Master's Research Project:
Dissecting Host Responses to Root Knot Nematode:
Utilizing Microarrays to Identify Genes Differentially Expressed upon Invasion by Meloidogyne spp.

Introduction

Root-knot nematodes (Meloidogyne spp.) are obligate sedentary root pathogens that form complex and intimate associations with their host plant. Root-knot nematodes infect over 2,000 plant species and essentially every economically important crop is susceptible to some species of root-knot nematode, costing growers over $50 billion in economic loss worldwide. The three main species in the US, Meloidogyne incognita, M. javanica and M. arenaria, have short life cycles (c. 4 weeks at 25oC), high fecundity (c. 1000 eggs/female) and reproduce by mitotic parthenogenesis. In cooler more temperate climates such as the Pacific northwest, other species of RKN are more prevalent. These include M. hapla, M. naasi and M. chitwoodi, which reproduce either by meiotic parthenogenesis or amphimixis. Other nematode species causing severe crop damage in the US include the soybean cyst nematode, Heterodera glycines. Management practices rely chiefly on the use of chemical nematicides. In 1982, this practice cost growers more than $1 billion dollars to distribute 109 million pounds of the nematicide active ingredient to US crops. Recent concerns of ground water contamination, toxicity to mammals and birds, and residues in food have caused tighter restrictions on the use of chemical nematicides, including deregulation of many of those traditionally employed in the US. Other management practices, such as crop rotation and host resistance, are more environmentally and economically sound, but are not feasible owing to the broad host range of root-knot nematode and limited sources of natural resistance for most crops. The key to nematode control will have to depend on understanding the biology and physiology of the host response to invasion by the root knot nematode.

Fig. 1

Figure 1a. Newly hatched M. incognita larva.
S: feeding stylet;
arrows: lipid storage granules.

Figure 1b. Toluidine blue stained trans-verse section of a root-knot nematode gall in tomato; four giant cells are evident.

Fig. 2

Root-knot nematodes hatch in the soil as second-stage juveniles (Fig. 1a) and infect plants by penetrating the root just behind the root cap. They then migrate towards the developing vascular tissue. Here, the nematode becomes sedentary and initiates a change that alters the fate of several procambium cells from normal developing root tissue to highly polyploid and metabolically active cells, termed giant cells, that serve as the obligate nutritive source for the developing nematode. (Fig. 1b).

In most plant species, giant cell formation coupled with the limited proliferation of the surrounding pericycle and cortical cells results in the characteristic root-knot gall.

I am interested in global changes in gene expression during nematode infection, migration, and initiation and maintenance of the giant cells. Are whole families of genes up- or down-regulated during infection and pathogenesis, or are changes in expression confined to a few specific genes? I would also like to elucidate how expression of these genes is temporally controlled. Recent publications have demonstrated that there are changes in auxin accumulation patterns associated with the formation of giant cells and/or galls (Hutangura et al, 1999), and that auxin transport is inhibited around newly formed nematode infection sites. This phenomenon raises numerous pertinent questions, such as does this temporary accumulation make auxin a key player in transcriptional control, and thus the initiation of the giant cells, or is it just a side effect? What are the differences between changes in induced gene expression by Meloidogyne incognita, which causes extensive galling in the host root system, and M. hapla, which induces little to no galling? Finally, how does expression of host resistance genes affect patterns of gene expression? We utilize two tomato varieties, Motelle and Moneymaker, which differ only in that Motelle has the Mi gene (in a very small introgressed region) which confers resistance to some root knot nematodes and not others. Differences in gene expression between these two varieties during infection by both compatible and incompatible root-knot nematode species may shed light on basic regulatory mechanisms involved in both feeding site formation and host resistance.

Building the Microarray

The Bird lab has previously used a subtractive, cDNA cloning approach to identify approximately 200 tomato genes differentially expressed in nematode feeding sites (Wilson et al, 1994 and Bird and Wilson, 1994). I routinely peruse the public databases for homologues from other plant species, and experimentally analyze genes with interesting identities (interesting with regard to potential function or localization). To find additional candidates I have initiated a microarray screen. I identified a set of EST clones defining approximately 4,300 unique genes expressed in tomato roots from the TIGR database. In most cases, each root gene is represented by a number of sequences organized into a cluster. Each cluster was examined for the longest sequence, and clones containing these sequences were obtained in a collection of 222 384-well microtiter plates. I am currently re-arraying these clones into the ToRuG (Tomato Root uniGene) set. ToRuG is a dynamic assembly, with new sequences being added as they are discovered. ToRuG clones will be merged with clones from the giant cell library, and this set will be used to construct the microarray.

Experiment Comparisons

The first experiment will be to hybridize the micro-arrays with healthy tomato root RNA to assess the quality, consistency, and efficacy of the target DNA and controls. Also, RNA from healthy tissues of different tomato varieties will have to be assessed before experiments of infected root tissue are performed. After controls are established, multiple series of experiments can be run using the arrays. Potential experiments will compare:

Functional analysis

The results of these experiments will dictate the next steps of the project. If there are a plethora of changes in gene expression (as we expect), the genes can be grouped or categorized. If there are only a few changes in gene expression, the genes can be manually analyzed. Individual genes or representatives of groups or families of genes that are highly up-regulated in response to nematode invasion will be potential candidates for gene ablation experiments. Because root-knot nematodes are root pathogens, I can exploit Agrobacterium rhizogenes, which genetically transforms prepared plant material and produces transgenic hairy roots. Roots appear in culture a week after transformation and can be inoculated with Meloidogyne sp. as soon as lateral branching occurs. Galls can be seen in the transgenic roots in as little as two days. This system provides a fast and effective way to screen genes whose roles in giant cell development provide a potential source in breaking plant-nematode compatible interactions.

Materials and Methods

Figure 2. Tomato seedlings
in growth-pouches.

Fig. 3In a typical experiment, two RNA samples are labeled with distinct fluorochromes, mixed and hybridized simultaneously to the DNA microarray. Specifically, total RNA will be harvested from tomato root tissue grown and inoculated in growth pouches (Fig. 2). PolyA fractions will be isolated using oligo(dT) linked Oligotex resin and 3ug of poly mRNA will be used as a template for each of six replicate cDNA reactions using SuperscriptII in the presences of fluorescently labeled Cy3 or Cy5-dCTP as essentially described by White et al, 1999.

The data values are expressed as ratios of the signals collected for the two samples. In conjunction with the Bioinfomatics department at NC State we are currently developing non-parametric methods to analyze our data.

By using the methods I have outlined above, I believe I will be able to identify numerous options that will lend themselves in conjunction, with genetic engineering and functional genomics, to develop suitable environmentally and economically sound methods for control of root-knot nematode.

References

Bird, D. M., Wilson, M. A. 1994. DNA Sequence and Expression Analysis of Root-Knot Nematode-Elicited Giant Cell Transcripts. Molecular Plant-Microbe Interactions. 7: 419-424

Hutangura, P., Mathesius, U., Jones, M.G.K., Rolfe, B.G. 1999. Auxin Induction Is a Trigger for Root Gall Formation Caused by Root-knot Nematodes in White Clover and Is Associated With the Activation of the Flavonoid Pathway. Austrailian Journal of Plant Physiology. 26: 221-231

White, K.P., Rifkin, S.A., Hurban, P., Hogness, D.S. 1999. Microarray analysis of Drosophila development during metamorphosis. Science 286: 2179-2184

Wilson, M.A., Bird, D. M., Knaap, E. 1994. A Comprehensive Subtractive cDNA Cloning Approach to Identigy Nematode-Induced Transcripts in Tomato. Molecular Plant Pathology 84: 299-303