xsgw¡ ,o a /kku es a jksxtudrk lca a/kh thu~l ds vfHkYk{k.k gsrq ckbiksysfjl lksjksdhfu;kuk ,o a ckbiksysfjl vksjk;t+h dh ØkWl&lØa kedrk ,o a rqyukRed thuksfeDl Cross infectivity and comparative genomics of Bipolaris sorokiniana and Bipolaris oryzae for characterization of pathogenicity related genes in wheat and rice KARTAR SINGH DIVISION OF PLANT PATHOLOGY ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTE, NEW DELHI – 110 012 (INDIA) 2016 Cross infectivity and comparative genomics of Bipolaris sorokiniana and Bipolaris oryzae for characterization of pathogenicity related genes in wheat and rice A Thesis By KARTAR SINGH Submitted to the Faculty of the Post-Graduate School, Indian Agricultural Research Institute, New Delhi, In partial fulfilment of the requirements For the award of the degree of DOCTOR OF PHILOSOPHY In PLANT PATHOLOGY 2016 Approved by the Advisory Committee: Chairman : Dr. Rashmi Aggarwal ________________________ Dr. S. C. Dubey ________________________ Co-Chairman : Dr. Bishnu M. Bashyal ________________________ Members : DIVISION OF PLANT PATHOLOGY ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTE, NEW DELHI-110 012 (INDIA) Dr. Rashmi Aggarwal E-mail: [email protected] Head and Principal Scientist Phone (office): 011-25840133 CERTIFICATE This is to certify that the thesis entitled “Cross infectivity and comparative genomics of Bipolaris sorokiniana and Bipolaris oryzae for characterization of pathogenicity related genes in wheat and rice” submitted to the Post-Graduate School, ICAR-Indian Agricultural Research Institute, New Delhi, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Pathology, embodies the results of bona fide research work carried out by Mr. Kartar Singh under my guidance and supervision. No part of the thesis has been submitted for any other degree or diploma. All the assistance and help received during the course of the investigation have been duly acknowledged by her. (Rashmi Aggarwal) Place: New Delhi Chairperson Dated: Advisory Committee ACKNOWLEDGEMENTS The work presented in this thesis would not have been possible without my close association with many people who were always there when I needed them the most. I take this opportunity to acknowledge them and extend my sincere gratitude for helping me make this Ph.D. thesis a possibility. My heartfelt gratitude to my chairman, Dr. Rashmi Aggarwal, Head, Division of Plant Pathology, ICAR-IARI, for her unwavering support, teaching, direction and valuable insight during my doctoral research program. She continually and convincingly conveyed a spirit of adventure with regard to research and scholarship. Without her guidance and persistent help this thesis would not have been possible. I take this opportunity to express my sincere thanks to the Co-chairman Dr. S.C. Dubey, Director (Acting) and Head Division of Plant Quarantine, National Bureau of Plant Genetic Resources, New Delhi, members of my Advisory Committee Dr. Bishnu Maya Bashyal, Scientist, Division of Plant Pathology, Dr. R. K. Sharma, Principal Scientist, Division of Genetics, Indian Agricultural Research Institute and Dr. Kishor Gaikwad, Principal Scientist, NRC, Plant Biotechnology, New Delhi for their active involvement in the present investigation and providing necessary support and guidance to carry out the present work successfully. I humbly place on record my respect and gratitude to Dr. R. K. Jain, Dean and Joint Director Education, ICAR-IARI, New Delhi for the help and cooperation received from him during the entire research programme. I am highly thankful to Dr. V. K. Baranwal, Professor, and Dr. Pratibha Sharma, Ex-Professor, Division of Plant Pathology, for their valuable suggestions, encouragement and for providing necessary facilities to meet the targets during the entire period of study. I am highly indebted to Officer Incharge, Dr. Akshya Talukdar, National Phytotron Facility, New Delhi, and Dr. Arun Kumar, NPF, New Delhi for providing the required facilities in the Phytotron to carry out the experiments. I am humbly and whole heartedly thankful to entire faculty members and scientist of Division of Plant Pathology, ICAR-IARI for their immense help, radiant tips and constant encouragement in successfully arranging out of this study. My acknowledgement will never be complete without the special mention of my lab seniors who have taught me the lab standards