University of Groningen Bacillus mycoides: novel tools for studying the mechanisms of its interaction with plants Yi, Yanglei

University of Groningen

Bacillus mycoides: novel tools for studying the mechanisms of its interaction with plants

Yi, Yanglei

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Publication date: 2018

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Yi, Y. (2018). Bacillus mycoides: novel tools for studying the mechanisms of its interaction with plants. University of Groningen.

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te zetten, bijvoorbeeld die het verwijderen en veranderen van genen in hun genoom mogelijk maken. In hoofdstuk 4 hebben we een methode ontwik-keld voor elektroporatie van milieu-afkomstige Bacillus mycoides stammen door factoren te optimaliseren die de elektroporatie efficiëntie beïnvloeden. Deze methode is reproduceerbaar en makkelijk te gebruiken. De hoge effi-ciëntie vergemakkelijkt geavanceerde genetische studies in deze bacterie. Visualisatie van fluorescent eiwit-gelabelde rhizobacteriën in de rhizo-sfeer en de endorhizo-sfeer is een belangrijke vereiste om gedetailleerd inzicht te krijgen in endofytisch gedrag en interacties tussen planten en bacteriën. Echter, moet de werking van fluorescerende eiwitten meestal worden geop-timaliseerd voor de bacteriële gastheer. In hoofdstuk 5 construeerden we een reeks mutanten van het ‘’superfolder’’ groene fluorescerende eiwit sf-GFP en het rood fluorescerende eiwit mKate2, in de bacterie B. mycoides. Na

in vivo selectie van de best presterende fluorescente eiwit varianten door

flu-orescentie-geactiveerde celsortering, werden de sfGFP-variant SPS6 en de mKate2-variant KPS12 met significant verhoogde helderheid geïdentificeerd. Beide geoptimaliseerde varianten bleken zeer geschikt voor in situ lokalisatie. De stam EC18 hechtte zich snel aan plantenwortels en vormde een multicellu-laire matrix op het oppervlak. Tijdens het kolonisatieproces zijn de wortelha-ren en de vertakkende gebieden hotspots voor plant-microbe interacties en vormen ze waarschijnlijk toegangsplaatsen voor B. mycoides om een endofy-tische levensstijl te beginnen. Hoewel zowel de endofyendofy-tische stam B. mycoides EC18 en de bodem stam B. mycoides SB8 werden gedetecteerd in de rhizosfeer, werden de wortels voornamelijk gekoloniseerd door EC18 nadat de plant be-ent was met een 1: 1 mengsel van de EC18- en SB8-cellen (hoofdstuk 3).

Een gebrek aan efficiënte hulpmiddelen voor genetische manipulatie heeft het onderzoek naar de plant-microbe interacties van het geslacht

Bacillus belemmerd. Het CRISPR-Cas9-systeem is een krachtige en

revo-lutionaire bewerkingsmethode voor eukaryotische genomen, maar het gebruik ervan in wilde Bacillus stammen is zeer beperkt. In hoofdstuk 6 hebben we het CRISPR-Cas9-systeem geïmplementeerd in rhizosfeer- geïsoleerde B. subtilis en B. mycoides om vervolgens hun plant-microbe interacties te bestuderen. We toonden aan dat de door B. mycoides gepro-duceerde sideroforen de groei van ijzer-gelimiteerde planten bevorderde. Mutanten deficient in siderofoor productie vertonen een verminderd ver-mogen tot kolonisatie van planten in vergelijking met het wildtype.

Samengevat werden er verschillende strategieën toegepast om de plant- microbe interacties van B. mycoides te onderzoeken. Sommige van de hier bes-chreven hulpmiddelen zijn ook bruikbaar in andere Bacillus soorten, bijvoor-beeld de fluorescente eiwit varianten en het CRISPR-Cas9 systeem. Er zijn echter nog steeds veel onbeantwoorde vragen en meer onderzoek is nodig om de relatie tussen B. mycoides en de gastheerplant volledig te begrijpen.

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ACKNOWLEDGEMENTS

This thesis is the outcome of a multi-year journey with many ups and downs, I am taking this opportunity to express my gratitude to everyone who have supported me throughout my Ph.D. First and foremost, I want to thank my supervisor Prof. Oscar P. Kuipers. Without your assistance and dedicated guidance in every step of the way, this thesis would have never been accomplished. I appreciate all your contributions of time and ideas to make my Ph.D productive and stimulating. The joy and enthusiasm you have for scientific research was contagious and motivational, even during tough times. You give me freedom in research and always have patience on me. Your trust and encouragement is the initiation for pursuing my scientific goal and motivation for writing of this thesis.

I would like to express my sincere gratitude to Prof. Jan Kok and Prof. Jan-Willem Veening for all kind suggestions and discussions during group meetings. My thanks also go to my reading committee, Prof. Arnold J.M. Driessen, Prof. Joana Falcao Salles and Prof. Jos Raaijmakers, for reading the draft and providing valuable comments.

The work on plant-associated Bacillus mycoides was initiated by Prof. Jan Dirk van Elsas, Prof. Theo Elzenga, and Jan Spoelder. After I came to Groningen in 2013, Oscar and I decided to participate in this interesting project. Back then, this project was extremely challenging for me as I had not work on Bacillus before and nobody in our group have related expe-rience. Thanks to our excellent collaborators for sharing the knowledge and expertise in the study of plant physiology and microbial ecology. I have learnt a lot from our B. mycoides meetings.

