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

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

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

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

On the role of dauer in the adaptation of nematodes to a parasitic lifestyle

Abstract Nematodes are presumably the most abundant Metazoa on Earth, and can even be found in some of the most hostile environments of our planet. Various types of hypobiosis evolved to adapt their life cycles to such harsh environmental conditions. The five most distal major clades of the phylum Nematoda (Clades 8–12), formerly referred to as the Secernentea, contain many economically relevant parasitic nematodes. In this group, a special type of hypobiosis, dauer, has evolved. The dauer signalling pathway, which culminates in the biosynthesis of dafachronic acid (DA), is intensively studied in the free-living nematode Caenorhabditis elegans, and it has been hypothesized that the dauer stage may have been a prerequisite for the evolution of a wide range of parasitic lifestyles among other nematode species. Biosynthesis of DA is not specific for hypobiosis, but if it results in exit of the hypobiotic state, it is one of the main criteria to define certain behaviour as dauer. Within Clades 9 and 10, the involvement of DA has been validated experimentally, and dauer is therefore generally accepted to occur in those clades. However, for other clades, such as Clade 12, this has hardly been explored. In this review, we provide clarity on the nomenclature associated with hypobiosis and dauer across different nematological subfields. We discuss evidence for dauer-like stages in Clades 8 to 12 and support this with a meta-analysis of available genomic data. Furthermore, we discuss indications for a simplified dauer signalling pathway in parasitic nematodes. Finally, we zoom in on the host cues that induce exit from the hypobiotic stage and introduce two hypotheses on how these signals might feed into the dauer signalling pathway for plant-parasitic nematodes. With this work, we contribute to the deeper understanding of the molecular mechanisms underlying hypobiosis in parasitic nematodes. Based on this, novel strategies for the control of parasitic nematodes can be developed

SolRgene: an online database to explore disease resistance genes in tuber-bearing Solanum species

Background The cultivated potato (Solanum tuberosum L.) is an important food crop, but highly susceptible to many pathogens. The major threat to potato production is the Irish famine pathogen Phytophthora infestans, which causes the devastating late blight disease. Potato breeding makes use of germplasm from wild relatives (wild germplasm) to introduce resistances into cultivated potato. The Solanum section Petota comprises tuber-bearing species that are potential donors of new disease resistance genes. The aim of this study was to explore Solanum section Petota for resistance genes and generate a widely accessible resource that is useful for studying and implementing disease resistance in potato. Description The SolRgene database contains data on resistance to P. infestans and presence of R genes and R gene homologues in Solanum section Petota. We have explored Solanum section Petota for resistance to late blight in high throughput disease tests under various laboratory conditions and in field trials. From resistant wild germplasm, segregating populations were generated and assessed for the presence of resistance genes. All these data have been entered into the SolRgene database. To facilitate genetic and resistance gene evolution studies, phylogenetic data of the entire SolRgene collection are included, as well as a tool for generating phylogenetic trees of selected groups of germplasm. Data from resistance gene allele-mining studies are incorporated, which enables detection of R gene homologs in related germplasm. Using these resources, various resistance genes have been detected and some of these have been cloned, whereas others are in the cloning pipeline. All this information is stored in the online SolRgene database, which allows users to query resistance data, sequences, passport data of the accessions, and phylogenic classifications. Conclusion Solanum section Petota forms the basis of the SolRgene database, which contains a collection of resistance data of an unprecedented size and precision. Complemented with R gene sequence data and phylogenetic tools, SolRgene can be considered the primary resource for information on R genes from potato and wild tuber-bearing relatives

Distinct roles for strigolactones in cyst nematode parasitism of Arabidopsis roots

Phytohormones play an essential role in different stages of plant-nematode interactions. Strigolactones (SLs) are a novel class of plant hormones which play an important role in plant development. Furthermore, certain soil-inhabiting organisms exploit this plant molecule as allelochemical. However, whether SLs play a role in plant parasitism by nematodes is as yet unknown. This prompted us to investigate the potential role of SLs in different stages of the nematode life cycle using the beet cyst nematode Heterodera schachtii and Arabidopsis as a model system. We analyzed the effect of SLs on cyst nematode hatching, host attraction and invasion, and the establishment of a feeding relation upon infection of the SL deficient mutant max4-1 and the SL signaling mutant max2-1. In addition, infection assays were performed under phosphate shortage to enhance SL production and in the presence of the synthetic SL analog GR24. From this study, we can conclude that SLs do not contribute to cyst nematode hatching at the levels tested but that they do play a role in host attraction and subsequent invasion in a MAX2 dependent manner. Furthermore, we observed that increased levels of exogenous and endogenous SLs change the root invasion zone. Upon root infection, cyst nematode development was enhanced in both the max2-1 and max4-1 mutants due to the formation of enlarged feeding cells. These data provide evidence for distinct roles of SLs during cyst nematode parasitism of plant roots