Valerie M. Williamson - US grants

The goal of this proposal is to initiate a study of plant resistance to aphids in a system consisting of tomato (Lycopersicon esculentum) and the potato aphid (Macrosiphum euphorbiae). Dr. Williamson has recently described a tomato gene, Meul, that confers resistance to potato aphid. Meul is tightly linked to Mi, a single, dominant locus that is present in some tomato lines and mediates resistance to root- knot nematodes. Mi-mediated nematode resistance is associated with a localized necrosis in the root within 24 hours of penetration by the nematode. The mechanism of Meul- mediated resistance to aphids has not yet been studied in detail. Preliminary observations, and comparison to the nematode resistance, suggest that specific recognition of the aphid by the plant is involved in the initial step of a signal transduction cascade that leads to localized necrosis and production of defense compounds. This would represent the first example of this mechanism of plant resistance to an insect. This proposal seeks to establish the groundwork for a future proposal on characterizing the recognition mechanism and signal transduction pathway of plant response to aphid.

9723679 Williamson The gene Meu1 confers resistance to the potato aphid in tomato. It is tightly linked to Mi, a single, dominant locus that mediates resistance to root-knot nemotodes. The projects starts from the recognition that the two genes are close, but separated by about 300 kb. The project will isolate Meu1 and characterize the gene product genetically and through transgenic plant analysis in a multidisciplinary approach. The gene will be obtained by positional cloning. Action of the gene will be analyzed through assessment of the aphid response to resistant plants and the response of resistant plants to aphid feeding. Included is an analysis of the effect of Meu1-mediated resistance on virus transmission by aphids. This approach unites two laboratories with emphasis on aphid biology and molecular biology.

Root-knot nematodes are tiny, parasitic worms that infect a wide range of plants and cause major yield losses to the world's crops. Changes to the plant after infection include the formation of galls or root-knots on root systems. Complex molecular signaling between the host and parasite mediates this interaction. Analysis of the roles of the nematode's genes in causing root galling and crop loss has not been possible because of the lack of genetic analysis of the nematode and the lack of knowledge of nematode's genome structure. A major constraint has been that several species of nematodes do not reproduce sexually, making genetic analysis very difficult. However, isolates of the nematode species, Meloidogyne hapla, do reproduce sexually, making this species an excellent model to gain information on what genes in the nematode are responsible for its destructive properties. The goal of the current research is to establish a genetic system for M. hapla. Nematode strains with differences in DNA markers and in ability to reproduce on particular plant lines have already been identified. In this study, the inheritance of ability to reproduce on a wild potato isolate with the gene Rmc1 will be investigated. A protocol to carry out controlled genetic crosses will be developed and segregation of DNA markers will be monitored. Segregation of markers and virulence in the presence of Rmc1 will be assessed to initiate a genetic linkage map. A DNA library representing the entire M. hapla genome will be produced. DNA hybridization to nematode chromosomes will be carried out to aid in determining the genome structure. The linkage map and markers will be a resource for cloning nematode genes associated with ability to parasitise different plant species. Information gained from these studies will facilitate understanding of mechanisms by which parasitic nematodes circumvent plant resistance genes and may lead to environmentally safer means of nematode control in agriculture.

The tomato gene, Mi, confers resistance against several species of root-knot nematodes (Meloidogyne spp.) as well as against specific isolates of potato aphid (Macrosiphum euphorbiae). It is the only major gene for resistance to an aphid to be cloned so far and the first discovered that can mediate gene-for-gene resistance to two unrelated pests. Mi is a member of a diverse class of plant resistance-genes (R-genes) involved in gene-specific resistance against pathogens including viruses, bacteria and fungi. Members of this class of plant proteins mediate specific pathogen recognition and signaling of host defense responses. More than 20 such genes have been identified, yet how the pathogen is recognized and resistance conferred is not known. The Mi clone constitutes a unique resource for gaining insights into R-gene functions associated with specific pest recognition and signaling in plants and for determining the mechanism(s) by which a single gene can mediate resistance to multiple organisms. Chimeric constructs of Mi that produce a lethal phenotype when transiently expressed in Nicotiana benthamiana leaves are already available. Specific in vitro mutations in Mi and in constitutively lethal constructs of Mi will be produced. The phenotypes of the mutations will be examined in two different assays to delineate the function in pathogen recognition and signal transmission of particular regions of the gene. These assays will determine the effects of mutated alleles on cell death in a leaf infiltration assay and on nematode resistance in transformed roots. The phenotypes of in vitro-generated alterations in Mi on the signalling of resistance will also be examined in transgenic plants to identify the functional domains associated with nematode and/or aphid recognition and downstream defense signaling. In addition, proteins that interact with Mi will be identified using the yeast 2-hybrid system and the role of these proteins in resistance will be examined.

Root- knot nematodes cause billions of dollars in damage to the world's crops. Current agricultural control measures include application of chemical pesticides or the use of resistant plants. A single gene present in some tomato varieties can confer effective resistance against nematodes as well as resistance against some aphids. This project explores the question of how a single gene, which was bred into tomato from a wild plant species, can mediate recognition of the nematode pest and trigger an effective defense. An increased understanding of how plants defend themselves from pathogens and pests should result from this work. Such understanding is essential for rational design of strategies to improve pest and pathogen resistance in plants.

ABSTRACT WILLIAMSON #052084

Genetic analysis of a root-knot nematode

Intellectual Merit: Root-knot nematodes are microscopic round worms that cause major yield losses to many of the world's crops. Efforts to understand how these pests recognize and interact with their hosts have been hampered by their parasitic life cycle and small size. The goal of the proposed research is to produce an integrated genetic map of the root-knot nematode Meloidogyne hapla. This species was selected because its unusual reproductive mode makes it possible to carry out genetic crosses and to produce large numbers of genetically uniform lines. Nematode strains that differ in attraction to specific plant species and in ability to reproduce on common bean carrying a resistance gene have already been characterized. The segregation of these traits as well as other traits affecting the nematode's ability to infect specific plants and DNA markers will be determined by examination of progeny from genetic crosses. This analysis will be coordinated with a project to sequence the M. hapla genome and will lead to the identification of genes that modulate how a nematode is attracted to and causes disease on specific crop plants.

Broader Impacts: Understanding the biology of these organisms is essential for the development of safer control measures. While the current research will focus on genes involved in host range, it will also establish a basis for other investigations of this complex parasitic interaction as well as for comparison of genome structure among nematode species. Insight into the novel genetic system of M. hapla is likely to stimulate general interest by evolutionary biologists and geneticists working on other organisms. The research will provide a rich source of projects for training students in genetic research approaches, and students at levels from high school to post graduate will participate in the proposed experiments.