Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
Song, Yin - \ 2017
University. Promotor(en): Bart Thomma; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - tomaten - receptoren - genen - tabak - aardappelen - katoen - transgene planten

Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
Zeijl, Arjan van - \ 2017
University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming

Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.

A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.

Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.

In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.

Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.

Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.

Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.

In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.

The role of strigolactones and the fungal microbiome in rice during drought adaptation
Andreo Jimenez, Beatriz - \ 2017
University. Promotor(en): Harro Bouwmeester, co-promotor(en): Carolien Ruyter-Spira. - Wageningen : Wageningen University - ISBN 9789463437028 - 205
drought resistance - drought - abiotic injuries - rice - oryza sativa - plant-microbe interactions - nutrient uptake - defence mechanisms - hormones - fungi - genes - droogteresistentie - droogte - abiotische beschadigingen - rijst - plant-microbe interacties - voedingsstoffenopname (planten) - verdedigingsmechanismen - hormonen - schimmels - genen

Rice is the most important food crop in the world, feeding over half the world’s population. However, rice water use efficiency, defined by units of yield produced per unit of water used, is the lowest of all crops. The aim of this thesis was to study the effect of plant hormones and the root microbiome on drought tolerance in rice. The new plant hormone, strigolactone, was shown to be upregulated under drought and to regulate drought tolerance in interaction with the drought-hormone abscisic acid. Using a large collection of rice genotypes grown in the field, we showed that the composition of the root associated fungal microbiome is determined by the rice genotype and can contribute to drought tolerance.

Mechanisms of vegetative propagation in bulbs : a molecular approach
Moreno-Pachón, Natalia - \ 2017
University. Promotor(en): Richard Immink, co-promotor(en): Henk Hilhorst. - Wageningen : Wageningen University - ISBN 9789463437011 - 178
ornamental bulbs - tulipa - lilium - vegetative propagation - flowering date - gene regulation - genes - transcriptomes - dna sequencing - regeneration - shoot apices - bloembollen - vegetatieve vermeerdering - bloeidatum - genregulatie - genen - transcriptomen - dna-sequencing - verjonging - scheuttoppen

Vegetative propagation is very important for the survival of species with long juvenile and adult vegetative phases, as it is the case for bulbous plants. Bulbous plants are ornamental geophytes with a bulb as an underground storage organ. Among flower bulbs, tulip and lily are the two commercially leading plants in The Netherlands. Tulip propagates vegetatively via axillary bud outgrowth, while lily propagates via adventitious bulblet formation. The vegetative propagation rate in tulip is very low due to the limited amount of axillary buds that will grow successfully. Moreover, tulip is very recalcitrant to in vitro regeneration. On the other hand, lily propagates efficiently via adventitious bulblet formation, either naturally from the underground portion of the stem of the apical bud, or artificially from detached bulb scales.

This thesis study aimed to understand how axillary bud outgrowth is controlled in tulip bulbs and how regeneration capacity is established in lily bulb scales. As a first step towards these goals, the state of the art of the molecular control of sexual and vegetative reproduction was reviewed for model species. Moreover, two approaches, “bottom-up” and “top-down”, to transfer the knowledge from model to non-model species were described (Chapter 2). In short, the “bottom-up” approach usually goes from individual genes to systems, assuming conservation of molecular pathways and using sequence homology searches to identify candidate genes. ”Top-down” methodologies go from systems to genes, and are based on large scale transcriptome profiling via e.g. microarrays or RNA sequencing, followed by the identification of associations between phenotypes, genes, and gene expression patterns and levels.

Next (Chapter 3), two sets of high quality transcriptomes, one for tulip and one for lily were generated from a collection of several tissues using the Illumina HiSeq 2000 platform. Several assembly filtering parameters were applied, to highlight the limitations of stringent but routinely used filtering in de novo transcriptome assembly. The final created transcriptomes were made publicly available via a user friendly Transcriptome browser (http://www.bioinformatics.nl/bulbs/db/species/index) and their usefulness was exemplified by a search for all potential transcription factors in lily and tulip, with special focus on the TCP transcription factor family.

One TCP member was of special interest because it has proven to integrate several pathways that control axillary bud outgrowth in a wide range of species. It is called TEOSINTE BRANCHED 1 (TB1) in monocots and BRNACHED 1 (BRC1) in dicots. A Tulipa gesneriana TB1 transcript was identified from the generated transcriptome and subsequently, tulip axillary bud outgrowth was studied through a “bottom-up” approach (Chapter 4). The degree of axillary bud outgrowth in tulip determines the success of their vegetative propagation. However the number of axillary meristems in one bulb is low –six on average– and not all of them seem to have the same growth capacity. The combination of physiological and targeted molecular experiments indicated that the first two inner located buds do not seem to experience dormancy (assessed by weight increase and TgTB1expression) at any point of the growth cycle, while mid-located buds enter dormancy by the end of the growing season. Moreover it was shown that TgTB1 expression in tulip bulbs can be modulated by sucrose, cytokinin and strigolactone, just as it has been reported for other species. However, the limited growth of mid-located buds even when their TgTB1 expression was naturally or artificially downregulated, pointed at other factors, probably physical, inhibiting their growth.

