UC Riverside UC Riverside Electronic Theses and Dissertations Title Root-Knot Nematode-Triggered Defense Responses in Arabidopsis thaliana During Early Stages of Parasitism Permalink https://escholarship.org/uc/item/40q0w285 Author Alves Teixeira, Marcella Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE Root-Knot Nematode-Triggered Defense Responses in Arabidopsis thaliana During Early Stages of Parasitism A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Plant Pathology by Marcella Alves Teixeira March 2017 Dissertation Committee: Dr. Isgouhi Kaloshian, Chairperson Dr. Phillip Roberts Dr. Hailing Jin Copyright by Marcella Alves Teixeira 2017 The Dissertation of Marcella Alves Teixeira is approved: Committee Chairperson University of California, Riverside Acknowledgments I am grateful for my supervisor Isgouhi Kaloshian for the support during the past years. Her trust extrapolated my specific research area and allowed me to explore themes I never considered I would be working on. I will forever remembeer her guidance during our research conduction and manuscripts writing, as well as all the suggestions she offered during these years. I am also deeply grateful to Barbara Jablonska, Gina Broitman-Maduro, James Peng, Ritu Chaudhary and Ming Wang for always being available during all the (several) times I needed help with my research. I thank my committee members, Dr. Philip Roberts and Hailing Jin for tailoring my research through their questionings and suggestions and specially for being so enthusiastic during our meetings. I am grateful for the Nematology Department members Scott and Matthew for sharing their knowledge, experience and inoculum every time I asked. I specially thank Scott for the contribution with photography and nematology techniques. I am grateful for having met Irma, Natalie, Hailey, Maggie, Jeanette and Jacob, inspiring colleagues who made some tougher moments somewhat easier. You are each very dear to me. I acknowledge my funding agency, CAPES for the financial support during my PhD and my employer for allowing me to pursuit my PhD in the first place. iv I was blessed to find close friends that became family and are always in my heart. I am thankful for having friends listening to all my theories and experimental plans, never showing how boring I was. I specially thank my children, Daniella and Eduardo, for all their patience and support during these years. Most and foremost, I am forever grateful to my parents for showing me life’s endless possibilities and teaching me to always question until I get a satisfactory answer. Finally, I thank my dear husband, Jansen, who participated in every single step of my PhD, giving me suggestions, actually working at the bench with me, supporting me with our family. You made it possible. v To my family for their love and support vi ABSTRACT OF THE DISSERTATION Root-Knot Nematode-Triggered Defense Responses in Arabidopsis thaliana During Early Stages of Parasitism. by Marcella Alves Teixeira Doctor of Philosophy, Graduate Program in Plant Pathology University of California, Riverside, March, 2017 Dr. Isgouhi Kaloshian, Chairperson Root-knot nematodes (Meloidogyne spp., RKN) are plant parasites responsible for great losses in agriculture worldwide. After penetrating host roots they establish feeding sites by modifying a few cells from the pericycle, which become multinucleated and enlarged, known as giant cells. Classical nematology research focuses on the characterization of nematode effectors and plant resistance genes. Therefore, little is known about basal immunity against plant parasitic nematodes. In Chapter One we use Arabidopsis thaliana and M. incognita interaction as a model system to investigate plant perception of parasitic nematodes. We show that RKNs can be perceived by plants irrespective of possible damage caused during migration and this perception relies on canonical immunity signaling partners. In addition, we show that RKN perception by Arabidopsis is mediated by BAK1- dependent and independent pathways. To best characterize the transcriptional responses induced by RKN in Arabidopsis roots we performed RNAseq analysis, which is described in Chapter Two. RNAseq analysis revealed induction of several genes 24h after inoculation with RKN in both wild type plants and bak1-5 mutant vii roots. To identify candidate nematode receptors, RNAseq data was searched for genes that were upregulated upon RKN inoculation and encoded proteins with predicted membrane localization and kinase domains. Screening Arabidopsis with mutations on a few of these mutants allowed identification of a negative regulator of immunity against RKN that has elevated basal