University of Dundee DOCTOR OF PHILOSOPHY Transcriptomic studies of the early stages of potato infection by Phytophthora infestans Kandel, Kabindra Prasad Award date: 2014 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 23. Sep. 2021 Transcriptomic studies of the early stages of potato infection by Phytophthora infestans Kabindra Prasad Kandel Doctor of Philosophy College of Life Sciences The University of Dundee and Cell and Molecular Sciences The James Hutton Institute June 2014 Declaration The results presented here are of investigations conducted by myself. Work other than my own is clearly identified with references to relevant researchers and/or their publications. I hereby declare that the work presented here is my own and has not been submitted in any form for any degree at this or any other university. Kabindra P. Kandel We certify that Kabindra Prasad Kandel has fulfilled the relevant ordinance and the regulations of the University Court and is qualified to submit this thesis for the degree of Doctor of Philosophy. Dr. Steve Whisson Professor Paul Birch Dr. Petra Boevink College of Life Sciences Cell and Molecular Sciences University of Dundee The James Hutton Institute I Table of contents Declaration……………………………….………………………………….………. I Table of contents…………………………………………………………………..… II List of abbreviations………………………………………………………………..... VI List of figures………………………………………………………………….…….. VIII List of tables………………………………………………………………….……… XI List of annexes in additional compact disc………………………………………….. XII List of appendices in additional compact disc………………………………………. XIII Acknowledgements…………………………………………………………….……. XIV Publications arising from this work…………………………………………………. XV Abstract…………………………………………………………………….………... XVI Chapter 1. Literature review……………………………………………………… 1 1.1 General introduction………………………………………………………. 1 1.2 Morphological characteristics and life cycle of oomycetes……………….. 1 1.3 Phylogenetic position of oomycetes………………………………………. 4 1.4 Diversity of oomycetes……………………………………………………. 5 1.5 Oomycete genomes………………………………………………………... 6 1.6 Genomic comparisons between oomycetes and fungi…………………….. 7 1.7 Background of Phytophthora infestans and its relationship with Solanaceae…………………………………………………...……………. 8 1.8 Population diversity of Phytophthora infestans……………...…………… 11 1.9 Disease impact…………………………………………………………….. 13 1.10 Cell biology of P. infestans infection………………………...……..…….. 15 1.11 Plant - pathogen interactions as an inter-molecular battle………………… 17 1.12 Plant immunity and disease resistance……………………...…………….. 17 1.13 PAMPs and elicitor molecules from Phytophthora……………………….. 23 1.14 Secreted effector proteins…………………………………………………. 25 1.14.1 Apoplastic effectors………………………………………...……………... 25 1.14.2 Cytoplasmic effectors………………………………………...…………… 29 1.15 Transcription factors and regulation of gene expression………………….. 30 1.16 Transcriptome studies of oomycetes………………………...……………. 32 1.17 Research scope of this thesis……………………………...………………. 34 Chapter 2. Materials and methods……………………………...………………… 36 2.1 Maintenance of P. infestans cultures……………………...………………. 3 6 2.2 Plant growth conditions…………………………………...………………. 36 2.3 Passage of P. infestans in potato microplants……………………………... 37 2.4 P. infestans 88069tdT10 inoculation on potato and sampling (infection time course) ………………………………………………………………. 37 2.4.1 Inoculum preparation……………………………………………...………. 37 2.4.2 Preparation of plant leaves for inoculation with P. infestans……...……… 38 II 2.4.3 Preparation of potato tubers for inoculation with P. infestans………...….. 39 2.4.4 Inoculation of leaf and tuber tissues………………………...….…………. 39 2.5 Digital imaging of infected leaves and tubers…………………...………... 40 2.6 Sampling of infected and mock inoculated samples for RNA extraction… 40 2.7 Sampling of P. infestans in vitro life cycle stages for RNA extraction.…... 41 2.7.1 Sampling of sporangia for RNA extraction…………………………….…. 41 2.7.2 Sampling zoospores for RNA extraction………………………….………. 41 2.7.3 Sampling germinating cysts for RNA extraction…………….…………… 42 2.7.4 Sampling vegetative mycelia for RNA extraction………………………… 42 2.8 Live cell imaging- confocal microscopy…………………………………. 42 2.9 Apoplastic fluid isolation………………………………………………….. 43 2.10 P. infestans inoculation of apoplastic fluid (N. benthamiana), pea-broth, and SDW and sampling for RNA isolation……………………………….. 44 2.11 Total RNA isolation………………………………………………………. 