Molecular analysis of the patatin gene familiy of potato (Solanum tuberosum L.)

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Chromosome identification and gene mapping in potato by pachytene, trisomic and half-tetrad analysis = [Chromosoomidentificatie en gen-kartering bij de aardappel door middel van pachyteen- trimosomen- en halftetraden-analyse]

The research described in this thesis deals with chromosome identification and gene mapping. In contrast to results from literature, in this study only three chromosomes (1, 2 and 12) could unambiguously be identified in mitotic cells using conventional staining, and four (1, 2, 3 and 4) in case of Giemsa C-banding. With both methods the chromosomes 1 and 2 could unambiguously be identified and are homologous to the chromosomes 1 and 2 as identified by pachytene analysis. Reliable chromosome identification in potato can be achieved by pachytene analysis.It was found in this study by using non-radioactive in situ hybridization that one basic chromosome of the potato contains rRNA genes. In contrast to a report in the literature about detection of one chromosome with gene(s) for patatin using a cDNA clone, hybridization with a genomic DNA clone used in this study detected more than one basic chromosome carrying genes related to patatin.The bivalents in S. phureja Juz. et Buk. were morphologically very similar to those of S. tuberosum L. ssp. tuberosum Hawkes cv. Gineke as identified by pachytene analysis. An interchange in S. phureja is described and the involvement of the chromosomes 3 and 12 in this interchange could clearly be demonstrated by pachytene analysis and the meiotic behaviour in F 1 hybrids. Trisomic descendants selected in the first selfed generation of the interchange heterozygote were primary trisomic being homozygous for the interchange or tertiary trisomic.Meiotic behaviour in 11 primary trisomics was investigated and female transmission of the extra chromosome determined. Triple synapsis of pachytene chromosomes was often found in the euchromatic parts of the chromosomes. In this study a significant correlation between the relative chromosome or euchromatin length and the coefficient of realization of a trivalent at metaphase 1 was found in the primary trisomics of the potato. In spite of this result no relationship could be established between female transmission and the length of the extra chromosome.By means of half-tetrad analysis the map distance relative to the centromere could be estimated of each of three dominant genes involved in resistance to potato viruses X and Y and to pathotype Ro1 from Globodera rostochiensis , and of the recessive gene for yellow leaf- margin. The gene for yellow margin was localized on chromosome 12 and that for topiary on chromosome 3 by means of trisomic analysis

The Medicago truncatula sucrose transporter family

In plants, long distance transport of sugars from photosynthetic source leaves to sink organs comprises different crucial steps depending on the species and organ types. Sucrose, the main carbohydrate for long distance transport is synthesized in the mesophyll and then loaded into the phloem. After long distance transport through the phloem vessels, sucrose is finally unloaded towards sink organs. Alternatively, sugar can also be transferred to non-plant sinks and plant colonization by heterotrophic organisms increases the sink strength and creates an additional sugar demand for the host plant. These sugar fluxes are coordinated by transport systems. Main sugar transporters in plants comprise sucrose (SUTs) and monosaccharide (MSTs) transporters which constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Although complete SUTs and MSTs gene families have been identified from the reference Dicot Arabidopsis thaliana and Monocot rice (Oriza sativa), sugar transporter families of the leguminous plant Medicago truncatula, which represents a widely used model for studying plant-fungal interactions in arbuscular mycorrhiza (AM), have not yet been investigated. With the recent completion of the M. truncatula genome sequencing as well as the release of transcriptomic databases, monosaccharide and sucrose transporter families of M. truncatula were identified and now comprise 62 MtMSTs and 6 MtSUTs. I focused on the study of the newly identified MtSUTs at a full family scale; phylogenetic analyses showed that the 6 members of the MtSUT family distributed in all three Dicotyledonous SUT clades; they were named upon phylogenetic grouping into particular clades: MtSUT1-1, MtSUT1-2, MtSUT1-3, MtSUT2, MtSUT4-1 and MtSUT4-2. Functional analyses by yeast complementation and expression profiles obtained by quantitative RT-PCR revealed that MtSUT1-1 and MtSUT4-1 are H+/sucrose symporters and represent key members of the MtSUT family. Conservation of transport capacity between orthologous leguminous proteins, expression profiles and subcellular localization compared to previously characterized plant SUTs indicate that MtSUT1-1 is the main protein involved in phloem loading in source leaves whilst MtSUT4-1 mediates vacuolar sucrose export for remobilization of intracellular reserve. The AM symbiosis between plants and fungi from the phylum Glomeromycota is characterized by trophic exchanges between the two partners. The fungus supplies the autotrophic host with nutrients and thereby promotes plant growth. In return, the host plant provides photosynthate (sugars) to the heterotrophic symbiont. Here, sugar fluxes from plant source leaves towards colonized sink roots in the association between the model leguminous plant M. truncatula and the arbuscular mycorrhizal fungus (AMF) Glomus intraradices were investigated. Sugar transporter candidates from both the plant and fungal partners presenting differential expression profiles using available transcriptomic tools were pinpointed. Gene expression profiles of MtSUTs and sugar quantification analyses upon high and low phosphorus nutrient supply and inoculation by the AMF suggest a mycorrhiza-driven stronger sink in AM roots with a finetuning regulation of MtSUT gene expression. Conserved regulation patterns were observed for orthologous SUTs in response to colonization by glomeromycotan fungi. In parallel, a non-targeted strategy consisting in the development of a M. truncatula - G. intraradices expression library suitable for yeast functional complementation and screening of symbiotic marker genes, similar to the approach that led to the identification of the first glomeromycotan hexose transporter (GpMST1), has been developed in this study. Taken together, with the identification, functional characterization and gene expression pattern of sugar transporter families, a more complete picture of sugar fluxes in the AM symbiosis has begun to emerge. This study opens new perspectives by identifying interesting candidate genes involved in sugar partitioning at both the plant and fungal levels and at the symbiotic interface in the widely used AM symbiosis model between M. truncatula and G. intraradices

