbrand zu bewältigen ist limitiert. Hilfreiche Strategien basieren hauptsächlich auf dem Zurückschneiden ganzer Obstanlagen, Behandlungen mit biologischen Kontrollorganismen, oder Chemikalien welche Kupfer oder Aluminium Sulfat enthalten oder Antibiotika. Eine umweltfreundlichere Methode ist die Verwendung von Feuerbrand resistenten Sorten. Obschon solche Sorten existieren, bevorzugen die Konsumenten und Produzenten die bekannten, Feuerbrand anfälligen Sorten, aufgrund ihrer Fruchtqualität und agronomischen Eigenschaften. Die Züchtung feuerbrandrobuster Sorten ist eines der Hauptziele der Apfelzüchtung in der Schweiz, aber bei einer Kreuzung entstehen jedes Mal neue Sorten, welche sich zuerst erfolgreich gegen die bereits etablierten Sorten durchsetzen müssen. Die Biotechnologie könnte dieses Problem umgehen, da sich damit sehr anfällige Sorten mit Resistenzgenen verbessern lassen, ohne weitere Sorteneigenschaften zu beeinflussen. Ein solcher Ansatz, bei dem im Endprodukt nur Apfel-Gene, unter Kontrolle ihrer entsprechenden, natürlichen regulatorischen Sequenzen vorhanden sind, wird als cisgener Ansatz bezeichnet. Kürzlich wurde von einem Kandidat Resistenzgen (FB_MR5) berichtet, welches für die Feuerbrand Resistenz des Wildapfels Malus ×robusta 5 verantwortlich sei. Obgleich bereits transgene Linien aus der Feuerbrand anfälligen Sorte 'Gala Galaxy', welche FB_MR5 tragen, erschaffen wurden, konnte bisher nicht nachgewiesen werden ob dieses Gen auch funktioniert. In einem ersten Experiment wurden die zuvor entwickelten, transgenen, FB_MR5 tragenden Linien im Gewächshaus auf ihre Feuerbrandresistenz getestet (Kapitel 1). Es konnte gezeigt werden, dass das Einführen von FB_MR5, in der sonst hoch Feuerbrand anfälligen Sorte 'Gala Galaxy' zu Feuerbrandresistenz führt. Zudem wurde gezeigt, dass FB_MR5 ein vielversprechendes Resistenzgen (ein sogenanntes „Cisgen“) ist, da es nebst Kontrolle durch einen starken Promotor auch unter Kontrolle durch seine natürlichen regulatorischen Elemente zu Feuerbrandresistenz führt. Folglich könnte dieses Gen verwendet werden um eine cisgene Apfelline zu entwickeln. Bei weiteren Experimenten, welche von unseren Partnern am