Next, the remarkable regeneration capacity of lily by initiating de novo shoot meristems from excised bulb scales without the addition of exogenous hormones or growth regulators was studied using a “top-down” approach (Chapter 5). An extensive and comprehensive transcriptome set was generated from lily bulb scales in a time-series using two cultivars and two explant types, all differing in regeneration capacity. This set up provided first insight in the key molecular process underlying pro-meristem induction and meristem initiation in lily. We found that wounding activates a very fast regeneration response, probably mediated by APETALA2/ETHYLENE RESPONSIVE FACTORS (AP2/ERF,) such as LoERF115 and WOUND INDUCED DEDIFFERENTIATION 2 (LoWIND2), which in turn might mediate polar auxin re-distribution, cell proliferation and de-differentiation. Moreover, the timing and level of induction of shoot meristem regulators, such as ENHANCER OF SHOOT REGENERATION 2 (LoESR2) and SHOOT MERISTEMLESS (LoSTM) correlated with the regeneration capacity of the scale.

Regardless the regeneration capacity of the different explants e.g. cultivar or position within the scale, regeneration occurs at the proximal-adaxial side of the bulb scale, right on top of the excision line. Thus the possible cellular and physiological factors granting lily bulb scales their competence to regenerate was investigated (Chapter 6). We found that the adaxial parenchyma tissue seems to be more competent than the abaxial tissue, partially because of higher number of secondary veins and larger cell population than the abaxial parenchyma region. It was proposed that upon explant excision, the polar auxin transport is disrupted, creating an auxin maximum at the excision line, which might create a gradient of cell divisions favouring the adaxial parenchyma tissue. The direction of this cell division gradient proved to be negatively affected by the absence of the adaxial epidermis. Moreover, explants without epidermis reduced dramatically their regeneration capacity, and lost the typical proximal-adaxial orientation of regeneration. Thus, a better understanding of the composition and physiology of the epidermis in lily bulb scales is essential to identify the regeneration stimulating signals originating from this tissue layer in Lilium sp.

Finally in Chapter 7, integration of all the results was done and I addressed how this may contributes to the fundamental and applied understanding of vegetative propagation in bulbous plants. Also, some challenges are discussed, for example, the complexity in the architecture of tulip bulbs and how this influences ways for improving its rate of axillary bud outgrowth. The challenge to prove the findings of this thesis through functional analysis is also discussed and the possibility of using transient virus-induced gene silencing is highlighted. Moreover, the potential of lily bulb scales as a model system to study some aspects of de novo regeneration, as well as to study the recalcitrance of in vitro propagation is highlighted, supporting the idea that more “omics” data and biotechnological tools for bulbous plant research are necessary.

Susceptibility genes : an additional source for improved resistance
Sun, Kaile - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Evert Jacobsen; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789463431415 - 174
solanum tuberosum - potatoes - solanum lycopersicum - tomatoes - genes - susceptibility - plant pathogenic fungi - phytophthora infestans - disease resistance - plant breeding - aardappelen - tomaten - genen - vatbaarheid - plantenziekteverwekkende schimmels - ziekteresistentie - plantenveredeling

Potato is affected by several diseases. Although, resistance can be obtained by introgression of major resistance genes from wild species, this has rarely been durable. Hence, other sources of resistance are highly needed. New research with a focus on loss of function mutations has led to the identification of disease susceptibility (S) genes in plants. The research in this thesis was aimed at the identification and characterization of potato S genes involved in the interaction with Phytophthora infestans and Botrytis cinerea. We selected 11 Arabidopsis thaliana S genes and silenced their potato orthologs by RNAi in the potato cultivar Desiree. The silencing of six genes resulted in resistance to P. infestans. Moreover, silencing of StDND1 reduced the infection of B. cinerea. Microscopic analysis showed that spore attachment and/or germination of P. infestans and B. cinerea was hampered on the leaf surface of StDND1-silenced potato plants. On StDMR1- and StDMR6-silenced potato plants, hyphal growth of P. infestans was arrested by the hypersensitive response-like cell death. Our results demonstrate that impairment of plant S genes may open a new way for breeding potatoes with resistance to pathogens like P. infestans and B. cinerea.

Ambient temperature‐directed flowering time regulation : the role of alternative splicing
Verhage, Dina Sara Leonie - \ 2017
University. Promotor(en): Gerco Angenent, co-promotor(en): Richard Immink; Guusje Bonnema. - Wageningen : Wageningen University - ISBN 9789462579705 - 161
plants - flowering date - flowering - temperature - alternative splicing - molecular biology - genes - planten - bloeidatum - bloei - temperatuur - alternatieve splitsing - moleculaire biologie - genen

As a consequence of a sessile lifestyle, plants are constantly facing a fluctuating environment. In order to both profit maximally and protect themselves from these environmental cues, plants evolved ways to sense and respond to signals.

Ambient temperature is one of the cues for which plants have acquired a strategy to enhance their chance of survival and reproduction. Small changes in ambient temperature can have major effects on plant architecture and development, such as the transition from the vegetative to the reproductive flowering phase. The moment of flowering is an important event in the life cycle of a plant, since reproductive success depends on it.