I have obtained lots of help from so many intelligent colleagues in the group. Siger Holsappel, Anne Hesseling, and Harma Karsens, thank you all for keeping the group running smoothly. Marielle, many thanks for in-troducing me to the lab. You have taught me a lot of lab skills required particularly in the beginning. Good luck with your defense later on! Anne de Jong, I am very appreciated for the kind help that you provided for the genome and transcriptome analysis in chapter 2 and 3. Ruud and Manolo, thank you for making me integrated to the group. Elrike, many thanks for the collaboration on the FP-optimization experiment. My gratefulness also goes to my students Evenlien and Vytenis. Thank you guys for work-ing with me. You guys definitely have a great future ahead.

In the last two years of my Ph.D, I feel less “lonely” at work because we gradually build up a plant-microbe team. Chunxu, it was always an enjoy-able experience for all the discussions with you. Zhibo, thank you for your generous help in chapter 5 and 6. Lu and Claudia, thanks for your company during conference traveling. Good luck with your Ph.D project. I must thank

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the Bacillus lab members for making the lab a pleasant place to work. Mirjam, thanks you for all the efforts in maintaining the lab crystal clean, and also for sharing your idears on various topics. Amanda, you are such a nice per-son who is always willing to help others. Thanks for translating the Dutch summary for me. Auke, thank you for bringing your cheerfulness to the lab and being my paranymph, as well as all the help you offered for my defense. Ard Jan, Barbara, and Luiza, thank you for all the help you offered to the lab. I am also grateful to all the former and present colleagues in Molgen including Jeroen, Rybon, Katrin, Morten, Martijn, Angel, Afzal, Irfan, Taketo, Yoshi, Xin, Tonia, Lieke, Dongdong, Liang, Sjoerd, Ruben, Rieza, Jakob, Jhonatan, Xinghong, Jingqi, Zhenhua, Fangfang, Anne-Marie, Patricia, Eduardo, Anna, I also thank the group members that have moved to Lausanne: Clement, Renske, Dimitra, Arnau, Lance, Stefano, and Xue. Thank you for all your support and help during these years.

Qian and Haojie, you deserve a special thank for many reasons. We came to Groningen at the same time and started our Ph.D project in Molgen on the same day. We share opinions, discuss scientific or personal- related topics, helping each other to overcome problems and difficulties. These memories will never be erased from my memory.

What’s more, I was so lucky to have amazing officemates: Maike, Marielle, Jingjing, and the previous member Sornchai. Thank you guys for creating a pleasant atmosphere. And thank you Maike for reading the chapter 1 and providing useful suggestions.

Getting through my dissertation required more than academic sup-port, and I have many people to appreciate for spending the leisure time with me. My first thankfulness goes to the table tennis club member: Jelle, Robin, Chenxi, and Zhibo, we had such enjoyable time. Secondly, the weekend basketball team member: Yuanze, Qihui, Hang He, Hengxu, Ling Liu, Lei Lv, I enjoyed a lot when we play together. My gratefulness also goes to all my friends outside the lab for your warmhearted company during weekends and holidays. Alberto, we met each other in an English course in the beginning Ph.D. Thank you for inviting me to your house and meet so many nice friends: Ajren, Jalmer, Roccio, and Simone. We had great time together. The fusion dinners we had, the barbecue and the paintball game we played, especially the escape room, will always be one of my best memories in Goningen. Martinus and Anna, what a fabulous time we had in Brazil! Most importantly, we had the “Yanglei-try” contin-ued in Amsterdam, Leiden, Groningen, and I believe it will be contincontin-ued on in the future. Bin Jiang, Lingyang, Boqun, I could not express my entire gratitude to your guys in a few lines here. The unforgettable cakes, and all the drinks and dinners has dramatically strengthened our friendship. Thank you for all the help at work and in daily life.

My gratitude also goes to all my Chinese friends. Youtao Liu, thank you for showing me around when I started my Ph.D, I am sure you will have a successful career after your post-doc in Canada. Jie, Chao, and Bing, we were in the same lab in China and now study in different countries, so nice that we are still able to catch up in Europe. Good luck with your Ph.D study. I also thank all the friends I met in Goningen. Nana Yu, Pu Yang, Miao Wang, Yingruo, Xiangfeng, Yuxiang, Jinfeng, Peiliang & Lijuan, Jing Wu, Yanjun & Peipei, Peng Wang, Min & Xuewen, Huifang, Gang Huang, Lianghui, Wei Qin, your company and presence has become an important part of my life in this city.

Last but not least, nobody has been more important to me in the pur-suit of this project than the members of my family. I would like to thank my dear parents, your love and guidance are with me wherever I go. My dear brother Yanghui, thank you for all the encouragement you provided by phone calls and messages. Most importantly, I would like to thank my loving and supportive wife, Fan, for providing me un-ended inspirations. It was very difficult for us as we were working in different countries, that you have to travel so many times between China and Netherlands. The holidays we had together in those beautiful European cities has been the best time in my life. I look forward to creating our new life together!