Debate on the Exploitation of Natural Plant Diversity to Create Late Blight Resistance in Potato

This paper reports on a debate on intriguing propositions relating to the scientific, agronomic, societal and economic impact of the BIOEXPLOIT project, focusing on late blight resistance in potato. It discusses (i) whether identifying pathogen effectors will facilitate selecting durable R genes, (ii) whether breeding for durable late blight resistance requires deploying Rpi (for Resistance to P hytophthora i nfestans) genes, (iii) whether breeding strategies and cultural practices determine the durability of new resistance genes, (iv) whether marker-assisted breeding for Phytophthora infestans resistance is already in the stage of adoption, (v) to what extent genetically-modified organism technology can advance realizing late-blight resistant potato cultivars, and (vi) whether modifying R genes will result in novel broad spectrum resistanc

Environmental control of the mucosal immune system

Mebius, R.E. [Promotor]Kraal, G. [Promotor

The BIOEXPLOIT Project

The EU Framework 6 Integrated Project BIOEXPLOIT concerns the exploitation of natural plant biodiversity for the pesticide-free production of food. It focuses on the pathogens Phytophthora infestans, Septoria tritici, Blumeria graminis, Puccinia spp. and Fusarium spp. and on the crops wheat, barley, tomato and potato. The project commenced in October 2005, comprises 45 laboratories in 12 countries, and is carried out by partners from research institutes, universities, private companies and small-medium enterprises. The project has four strategic objectives covered in eight sub-projects. These objectives relate to (i) understanding the molecular components involved in durable disease resistance, (ii) exploring and exploiting the natural biodiversity in disease resistance, (iii) accelerating the introduction of marker-assisted breeding and genetic engineering in the EU plant breeding industry, and (iv) coordinating and integrating resistance breeding research, providing training in new technologies, disseminating the results, and transferring knowledge and technologies to the industry

An Outlook on the Localisation and Structure-Function Relationships of R Proteins in Solanum

The co-evolution of plants and plant-pathogens shaped a multi-layered defence system in plants, in which Resistance proteins (R proteins) play a significant role. A fundamental understanding of the functioning of these R proteins and their position in the broader defence system of the plant is essential. Sub-project 3 of the BIOEXPLOIT programme studies how R proteins are activated upon effector recognition and how recognition is conveyed in resistance signalling pathways, using the solanaceous R proteins Rx1 (from S. tuberosum spp. andigena; conferring extreme resistance against Potato Virus X), I-2 (from S. lycopersicon; mediating resistance to Fusarium oxysporum) and Mi-1.2 (from S. lycopersicon; conferring resistance to Meloidogyne incognita) as model systems. The results obtained in this project will serve as a model for other R proteins and will be translated to potential applications or alternative strategies for disease resistance. These include the modification of the recognition specificity of R proteins with the aim to obtain broad spectrum resistance to major pathogens in potato

Redirection of auxin flow in Arabidopsis thaliana roots after infection by root-knot nematodes

Plant auxin efflux and influx proteins redirect the plant hormone auxin towards the feeding site upon root-knot nematode infection in Arabidopsis thaliana roots.Plant-parasitic root-knot nematodes induce the formation of giant cells within the plant root, and it has been recognized that auxin accumulates in these feeding sites. Here, we studied the role of the auxin transport system governed by AUX1/LAX3 influx proteins and different PIN efflux proteins during feeding site development in Arabidopsis thaliana roots. Data generated via promoter-reporter line and protein localization analyses evoke a model in which auxin is being imported at the basipetal side of the feeding site by the concerted action of the influx proteins AUX1 and LAX3, and the efflux protein PIN3. Mutants in auxin influx proteins AUX1 and LAX3 bear significantly fewer and smaller galls, revealing that auxin import into the feeding sites is needed for their development and expansion. The feeding site development in auxin export (PIN) mutants was only slightly hampered. Expression of some PINs appears to be suppressed in galls, probably to prevent auxin drainage. Nevertheless, a functional PIN4 gene seems to be a prerequisite for proper nematode development and gall expansion, most likely by removing excessive auxin to stabilize the hormone level in the feeding site. Our data also indicate a role of local auxin peaks in nematode attraction towards the root