levels of defense marker genes and respond to elicitor treatment with stronger and faster ROS burst. Interestingly, this negative regulator belongs to a family of proteins that has not been extensively characterized, the G-type lectin receptor kinases (G-LecRKs). The Chapter Three shows an update on the characterization of Arabidopsis G-LecRKs as well as the first characterization of tomato G-LecRKs by using a methodology well established for characterization of other lectin receptor kinases family. Our analysis shows an expansion of G-LecRKs family in tomato as compared to Arabidopsis and organization of genes in clusters throughout each species genome. Motif enrichment analysis shows conservation of motifs among members of G-LecRKS of Arabidopsis as well as among members of Arabidopsis, tomato and the previously characterized rice G-LecRKs. viii Table of Contents Introduction ………………….……………………………………………1 References…………………………………………………………..24 Tables………………………………………………………………...37 Chapter 1 – Root-knot nematodes induce pattern-triggered immunity in Arabidopsis thaliana roots Abstract……………………………………………………………….40 Introduction…………………………………………………………...41 Materials and Methods………………………………………………46 Results………………………………………………………………...51 References……………………………………………………………66 Figures and Tables…………………………………………………..77 Chapter 2 – G-LecRK-VI.13 is a negative regulator of immunity against root-knot nematodes Abstract……………………………………………………………..86 Introduction………………………………………………………….88 Materials and Methods…………………………………………......93 Results……………………………………………………………….100 References……………………………………………………….…116 Figures and Tables…………………………………………………123 ix Chapter 3 – Characterization of G-type LecRKS in Arabidopsis and tomato Abstract……………………………………………….....................150 Introduction………………………………………..........................151 Materials and Methods……………………………………….........156 Results……………………………………….................................159 References………………………………………...........................176 Figures and Tables………………………………………..............182 Conclusion……………………………………….....................................207 References………………………………………...........................212 Appendix A…………………………………………………………………215 Appendix B…………………………………………………………………216 Appendix C…………………………………………………………………217 Appendix D…………………………………………………………………218 Appendix E…………………………………………………………………225 Appendix F…………………………………………………………………239 x List of figures Figures Page Figure 1.1. BAK1 is involved in nematode resistance. 77 Figure 1.2. Pretreatment with the defense inducer flg22 enhances resistance to RKN. 78 Figure 1.3. Nematode recognition depends on canonical PTI signaling partners. 79 Figure 1.4. Nematodes trigger expression of PTI marker genes. 80 Figure 1.5. Basal defense, glucosinolate and camalexin participate in nematode resistance. 81 Figure 1.6. RKNs are not perceived by the tested MAMP or DAMP receptors. 82 Figure 1.7. RKNs penetrate equally roots of A. thaliana wild type Col-0 and the tested MAMP and DAMP receptor mutants. 83 Figure 2.1. Overview of the constitutive differentially expressed genes and the cellular localization of their encoded proteins in naïve roots of the Arabidopsis mutant bak1-5 as compared to the WT Col-0. 123 Figure 2.2. Overview of differentially expressed genes in RKN-infected Arabidopsis WT Col-0 and bak1-5 mutant classified into functional categories. 124 xi Figure 2.3. Biological process classification of differentially expressed genes (DEG) involved in signaling and response to stimuli using singular enrichment analysis (SEA) from agriGO. 125 Figure 2.4. Biological process classification of differentially expressed genes (DEGs) involved in lipid metabolic process using singular enrichment analysis (SEA) from agriGO. 127 Figure 2.5. Changes in DEG during RKN infection of Arabidopsis roots. 129 Figure 2.6. Overview of differentially expressed genes in RKN-infected Arabidopsis Col-0 and bak1-5 mutant classified into overview and biotic stress functional categories. 130 Figure 2.7. Overview of differentially expressed genes in RKN-infected Arabidopsis Col-0 and bak1-5 mutant classified into different cellular compartments with a statistical significance. 131 Figure 2.8. Biological process classification of differentially expressed genes (DEGs) in WT Col-0 and mutant bak1-5 roots in response to RKN using singular enrichment analysis (SEA) from agriGO. 132 Figure 2.9. Biological process classification of differentially expressed genes (DEGs) in WT Col-0 roots in response to TKN using singular enrichment analysys (SEA) from agriGO. 133 Figure 2.10. Biological process classification of differentially expressed genes (DEGs) in mutant bak1-5 roots in response to RKN using singular