Characterisation of the processes of nutrient flow from a host plant to a parasitic nematode

Lead Research Organisation: University of Leeds
Department Name: Inst of Integrative & Comparative Biolog

Abstract

Cyst nematodes are small with a length of only 0.5mm but there can be thousands of millions in one hectare of field soil. Each nematodes moves from the soil into the roots of its host crop plant. Once inside the plant it loses the ability to move and feeds at just one place. Approximately 75 % of the nematode population becomes female. Females feed for about 8 weeks growing to about 1000 times their original size and laying up to 700 eggs. They modify one plant cell. This provides all the nematode's food for several weeks. This cell is highly specialised structure and transport nutrients from the plant flow though this cell to the feeding nematode. We have identified the plant molecules that transport the essential nutrients the feeding nematode needs. This project will define how these molecules are distributed around the specialsed plant cell that gathers nutrients to supply to the nematode parasite. We will determine how the walls of this cell are changed to allow it to merge with other cells, grow in size and also support the flow of nutrients through it to the feeding nematode. We will use antibodies designed to recognise components of the cell wall to understand the structure of the wall of this cell. The nematode must ensure its feeding does not kill the plant cell from which it feeds. If that happened the nematode would die. The nematode makes a feeding tube that has holes all the way along its length, not just at the end. We will find out if this allows the nematode to feed by without damaging the internal structure of the plant cell. If the nematode took the contents of the cell through just a single hole in the feeding tube (like a straw) the cell might be badly damaged. We want to understand how these plant parasites feed from plant cells. No other animals feed in this way. Nematodes damage many crop plants throughout the world. Cyst nematodes cause losses of approximately £43 million each year just to UK potato crops. This new knowledge may help to fnd a way to stop the nematodes from feeding. This would protect plants from the damage they cause. Currently farmers often use pesticides that harm the environment and can damage the health of farm workers. We have shown before that new knowledge can help develop better ways of controlling nematodes in the future.

Technical Summary

Cyst nematodes invade plant roots from soil and become obligate endoparasites. A single plant cell is selected by each nematode. It undergoes cellular redevelopment and forms a syncytium with other adjacent cells. The female nematode feeds exclusively from this single syncytium throughout its development. They feed for approximately 8 weeks growing to c.100x their original size and draining the plant of vital resources when many establish concurrently on a root system. This work characterises three important and inter-related aspects of this unique host/pathogen interaction that characterise nutrient flow from the plant to the parasite: 1) the distribution of transporter proteins in the plasma membrane of the syncytium and their role in replacing the nutrients withdrawn from the cell by the nematode, 2) the change in plant cell architecture that accommodates the effective functioning of these cells and 3) the ability of the nematode to feed continually without lethal consequences for the cell. We have carried out microarray analysis using syncytial material within c. 2 mm root lengths thereby minimising dilution of that mRNA originating from the syncytium. This has allowed us to identify a number of transporter proteins that are up regulated in the infected sample. In this project we will determine the pattern of expression of these transporters in the syncytium. We will make translation fusions of these proteins with Green Fluorescent Protein and use confocal imaging to determine their distribution around the plasma membrane of the syncytium. It is highly likely that there will be a greater concentration of these transporters where cell wall invaginations provide an enhanced surface area in contact with the vasculature. The cell wall of the syncytium is altered in ways that presumably favour syncytial expansion and high nutrient influx. We will use antibodies designed to recognise components of the cell wall to understand the structure of the wall around the circumference of the syncytium. We have observed carbohydrate moieties of the syncytial wall in preliminary work and gained evidence that its carbohydrate components are significantly different from those of other plant cell walls. The nematode makes a feeding tube, a unique structure within the syncytium. Our work to-date suggests that feeding tubes are ultrafilters. We propose to study if their role is to ensure continual uptake of permeate is achieved without perturbing the internal structure of the cell. We seek to define why each feeding tube has a functional life of only a few hours. Commercially produced ultrafiltration devices provide a preliminary understanding of the problems associated with their use, such as blocking and gel formation on the outer surface. The attributes of the nematode feeding tube will be analysed and used to determine the biological role of this unique structure.
 