and have lived by example to make me understand the hard facts of life. I would like to acknowledge, Dr. V. Shanmugham, Dr. Malkhan Singh Gurjar, Dr. Sangeeta Sharma, Dr. Sapna Sharma, Dr. Manjunatha, Deepika, Kirti, Abhimanyu, Devendra, Rahul, Sunil, Veena, Jagdish, Kavi, Rihan, Dr. Valarmati, Abhinav, Sanskriti, Pooja, Shekhar, Gangaji, Manoj and Rajesh for all their support and motivation during my stay in the lab. I avail this opportunity to express my deep sense of reverence and everlasting gratitude to Dr. Praveen Verma, Dr. Sandhya Verma, Dr. Vikash, Chandra, Ratnesh, Kanchan, Shrinivas, Manisha of National Institute of Plant Genome Research, New Delhi for their help during the course of investigation. I wish to accord cordial thanks to all my batchmates Bharat, Damaram, Jeevan, Balram, Abhijeet, Abhishek, Vikash, Madhusudan sir and Seshakiran sir, friends, seniors, juniors and all my colleagues from different disciplines and special words of gratitude to Manoj Choudhary, Laxman, Sanjeev, Rakesh, Prakash, Hanuman, Mukesh and Kailash naga for all the help that they have extended to me throughout. I am grateful to DST- Government of India and ICAR-IARI, New Delhi for providing financial assistance and necessary facilities for carrying out my research work and for successful completion of my study at this institute. Without the support and encouragement of my family members, it would not have been possible to complete the research. Love, prayers, patience, understanding and support at all times of my parents, support of my family members and relatives for which my mere expression of thanks likewise does not suffice. Kartar Singh Date: New Delhi CONTENTS Chapter No. Title Page No. 1 Introduction 1-4 2 Background 5-33 2.1 Major diseases of wheat and Rice 5 2.2.1 The pathogen 7 2.2.2 Symptoms 11 2.2.3 Disease cycle 12 2.2.4 Host range and cross infection 16 2.3 Synergisms between fungal pathogens 18 2.4 Comparative genomics 20 2.5.1 Pathogen effectors 24 2.5.2 Effectors of biotrophic pathogens 25 2.5.3 Function of biotrophic effectors 26 2.5.4 Effectors of necrotrophic pathogens 27 2.5.5 Function of necrotrophic effectors 28 2.6 Pathogenicity related genes 29 2.7 Melanin synthesis 32 3 Materials and Methods 34-58 3.1 Establishing cross infectivity of Bipolaris sorokiniana and 34 Bipolaris oryzae through phenotyping 3.1.1 Collection of pathogen isolates 34 3.1.2 Establishing pure cultures of different isolates of B. 35 sorokiniana and B. oryzae and maintaining mass culture 3.1.3 Confirmation of B. oryzae isolates using ITS-PCR 36 3.1.3.1 Extraction of Genomic DNA 36 3.1.3.2 DNA quantification 36 3.1.3.3 Amplification and sequencing of the ITS regions 36 3.1.3.4 Analysis of ITS sequences of B. oryzae 37 3.1.4 Virulence analysis of B. sorokiniana and B. oryzae on 37 their respective hosts 3.1.4.1 Raising of wheat and rice genotypes for phenotyping in 37 glass house 3.1.4.2 Inoculation 37 3.1.4.3 Disease scoring 38 3.1.5 Cross infectivity study of B. sorokiniana and B. oryzae on 39 rice and wheat respectively 3.1.6 Synergistic effect of pathogens, B. sorokiniana and B. 39 oryzae on rice and wheat 3.1.7 Confirmation of cross infectivity and synergism using 39 PCR based diagnostic marker 3.1.8 Validation of cross infectivity under natural conditions 40 3.2 Comparative analysis of Bipolaris sorokiniana and 42 Bipolaris oryzae genome sequences and characterization of some selected genes having role in pathogenesis 3.2.1 Identification of the secretome 42 3.2.2 Specificity of small secreted proteins for B. oryzae and B. 42 sorokiniana 3.2.3 Functional annotation of predicted small secretory 42 proteins 3.2.4 Analysis of the secretory proteins containing tandem 43 repeats 3.2.5 Cysteine rich secreted proteins 43 3.2.6 Analysis of sequence motifs associated with fungal and 43 oomycete effectors 3.2.7 In planta localization of effectors and NLS prediction 43 3.2.8 Gene families involved in pathogenicity 44 3.3 Expression analysis of putative effector proteins 44 3.3.1 Selection of functionally annotated SSPs 44 3.3.2 Designing of real time – PCR primers 44 3.3.3 Isolation of fungal genomic DNA 44 3.3.4 Characterization of shortlisted SSPs 44 3.3.5 Optimization of PCR condition 45 3.3.6 Cloning of PCR products 45 3.3.6.1 Preparation of competent cells for transformation 45 3.3.6.2 Transformation of competent cells and selection of 46 