Sucrose distribution in peach fruit during development and role of three distinct sucrose transporters

The carbohydrate distribution is a complex system governed by several physiological processes, including phloem loading in the source leaf, long-distance translocation in the phloem, phloem unloading in sink organs, post-phloem transport and metabolism of imported sugars in sink cells. The pathway of phloem unloading and post-phloem transport has been analyzed in several sink organs, including fruits, but is not still elucidated in peach fruit. Therefore, aim of this work was to characterize the mechanism of phloem unloading and post-phloem transport in two different types of peach tissues (mesocarp and seed) using a specific fluorescent marker of phloem transport, the carboxyfluorescein diacetate (CFDA), and analyzing the role of different sucrose transporters. The availability of the complete sequence of peach genome, allowed the identification of genes encoding proteins involved in sucrose transport, and several approaches, such as Real-Time PCR, laser capture microdissection and in situ hybridization, have been adopted in order to analyze their transcriptional regulation during different stages of seed and fruit growth. Finally, SUT proteins have been functional characterized in heterologous system, and localized at subcellular level through expression of tag fused protein by transient transformation of Nicotiana benthamiana leaves mediated by agrobacterium tumefaciens.openDottorato di ricerca in Scienze e biotecnologie agrarieembargoed_20151002Zanon, Laur

Functional genomics of photoperiodic bulb initiation in onion (Allium cepa)

Bulb initiation is a process which is photoperiodically driven, drawing parallels with flowering. Photoperiodic flowering is well characterised at molecular and genetic levels and occurs when photoreceptors interact with the circadian clock, regulating the expression of CONSTANS (CO), which itself regulates the expression of floral pathway integrating genes such as FLOWERING LOCUS T (FT), leading to floral initiation. Two genes which regulate CO transcription are FLAVIN-BINDING, KELCH REPEAT, F-BOX (FKF1) and GIGANTEA (GI). The onion genome is very large with a high level of duplication, presenting challenges for any molecular-based study. The aim of this study was to test the hypothesis that genes controlling daylength response are conserved between the model plant Arabidopsis and onion and hence between the different end-processes bulbing and flowering. Bulbing ratios were used to measure the response of onion plants to long day (LD) and short day (SD) conditions and the reversibility of the bulbing process. It was shown that bulbing is reversible, with a delay when plants are transferred from SDs to LDs, suggesting the accumulation of an inhibitor. Diurnal expression patterns of onion genes homologous to Arabidopsis flowering time genes were examined using quantitative RT-PCR. Phylogenetic analyses were conducted to validate the identity of the homologues. Molecular and phylogenetic data suggests that an onion GIGANTEA (GI) homologue was isolated. Peaks of expression of ZT10 in LDs and ZT7 in SDs mirror the expression of Arabidopsis GI. Homologues of FKF1 and the circadian clock gene ZEITLUPE (ZTL) were also characterised. The putative FKF1 homologue showed different expression patterns in varieties exhibiting different daylength responses. These differences may contribute to the different daylength responses. A CO-like gene, which is closely related to Arabidopsis COL4, and three FT-like genes were also characterised. It appears that many of the genes controlling daylength response are conserved in onion

Profiling the expression of grain quality related genes in developing hybrid rice seeds.

