PLANT IMMUNE DEFENSES AGAINST THE HUMAN PATHOGENS ESCHERICHIA COLI O157:H7 AND SALMONELLA ENTERICA by DEBANJANA ROY Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON May 2015 Copyright © by Debanjana Roy 2015 All Rights Reserved ii Acknowledgements It would not have been possible to write this doctoral thesis without the help and support of a lot of people around me, to only some of whom it is feasible to give particular mention here. First of all, I would like to express my sincere gratitude to my supervisor and committee chair Dr. Maeli Melotto. Her guidance, wisdom, knowledge and commitment to high standards inspired and challenged me all throughout. One simply could not wish for a better supervisor. Alongside my advisor I would like to thank rest of my committee members, Dr. Julian Hurdle, Dr. Jorge Rodrigues, Dr. Laura Mydlarz, and Dr. Michael Roner for being a valuable part of my committee and their insightful comments and advices. Beside my advisor and my committee members my warmest thanks to my wonderful lab mates Nisita, Shweta, Paula, Cleverson, Lenore, Giselle, and all the undergraduate students for making the basement room a fun workplace. In my personal life I have been blessed to have friends like Aditya, Nisita, Shweta, Saket, Shubham, Dheeral, Ami, Utpal, who made my life away from home really worth enjoyable. Very special thanks to Sarthak, who often had to bear the brunt of my frustration and rages but cheered me up every single time. I can’t thank him enough for being there at good times and specially at bad times and encouraging me every single moment for all good things. This won’t be complete if I miss thanking world’s most awesome best friend Tapojita and Ankur for their priceless presence in my life. Above all, I am truly grateful to my family. Without my Father’s belief in me, Mother’s immense support, Uncle’s guidance, brother’s encouragement this journey would have been really tough and my story might be different. January 20, 2015 iii Abstract PLANT IMMUNE DEFENSES AGAINST THE HUMAN PATHOGENS ESCHERICHIA COLI O157:H7 AND SALMONELLA ENTERICA Debanjana Roy, PhD The University of Texas at Arlington, 2015 Supervising Professor: Maeli Melotto Several pathogenic and non-pathogenic microorganisms reside on plant leaf surfaces while bacteria being the most abundant of them all. Often bacteria not only use the plant leaf surface as their habitat but also penetrate through stomatal pore on leaf epidermis to survive in the plant’s intercellular space also known as the apoplast. To fight against infection caused by bacteria plants rely on their innate immunity system which consists of pathogen-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is considered to be the first line of defense response and it is effective against a broad range of pathogens. Nonetheless, some microbial species are able to overcome and /or avoid plant defenses and colonize the apoplast. In particular, there have been frequent cases of association of human pathogens, such as Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium SL1344, with fresh fruits and vegetables that can cause illness or death among consumers who eat contaminated produce. The economic consequences of produce-associated outbreaks including medical costs, damage control cost, produce recall cost, and production time are substantial. At this point the underlying mechanism of this intriguing phenomenon of human pathogens and plant interaction is not well understood and this is the area of my study. In the first chapter of my dissertation I introduce the newly developed field of human pathogen on plants following innate immunity of plant against human pathogens in the second chapter. iv In the third chapter, the defense pathway(s) playing significant role in diminishing human pathogens populations in plant tissue is explored. Laying emphasis on these puzzling areas of plant pathology, my study will contribute to understanding the initial contamination process, effect of environmental factors on human pathogen infection of plants, and important defense pathway(s) in plants which are paramount in preventing food borne diseases. v Table of Contents Acknowledgements ........................................................................................................... iii Abstract ............................................................................................................................. iv List of Illustrations ............................................................................................................. ix Chapter 1 Introduction to Human pathogen and plant interaction .....................................1 1.1 Overview……………………………………………………………………………….…1 1.1.1 Arabidopsis is an established model plant for studying plant-pathogen interaction .......................................................................................1 1.1.2 Salmonella enterica and Escherichia coli can be used to study plant-human pathogeninteraction………………………………………………………………………….2 1.1.3 Plant surface is the first barrier for bacterial invaders ..........................................5 1.1.4 Plant immune system and perception of human pathogens ................................9 1.1.5 Plant intracellular response to human pathogens infection…………………..