Molecular analysis of Botrytis cinerea infection and Trichoderma-induced systemic resistance in Arabidopsis thaliana and Lactuca sativa.

Botrytis cinerea is a necrotrophic fungal pathogen and the causal agent of grey mould on numerous economically important crops. Development of this broad-spectrum disease can be antagonized by the presence of so-called biocontrol organisms (BCOs). In this respect, the genus Trichoderma constitutes a large pool of such BCOs that can have beneficial effects on the plant by enhancing its general fitness and/or by suppressing plant diseases and pests. Research on these BCOs has expanded during the last decades and their importance increased as part of integrated pest and productivity management practices to reduce the use of hazardous chemical pesticides. Early research was mainly focused on Trichoderma spp. that exert their protective effect by direct interaction with the pathogen. Interestingly, some strains were found to be also effective indirectly against pathogens through interactions mediated by the plant, which is referred to as induced systemic resistance (ISR). In the first part of this doctoral thesis, the reported potential of Trichoderma species regarding control of B. cinerea disease in plants is reviewed.In a second part of this study, the molecular basis of the ISR induced in Arabidopsis thaliana by the BCO Trichoderma hamatum T382 against the phytopathogen B. cinerea was unravelled by microarray-based transcriptome analysis both before and after additional pathogen inoculation, termed ISR-prime and ISR-boost , respectively. The observed high numbers of differentially expressed genes allowed us to classify them according to the biological processes in which they are involved based on standard Gene Ontology annotation. By focusing on processes instead of genes, a holistic picture of the mechanisms underlying ISR emerged. In general, a close resemblance is observed between ISR-prime and systemic acquired resistance (SAR), the systemic defence response that is triggered in plants upon pathogen infection leading to increased resistance towards secondary infections. Additionally, we observed that treatment with T. hamatum T382 primes the plant, resulting in an accelerated activation of the defence response against B. cinerea during ISR-boost and a subsequent moderation of the B. cinerea-induced defence response (BIDR). The involvement of various defence-related pathways was confirmed by phenotypic analysis of mutants affected in these pathways, thereby proving the validity of our approach. Combined with additional anthocyanin analysis data these results all point to the involvement of the phenylpropanoid pathway in T. hamatum T382-induced ISR.In the next part, a limited number of Trichoderma strains was used in first instance to select strains with and without ISR-inducing capacity against B. cinerea in A. thaliana. The expression levels of a set of potential markers genes for this ISR, based on the transcriptome analysis of T. hamatum T382-treated A. thaliana, were monitored in leaves of A. thaliana plants root-inoculated with the different Trichoderma strains. Next, transgenic A. thaliana promoter-GUS lines corresponding to the selected ISR marker genes were constructed and their potential as screening tool for identification of ISR-triggering BCOs was validated. We successfully identified both known and unknown ISR-inducing Trichoderma spp., without inclusion of any false positive. In preliminary experiments, these results were extrapolated to (i) another type of fungal BCO, being the basidiomycete Piriformospora indica, for which the biocontrol potential against several plant pathogens, but not B. cinerea, has been demonstrated and (ii) a crop plant, being Lactuca sativa (lettuce).Subsequently, to provide additional background for future understanding of ISR against B. cinerea in lettuce on a genome-wide scale, global expression profiling of this two-player interaction using RNA-sequencing and the newly sequenced lettuce genome was performed. A complex network of genes involved at multiple time points of the lettuce B. cinerea interaction was revealed. Most pronounced are the induction of the phenylpropanoid pathway and terpenoid biosynthesis, whereas photosynthesis was globally down-regulated. Large-scale comparison with data available on the interaction of B. cinerea with the model plant A. thaliana revealed both general and specific plant responses to infection with this pathogen. Surprisingly, expression analysis of a limited number of selected genes could not detect significant systemic transcriptional alterations in lettuce leaves distant from the inoculation site. Additionally, we assessed the response of these lettuce genes to a biotrophic pathogen, Bremia lactucae, revealing that similar pathways are induced during compatible interactions of lettuce with necrotrophic and biotrophic pathogens.Finally, the results obtained during this PhD research are discussed in a more general context of recently reported data on plant interactions with either the pathogen B. cinerea and/or with Trichoderma spp. and other ISR-inducers. This study not only provides extensive insight in the plant s defence response to one of the most important pathogens worldwide, but additionally characterizes the Trichoderma-induced ISR against this pathogen.Table of Contents Dankwoord i Table of Contents iii Summary vii Samenvatting ix List of Abbreviations xi Chapter I Potential of