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Regulation of Plant Defense Genes Against Bacterial Pathogens

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Regulation of Plant Defense Genes Against Bacterial Pathogens DEGREE PROJECT IN BIOTECHNOLOGY, SECOND CYCLE, 30 CREDITS STOCKHOLM, SWEDEN 2021 Regulation of Plant Defense Genes Against Bacterial Pathogens JENNY SJÖSTRÖM KTH ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH Abstract Several factors contribute to the demand of new, sustainable solutions to bring food security to the world population. The United Nations predicts, with a confidence of 95%, that the world population will be between 9.9 and 12.7 billion by year 2100. At the same time plant agriculture as seen today is threatened by climate changes e.g., rising temperatures and more extreme weather conditions. In addition, plant bacterial pathogens reduce yields, and cause losses of over $1 billion dollars worldwide every year to the food production chain. The currently most used and effective treatment against bacterial infections on crops is antibiotics, but this is not a viable alternative for most growers due to increasing antibiotic resistance and the high development, production, and distribution cost. During the upcoming years development of new approaches against bacterial infections on crops is of high importance but currently there are information gaps in the field of plant defense regulation systems. This study was aimed to provide knowledge about the transcriptional regulation of genes that are included in plant immune system towards bacteria. To investigate this, conserved regulatory elements of the upstream sequences of two defense-related plant receptor kinases, FLS2 and SERK1, was searched for in different species. FLS2 is a surface receptor that recognizes a peptide derived from the bacterial flagellin protein, and is part of the pathogen-triggered immunity response of most of higher plants. In FLS2 no conserved module was found but a single motif, CAACTTG, is conserved in all chosen species. In SERK1 a strikingly long and conserved module was found. Both the FLS2 motif and two motifs in the SERK1 module are recognition motifs with MYC2, a transcription factor involved in different plant mechanisms and the regulation of phytohormones like abscisic acid and auxin. To address whether MYC2 is involved in the transcriptional regulation of FLS2, an experimental approach is described, involving transactivation by MYC2 of FLS2 reporter constructs, studies using agroinfiltration in Nicotiana benthamiana. An increased knowledge about the different components and mechanisms of plant defense regulation will help the research towards new bactericides, transgenic plants, and other ways to secure food for upcoming generations. KEYWORDS: PLANT DEFENSE, PTI, FLS2, SERK1, MYC2, REGULATION, TRANSCRIPTION FACTOR i Sammanfattning Sjukdom på grödor orsakad av bakterier kan bidra till ekonomiska förluster för bönder samt brist på mat, därför är det viktigt att utveckla nya hållbara sätt att motverka och behandla grödor mot bakterier. Idag är det mest vanliga tillvägagångssättet antibiotika men detta är inte hållbart p.g.a uppkomst av antibiotikaresistens. Antibiotika är inte heller tillgängligt för alla bönder och grödor då kostnaden blir för hög. Världsbefolkningen växer och om 80 år beräknas det bo mellan 9.9 och 12.7 biljoner (95% konfidens) människor på jorden. Växande befolkning samt ökande klimatförändringar, som torka och höjda temperaturer kräver nya bekämpningsmetoder mot bakterier för att tillgodose behoven i framtiden. Det saknas information om hur växter hanterar och reglerar bakteriella hot, därför är målet med denna studie att bidra med kunskap kring den transkriptionella regleringen av växters immunsystem mot bakterier. För att göra detta har promotorsekvenser hos gener som är förknippade med immunförsvaret i växter undersökts efter konserverade regulatoriska element. En känd receptor, FLS2 har en stor roll i växters försvar mot bakterier och känner igen en peptid från bakteriers flagell. Denna studie har undersökt FLS2 och den sammankopplade receptorn SERK1. Hos FLS2 kunde ingen konserverad modul hittas i uppströmsekvensen, däremot observerades ett 8 bp långt motiv, CAACTTG, i alla undersökta arter. I SERK1 hittades en lång konserverad modul bestående av flera motiv. Både FLS2-motifet och två motiv i SERK1-modulen binds av transkriptionsfaktorn MYC2. För att testa hypotesen att MYC2 bidrar till den transkriptionella regleringen av FLS2 och SERK1 har en experimentell plan utformats, där Nicotiana benthamiana transfekteras av Agrobacterium tumefaciens innehållandes promotorsekvenserna samt generna till transkriptionsfaktorn MYC2. En ökad förståelse kring de olika delarna och mekanismerna som medverkar inom växters immunförsvar kan bidra till den fortsatta forskningen mot hållbara lösningar till att säkra mat i framtiden. NYCKELORD: VÄXTFÖRSVAR, PTI, FLS2, SERK1, REGULERING, TRANSKRIPTIONFAKTOR ii Table of contents Abstract .................................................................................................................................................... i Sammanfattning .......................................................................................................................................ii Preface ..................................................................................................................................................... 1 Abbreviations .......................................................................................................................................... 2 Introduction ............................................................................................................................................. 3 Background and theory ........................................................................................................................... 4 The plant defense is a two-tier system ............................................................................................... 4 Antibiotics are not a sustainable control of infections ....................................................................... 4 Phytohormones regulate plant defense in complex networks ........................................................... 6 The bacterial peptide flg22 is perceived by FLS2 and a defense response is activated ...................... 7 FLS2 is a receptor kinase with leucine rich repeats and oligomerize with BAK1 upon activation ...... 8 PAMP-responses ............................................................................................................................... 10 FLS2 regulation .................................................................................................................................. 11 Ethylene has a central role in the positive feedback loop of FLS2 regulation .............................. 11 Transcription factor MYC2 may have an indirect role in FLS2 regulation ..................................... 11 Methodology ..................................................................................................................................... 11 Conserved cis-regulatory modules ................................................................................................ 11 Agrobacterium and Agrobacterium vectors .................................................................................. 12 Transactivation studies in plants with reporter and effector constructs ...................................... 13 Materials and Methods ......................................................................................................................... 15 Data analysis ...................................................................................................................................... 15 Description of experimental verification........................................................................................... 16 Cloning ........................................................................................................................................... 16 Agroinfiltration and transactivation study .................................................................................... 23 Results ................................................................................................................................................... 25 Motif search ...................................................................................................................................... 25 Expression analysis ............................................................................................................................ 28 Discussion .............................................................................................................................................. 30 Conclusion ......................................................................................................................................... 32 Future perspectives ............................................................................................................................... 33 References ............................................................................................................................................. 34 Preface This study was performed as my degree project of 30 credits to complete my Master´s degree in Industrial and environmental Biotechnology and thereby also my engineering degree at the Royal Institute of Technology. The project was performed at the department of Industrial Biotechnology at the CBH-school. I would like to thank my supervisor Ines Ezcurra for her guidance
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