Cyst nematode-induced changes in plant development

This thesis describes a first attempt to investigate the biological activity of cyst nematode secretions on plant cell proliferation and the molecular mechanisms underlying feeding cell development in plant roots upon cyst nematode infection.To investigate the role of nematode secretions in feeding cell development, the in vitro induction and collection of putative nematode signalling molecules is needed. Chapter 2 describes the specific induction of nematode secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis upon exposure to potato root diffusate (PRD). In pre-parasitic juveniles (J2) of Globodera rostochiensis , six proteins with molecular weights of 30, 31a/b, 32, 39, and 49 kDa were recognized on western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All these subventral gland proteins ( svp s) focused in the basic range (pI 6.8-8.6) of an immobilized pH gradient. Western blotting showed that the svp s were present in pre-parasitic and in parasitic J2, and not in following juvenile stages and adult females. Minor svp quantities were also observed in adult males. Immunogold labelling of pre-parasitic (J2) showed that the svp s were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of the svp s through the stylet and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate (DMT) was shown to have only a quantitative, additional effect. The forward flow of the svp s through the metacorporal pump chamber was confirmed by the presence of svp s in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.Using PRD as a host stimulus, relatively large quantities of naturally-induced secretions could be collected from infective juveniles. In Chapter 3, we describe the detection of an oligopeptide(s) in nematode secretions, which stimulate(s) the proliferation of plant cells using a bioassay based on tobacco leaf protoplasts. Naturally-induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones NAA and BAP. Using a protoplast-based bioassay, a low molecular weight peptide(s) (The analysis of early changes in gene expression in response to cyst nematode infection is strongly supported by the use of an in vivo reporter gene system. Chapter 4 describes the use of the green fluorescent protein (GFP) to monitor the transcriptional regulation of respectively the viral CaMV 35S and the bacterial TR2' promoter in early feeding cell development in transgenic potato roots infected with G. rostochiensis . Under the control of either the constitutive CaMV 35S or the mannopine synthase TR2' promoter, the Green Fluorescent Protein (GFP ) from the jellyfish Aequorea victoria , was expressed in transgenic potato ( Solanum tuberosum ) plants. Confocal laser scanning microscopy (CLSM) was applied to observe GFP in planta and, subsequently, to investigate promoter activity in developing feeding cells upon potato cyst nematode ( Globodera rostochiensis ) infection. Both the CaMV 35S and the TR2' promoter were strongly up-regulated in young feeding cells in less then 4 days upon infection by G. rostochiensis whereas the GFP level in the surrounding tissues remained low. Optical sectioning revealed intense green fluorescence in the dense cytoplasm of the entire syncytial cell, including the most distal cell. Furthermore, GFP was observed within the digestive system of the feeding nematode, showing that proteins with an apparent molecular weight of 32 kDa can be taken up by parasitic juveniles of G. rostochiensis . Provided CLSM is used, GFP was shown to be a powerful tool that allows in vivo monitoring of gene expression inside young developing feeding cells. Finally, the transcriptional regulation of the CaMV 35S and TR2' promoter in plant-nematode interactions is discussed. Unfortunately, the use of GFP in combination with the more subtle Arabidopsis cell cycle promoters cycB1;1 and cdc2a was complicated by inadequate expression levels necessary for proper GFP detection in infected potato roots. This obstacle was overcome by using the firefly luciferase gene luc as an in vivo reporter gene (Chapter 6).For decades, it is hypothesised that phytohormones could be involved in syncytium formation. In Chapter 5, a molecular genetic approach was used to study the role of auxin in cyst nematode-induced feeding cell development. The infection of the auxin-insensitive tomato mutant diageotropica and several A. thaliana auxin response mutants suggest that cyst nematodes manipulate the local auxin balance upon early feeding cell induction. Infection of the extreme auxin-insensitive tomato mutant diageotropica ( dgt ) and a number of Arabidopsis auxin-response mutants with the potato cyst nematode Globodera rostochiensis and the beet cyst nematode Heterodera schachtii , respectively, strongly point at a role for auxin in early feeding cell development. Nematode development was significantly reduced in the single gene, recessive mutant dgt and was accompanied by abnormal feeding cell development. For the Arabidopsis auxin-insensitive mutants, only a significant reduction of the infection rate was observed for the mutant axr2 . However, the majority of the mutants showed a reduction in feeding cell hypertrophy - like in dgt - and a reduction in nematode-induced lateral root formation. The induction of gus expression in expanding syncytia of the auxin-responsive Arabidopsis promoter trap line 5-1E1 upon infection with H. schachtii suggested that early feeding cell development is accompanied by a local auxin accumulation. In addition, the obstruction of polar auxin transport in the root either by the application of N -(1-naphthyl)phtalamic acid (NPA) or by the use of the Arabidopsis auxin efflux mutants pin1-1/ttg1 and eir1-1 resulted in a substantial reduction of cyst nematode infections. Moreover, abnormal feeding cell development was observed in the presence of NPA, which was accompanied by the disruption of radial feeding cell expansion, disorganized cell division and metaxylem formation.Chapter 6 gives an overview of cell cycle activation by plant-parasitic nematodes; especially cyst and root knot nematodes, and links the possible role of nematode secretions and auxin in feeding cell formation. Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialised feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific plant cells finally resulting in a transfer cell-like feeding site. For growth and development the nematodes fully depend on these cells. The mechanisms underlying the intriguing ability of these nematodes to manipulate a plant for its own benefit are unknown. Nematode secretions are thought to play a key role both in plant penetration and feeding cell induction. Research on plant-nematode interactions is hampered by the minute size of cyst and root knot nematodes, their obligatory biotrophic nature and their relatively long life cycle. Recently, insights in cell cycle control in Arabidopsis thaliana in combination with reporter gene technologies showed the differential activation of cell cycle gene promoters upon infection with cyst or root knot nematodes. In this review, we integrate the current views of plant cell fate manipulation by these sedentary nematodes and made an inventory about possible links between cell cycle activation and local, nematode-induced changes in auxin levels.</p