In Chapter 1, I introduced the concept of alternative splicing, a molecular mechanism with a pivotal role in ambient temperature regulation of flowering time. In the model plant Arabidopsis thaliana, approximately 60% of the intron-containing genes show alternative splicing. Gene splicing varies depending on developmental stage and tissue type, but also environmental changes trigger differential splicing. Splicing is conducted by a large cellular machinery called the spliceosome, which recognizes intron-defining sequences and other cis-regulatory elements acting as splicing enhancers or silencers. Moreover, factors like chromatin structure, histone marks, RNA polymerase II (polII) elongation speed and the secondary structure of the pre-mRNA all play a role in the splicing outcome. Due to alternative splicing, a single gene can yield various transcripts. However, this does not cause an equal expansion of the proteome. Part of the transcripts are targeted for nonsense-mediated decay, or will be translated into unstable proteins. This is a way of regulating gene expression at the post-transcriptional or –translational level. Other transcripts will be translated into functional proteins that may be structurally and functionally different. Hence, alternative splicing creates additional complexity in the transcriptome, providing plants with molecular tools to respond to their environment, including the translation of ambient temperature alterations into a flowering time response.
In Chapter 2, we reviewed the current knowledge on molecular mechanisms that control the ambient-temperature directed flowering time pathway in the plant model species Arabidopsis thaliana. Several different mechanisms have been proposed, like alternative splicing of FLOWERING LOCUS M (FLM) (described in Chapter 4) and protein degradation of SHORT VEGETATIVE PHASE (SVP), two mechanisms that probably work in a cooperative manner to release floral repression at higher ambient temperatures. Another mechanism that is involved at high ambient temperature is the replacement of the canonical histone H2A by the variant H2A.Z. As a consequence of this replacement, chromatin becomes less tightly wrapped around the nucleosomes, which allows transcription of flowering time activators, such as PHYTOCHROME INTERACTING FACTOR 4 (PIF4). Lastly, we discuss microRNAs (miRNA) that can either repress or activate flowering (miR156 and miR172, respectively). These miRNAs have been proposed to be regulated by low and high ambient temperature. However, due to the lack of mutant analyses, more research is necessary to show the true involvement of these factors. Altogether, there are several mechanisms acting partly in cooperation to regulate thermosensitive floral timing.
In Chapter 3, we analysed ambient temperature-directed alternative splicing events that occur after a temperature shift by RNAseq. We performed the experiment in two different accessions of A. thaliana, and in one variant of B. oleracea (cauliflower). We showed that flowering time genes are overrepresented amongst the ambient temperature induced alternatively spliced genes, but also genes encoding components of the splicing machinery itself, indicating that alternative splicing is one of the potential mechanisms by which plants are able to sense temperature and adapt floral timing. Analysis of a mutant for one of these alternatively-spliced splicing related factors, ATU2AF65A, showed a temperature-dependent flowering time phenotype, confirming its proposed role in the flowering time response upon temperature fluctuations. Based on these findings, we proposed a two-step model in which splicing related genes are targeted for differential splicing upon ambient temperature fluctuations, which results in changes in the composition of the spliceosome, causing differential splicing of downstream genes that affect the development and architecture of the plant, including flowering time.
In Chapter 4, we investigated the molecular mode-of-action of FLM, one of the differentially spliced flowering time regulating genes that we identified in Chapter 3. We showed that in A. thaliana Col-0, the main splice forms of FLM are FLMβ and FLMδ. FLMβ forms an obligate heterodimer with SVP, and this complex represses floral integrators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) by binding to the regulatory regions of these genes. FLMδ also dimerizes with SVP, but this complex is not able to bind to DNA. When temperature rises, more FLMδ is produced at the cost of FLMβ. Hence, less repressive complexes can be formed. However, the fact that FLMδ is still able to binds SVP makes it function as a dominant negative form, titrating out SVP and preventing repressive SVP/FLMβ-complex formation.
Chapter 5 is a short comment written to clarify the concept of thermoplasticity in flowering time control. Occasionally, this concept is confused with adaptation to different ambient temperature environments on the long term. Thermoplasticity is the ability to adapt flowering time to fluctuations in ambient temperature within one life cycle. Furthermore, some genes have been marked as players in the ambient temperature response, whereas these appear to be general flowering repressors or activators, affecting flowering time in a similar manner at low and high ambient temperature. In order to interpret novel findings on thermosensitive flowering time control, it is essential to distinguish between these various concepts.
In chapter 6, we unveiled the first indications that differential splicing of FLM can be caused by differences in polymerase II elongation rate. We mimicked a situation in which FLM is transcribed at a higher rate, by expressing the genomic FLM gene under a strong artificial promoter. Preliminary results showed that plants harbouring this construct have altered flowering time and temperature-responsiveness, which can be explained by the altered FLMβ/FLMδ ratio that we observed.
In chapter 7, we assessed the functional conservation between FLM and closely related genes at the intraspecific level in A. thaliana. FLM (also called MAF1) is a member of the FLC-clade, that consists of FLC, FLM and MAF2-5. FLC is widely known for its function in the vernalization pathway, whereas MAF2 has been shown to regulate flowering time through alternative splicing in a way very similar to FLM. For the other MAF genes, not much is known. We showed that all of these genes produce splicing isoforms that function in a more or less similar way to FLM and MAF2. Despite the high functional conservation at the intraspecific level, FLM and MAF orthologues are not widely present. Through synteny analysis, we showed that FLM and MAF2 are very recent genes, which are only present in a small group of Brassicaceae species. MAF3-5 originated less recently, but are not present outside the Brassicaceae. For FLC, it was previously shown that it originated from an ancestor of the seed plants, and in many plant species belonging to other families, presence of more than one FLC-like gene has been reported. This raises the question what the function of these genes is. In tomato, we showed that the FLC-like gene MBP8 becomes differentially spliced upon temperature changes, suggesting a function in the ambient temperature pathway. A binding assay showed high similarities of the different MBP8 isoforms to FLM and MAF isoforms, but suggests a slightly different functionality, since all three isoforms showed binding to the DNA. Further research is necessary to confirm the role of MBP8 in thermosensitive flowering time control, and elucidate the functionality of the different splice forms.
In Chapter 8, I discussed the finding of this thesis in a broader perspective, and make suggestions for future research. Over the last few years, several mechanisms that act in the temperature-directed floral pathway have been revealed. In this thesis, we showed that alternative splicing plays an important role, and we demonstrated how temperature may affect the splicing outcome directly through the effect of temperature on transcription elongation rate. It is becoming clear that most likely a single thermosensor does not exist in plants, and a model in which temperature is sensed through thermodynamic properties of DNA, RNA and proteins, is gaining support. Future research is assigned to the exiting task to elucidate the exact mechanisms by which temperature-sensing is achieved in different plant species and to determine how conserved the currently identified molecular mechanisms are.