44 2.11.1 Leaf samples………………………………………………………………. 44 2.11.2 In vitro P. infestans stages………………………………………………… 45 2.11.3 Tuber samples…………………………………………………..…………. 45 2.12 DNase treatment………………………………………………..…………. 46 2.13 Quality control of the extracted RNA (Bioanalyzer analysis)…………….. 47 2.14 Microarray hybridization and data analysis……………………………….. 47 2.15 General bioinformatics and data analysis…………………………………. 50 2.16 cDNA synthesis…………………………………...………………………. 51 2.17 Microarray validation: quantitative real-time reverse transcribed polymerase chain reaction (QRT-PCR)…………………………………… 51 Chapter 3. Transcriptional change in P. infestans during leaf infection.………. 59 3.1 Introduction…………………………………………………….…………. 59 3.2 Results………………………………………………………….…………. 60 3.2.1 Macroscopic symptoms on potato leaves………………………….……… 61 3.2.2 Confocal microscopic examination of the P. infestans infection cycle….... 62 3.2.3 RNA extraction and quality control (spectrophotometry and bio-analyser), and cDNA synthesis………………………..…………………..….…...…. 65 3.2.4 Microarray analysis of P. infestans transformant 88069tdT10 leaf infection time course in S. tuberosum……………………….……………. 65 3.2.5 Flag values represent the detection of the individual genes at each different time point……………………………………….……………….. 7 0 3.2.6 Volcano plot analysis of transcriptional change during each infection stage….……………………………………………………….…..………. 74 3.2.7 Heatmap analysis of transcriptional change during infection….…………. 79 3.2.8 Co-expressed transcripts during infection by P. infestans…….………….. 96 3.2.8.1 Sporulation marker (Cdc14) co-expressed genes……………….………… 96 3.2.8.2 Biotrophic marker (Hmp1) co-expressed genes……………….………….. 98 3.2.8.3 Necrotrophic marker (NPP1) co-expressed genes…………….………….. 101 3.2.9 In planta infection-specific genes…………………….…………………... 104 3.2.10 Microarray expression validation by QRT-PCR………….………………. 10 5 III 3.2.10.1 Selection and validation of differentially expressed genes from the leaf infection time course (gene tree groups)………………….…………… … 105 3.2.10.2 Expression validation of co-expressed genes…………….……………….. 111 3.2.10.3 Expression validation of selected infection-specific genes…….…………. 114 3.2.11 Gene expression analysis of selected genes detected in the microarray of leaf infection (not differentially expressed)………………………………. 115 3.3 Discussion.……………………………………………….………………... 117 3.3.1 Study of cell biology revealed early biotrophic interaction with the host.... 1 17 3.3.2 A whole-transcriptome microarray approach reveals transcriptional changes during P. infestans infection of potato leaves………………..…... 1 18 3.3.3 Comparative studies of the differentially expressed transcripts revealed biotrophy as an essential stage in host colonization………….…………… 1 18 3.3.4 Genes co-expressed with stage-specific marker genes confirm stages during the infection process………………………………………..…..….. 121 3.3.5 The microarray experiment revealed infection-specific genes….………… 124 Chapter 4. Comparison of leaf and tuber infection by Phytophthora infestans in Solanum tuberosum (cv. Bintje)…………….…………… 126 4.1 Introduction………………………………………….……………………. 126 4.2 Results……………………………………………….……………………. 129 4.2.1 Macroscopic symptoms on potato tubers…………….…………………… 129 4.2.2 Microscopic examination of P. infestans infection in potato tuber slices.... 130 4.2.3 RNA extraction, quality control (spectrophotometry), and cDNA synthesis………………………………………………………..……….…. 134 4.2.4 Gene expression analysis of P. infestans PAMPs, elicitors and necrosis inducing proteins, and cytoplasmic effectors during tuber infection time course……………………………………………………………..….……. 13 4 4.2.5 Gene expression analysis of selected infection-specific genes identified from the leaf infection microarray………………………………..……….. 1 38 4.3 Discussion………………………………………………..……..…………. 139 4.3.1 The cell biology of tuber infection revealed a biotrophic interaction..……. 1 39 4.3.2 Down-regulation of expression for the PAMP (CBEL) and sporulation marker gene (Cdc14) during early initiation of tuber infection…..……….. 14 0 4.3.3 A similar pattern of gene expression in both leaf and tuber infection suggests a biotrophic stage is also important in tuber tissue infection..…... 14 1 4.3.4 Comparative studies of previously identified infection-specific transcripts revealed leaf infection-specific genes……………………..……….……… 14 1 Chapter 5. Transcriptional change in Phytophthora infestans