Duan Meijuan.Thesis submitted in: August 2003.Thesis (M.Phil.)--Chinese University of Hong Kong, 2004.Includes bibliographical references (leaves 170-194).Abstracts in English and Chinese.Acknowledgements --- p.iAbstract --- p.iiiChinese abstract --- p.viList of Tables --- p.viiiList of Figures --- p.ixList of Abbreviations --- p.xivChapter Chapter 1. --- General Introduction --- p.1Chapter Chapter 2. --- Literature Review --- p.3Chapter 2.1 --- Hybrid rice: Genetics and breeding --- p.3Chapter 2.1.1 --- Classification in rice --- p.5Chapter 2.1.2 --- Heterosis in rice --- p.6Chapter 2.1.2.1 --- Performance of heterosis in rice --- p.6Chapter 2.1.2.2. --- Genetic mechanism of heterosis in rice --- p.7Chapter 2.1.3 --- Utilization of heterosis in rice --- p.9Chapter 2.2 --- Grain quality in rice --- p.11Chapter 2.2.1 --- Composition of rice grain quality --- p.11Chapter 2.2.1.1 --- Appearance quality --- p.11Chapter 2.2.1.2 --- Milling quality --- p.11Chapter 2.2.1.3 --- Nutritional quality --- p.12Chapter 2.2.1.4 --- Cooking and eating quality --- p.17Chapter 2.2.2 --- Genetic and breeding for high nutritional quality rice --- p.17Chapter 2.2.3 --- "Structural, physiological and biochemical changes during rice seed development" --- p.18Chapter 2.3 --- Molecular biological characteristics of rice seed storage protein --- p.20Chapter 2.3.1 --- "Property, classification and structure" --- p.20Chapter 2.3.1.1 --- Property and classification --- p.20Chapter 2.3.1.2 --- Composition structure --- p.20Chapter 2.3.1.2.1 --- Glutelin --- p.21Chapter 2.3.1.2.2 --- Prolamin --- p.22Chapter 2.3.1.2.3 --- Globulin and Albumin --- p.23Chapter 2.3.2 --- "Structure, expression and regulation of genes encoding rice seed storage protein genes" --- p.24Chapter 2.3.2.1 --- Structure --- p.24Chapter 2.3.2.1.1 --- Glutelin gene family --- p.24Chapter 2.3.2.1.2 --- Prolamin gene family --- p.26Chapter 2.3.2.1.3 --- Albumin and globulin gene family --- p.27Chapter 2.3.2.2 --- Expression of storage proteins in rice seed development --- p.28Chapter 2.3.2.3 --- Regulation of expression of seed storage protein genes --- p.29Chapter 2.3.2.3.1 --- Regulation at transcriptional level --- p.29Chapter 2.3.2.3.2 --- Regulation at post-transcriptional level --- p.31Chapter 2.3.2.3.3 --- Regulation at translational level --- p.31Chapter 2.3.3 --- "Synthesis, processing and deposition of rice seed storage proteins" --- p.32Chapter 2.4 --- Molecular characteristics of starch in rice grain --- p.34Chapter 2.4.1 --- Property of rice starch --- p.34Chapter 2.4.2 --- Starch biosynthesis in rice --- p.37Chapter 2.4.3 --- Enzymes involved in starch biosynthesis in rice --- p.39Chapter 2.4.3.1 --- ADP-glucose pyrophosphorylase (AGPase) --- p.39Chapter 2.4.3.2 --- Soluble starch synthase (SSS) --- p.41Chapter 2.4.3.3 --- Granular-bound starch synthase (GBSS) --- p.42Chapter 2.4.3.4 --- Starch branching enzyme (SBE) --- p.44Chapter 2.4.3.5 --- Starch debranching enzyme (SDE) --- p.46Chapter 2.5 --- Aspartate family amino acid biosynthetic pathway in rice --- p.48Chapter 2.5.1 --- Introduction --- p.48Chapter 2.5.2 --- Two key regulatory enzymes involved in lysine synthesis pathway --- p.50Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.50Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.51Chapter 2.5.2.3. --- Regulation of lysine and the other end products of AK pathway --- p.52Chapter 2.6 --- Proteomics in plants --- p.53Chapter 2.7 --- Approaches for grain quality improvement in rice --- p.56Chapter 2.7.1 --- Improvement of nutrition quality --- p.56Chapter 2.7.2 --- Improvement of eating and cooking quality --- p.57Chapter 2.8 --- Objectives of my project --- p.58Chapter Chapter 3. --- Materials and Methods --- p.60Chapter 3.1 --- Materials --- p.60Chapter 3.1.1 --- Chemicals --- p.60Chapter 3.1.2 --- Apparatus and commercial kits --- p.60Chapter 3.1.3 --- Plant materials --- p.61Chapter 3.1.4 --- DNA