transformants 3.3.6.3 Selection of transformants 46 3.3.6.4 Blue/white colony screening 46 3.3.6.5 Colony PCR 46 3.3.6.6 Isolation of recombinant plasmid DNA by miniprep spin 47 method 3.3.6.7 Restriction analysis of recombinant plasmid DNA 47 3.3.6.8 Sequencing and sequence based analysis 48 3.3.7 Quantitative PCR for in-vivo/ constitutive gene expression 48 study 3.3.7.1 Total RNA extraction 48 3.3.7.2 RNA quantification 49 3.3.7.3 First strand cDNA synthesis 49 3.3.7.4 Optimization of qPCR conditions 49 3.3.7.5 Quantitative real time PCR 49 3.3.8 Quantitative PCR for in-planta analysis of SSPs in wheat 50 and rice at different time point of inoculation 3.4 Characterization of melanin synthesis genes in B. 50 sorokiniana and B. oryzae 3.4.1 Extraction, purification and quantification of melanin 51 3.4.2 Expression study for melanin synthesis pathway genes 51 3.4.2.1 Development of primers 51 3.4.2.2 Optimization of PCR conditions, cloning and sequencing 51 3.4.2.3 Sequence analysis 52 3.4.2.4 Conserved domain analysis 52 3.4.3 Constitutive gene expression study of BRN1 and SCD1 52 genes with qRT-PCR 3.4.3.1 Designing of qRT-PCR primers 52 3.4.3.2 Optimization of qPCR conditions 53 3.5 Functional analysis of scytalone dehydratase gene (SCD1) 53 in Bipolaris sorokiniana 3.5.1 Fungal strain used in study 53 3.5.2 Primer synthesis 53 3.5.3 Isolation of 5’ and 3’ flanking region of SCD1 by PCR 54 based cloning 3.5.4 Generation of SCD1 knockout construct 55 3.5.5 Restriction digestion of DNA molecules 55 3.5.6 Colony PCR 56 3.5.7 Sensitivity to hygromycin tolerance 56 3.5.8 Fungal transformation 56 3.5.9 Protoplast preparation 56 3.5.10 Transformation 57 3.5.11 Confirmation of knockout of SCD1 57 3.5.12 Melanin extraction from WT and ΔSCD1 strain of B. 58 sorokiniana 3.5.13 Analysis of fungal morphology of WT and ΔSCD1 58 3.5.14 Pathogenicity assay of WT and ΔSCD1 on wheat plants 58 4 Results 59-90 4.1.1 Establishment of Bipolaris oryzae isolates 59 4.1.2 PCR amplification and analysis of ITS region of B. oryzae 59 4.2 Pathogenicity test 60 4.3.1 Cross infectivity of Bipolaris species on wheat and rice 60 4.3.2 Confirmation of cross infectivity using PCR based 62 markers 4.3.3 Testing of cross infectivity in natural conditions 62 4.4 Comparative genome analysis of Bipolaris sorokiniana 62 and Bipolaris oryzae 4.4.1 Secretomics of B. sorokiniana and B. oryzae 62 4.4.2 Analysis of the attributes of the C. sativus and C. 64 miyabeanus secretome 4.4.2.1 Analysis of the secretory proteins with no predicted 69 functions 4.4.2.2 Identification of tandem repeat containing proteins 69 4.4.2.3 Cysteine rich secreted proteins 70 4.4.2.4 Analysis of sequence motifs associated with fungal and 70 oomycete effectors 4.4.2.5 In planta localization of effectors and NLS prediction 72 4.4.2.6 Important gene families involved in pathogenicity 74 4.4.3 Expression analysis of small secreted proteins 76 4.4.3.1 Selection of candidate effectors protein 76 4.4.3.2 Designing of real time PCR primers 76 4.4.3.3 Characterization of shortlisted SSPs 77 4.4.3.4 Constitutive expression pattern of SSPs through qRT-PCR 78 4.4.3.5 Quantitative PCR for in-planta analysis of SSPs in wheat 78 and rice at different time point of inoculation 4.5 Extraction, purification and quantification of melanin in 79 B. oryzae and B. sorokiniana 4.5.1 Characterization of melanin synthesis genes in B. oryzae 81 and B. sorokiniana 4.5.2 Sequence analysis 81 4.5.3 Phylogenetic analysis of BRN1 and SCD1 83 4.5.4 Conserved domain analysis 84 4.5.5 Constitutive gene expression study of BRN1 and SCD1 85 genes with qRT-PCR 4.6 Functional analysis of scytalone dehydratase gene (SCD1) 86 in Bipolaris sorokiniana 4.6.1 PCR based amplification and cloning of 5’ and 3’ flanking 86 region of SCD1 4.6.2 Generation of SCD1 knockout construct 86 4.6.3 Preparation of fungal protoplast 87 4.6.4 Isolation of DNA for protoplast transformation 87 4.6.5 Sensitivity of hygromycin 88 4.6.6 Fungal protoplast transformation 88 4.6.7 Confirmation of knockout of SCD1 88 4.6.8 Melanin extraction from WT and ΔSCD1 strain B. 89 sorokiniana 4.6.9 Analysis of fungal morphology of WT and ΔSCD1 89 4.6.10 Pathogenicity assay of WT and ΔSCD1 on wheat plants 89 5 Discussion 91-107 6 Summary and Conclusions 108-112 7 Abstract in English 113-114 Abstract in Hindi 115-116 8 Bibliography i-xxxv 9 Annexure I i-x 10 Annexure II i-x