5.4.12
INFECTION-INDUCED EXPRESSION OF THE AVIRULENCE GENE Avr9 IN TRANSGENIC CF9 TOMATO PLANTS CONFERS RESISTANCE TO FUNGAL PATHOGEN ATTACK

R WEIDE1, G HONEE1, M STUIVER2, H TIGELAAR2, LS MELCHERS2 and PJGM DE WIT1

1Department of Phytopathology, Agricultural University Wageningen, Binnenhaven 9, 6709 PD Wageningen, The Netherlands; 2Zeneca Mogen, Einsteinweg 97, 2333 CB Leiden, The Netherlands

Background and objectives
The most effective defence response of plants against pathogen attack is probably the hypersensitive response (HR), which is a localized collapse of tissue surrounding the infection site. The genetic basis of the HR-mediated disease resistance in tomato to the fungal pathogen Cladosporium fulvum is described by the gene-for-gene model [1]. Tomato plants carrying the Cf-9 resistance gene are resistant against attack of C. fulvum races which contain the avirulence gene Avr9. The Avr9 gene encodes a pre-pro-protein of 63 amino acids which, after secretion, is converted by proteases into a mature elicitor peptide of 28 amino acids. The Cf-9 gene encodes a putative membrane-anchored extracytoplasmic glycoprotein which consists of 28 imperfect leucine-rich repeats [2]. Injection of the AVR9 peptide into leaves of tomato plants carrying the Cf-9 resistance gene results in HR confined to the injected area. Constitutive expression of the Avr9 gene in transgenic Cf-9 plants results in delayed growth, necrosis and complete plant death [3]. Our objective is to investigate whether infection-induced expression of the avirulence gene Avr9 in transgenic Cf-9 tomato plants confers resistance to fungal pathogen attack.

Results and conclusions
Promoter activity of the defence-related gene gstl from potato is induced upon pathogen attack [4]. Constructs were designed containing the Avr9 coding region under the transcriptional control of a gstl promoter fragment. These constructs were used for Agrobactenum-mediated transformation of Cf-9 leaf discs, which resulted in transgenic plants showing a normal phenotype. Transgenic plants were selfed and progeny plants were used for inoculation with races of C. fulvum lacking the Avr9 gene. Several lines showed resistance to C. fulvum. Microscopical observations showed restricted fungal growth in transgenic leaves, while in wild-type Cf-9 leaves growth was abundant. Resistance was stably inherited and co-segregated with the presence of the Avr9 transgene. Transgenic lines which showed resistance against C. fulvum attack were also found to be resistant against other fungal pathogens, including Oidium lycopersicum. At the infection sites of resistant plants, cell death, unsuccessful colonization and restricted fungal growth were observed. Thus resistance mediated by the two well-defined components Avr9 and Cf-9 results in broad-spectrum pathogen control.

References
1. de Wit PJGM, 1992. Annual Review of Phytopathology 30, 391-418.
2. Jones DA, Thomas CM, Hammond-Kosack KE et al., 1994. Science 266, 789-93.
3. Honee G, Melchers LS, Vleeshouwers VGM et al., 1995. Plant Molecular Biology 29, 909-920.
4. Strittmater G, Gheysen G, Gianinazzi-Pearson V et al., 1996. Molecular Plant-Microbe Interactions 9, 68-73.