sequencing --- p.61Chapter 3.1.5 --- Software --- p.61Chapter 3.2 --- Methods --- p.62Chapter 3.2.1 --- Search for protein and DNA sequences of all genes --- p.62Chapter 3.2.1.1 --- Genes encoding rice glutelin family --- p.62Chapter 3.2.1.2 --- Genes encoding rice prolamin family --- p.63Chapter 3.2.1.3 --- Genes encoding rice albumin family --- p.63Chapter 3.2.1.4 --- Genes encoding rice globulin family --- p.64Chapter 3.2.1.5 --- Genes encoding rice starch synthesis enzymes --- p.64Chapter 3.2.2 --- Alignment of homologous DNA sequence between family member genes --- p.65Chapter 3.2.2.1 --- Seeds storage protein gene families of rice seeds --- p.65Chapter 3.2.2.2 --- Rice starch synthase gene families --- p.67Chapter 3.2.3 --- Primer design --- p.69Chapter 3.2.4 --- Collection of developing hybrid rice seeds --- p.71Chapter 3.2.5 --- Total RNA extraction --- p.75Chapter 3.2.6 --- Quantification of total RNA and determination of internal control --- p.75Chapter 3.2.7 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.77Chapter 3.2.8 --- Northern blot analysis --- p.78Chapter 3.2.9 --- DNA sequencing --- p.79Chapter 3.2.10 --- Protein extraction --- p.80Chapter 3.2.10.1 --- Extraction of four kinds of storage proteins --- p.80Chapter 3.2.10.2 --- Extraction of the Wx protein --- p.81Chapter 3.2.11 --- Tricine SDS-PAGE --- p.82Chapter 3.2.12 --- "Determination of crude protein and amylose content in P64S,9311 and F1 hybrid" --- p.83Chapter 3.2.12.1 --- Determination of crude protein --- p.83Chapter 3.2.12.2 --- Determination of amylose content --- p.84Chapter 3.2.13 --- Two-dimension gel electrophoresis --- p.85Chapter 3.2.13.1 --- Clean up of protein sample for 2-D gel --- p.85Chapter 3.2.13.2 --- Quantification of protein samples --- p.86Chapter 3.2.13.3 --- First-dimension IEF (isoelectric focusing) --- p.86Chapter 3.2.13.4 --- IPG strips equilibration --- p.87Chapter 3.2.13.5 --- Second-dimension SDS PAGE --- p.87Chapter 3.2.13.6 --- Silver staining of 2-D gel --- p.88Chapter 3.2.14 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.88Chapter 3.2.14.1 --- Sample destaining --- p.88Chapter 3.2.14.2 --- In-gel digestion with trypsin enzyme --- p.89Chapter 3.2.14.3 --- Desalination of the digested sample with Zip Tip --- p.90Chapter 3.2.14.4 --- Mass spectrometry --- p.90Chapter Chapter 4. --- Results --- p.91Chapter 4.1 --- Quantification of the total RNA from developing seeds at different developingstages --- p.91Chapter 4.2 --- Determination of internal control --- p.92Chapter 4.3 --- DNA sequence analysis --- p.95Chapter 4.4 --- Profiling the expression of genes encoding rice seed storage proteins --- p.97Chapter 4.4.1 --- The glutelin genes --- p.97Chapter 4.4.1.1 --- The Gtl (GluA-2) gene --- p.100Chapter 4.4.1.2 --- The Gt2 (GluA-1) gene --- p.100Chapter 4.4.1.3 --- The Gt3 (GluA-3) gene --- p.101Chapter 4.4.1.4 --- Comparison of the expression profiles of GluA subfamily genes --- p.101Chapter 4.4.1.5 --- The GluB-1 gene --- p.101Chapter 4.4.1.6 --- The GluB-2 gene --- p.102Chapter 4.4.1.7 --- The GluB-4 gene --- p.102Chapter 4.4.1.8 --- Comparing of the expression profiles of GluB subfamily genes --- p.102Chapter 4.4.1.9 --- Profiling the expression of glutilin family genes in developing hybrid rice seeds --- p.103Chapter 4.4.1.10 --- Profiling glutelin gene expression in developing seeds at protein level --- p.103Chapter 4.4.2 --- Profiling the expression of genes encoding prolamin familyin developing hybrid rice seeds --- p.105Chapter 4.4.2.1 --- The 10-kDa prolamin gene --- p.105Chapter 4.4.2.2 --- The RP5 gene --- p.108Chapter 4.4.2.3 --- The RP6 gene --- p.108Chapter 4.4.2.4 --- The Prol 7 gene --- p.109Chapter 4.4.2.5 --- The Prol 14 gene --- p.109Chapter 4.4.2.6 --- The Prol 17 gene --- p.109Chapter 4.4.2.7 --- Expression profiles of prolamin family genes --- p.110Chapter 4.4.2.8 --- Expression profiles of prolamin genes in developing hybrid rice