Susceptibility pays off: insights into the mlo-based powdery mildew resistance
Appiano, Michela - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Anne-Marie Wolters. - Wageningen : Wageningen University - ISBN 9789462579484 - 265
solanum lycopersicum - tomatoes - disease resistance - susceptibility - oidium neolycopersici - genes - gene expression - genomics - molecular breeding - plant breeding - tomaten - ziekteresistentie - vatbaarheid - genen - genexpressie - genomica - moleculaire veredeling - plantenveredeling

Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance.

A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium.

After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs.

In Chapter 2, we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role.

Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function.

In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species.

In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein.

In Chapter 5, we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution.

In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber.

In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T à A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2.

Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.

An evolutionary and functional genomics study of Noccaea caerulescens, a heavy metal hyperaccumulating plant species
Wang, Y. - \ 2016
University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462578562 - 190 p.
brassicaceae - genomics - hyperaccumulator plants - heavy metals - genes - genetic variation - genomica - hyperaccumulerende planten - zware metalen - genen - genetische variatie

Noccaea caerulescens is the only known Zn/Cd/Ni hyperaccumulator. The Ganges accession (2n = 14) has an, yet unpublished, genome size of ~319 Mb, with 29,712 predicted genes representing 15,874 gene families. This species is distributed mainly in Europe. Three ecotypes can be distinguished: two metallicolous ecotypes, resident to serpentine soil (Ni enriched) and calamine soil (Zn/Cd enriched), and a non-metallicolous ecotype, growing on regular, non-metalliferous soils. The physiological differences that underlie variation in heavy metal accumulation and tolerance are well-understood, and the molecular basis of hyperaccumulation and tolerance has been explored by transcript profiling in the presence of metals and by comparative transcriptome analysis using N. caerulescens and non-hyperaccumulators such as Arabidopsis thaliana. The genetic variation which emerged during the evolution of metal hyperaccumulation has not yet been investigated. The work described in this thesis considers the identification of genetic variation under selection for Zn/Cd hyperaccumulation and tolerance by next generation resequencing of the wild metallicolous (calamine) and non-metallicolous populations and the generation of a mutant N. caerulescens library for functional analysis. The regulation of flowering time was also investigated, using early flowering mutants selected from the mutant library.

Mechanistic dissection of plant embryo initiation
Radoeva, T.M. - \ 2016
University. Promotor(en): Dolf Weijers, co-promotor(en): Sacco de Vries. - Wageningen : Wageningen University - ISBN 9789462578135 - 183 p.
embryogenesis - embryos - plants - auxins - genes - genomics - arabidopsis - cell suspensions - in vivo experimentation - zygotes - monozygotic twins - embryogenese - embryo's - planten - auxinen - genen - genomica - celsuspensies - in vivo experimenten - zygoten - monozygote tweelingen

Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in many plant species. These non-zygotic embryos go through analogous developmental phases and are morphologically similar to the zygotic embryo. Despite its fundamental importance and enormous application potential, the mechanisms that alter cell fate from non-embryonic to embryonic are elusive. In the past decades, a variety of different model systems have been used to identify regulators of embryo induction, but it is unclear if these act in a common network. We recently found that inhibition of auxin response in the extra-embryonic suspensor cells cell-autonomously and predictably triggers a switch towards embryo identity. In my thesis I have used the suspensor-derived embryogenesis as a uniform model system to study the crucial first reprogramming step of embryo initiation process.

Through genome-wide transcriptional profiling upon local (suspensor-specific) auxin response inhibition (Chapter 2) and through testing the ability of fifteen known embryogenesis inducers to promote embryo formation in suspensor cells (Chapter 3), we suggest that suspensor to embryo transformation requires a defined set of genetic regulators. The results obtained in my thesis provide essential tools and basis for further research and are a step forward to understanding the first step of embryo initiation process and to unravel the mystery of totipotency in plants.

Role of MLO genes in susceptibility to powdery mildew in apple and grapevine
Pessina, Stefano - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Henk Schouten; M. Malnoy; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789462576209 - 222 p.
malus domestica - apples - vitis vinifera - grapes - plant pathogenic fungi - podosphaera leucotricha - erysiphe necator - disease resistance - susceptibility - genes - gene expression - gene knock-out - resistance breeding - appels - druiven - plantenziekteverwekkende schimmels - ziekteresistentie - vatbaarheid - genen - genexpressie - inactivering van genen - resistentieveredeling

Powdery mildew (PM) is a major fungal disease that threatens thousands of plant species. PM is caused by Podosphaera leucotricha in apple and Erysiphe necator in grapevine. Powdery mildew is controlled by frequent applications of fungicides, having negative effects on the environment, and leading to additional costs for growers. To reduce the amount of chemicals required to control this pathogen, the development of resistant apple and grapevine varieties should become a priority.