…12 1.1.6 Are human pathogens able to induce plant ETI? .............................................. 15 1.1.7 Plant-Salmonella and plant- E. coli interactions depends on genotypic variety of plant and bacteria………………………………………………………………………..19 1.1.8 Future perspective…………………………………………………………………...20 1.1.9 Research goal………………………………………………………………………..21 Chapter 2 Salmonella enterica serovar Typhimurium induces weaker immunity in plants compared to Escherichia coli O157:H7……………………………………………...23 2.1 Abstract………………………………………………………………………………….23 2.2 Introduction……………………………………………………………………………..24 2.3 Materials and Methods………………………………………………………………..27 2.3.1 Plant Material and Growth Conditions……………………………………....27 2.3.2 Bacterial Strains and Growth Conditions…………………………………....28 vi 2.3.3 Stomatal Assay………………………………………………………………...28 2.3.4 Bacterial Inoculations …………………………………………………………29 2.3.5 Gene expression analysis…………………………………………………….30 2.3.6 Statistical Analysis……………………………………………………………..31 2.4 Results…………………………………………………………………………………..31 2.4.1 Human pathogens trigger unique stomatal movements…………………...31 2.4.2 SL1344 cannot actively open stomata………………………………………35 2.4.3 Stomatal immunity diminish human pathogen penetration into leaves…..38 2.4.4 O157:H7 induces higher PR1 gene expression than SL1344…………….40 2.5 Discussion……………………………………………………………………………….41 Chapter 3 Assessing molecular components responsible for defense mechanism of plants against human pathogens inside……………………………………………………..45 3.1 Abstract………………………………………………………………………………….45 3.2 Introduction……………………………………………………………………………...46 3.2.1 Plants general immune system against bacteria………………….………..47 3.2.2 Human enteric pathogens on phyllosphere .............................................. 48 3.2.3 FLS2 receptor is important for bacterial perception before infection……..49 3.2.4 Salicylic acid is crucial for plant defense against bacterial infection……..51 3.2.5 Research goals………………………………………………………………...54 3.3 Methods……………………………………………………………………………..…..54 3.3.1 Stomatal Assay………………………………………………………………..55 3.3.2 Pathogenesis Assay………………………….…………………………….....55 3.3.3 Swarming Assay…………………………………………………………….....55 3.3.4 Chemotaxis Assay…………………………………………………….……....56 3.3.5 Statistical analysis………………………………………………………..…....57 vii 3.4 Results…………………………………………………………………………………..57 3.4.1 FLS2 is crucial for plant immunity against human pathogens……………………………………………………………………………..57 3.4.2 Salicylic acid might act as an important factor in plant immunity against SL1344 and O157:H7……………………………………………………………..…60 3.4.3 NPR1 contributes in controlling bacterial population inside plant apoplast……………………...………………………………………………………..62 3.4.4 SL1344 and O157:H7 show efficient swarming motility at 30ºC and 25ºC rather than 22ºC………….. ………………………………………………………….65 3.4.5 Human pathogenic strain SL1344 and O157:H7 showed chemotactic ability towards plant extract….………………………………………………………66 3.5 Discussion …………………………………………………………………..………….70 3.6 Conclusion………………………………………………………………………………79 References………………………………………………………………………………..….…81 Biographical Information……..………………………………………………………...........102 viii List of Illustrations Figure 1-1 Diagramatic representation of humna pathogen (HP) inteaction and Colonization in plants………………………………………………………………..…………...8 Figure 1-2 Plant cellular defense respose against human pathogens…………………….17 Figure 2-1 Human pathogen trigger unique stomatal ………………………………………32 Figure 2-2 High RH supports penetration as well as survival movement of SL1344 but not O157:H7………………………………………………………………………………………….34 Figure 2-3 SL1344 cannot actively open stomata…………………………………………...35 Figure 2-4 Strong pathogen-triggered immunity overrides the effect of high relative humidity (RH) in opening stomata………………………………………………………….….37 Figure 2-5 Stomatal immunity reduces penetration of human pathogens through the leaf epidermis…………………………………………………………………………………………39 Figure 2-6 O157:H7 triggers higher level of PR1 gene expression than SL1344………..40 Figure3-1 Stomatal response in fls2 plants at 2hr and 4hr post inoculation……………...58 Figure 3-2 Bacterial enumeration in the leaf apoplast at different days (d) after dip- inoculation with O157:H7 or SL1344 of Col-0 and fls2 mutant plants……………………..60 Figure 3-3 Mutant sid2 plants apoplast favors better survival of human pathogenic strains than wild type plants…………………………………………………………….………………62 ix Figure 3-4 Bacterial enumeration in the leaf apoplast at different days (d) after inoculation of Col-0 and npr1 mutants…………………………………………………….………………..64 Figure 3-5 SL1344 (B) and O157:H7 (A) swarm on agar surface .………………….…….66 Figure 3-6 Chemotactic movement of SL1344 and O157:H7 at 15mins post inoculation on swim agar plate………………………………………………………………………………68 Figure 3-7 Chemotaxis of SL1344 and O157:H7………………..…………………………..69 Figure 3-8 Indicated importance of FLS2, NPR1 and SID2 in plant defense against SL1344 infection…………………………………………………………………………………75 Figure 3-9 Indicated importance of FLS2, NPR1 and SID2 in plant defense against O157:H7 infection………………………………………..……………………………………..76 x