seeds at protein level --- p.111Chapter 4.4.3 --- Profiling the expression of genes encoding globulin familyin developing hybrid rice seed --- p.113Chapter 4.4.3.1 --- The 26-kDa globulin (alpha-globulin) gene --- p.113Chapter 4.4.3.2 --- The globulin 1 gene --- p.113Chapter 4.4.3.3 --- The globulin 2 gene --- p.115Chapter 4.4.3.4 --- The Low molecular weight (LMW) globulin gene --- p.115Chapter 4.4.3.5 --- Profiling the expression of the globulin family genes --- p.115Chapter 4.4.3.6 --- Expression profiles of globulin proteins in developing hybrid rice seeds at protein level --- p.117Chapter 4.4.4 --- Profiling the expression of genes encoding rice albumin familyin developing hybrid rice seeds --- p.118Chapter 4.4.4.1 --- The RA5 gene --- p.118Chapter 4.4.4.2 --- The RA 14 gene --- p.119Chapter 4.4.4.3 --- The RA 17 gene --- p.119Chapter 4.4.4.4 --- Profiling the expression of the albumin family genes --- p.121Chapter 4.4.4.5 --- Albumin gene expression in developing hybrid rice seeds at protein level --- p.121Chapter 4.4.5 --- Comparison of expression pattern of all genes encoding rice seed storage proteins in developing hybrid rice seeds --- p.123Chapter 4.4.6 --- Profiling the total proteins in developing hybrid rice seeds --- p.126Chapter 4.5 --- Profiling the expression of genes encoding rice starch synthasesin developing hybrid rice seeds --- p.127Chapter 4.5.1 --- Rice ADP-glucose pyrophosphorylase (AGPase) genes --- p.127Chapter 4.5.1.1 --- The AGPase large subunit gene --- p.127Chapter 4.5.1.2 --- The AGPase small subunit gene --- p.127Chapter 4.5.2 --- "The Wx (Granule bound starch synthase, GBSS) gene" --- p.129Chapter 4.5.3 --- Genes encoding rice SSS (Soluble starch synthase) family --- p.132Chapter 4.5.3.1 --- The SSS1 gene --- p.132Chapter 4.5.3.2 --- The SSS II-1 gene --- p.132Chapter 4.5.3.3 --- The SSS II-2 gene --- p.132Chapter 4.5.3.4 --- The SSS II-3 gene --- p.135Chapter 4.5.3.5 --- The SSS III-2 gene --- p.135Chapter 4.5.3.6 --- The SSS IV-1 gene --- p.135Chapter 4.5.3.7 --- The SSS IV-2 gene --- p.135Chapter 4.5.3.8 --- Profiling the expression of SSS family genes --- p.136Chapter 4.5.4 --- Genes encoding rice starch branching enzyme (SBE) family --- p.138Chapter 4.5.4.1 --- The SBE-1 gene --- p.138Chapter 4.5.4.2 --- The SBE-3 gene --- p.138Chapter 4.5.4.3 --- The SBE-4 gene --- p.138Chapter 4.5.4.4 --- Profiling the expression of SBE family genes --- p.140Chapter 4.5.5 --- Genes encoding rice starch debranching enzyme (SDE) family --- p.141Chapter 4.5.5.1 --- The isoamylase gene --- p.141Chapter 4.5.5.2 --- The pullulanase gene --- p.141Chapter 4.5.5.3 --- Difference between isoamylose and pullulanase --- p.141Chapter 4.5.6 --- Comparison of the expression patterns of genes encoding the enzymes involved in starch synthesis in developing hybrid rice seeds --- p.143Chapter 4.6 --- Profiling the expression of genes encoding aspartate family amino acid biosynthetic pathway in rice in developing hybrid rice seeds --- p.145Chapter 4.6.1 --- Rice AK (aspartate kinase) gene --- p.145Chapter 4.6.2 --- The DHPS gene --- p.145Chapter 4.7 --- Two-dimension gel electrophoresis and MALDI-ToF seed proteins analysis of rice --- p.147Chapter Chapter 5. --- Discussion --- p.152Chapter 5.1 --- Super hybrid rice as experimental material and its significance --- p.152Chapter 5.2 --- RT-PCR and northern blotting as methods to profile gene expression --- p.153Chapter 5.3 --- Regulation of genes related to nutritional quality in rice --- p.155Chapter 5.3.1 --- Storage protein genes --- p.155Chapter 5.3.2 --- Lysine synthesis enzymes --- p.158Chapter 5.4 --- Regulation of genes related to cooking and eating quality in rice --- p.159Chapter 5.5 --- Heredity of genes expression in F1 hybrid --- p.161Chapter 5.6 --- Application of 2-D gel electrophoresis --- p.162Chapter 5.7 --- Future perspectives --- p.163Chapter Chapter 6. --- Conclusion --- p.164References --- p.17