PM pathogenesis is associated with up-regulation of specific MLO genes during early stages of infection, causing down-regulation of plant defense pathways. These up-regulated genes are responsible for PM susceptibility (S-genes) and their knock-out causes durable and broad-spectrum resistance. All MLO S-genes of dicots belong to the phylogenetic clade V. In grapevine, four genes belong to clade V. VvMLO7, 11 and 13 are up-regulated during PM infection, while VvMLO6 is not.

Chapter 2 reports the genome-wide characterization and sequence analysis of the MLO gene family in apple, peach and woodland strawberry, and the isolation of apricot MLO homologs. Twenty-one homologues were found in apple, 19 in peach and 17 in woodland strawberry. Evolutionary relationships between MLO homologs were studied and syntenic blocks constructed. Candidate genes for causing PM susceptibility were inferred by phylogenetic relationships with functionally characterized MLO genes and, in apple, by monitoring their expression following inoculation with the PM causal pathogen P. leucotricha. In apple, clade V genes MdMLO11 and 19 were up-regulated, whereas the two other members of clade V, MdMLO5 and 7, were not up-regulated. The clade VII gene MdMLO18 was also up-regulated upon P. leucotricha infection.

Chapter 3 reports the knock-down, through RNA interference, of MdMLO11 and 19, as well as complementation of the mutant phenotype by expression of the MdMLO18 gene in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 resulted in a reduction of PM disease severity up to 75%, whereas the knock-down of MdMLO11, alone or combined with MdMLO19, did not cause any reduction or additional reduction of susceptibility compared to MdMLO19 alone. Complementation by MdMLO18 did not restore susceptibility. Cell wall appositions (papillae), a response to PM infection, were found in both susceptible plants and PM resistant plants where MdMLO19 was knocked-down, but were larger in resistant lines. The expression analysis of 17 genes related to plant defense, and quantification of phenolic metabolites in resistant lines revealed line-specific changes compared to the control.

Chapter 4 evaluates the presence of non-functional alleles of the MdMLO19 S-gene in apple germplasm. The screening of the re-sequencing data of 63 apple genotypes led to the identification of 627 SNP in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18 and MdMLO19). Insertion T-1201 in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino-acids and the calmodulin-binding domain. The presence of the insertion was evaluated in a collection of 159 apple genotypes: it was homozygous in 53 genotypes, 45 of which were resistant or very resistant to PM, four partially susceptible and four not assessed. These results strongly suggest that this insertion is causative for the observed PM resistance. The absence of a clear fitness cost associated to the loss-of-function of MdMLO19, might have contributed to the high frequency of the mutation in breeding germplasm and cultivars. Among the genotypes containing the homozygous insertion, ‘McIntosh’ and ‘Fuji’ are commonly used in apple breeding. After barley and tomato, apple is the third species with a reported natural non-functional mlo allele in its germplasm, with the important difference that the allele is present in a relatively large number of apple genotypes, most of which not related to each other.

Chapter 5 reports the knock-down through RNA interference of four grapevine MLO genes, all members of clade V. VvMLO7, 11 and 13 are up-regulated in early stages of infection, whereas VvMLO6 is not responsive to the pathogen. Knock-down of VvMLO6, 11 and 13, alone or combined, did not decrease PM severity, whereas the knock-down of VvMLO7, alone or in combination with VvMLO6 and VvMLO11, caused a reduction of severity of 77%. Cell wall appositions (papillae), a response to PM attack, were present in both resistant and susceptible lines, but were larger in resistant lines. Thirteen genes involved in defense were less up-regulated in resistant plants, highlighting the reduction of PM disease severity.

In Chapter 6 we discuss the results presented in this thesis. The pivotal role of MLO genes in the interaction of PM pathogens with apple and grapevine is described and further experiments aimed at addressing open questions are proposed. The results described in this thesis open interesting avenues in MLO genes research, particularly the finding that a natural mlo mutation in apple appeared to be more common than expected. This mutation is directly applicable in marker assisted breeding for durable PM resistance in apple.

The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum
Griffiths, S.A. - \ 2015
University. Promotor(en): Pierre de Wit; Pedro Crous, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789462575813 - 167
passalora fulva - secundaire metabolieten - metabolomen - genen - genomica - biologische activiteit - biosynthese - natuurlijke producten - secondary metabolites - metabolomes - genes - genomics - biological activity - biosynthesis - natural products

Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised
by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other
organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal
growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived
from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats.
Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous
fungi contain very large numbers of genes related to SM production. Yet these genes are typically
silent under laboratory conditions. There are now many tools and strategies available to activate
or clone silent SM genes. This thesis details our efforts to apply various methods to define and
then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato
containing many silent SM genes and gene clusters.

In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi
are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM
biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and
increase the chemical diversity of fungi are detailed. These include the deletion or over-expression
of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation
of growth conditions. Heterologous expression of silent SM genes in a production host is also
discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced
as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis
showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10
polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid
and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the
majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole
detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to
apply the aforementioned strategies to induce these silent genes and obtain new compounds. The
importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential
medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential
source of novel biosynthetic enzymes.

In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was
analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic
genes linked to SM production, such as those encoding decorating enzymes and regulators. Products
of these SM genes or gene clusters were speculated, based on their similarity to those characterized
in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other
fungal species. Remarkably, two predicted functional gene clusters were linked to the production
of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive
oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum
under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR
and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous
findings, the majority of SM genes remained silent and only cladofulvin was detected. During
growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were
significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We
confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into
their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our
studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent
another mechanism associated with a biotrophic lifestyle.

