Proteomic Responses of Uninfected Tissues of Pea Plants Infected by Root-Knot Nematode, Fusarium and Downy Mildew Pathogens Al-S
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PROTEOMIC RESPONSES OF UNINFECTED TISSUES OF PEA PLANTS INFECTED BY ROOT-KNOT NEMATODE, FUSARIUM AND DOWNY MILDEW PATHOGENS AL-SADEK MOHAMED SALEM GHAZALA A thesis submitted in partial fulfilment of the requirements of the University of the West of England, Bristol for the degree of Doctor of Philosophy. Department of Applied Sciences, University of the West of England, Bristol. December 2012 This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgment. Al-Sadek Mohamed Salem Ghazala December 2012 Abstract Peas suffer from several diseases, and there is a need for accurate, rapid in-field diagnosis. This study used proteomics to investigate the response of pea plants to infection by the root knot nematode Meloidogyne hapla, the root rot fungus Fusarium solani and the downy mildew oomycete Peronospora viciae, and to identify potential biomarkers for diagnostic kits. A key step was to develop suitable protein extraction methods. For roots, the Amey method (Chuisseu Wandji et al., 2007), was chosen as the best method. The protein content of roots from plants with shoot infections by P. viciae was less than from non-infected plants. Specific proteins that had decreased in abundance were (1->3)-beta-glucanase, alcohol dehydrogenase 1, isoflavone reductase, malate dehydrogenase, mitochondrial ATP synthase subunit alpha, eukaryotic translation inhibition factor, and superoxide dismutase. No proteins increased in abundance in the roots of infected plants. For extraction of proteins from leaves, the Giavalisco method (Giavalisco et al., 2003) was best. The amount of protein in pea leaves decreased by age, and also following root infection by F. solani and M. hapla at six weeks post-inoculation. F. solani caused a decrease in abundance of isocitrate dehydrogenase, glycerate dehydrogenase, carbonic anhydrase, oxygen evolving enhancer protein 2 (OEE2), phosphoglycerate kinase, chloroplastic and one unknown protein. Some leaf proteins increased in abundance, and included heat shock-related proteins (HSP70) and two unknown proteins. Proteins that decreased in leaves following root infection by M. hapla six week post-inoculation were RuBisCo large subunit, fructose bisphosphate aldolase 2, carbonic anhydrase, OEE1, OEE2, OEE3, RuBisCo small subunit and a 28KDa ribonucleoprotein. Some proteins increased in abundance, such as HSP70, fructose bisphosphate aldolase 1 and trypsin. In contrast i to the decrease in protein observed at six weeks post-inoculation, the amount of protein increased in leaves three weeks after inoculation of roots with M. hapla. Root infection by both M. hapla and F. solani caused a reduction in leaf area, and also a reduction in fresh and dry weight of the shoot and root systems. The use of digital imaging and visible and infra-red light to study the changes in leaves was explored in this study. A clear difference was visible between leaves from healthy plants and between those from M. hapla and F. solani infected plants when imaged using a normal digital camera. In contrast, no clear differences were noticed between leaves of healthy, M. hapla and F. solani infected plants when using an infra-red camera with 850 nm wavelength light. This study indicates that specific proteins are altered in abundance in leaves following root infection, and provides the basis for future studies to develop rapid diagnostic tests. ii Declaration I declare that the fulfilling of this thesis entitled proteomic responses of uninfected tissues of pea plants infected by root knot nematode, Fusarium and downy mildew pathogens, is a result of original research work, and that I acknowledge anyone who gave me any help during this work. I confirm that this work has not been submitted for a degree or as part of the requirement for a degree at any university or institution other that the University of the West of England. Al-Sadek Ghazala December 2012 iii Acknowledgments I would like to express my deep gratitude to my supervisor Dr Peter Spencer- Phillips, the Head of the Department of Applied Sciences for his help and support. I am also grateful to my second supervisor Dr Heather Macdonald, for her important support throughout this work. I would also like to thank: Professor Valerie Moroz Williamson, Department of Nematology and University of California Davis for providing Meloidogyne hapla; Dr Jane Thomas from the National Institute of Agriculture Botany (NIAB, Cambridge) for providing Peronospora viciae; CABI for providing Fusarium solani f. sp. pisi. I owe my most sincere gratitude to Dr Neil Willey for his help and valuable advice in data analysis, Dr Amanda Burridge for her help in PDQuest analysis, and Jenna Slinn and Mervyn Lewis for MS analysis. During this work I have collaborated with many colleagues for whom I have a high regard, and I want to extend my warmest thanks to all those who have helped me with my work in the Department of Applied Sciences, especially Dr Josiane Chuisseu and all the people in the BGRI and OJ3 labs, and my colleagues in 1M010. I owe my loving thanks to my wife and children Mohammed, Waad and Dana. They have lost much due to the time I have been away from them during my research. Without their encouragement and understanding it would have been impossible for me to finish this work. I would like to thank the Libyan Government for financial support during my study. My very special gratitude is due to all my family for their loving support. iv Dedication “To the spirits of my departed Father and Brother”. v Table of contents Abstract .............................................................................................................................. i Declaration ....................................................................................................................... iii Acknowledgments ............................................................................................................ iv Dedication ......................................................................................................................... v Table of contents .............................................................................................................. vi List of figures ................................................................................................................. xiii List of Tables.................................................................................................................. xxi 1. Introduction ................................................................................................................... 1 1.1. Background ............................................................................................................ 2 1.2. Pea diseases ............................................................................................................ 4 1.2.1. Pea downy mildew .................................................................................................. 6 1.2.2. Fusarium root rot .................................................................................................. 10 1.2.3. Nematode diseases ................................................................................................ 14 1.3. Diagnosis of plant diseases ................................................................................... 19 1.4. Plant disease diagnostics for field use .................................................................. 22 1.5. Biosensors for diagnosis of pea diseases .............................................................. 24 1.6. Proteomics ............................................................................................................ 27 1.7. Early detection of pea root disease using infrared imaging ................................. 29 vi 1.8. Estimation of leaf area, fresh and dry weight of pea shoot and root system from plants infected by F. solani f. sp. pisi and M. hapla ........................................... 30 1.9. Plant disease control ............................................................................................. 31 1.10. Methods of disease control ................................................................................. 31 1.11. Aims of the project ............................................................................................. 36 2. Materials and Methods ............................................................................................... 38 2.1. Pisum sativum L. (pea) cultivars .......................................................................... 39 2.2. Solanum lycopersicon (tomato) cultivars ............................................................. 39 2.3. Inoculation of pea plants with Peronospora viciae f. sp. pisi .............................. 40 2.3.1. Leaf inoculation .................................................................................................... 40 2.4. Inoculation of pea plants with Fusarium solani f. sp. pisi ................................... 41 2.4.1. Soil inoculation method of Clarkson (1978) ......................................................... 41 2.4.2. Seed inoculation method of Kraft and Kaiser (1993) ........................................... 42 2.5. Inoculation of pea plants with Meloidogyne hapla .............................................. 42 2.6. Methods of protein extraction .............................................................................. 43 2.6.1.