Identifying signal transduction components acting downstream of reactive oxygen species (ROS) in Arabidopsis thaliana

Traditionally, reactive oxygen species (ROS) have been regarded as toxic by-products of aerobic metabolism. However, in recent years it has become apparent that plants actively produce ROS as signalling molecules. ROS are able to mediate adaptive responses to various environmental stresses as well as processes such as stomatal closure and development. Downstream signalling events that are modulated by ROS include calcium mobilisation, protein phosphorylation and gene expression. This study investigated signalling proteins acting downstream of ROS, in order to understand how ROS are perceived and transduced to elicit such a wide range of responses. To establish a molecular profile provoked by ROS, a microarray experiment of Arabidopsis plants exposed to exogenous H(_2)O(_2) was analysed. Of the 895 differentially expressed transcripts, a substantial proportion had predicted functions in cell rescue and defence, including heat shock, disease resistance and antioxidant genes. Genes encoding candidate H(_2)O(_2) signalling components were identified from this microarray experiment and their H(_2)O(_2) - induced expression was verified by northern RNA-blot analysis. Two transcription factors of the ethylene response factor (ERF) family (AtERFS [At5g47230]) and AtERF6 [At4g17490])and an ankyrin protein kinase (APK [At4g18950]) were selected for further study. Northern blot analysis and comparison with publicly available transcriptome data sets demonstrated that the expression of these three genes was induced by various stress treatments, such as UV-B irradiation, cold and elicitor challenge. To unravel the potential in vivo function of these proteins, loss- and gain-of-function lines were generated and analysed. No abnormal plant phenotypes were observed during development or in response to the stress and hormone treatments tested. A high level of functional redundancy may exist between AtERFS and AtERF6. Microarray analyses were performed on the over-expression lines. Genes that were differentially regulated in APK over-expressor lines gave no indication of its function. However, the microarray analyses revealed that AtERFS and AtERF6 have roles in the plant pathogen defence response, since their over-expression induced defence gene expression. Analysis of cis elements in the promoters of the ERF-differentially regulated genes revealed that both transcription factors displayed GCC box binding activity. However, the GCC box was not over-represented in the promoters of H202-differentially regulated genes, which suggests that this element has a ROS independent regulation