In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim
of discovering new compounds. Many Ascomycete-specific global regulators of SM production
and morphological development in other fungi were identified in C. fulvum. We investigated
three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes
encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis
was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of
cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild
type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was
affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly
repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of
VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was
not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin.
This suggests that cladofulvin production is not required for asexual reproduction. The main
finding of this chapter is that global regulator manipulation cannot considered to be a universal
tool to discover new fungal natural products.

In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines
and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that
is produced by many fungi and plants. Once synonymous only with constipation, this former
laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production
and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided
by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans.

We predicted early acting cladofulvin genes and cloned them for heterologous expression in A.
oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin
biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the
point at which the pathways to cladofulvin and monodictyphenone production diverge. It was
speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK
and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by
claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin
but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery
of functionally similar genes and enzymes, providing a template to engineer enzymes capable of
forming novel dimers from existing monomers.

In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by
C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most
active during early and late stages of infection of tomato, but are down-regulated during biotrophic
growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild
type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether
or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum
transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused
to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin
once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this
way, we aimed to test the effect of cladofulvin over-production on disease symptom development.
The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post
inoculation (dpi). At each time point the infections were inspected microscopically to detect any
phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection.
Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves.
In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between
8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation
of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible
reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the
Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the
absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet
(UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less
resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and
repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these
results suggested that cladofulvin confers resistance to abiotic stress.

In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly,
the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might
enable discovery of homologous genes encoding enzymes with different specificities. Combining
bioinformatic and functional analyses should prove to be a powerful strategy for discovering
compounds with new biological activities, or enzymes relevant to metabolic engineering.

