1.1.37
STRUCTURAL ANALYSIS OF THE AVR4 ELICITOR OF CLADOSPORIUM FULVUM AND ITS PERCEPTION BY TOMATO PLANTS CARRYING THE MATCHING Cf-4 RESISTANCE GENE LOCUS

R ROTH1*, N WESTERINK1*, E WOESTENENK1, P VOSSEN1, C GOLSTEIN2, CM THOMAS2, JDG JONES2, MHAJ JOOSTEN1 and PJGM DE WIT1

1Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6709 PD Wageningen, The Netherlands; 2Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
*Equal author contribution.

Background and objectives
The interaction between tomato and the fungal pathogen Cladosporium fulvum is used as a model system to study the molecular basis of communication between plants and their pathogens. Strains of C. fulvum that produce the race-specific elicitor AVR4 induce an active resistance response in tomato genotypes that carry the matching resistance gene, Cf-4. AVR4 is produced as a pre-pro-protein that is processed by fungal and plant proteases to yield a mature elicitor peptide of 86 amino acids. Strains of C. fulvum that circumvent Cf4-specific resistance have been shown to contain single point mutations in the coding region of the Avr4 gene [1]. AVR4 isoforms could not be detected in apoplastic fluids isolated from colonized MM-Cf4 plants. Interestingly, as some of the AVR4 isoforms still induced necrosis when expressed in plants by recombinant potato virus X (PVX: :avr4), it was concluded that instability of the AVR4 isoforms produced by virulent strains of C. fulvum is a crucial factor in circumventing Cf-4-mediated resistance. Although the complete amino-acid sequence of AVR4 is known, its three-dimensional structure has not yet been determined. This has partly been due to the inability to obtain adequate amounts of the AVR4 protein. To this end, AVR4 elicitor protein will be overproduced in the methylotrophic yeast Pichia pastoris. In order to determine how AVR4 is perceived in Cf-4 tomato genotypes, binding studies will be performed with 125I-AVR4 elicitor, whilst insight into the three- dimensional structure of AVR4 will be obtained through lH-NMR spectroscopy.

Results and conclusions
Preliminary analysis of avr4 mutant alleles (in which single cysteine residues were replaced by an alanine residue) has shown that four of the eight cysteine residues (Cys 40, 70, 87 and 101) are essential for AVR4 elicitor activity. This suggests that disulfide bonds between these cysteine residues are crucial for correct folding and/or stability of the AVR4 elicitor. PVX::avr4 constructs with single point mutations in the codons for the Cys residues 50, 56, 64 and 109, still produced an active AVR4 elicitor. However, activity was lower as compared to wild-type, indicating that these Cys residues are also involved in disulfide bonds. Simultaneous substitution of two Cys residues indicated that Cys 50 and 56, and Cys 64 and 109, interact to form disulfide bonds. Additional amino acids will be analysed in a similar manner to investigate the importance for stability and/or specific AVR4 elicitor activity.

To date, expressing the ORF encoding the AVR4 pre-pro-protein in P. pastoris and subsequent purification of active AVR4 by chromatography have been successtul. Both the mature glycosylated and non-glycosylated AVR4 proteins are currently being expressed in P. pastoris.

The Cf-4 gene has been cloned and sequence analysis has revealed high homology of the encoded protein with CF-9 and CF-2 proteins. The Cf-4 locus has been shown to comprise tandem arrays of closely linked gene homologues, some of which are able to confer novel specificities [2]. Apart from the Avr4/Cf-4 gene pair, another member of the Cf-4 locus confers additional resistance specificity to C. fulvum races that produce an as-yet unknown elicitor protein (AVR4A). To confirm the presence of Avr4A in these C. fulvum races, the Avr4 gene will be deleted, which should result in strains that are still avirulent on MM-Cf4 plants. Progress on the isolation and characterization of AVR4A elicitor protein will be presented.

References
1. Joosten MHAJ, Vogelsang R, CozUnsen TJ et al., 1997. Plant Cell 9, 367-379.
2. Takken, FLW, Schipper D, Nijkamp HJJ et al., 1998. Plant Journal (in press).