Genome structure and pathogenicity of the fungal wheat pathogen Mycosphaerella graminicola

The phytopathogenic fungus Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn (asexual stage: Zymoseptoria tritici (Desm.) Quaedvlieg & Crous) causes septoria tritici leaf blotch (STB) in wheat and is one of the most important diseases of this crop worldwide. However, STB control, mainly based on the use of resistant cultivars and fungicides, is significantly hampered by the limited understanding of the genetic and biochemical bases of pathogenicity, and mechanisms of infection and resistance in the host. M. graminicola has a very active sexual cycle under field conditions, which is an important driver of STB epidemics. Moreover, it results in high genetic diversity of field populations that causes a major challenge for the development and sustainable management of resistant cultivars and the discovery of new antifungal compounds. Understanding the role of the sexual and asexual life cycles on genome composition of this versatile pathogen and its infection strategy is crucial in order to develop novel control methods. Chapter 1 is an introduction to the biology and pathogenicity of M. graminicola. In addition, it shortly describes the impact of improved and novel technologies on the speed, scope and scale of comparative genomics research. Chapter 2 provides detailed genetic analyses of two M. graminicola mapping populations, using mainly DArT markers, and the analysis of the meiotic transmission of unequal chromosome numbers. Polymorphisms in chromosome length and number were frequently observed in progeny isolates, of which 15–20% lacked one or more chromosomes despite their presence in one or both parents, but these had no apparent effect on sexual and pathogenic fitness. M. graminicola has up to eight so called dispensable chromosomes that can be easily lost - collectively called the dispensome - which is, so far, the highest number of dispensable chromosomes reported in filamentous fungi. They represent small-sized chromosomes and make up 38% of the chromosome complement of this pathogen. Much of the observed genome plasticity is generated during meiosis and could explain the high adaptability of M. graminicola in the field. The generated linkage map was crucial for finishing the M. graminicola genome sequence. Chapter 3 describes the M. graminicola genome sequence with highlights on genome structure and organization including the eight dispensable chromosomes. The genome comprises a core set of 13 chromosomes and a dispensome, consisting of eight chromosomes that are distinct from the core chromosomes in structure, gene and repeat content. The dispensome contains a higher frequency of transposons and the genes have a different codon use. Most of the genes present one the dispensome are also present on the core chromosomes but little synteny is observed neither between the M. graminicola dispensome and the core chromosomes nor with the chromosomes of other related Dothideomycetes. The dispensome likely originates from ancient horizontal transfer(s) from (an) unknown donor(s). Chapter 4 shows a global analysis of proteins secreted by M. graminicola in apoplastic fluids during infection. It focuses mainly on fungal proteins secreted in a compatible interaction. The study showed that many of the annotated secreted proteins have putative functions in fungal pathogenicity, such as cell wall degrading enzymes and proteases, but the function of a substantial number of the identified proteins is unknown. During compatible interactions proteins are primarily secreted during the later stages. However, many pathogenesis-related host proteins, such as PR-2, PR-3 and PR-9, accumulated earlier and at higher concentrations during incompatible interactions, indicating that fungal effectors are recognized by resistant plants and trigger resistant gene-mediated defence responses, though without a visible hypersensitive response. Chapter 5 further details the initial identification and characterization of necrosis-inducing proteins that are produced in culture filtrates (CFs) of M. graminicola. The necrosis-inducing activity of CFs is light dependent and inactivated by proteinase K and heat treatment (100C). This is reminiscent of the necrosis-inducing properties of host selective toxins of other Dothideomycete pathogens such as Stagonospora nodorum and Pyrenophora tritici-repentis. Subsequent purifications of CFs and mass spectrometry identified several candidate proteins with necrosis-inducing activity. Heterologous expression of the two most prominent proteins in Pichia pastoris produced sufficient quantities for infiltration assays in a panel of wheat cultivars that showed differential responses, suggesting specific recognition. Chapter 6 provides a general discussion of the thesis and puts the results obtained in a broader perspective with a focus on the genome structure of M. graminicola and its function. In addition, aspects of the hemi-biotrophic lifestyle, the relevance of secreted proteins for the wheat-M. graminicola pathosystem in relation to gene-for-gene models and the potential implications for resistance breeding strategies are discussed. </p