Summary In this study two plant resistance genes, Cf‐4 and Cf‐9 , were expressed in transgenic plants together with their corresponding pathogen avirulence genes Avr4 and Avr9. Three nematode responsive promoters (4xB5+A, A0.3TobRB7 and 35S) in different combinations were used to control the expression of these transgenes. According to previous descriptions of the activity of these promoters, for the promoter combinations used, co‐expression would be expected to occur only in the feeding sites of root‐knot nematodes, and not in uninfected plants. This being the case, it was predicted that transgenic plants which express a hypersensitive response, induced by nematode feeding, specifically at the feeding site could be engineered. However, transgenic plants that harbored both resistance and avirulence gene constructs underwent spontaneous necrosis, revealing new features of promoter activity. We show that the pattern of hypersensitive response was dependent upon the combination of promoters used to control the expression of the Cf and Avr genes, transgene position effects and growth conditions. Although activity of the Cf genes has only previously been reported from aerial parts of the plant, here we present evidence that both Cf‐9 and Cf‐4 are active in tobacco roots.

Root-knot and cyst nematodes are obligate plant parasites that induce complex biotrophic feeding structures in host roots. The mechanisms by which nematodes regulate host gene expression to produce feeding sites are unknown. The cauliflower mosaic virus (CaMV) 35S promoter has been reported to be repressed strongly in the feeding sites of both root-knot and cyst nematodes. In contrast, other work has indicated that this promoter is partially active in some feeding sites. Considering the importance of the 35S promoter in biotechnology, we have defined the nematoderesponsive nature of this promoter in more detail. Transgenic tobacco harboring various 35S-uidA constructs was assayed for β-glucuronidase (GUS) activity after infection by root-knot nematodes (Meloidogyne incognita) and cyst nematodes (Globodera tabacum subsp. tabacum). The entire 35S promoter (-343 to +8) was active in giant cells induced by M. incognita and, to a lesser extent, the syncytia of G. tabacum subsp. tabacum. In the latter case, activity decreased as the feeding sites matured. Subdomains of the 35S promoter were also active in feeding sites, particularly B4 and B5 in giant cells. However, subdomain B3 was strongly down-regulated in gall tissue and syncytia. In total, 14 constructs were studied and nematode-responsive expression was always stronger and more consistent with the root-knot nematode than the cyst nematode.

Due to increasing restrictions on the use of toxic and expensive nematicides, there is now a greater than ever need for crop cultivars that are resistant to plant-parasitic nematodes. Genetically engineered nematode resistance is not as well developed as other engineered traits but, even so, the first genetically modified plants with enhanced nematode resistance have been produced and tested. Plant-derived enzyme inhibitor and lectin genes are being evaluated for their ability to confer broad-spectrum nematode resistance in transgenic crop plants. Early indications are that these are likely to be effective. Gene pyramiding has potential to increase field durability and to widen the spectrum of nematodes controlled by any one transgenic line. (C) 1998 SCI.

A method is described for identifying strains of Metarhizium suitable for use in field experiments. It involves the restriction endonuclease digestion of a PCR product derived from the PrI protease gene and the analysis of the fragments by electrophoresis. Using this technique, 40 Metarhizium strains produced 15 different profile types and were clustered into four groups. Correlation between the profile of restriction fragments and geographic origin was observed for certain groups of strains. This PCR strategy allowed the identification of fungal strains, using as samples spores scraped from the surface of single insects killed by the fungus or single whole dead insects with external mycelium. The sequence of the PrI PCR product from two strains revealed that Prl gene has at least three introns.

We have established culture conditions for successful infection and development of several economically important cyst-forming and root-knot nematodes on Arabidopsis thaliana under monoxenic conditions. Complete life cycles were obtained with the sedentary cyst nematodes Heterodera schachtii, H. trifolii, H. cajani and the root-knot nematodes Meloidogyne incognita and M. arenaria as well as with the migratory nematode Pratylenchus penetrans. In contrast, H. goettingiana and Globodera rostochiensis were unable to develop on Arabidopsis roots. Tissue-culture quality agar and medium conditions optimized for hydroponic root culture were essential for successful infections. Detailed in-vivo observations were made inside Arabidopsis roots during the early infection stages of M. incognita and during complete development of H. schachtii. Seventy-four different ecotypes of Arabidopsis were screened for their susceptibility towards H. schachtii resulting in a range of infection rates. None of the ecotypes tested showed complete resistance in vitro. The use of Arabidopsis as a host for plant-parasitic nematodes will provide a new model system for the molecular genetic analysis of this interaction.

Faced with the conservative morphology of many plant nematode groups, taxonomy has focused much attention on a biochemical approach. Biochemical systematics is dependant on stable, subtle molecular differences which underlie taxonomic variation. Characterisation of carbohydrate, lipid, and especially proteins, have al1 found applications; however, like serological investigations these have met with mixed degrees of success (Hussey, 1979). Recent advances in DNA technology now make possible the rapid and reliable analysis of the nematode genome using restriction endonucleases (Curran, Baillie & Webster, .1985). The size and number of restriction fragments formed will be a direct reflection of the genomic base sequence. This offers the potential of a unique (( fïnger print )) of a genome independent of a phenotype.