Trichoderma species in controlling disease caused by Botrytis cinerea 1.1 Introduction 2 1.2 Direct interaction 4 1.2.1 Antagonism 4 1.2.1.1 Cell wall degrading enzymes 4 1.2.1.2 Other enzymes 5 1.2.1.3 Antibiosis and secondary metabolites 6 1.2.2 Mycoparasitism 8 1.2.3 Competition 10 1.3 Indirect interaction 11 1.3.1 Formation of the Trichoderma spp. – plant interaction and recognition of the BCO by the plant 12 1.3.2 Plant responses induced by Trichoderma spp. 14 1.3.2.1 Signal transduction pathways 14 1.3.2.2 Phytoalexin production 16 1.3.2.3 Control of ROS-damage 17 1.3.3 Responses induced by B. cinerea in Trichoderma-triggered plants 18 1.4 Objectives and rationale of the doctoral project 19 1.5 References 22 Chapter II Genome-wide characterization of ISR induced in Arabidopsis thaliana by Trichoderma hamatum T382 against Botrytis cinerea infection 2.1 Introduction 34 2.2 Results 36 2.2.1 T. hamatum T382 suppresses disease symptoms in A. thaliana infected with B. cinerea... 36 2.2.2 Gene-specific determination by qRT-PCR of relevant time points for a further ISR transcriptome study 37 2.2.3 Transcriptome analysis of A. thaliana after interaction with T. hamatum T382 and/or B. cinerea using microarrays 38 2.2.4 Biological processes involved in ISR-prime 42 2.2.5 Biological processes involved in ISR-boost as compared to BIDR 42 2.2.6 Validation of microarray results 43 2.2.6.1 Validation of microarray results using qRT-PCR 43 2.2.6.2 Validation of microarray results using GUS staining 45 2.2.7 Confirmation of the involvement of various defence-related pathways in ISR 46 2.2.7.1 Confirmation of the involvement of defence-related pathways in ISR using mutants... 46 2.2.7.2 Confirmation of the involvement of the phenylpropanoid pathway in ISR using anthocyanin measurements 49 2.3 Discussion 51 2.3.1 T. hamatum T382 induces a MAMP-triggered defence reaction in the plant 51 2.3.1.1 The SA-pathway as a first key player in T. hamatum T382-induced ISR 59 2.3.1.2 The JA-pathway as a second important player in T. hamatum T382-induced ISR ......... 60 2.3.1.3 The Et-pathway shows no or minimal involvement in T. hamatum T382-induced ISR 60 2.3.1.4 The phenylpropanoid pathway is involved in T. hamatum T382-induced ISR...61 2.3.2 T. hamatum T382 primes the plant to respond more quickly to pathogen infection.... 62 2.3.3 T. hamatum T382 restrains the defence response after B. cinerea-inoculation 62 2.4 Conclusion 65 2.5 Materials and Methods 66 2.5.1 Biological materials 66 2.5.2 Disease assays 66 2.5.3 Determination of the level of Trichoderma hamatum T382 67 2.5.4 qRT-PCR 67 2.5.5 Microarrays 68 2.5.6 GUS staining 69 2.5.7 Anthocyanin analysis 69 2.6 Supporting Information 70 2.7 Acknowledgments 70 2.8 References 71 Chapter III Development of a screening system for Trichoderma-induced ISR against Botrytis cinerea 3.1 Introduction 82 3.2 Results 84 3.2.1 Selection of Trichoderma spp. with and without capacity to induce ISR against B. cinerea in A. thaliana 84 3.2.2 Selection of potential marker genes for Trichoderma-induced ISR 86 3.2.3 Development of an ISR-screening system 89 3.2.4 Validation of the screening system 92 3.2.5 Piriformospora indica-induced ISR against B. cinerea 95 3.2.6 Potential of Trichoderma spp. in triggering ISR in lettuce 96 3.3 Discussion 99 3.4 Materials and Methods 102 3.4.1 Biological materials 102 3.4.2 In vitro assay with A. thaliana 102 3.4.3 Hydroponics assay with lettuce 102 3.4.4 qRT-PCR and annotation of lettuce genes 103 3.4.5 Isolation of genomic DNA 104 3.4.6 Construction of plasmids 105 3.4.7 Plant transformation and selection of transformants 105 3.5 References 106 Chapter IV RNAseq-based transcriptome analysis of Lactuca sativa infected by the fungal necrotroph Botrytis cinerea 4.1 Introduction 110 4.2 Results 113 4.2.1 Analysis of RNAseq data of lettuce after inoculation with B. cinerea 113 4.2.2 Biological responses of lettuce after inoculation with B. cinerea 116 4.2.3 Comparison of the locally induced response to B. cinerea in lettuce and A. thaliana.. 119 4.2.4 Comparison of the local and systemic response of lettuce to inoculation with B. cinerea.... 121 4.2.5 Comparison between the local response of lettuce to B. cinerea and B. lactucae 123 4.3 Discussion 125 4.3.1 Local responses of lettuce after inoculation with B. cinerea 125 4.3.1.1 Pathogen recognition 125 4.3.1.2 ROS 126 4.3.1.3 Terpenoid biosynthesis... …………………………………………………….. 127 4.3.1.4 Down-regulation of photosynthesis 128 4.3.2 Systemic response of lettuce to inoculation with B. cinerea 129 4.3.3 Comparison of genes locally induced by the necrotroph B. cinerea versus the biotroph B. lactucae 129 4.4 Materials and Methods 131 4.4.1 Biological materials 131 4.4.2 B. cinerea disease assays 131 4.4.3 B. lactucae disease assays 131 4.4.4 Illumina sequencing and data processing 132 4.4.5 Data analysis 132 4.4.6 qRT-PCR 133 4.5 Supporting Information 134 4.6 Acknowledgments 135 4.7 References 136 Chapter V General discussion and future perspectives 5.1 B. cinerea-induced defence response in A. thaliana and lettuce 144 5.1.1 BIDR in A. thaliana: local vs. systemic responses 144 5.1.2 BIDR in lettuce 147 5.2 Trichoderma-induced ISR against B. cinerea in A. thaliana 149 5.2.1 Comparison of Trichoderma-induced ISR with BIDR 149 5.2.2 Role of plant hormones during ISR 149 5.2.3 Priming effect 151 5.2.4 Screening system for effective ISR 152 5.2.3 Concluding remarks 153 Appendix xv List of Publications xixnrpages: 181status: publishe