The Sw-5 gene cluster : analysis of tomato resistance against tospoviruses
Silva de Oliveira, A. - \ 2015
University. Promotor(en): Monique van Oers; R. de Oliveira Resende, co-promotor(en): Richard Kormelink. - Wageningen : Wageningen University - ISBN 9789462575769 - 158
solanum lycopersicum - tomaten - ziekteresistentie - plantenvirussen - tospovirus - genen - tomatenbronsvlekkenvirus - plantenveredeling - resistentieveredeling - tomatoes - disease resistance - plant viruses - genes - tomato spotted wilt virus - plant breeding - resistance breeding
Wat is erfelijkheid?
Maurice - Van Eijndhoven, M.H.T. ; Oldenbroek, Kor - \ 2015
Zeldzaam huisdier 40 (2015)3. - ISSN 0929-905X - p. 10 - 12.
heritability - rassen (dieren) - dierveredeling - dna - eigenschappen - spermatozoön - eicellen - bevruchting - genen - allelen - homozygoten - heterozygoten - mutaties - genetische merkers - breeds - animal breeding - properties - spermatozoa - ova - fertilization - genes - alleles - homozygotes - heterozygotes - mutations - genetic markers
Eigenschappen van dieren zijn in meer of mindere mate erfelijk. Ze gaan over van ouders op nakomelingen. Maar ervaren fokkers weten dat in de fokkerij 1+1 geen 2 is. Welke wetmatigheden en welke toevalligheden spelen een rol in de erfelijkheid? Wat heeft het DNA-onderzoek ons daar recentelijk over geleerd en wat kunnen we daarmee?
Immune-relevant thrombocytes of common carp undergo parasite-induced nitric oxide-mediated apoptosis
Fink, I.R. ; Ribeiro, C.M.S. ; Forlenza, M. ; Taverne-Thiele, J.J. ; Rombout, J.H.W.M. ; Savelkoul, H.F.J. ; Wiegertjes, G. - \ 2015
Developmental and Comparative Immunology 50 (2015)2. - ISSN 0145-305X - p. 146 - 154.
lymphocyte maturation factor - cyprinus-carpio - expression analysis - human platelets - functional-characterization - monoclonal-antibodies - cell-differentiation - teleost fish - genes - l.
Common carp thrombocytes account for 30–40% of peripheral blood leukocytes and are abundant in the healthy animals' spleen, the thrombopoietic organ. We show that, ex vivo, thrombocytes from healthy carp express a large number of immune-relevant genes, among which several cytokines and Toll-like receptors, clearly pointing at immune functions of carp thrombocytes. Few studies have described the role of fish thrombocytes during infection. Carp are natural host to two different but related protozoan parasites, Trypanoplasma borreli and Trypanosoma carassii, which reside in the blood and tissue fluids. We used the two parasites to undertake controlled studies on the role of fish thrombocytes during these infections. In vivo, but only during infection with T. borreli, thrombocytes were massively depleted from the blood and spleen leading to severe thrombocytopenia. Ex vivo, addition of nitric oxide induced a clear and rapid apoptosis of thrombocytes from healthy carp, supporting a role for nitric oxide-mediated control of immune-relevant thrombocytes during infection with T. borreli. The potential advantage for parasites to selectively deplete the host of thrombocytes via nitric oxide-induced apoptosis is discussed.
Diversity of acid stress resistant variants of Listeria monocytogenes and the potential role of ribosomal protein S21 encoded by rpsU
Metselaar, K.I. ; Besten, H.M.W. den; Boekhorst, J. ; Hijum, S.A.F.T. van; Zwietering, M.H. ; Abee, T. - \ 2015
Frontiers in Microbiology 6 (2015). - ISSN 1664-302X - 12 p.
high hydrostatic-pressure - gamma-aminobutyric-acid - microtiter plate assay - glutamate-decarboxylase - bacillus-subtilis - escherichia-coli - low ph - genes - strains - inactivation
The dynamic response of microorganisms to environmental conditions depends on the behavior of individual cells within the population. Adverse environments can select for stable stress resistant subpopulations. In this study, we aimed to get more insight in the diversity within Listeria monocytogenes LO28 populations, and the genetic basis for the increased resistance of stable resistant fractions isolated after acid exposure. Phenotypic cluster analysis of 23 variants resulted in three clusters and four individual variants and revealed multiple-stress resistance, with both unique and overlapping features related to stress resistance, growth, motility, biofilm formation, and virulence indicators. A higher glutamate decarboxylase activity correlated with increased acid resistance. Whole genome sequencing revealed mutations in rpsU, encoding ribosomal protein S21 in the largest phenotypic cluster, while mutations in ctsR, which were previously shown to be responsible for increased resistance of heat and high hydrostatic pressure resistant variants, were not found in the acid resistant variants. This underlined that large population diversity exists within one L. monocytogenes strain and that different adverse conditions drive selection for different variants. The finding that acid stress selects for rpsU variants provides potential insights in the mechanisms underlying population diversity of L. monocytogenes.
Varied responses by yeast-like symbionts during virulence adaptation in a monophagous phloem-feeding insect
Ferrater, J.B. ; Naredo, A.I. ; Almazan, M.L.P. ; Jong, P.W. de; Dicke, M. ; Horgan, F.G. - \ 2015
Arthropod-Plant Interactions 9 (2015)3. - ISSN 1872-8855 - p. 215 - 224.
resistant rice varieties - nilaparvata-lugens homoptera - brown planthopper resistance - xylem ingestion - uric-acid - delphacidae - selection - populations - genes - aphid
This study examines the three-way interaction between symbionts, insect herbivores and their host plants during adaptation to resistant crop varieties. We conducted a long-term selection study (20 generations of continuous rearing) with a monophagous phloem-feeder, the brown planthopper [Nilaparvata lugens (Stål)], on several resistant rice (Oryza sativa L.) varieties. Planthopper fitness and the abundance of yeast-like symbionts (YLS) were monitored throughout the selection process. N. lugens populations collected from six regions in the Philippines adapted to the resistant varieties as noted by increasing body size and increased egg-laying. Adaptation was partially through physiological and behavioral changes apparent during feeding: Planthoppers on resistant plants had relatively high levels of xylem feeding compared with planthoppers on susceptible plants. YLS densities were highly dependent on the host rice variety. However, there were no consistent trends in YLS density during host plant switching and virulence adaptation: Compared to densities in planthoppers on the standard susceptible variety Taichung Native 1 (TN1), YLS densities were consistently higher on PTB33 (resistant), similar on IR62 (resistant) and IR65482 (moderately resistant) but lower on IR22 (susceptible). Furthermore, YLS densities often remained the same despite improved planthopper fitness over generations. Our results do not support the hypothesis that changes in YLS density mediate planthopper adaptation to resistant varieties. However, slight reductions in YLS densities toward the end of selection on TN1, IR22 and IR62 may indicate that YLS have lower functional significance where varieties and environmental conditions are constant between generations.
Biological processes induced by ZnO, Amoxicillin, Rye and Fructooligosaccharides in cultured Intestinal Porcine Epithelial Cells : VDI-4; In-vitro tests 2013-2014
Hulst, M.M. ; Hoekman, A.J.W. ; Wijers, I. ; Schokker, D. ; Smits, M.A. - \ 2015
Wageningen : Wageningen UR Livestock Research (Livestock Research report 882) - 42
in vitro - bioassays - epithelium - livestock - feed additives - genes - immunology - biotesten - epitheel - vee - voedertoevoegingen - genen - immunologie
The objective of this study was to develop an in-vitro bioassay using cultured Intestinal Porcine Epithelial Cells (IPEC-J2) and evaluate the capability of this assay to predict enterocyte-specific physiological and immunological processes induced by nutrients/additives in the intestines of farm animals. Responses to five nutrients/feed-additives, similar to those studied in animal trials, performed in the Feed4Foodure framework, were measured by gene expression analysis of IPEC-J2 cells either under stressed (Salmonella) or non-stressed conditions. Response genes were analysed using bioinformatics web-tools in order to identify dominant biological processes induced by these nutrients/feed-additives and to predict key-genes/proteins important for regulation of these biological proc
Drought stress affects plant metabolites and herbivore preference but not host location by its parasitoids
Weldegergis, B.T. ; Zhu, F. ; Poelman, E.H. ; Dicke, M. - \ 2015
Oecologia 177 (2015)3. - ISSN 0029-8549 - p. 701 - 713.
volatile emissions - water-stress - abiotic factors - oviposition - genes - biosynthesis - consequences - lepidoptera - complexity - expression
One of the main abiotic stresses that strongly affects plant survival and the primary cause of crop loss around the world is drought. Drought stress leads to sequential morphological, physiological, biochemical and molecular changes that can have severe effects on plant growth, development and productivity. As a consequence of these changes, the interaction between plants and insects can be altered. Using cultivated Brassica oleracea plants, the parasitoid Microplitis mediator and its herbivorous host Mamestra brassicae, we studied the effect of drought stress on (1) the emission of plant volatile organic compounds (VOCs), (2) plant hormone titres, (3) preference and performance of the herbivore, and (4) preference of the parasitoid. Higher levels of jasmonic acid (JA) and abscisic acid (ABA) were recorded in response to herbivory, but no significant differences were observed for salicylic acid (SA) and indole-3-acetic acid (IAA). Drought significantly impacted SA level and showed a significant interactive effect with herbivory for IAA levels. A total of 55 VOCs were recorded and the difference among the treatments was influenced largely by herbivory, where the emission rate of fatty acid-derived volatiles, nitriles and (E)-4,8-dimethylnona-1,3,7-triene [(E)-DMNT] was enhanced. Mamestra brassicae moths preferred to lay eggs on drought-stressed over control plants; their offspring performed similarly on plants of both treatments. VOCs due to drought did not affect the choice of M. mediator parasitoids. Overall, our study reveals an influence of drought on plant chemistry and insect-plant interactions.
Genomics 4.0 : syntenic gene and genome duplication drives diversification of plant secondary metabolism and innate immunity in flowering plants : advanced pattern analytics in duplicate genomes
Hofberger, J.A. - \ 2015
University. Promotor(en): Eric Schranz. - Wageningen : Wageningen University - ISBN 9789462573147 - 142
genomica - planten - metabolisme - bloeiende planten - genomen - genen - next generation sequencing - genomics - plants - metabolism - flowering plants - genomes - genes