Description Cyst nematodes invade host roots and modify plant cells into a feeding site from which females feed continually for up to 8 weeks. Females grow, their body swells to a sphere of about 0.8mm diameter and subsequently act to contain the new eggs the female makes. When reproduction is finished the body wall tans to form a cyst that protects the eggs. Large numbers of females can occur on a root system of host crops such as potato and if not controlled they can reduce yield to less than 20% of the normal harvest.
The key question we address is; how does the plant cell complex induced by the parasite cope with continual and intensive removal of nutrients from it by the nematode?
The starting point for our research was plant genes we identified as being active at the feeding sites of a cyst nematode in previous work. They are likely to be important in transporting nutrients into the modified plant cells. The protein encoded by one gene is involved in transporting simple sugars between plant cells and so may replenish these important sources of energy to the plant cell after the parasite feeds. A second allows amino acids, the constituent parts of proteins, to move between plant cells. A third gene has a similar role for peptides (small proteins) and the fourth transports sulphate. Initial molecular analysis of gene activity showed that peptide and sulphate transporters were more abundant when the parasite first enters the plant root, the sugar transporter was more abundant when the feeding cell was expanding and the amino acid transporter were less abundant at that time.
We isolated the promoter from each gene of interest to determine if these changes occurred within the modified plant cells or the cells surrounding it that may supply nutrients. These promoters act as switches for genes. The isolated promoter can be made to control the activity of a reporter gene, the product of which can be visualised - this protein is not normally present in plants. Any cell in which the promoter is active emits fluorescence from this protein when viewed under a microscope. This approach confirmed that the sugar transporter was active in the feeding cell whereas the amino acid transporter was active in the surrounding cells.
We then investigated the importance of the change in activity of these genes for nutrient supply to the parasite. The first (RNA interference) reduce the activity of each of the 4 genes when they were specifically targeted. The number of females that developed on roots was reduced when the sugar transporter gene was prevented from increasing expression as normally caused by the parasite. Equally interesting, when the amino acid transporter that the parasite causes to be less present was targeted to lower its activity the parasites were more successful. These effects were also obtained when seeds of mutant plants, known not to express one of each of these two genes were grown and the parasite allowed to develop on them. These effects were not absolute suggesting the nematode may alter expression of many genes which help its parasitism but are not totally essential for it.
When the nematode modifies plant cells their cell walls must change to allow development of this large feeding cell made up of the contents of several cells. We studied change in plant cell wall components using a set of antibodies to specific plant cell wall components. Some of the changes in plant cell walls were common to potato, soybean and Arabidopsis each parasitised by a different host-specific cyst nematode. Other changes in cell wall components were specific to one of the plants.
We also investigated how the feeding tube of the nematode, a renewable structure through which plant cell contents are ingested, imposes a size selection on the proteins that can be ingested from the feeding site.
Exploitation Route A number of resources were generated in this project that are available for further study by us and other researchers. These will be of use not just to those working with plant parasitic nematodes, but for many aspects of plant biology.
A resource of transgenic Arabidopsis reporter lines was generated for the project to study the temporal and spatial expression patterns of plant transporter proteins during infection with the cyst nematode Heterodera schachtii. These lines are now available for use in further work to investigate the impact on transporter gene expression of other plant parasitic nematodes, with diverse feeding site structures and modes of parasitism and, indeed, other plant pathogens.
A collection of fixed, embedded and sectioned roots containing nematode feeding sites has been established. As the material can be stored in good condition for a number of years this provides a valuable resource for future work to investigate immunochemically the distribution of a range of proteins in the feeding cells of cyst and root knot nematodes parasitising different host plants. The collection includes feeding site sections for both Heterodera glycines and Meloidogyne incognita infecting soybean, Globodera pallida infecting potato and Heterodera schachtii infecting sugar beet.
New monoclonal antibodies to plant cell wall epitopes were generated concurrent with the project and used in the project (LM15 xyloglucan, LM18 pectic homogalacturonan, LM19 pectic homogalacturonan, LM20 pectic homogalacturonan). These have subsequently been made available to the wider research community through PlantProbes. This is a distribution service run by the Paul Knox Cell Wall Lab in collaboration with University of Leeds Consulting Limited.
Much of our fundamental work such as in this grant allows us to design novel defences to suppress nematodes. We hope in the future to extend the current research in that direction particularly to control potato cyst nematode which is now the most serious pest problem for UK potato farmers.
Sectors Agriculture, Food and Drink

 
Description The work carried out in this project produced new insights into how the nematode pathogen extracts nutrients from the plant. In particular it determined the changes in the plant cell walls that are important in allowing cyst nematodes to feed successfully. The work has the long term potential to engineer new crop varieties that impair the ability of the nematode to gain essential nutrition from the plant. In the shorter term, the work from this project has led directly to two PhD studentships focussed on in depth characterisation of the cell walls of nematode feeding sites for a wider range of nematode and host plant species. The new monoclonal antibodies used in the project have already been distributed to many industrial and academic researchers around the world. Work in this project to investigate the function of cyst nematode feeding tubes led to the development of improved software for the prediction of protein size. This software, which has utility in many areas of protein research, has been made publically available and has been downloaded more than 300 times for use by other researchers.
First Year Of Impact 2011
Sector Agriculture, Food and Drink
 
Title Rotamol 
Description A short script, written in Autoit, designed to work in conjunction with PyMOL to predict the Rotationally averaged Collision Cross Section (CCS) of protein models (PDB files). It also allows measurement of protein external electrostatics for a combined measure of size and external charge. 
Type Of Technology Software 
Year Produced 2011 
Open Source License? Yes  
Impact The new software was instrumental in resolving previous discrepancies in the apparent size of proteins that can be ingested from plants by plant parasitic cyst nematodes. It has been downloaded for use by more than 300 researchers 
URL https://code.google.com/p/rotamol/
 
Description Worksop College (Nottingham) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Generate understanding an critical thinking about new biotechnological advances

All girl college: students reported a greater interest in continuing in the science field
Year(s) Of Engagement Activity Pre-2006