1.14.4S
EARLY CHANGES IN GENE EXPRESSION AFTER INFECTION OF TOMATO WITH ROOT-KNOT NEMATODES

VM WILLIAMSON, BJ FERRIE and ED BRENNER

Department of Nematology, University of California, Davis, CA 95616, USA

Background and objectives
Root-knot nematodes are important pathogens of many crop plants [1]. Second-stage juveniles penetrate the host near root tips then migrate intercellularly to the vascular cylinder. In response to signals from the nematode, plant cells adjacent to the head of the nematode enlarge to form giant cells - large multinucleate, feeder cells for the developing nematode. Concurrent hyperplasia and hypertrophy in the surrounding tissues leads to the formation of characteristic galls. The gene Mi, present in many lines of tomato, confers effective resistance to several species of root-knot nematodes. Resistance is characterized by a localized necrosis near the anterior end of the invading nematode.

Our goal is to identify transcripts that are crucial to the early stages of the resistant and susceptible host responses and to determine the roles of these genes. We have focused on the first 24 h of infection, as it is during this period that the first visible changes occur that lead to resistant and susceptible responses.

Materials and methods
A PCR protocol [2] was used to produce a cDNA library from nematode-infected roots of resistant tomato seedlings. Differential screening was done on 1100 clones by spotting the inserts on nylon membranes and hybridizing these with labelled first-strand cDNA probes made from total RNA from resistant and susceptible tomato roots, each inoculated with nematodes or mock-inoculated. Clones which appeared to be induced by nematodes and at higher levels in resistant than in susceptible tomato were further studied.

Results and conclusions
DNA sequences of clones that showed induction upon nematode infection were compared to sequences available in Genbank. In many cases, similarity was found to genes known to be induced by pathogens and/or wounding in other systems (chitinase, peroxidase, proteinase-inhibitor, and extracellular dermal glycoprotein). Several clones had no homology to known genes. All genes characterized so far are induced in both resistant and susceptible root tips after nematode infection, though some appear to be more highly induced in the resistance response. Variable results in replicated experiments suggest that expression is transient and timing is very important. Several of these genes are induced upon wounding in roots but not in the upper parts of plants. This result suggests that the defence systems of roots may be distinct from those in the upper parts of plants. Not all the genes that we identified as induced by nematodes were induced by wounding, indicating that other triggers are involved. These triggers may represent other defence pathways or very early events in the susceptible response, including initiation of the elaborate feeding site, that are induced before the resistance response stops the process.

One gene identified in our screen shows high sequence similarity to a protein called miraculin that causes sour substances to taste sweet to higher primates. This gene, which we have named LeMir, encodes a member of the Kunitz trypsin inhibitor family. The protein is expressed constitutively in roots with highest level near the crown, a common site of ingress of pathogens. Analysis of root washes from seedlings reveals that lemir is released into the medium. The tissue localization and the induction by wounding suggests that lemir may have a role in defence

References
1. Williamson VM, Hussey RS, 1996. Plant Cell 8, 1735-1745.
2. Lambert KN, Williamson VM, 1996. In Cowell I, Austin CA, eds, Protocols for cDNA Libraries. Humana Press, Totowa, NJ, pp. 1-12.