Genomics 4.0 - Syntenic Gene and Genome Duplication Drives Diversification of Plant Secondary Metabolism and Innate Immunity in Flowering Plants

Johannes A. Hofberger1, 2, 3

1 Biosystematics Group, Wageningen University & Research Center, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands (August 2012 – December 2013)

2 Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands (December 2010 – July 2012)

3 Chinese Academy of Sciences/Max Planck Partner Institute for Computational Biology, 320 Yueyang Road,

Shanghai 200031, PR China (January 2014 – December 2014)

TWO-SENTENCE SUMMARY

Large-scale comparative analysis of Big Data from next generation sequencing provides powerful means to exploit the potential of nature in context of plant breeding and biotechnology. In this thesis, we combine various computational methods for genome-wide identification of gene families involved in (a) plant innate immunity and (a) biosynthesis of defense-related plant secondary metabolites across 21 species, assess dynamics that affected evolution of underlying traits during 250 Million Years of flowering plant radiation and provide data on more than 4500 loci that can underpin crop improvement for future food and live quality.

GENERAL ABSTRACT

As sessile organisms, plants are permanently exposed to a plethora of potentially harmful microbes and other pests. The surprising resilience to infections observed in successful lineages is due to a complex defense network fighting off invading pathogens. Within this network, a sophisticated plant innate immune system is accompanied by a multitude of specialized biosynthetic pathways that generate more than 200,000 secondary metabolites with ecological, agricultural, energy and medicinal importance. The rapid diversification of associated genes was accompanied by a series of duplication events in virtually all plant species, including local duplication of short sequences as well as multiplication of all chromosomes due to meiotic errors (plant polyploidy). In a comparative genomics approach, we combined several bioinformatics techniques for large-scale identification of multi-domain and multi-gene families that are involved in plant innate immunity or defense-related secondary metabolite pathways across 21 representative flowering plant genomes. We introduced a framework to trace back duplicate gene copies to distinct ancient duplication events, thereby unravelling a differential impact of gene and genome duplication to molecular evolution of target genes. Comparing the genomic context among homologs within and between species in a phylogenomics perspective, we discovered orthologs conserved within genomic regions that remained structurally immobile during flowering plant radiation. In summary, we described a complex interplay of gene and genome duplication that increased genetic versatility of disease resistance and secondary metabolite pathways, thereby expanding the playground for functional diversification and thus plant trait innovation and success. Our findings give fascinating insights to evolution across lineages and can underpin crop improvement for food, fiber and biofuels production

AIL and HDG proteins act antagonistically to control cell proliferation
Horstman, A. ; Fukuoka, H. ; Muino Acuna, J.M. ; Nitsch, L.M.C. ; Guo, Changhao ; Passarinho, P.A. ; Sanchez Perez, G.F. ; Immink, R.G.H. ; Angenent, G.C. ; Boutilier, K.A. - \ 2015
Development 142 (2015). - ISSN 0950-1991 - p. 454 - 464.
arabidopsis-thaliana - transcription factors - plant transformation - ectopic expression - quantitative pcr - chip-seq - differentiation - genes - plethora - growth
AINTEGUMENTA-LIKE (AIL) transcription factors are key regulators of cell proliferation and meristem identity. Although AIL functions have been well described, the direct signalling components of this pathway are largely unknown.We show that BABY BOOM(BBM) and other AIL proteins physically interact with multiple members of the L1-expressed HOMEODOMAIN GLABROUS (HDG) transcription factor family, including HDG1, HDG11 and HDG12. Overexpression of HDG1, HDG11 and HDG12 restricts growth due to root and shoot meristem arrest, which is associated with reduced expression of genes involved in meristem development and cell proliferation pathways, whereas downregulation of multiple HDG genes promotes cell overproliferation. These results suggest a role for HDG proteins in promoting cell differentiation. We also reveal a transcriptional network in which BBM andHDG1regulate several common target genes, and whereBBM/AIL and HDG regulate the expression of each other. Taken together, these results suggest opposite roles for AIL and HDG proteins, with AILs promoting cell proliferation and HDGs stimulating cell differentiation, and that these functions are mediated at both the protein-protein interaction and transcriptional level.
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