J JONES, CM THOMAS, J PARKER, K HAMMOND-KOSACK, M COLEMAN, E VAN DER BIEZEN, W DURRANT, T ROMEIS and P PIEDRAS

Sainsbury Laboratory, John lnnes Centre, Colney Lane, Norwich NR4 7UH, UK

Background and objectives
Various mechanisms are used by plants to resist pathogens. One of the most interesting seems to involve recognition of pathogen-specified molecules by the plant. Disease resistance (R) genes confer recognition of pathogen races carrying the corresponding 'avirulence' (Avr) genes. This observation has prompted research into the basis of R gene-dependent recognition, and the subsequent signalling and defence responses. Since 1993, four classes of 'gene-for-gene' R genes have been isolated. All except the protein kinase-encoding Pto gene encode leucine-rich repeats (LRRs), a protein motif associated with protein/protein interactions. The largest class appears to be cytoplasmic, with LRRs at the C terminus and a consensus nucleotide-binding site (NBS) for interaction with ATP or GTP towards the N terminus.

NBS/LRR genes confer resistance to viruses, bacteria, fungi and nematodes. This class probably recognizes Avr gene-dependent molecules that enter the plant cell. Two other classes of R gene carry extracytoplasmic (e) LRRs and probably recognize Avr gene-specified molecules delivered from outside the plant cell. Xa21 encodes LRRs and a cytoplasmic protein kinase domain, whereas Cf-2, Cf-4, Cf-5 and Cf-9 carry membrane-anchored eLRRs. Since many R genes have been isolated, attention has shifted to what and how their products recognize, and how signals are transduced to initiate defence mechanisms.

We study tomato Cf-2, Cf-4, Cf-5 and Cf-9 genes conferring resistance to specific races of Cladosporium fulvum. Cf-4 and Cf-9 genes also confer responsiveness to the C. fulvum Avr4 and Avr9 peptides, respectively.

Results and conclusions
Sequence analysis at the Cf-9, Cf-4 and orthologous CfO loci has permitted comparison of 11 Cf-9 homologues, five at Cf-4 and Cf-9, and one at CfO. Most variation is seen at the amino acids that flank the conserved leucines in the LXXLXLXX beta-strand/beta-turn region. These amino acids are believed to be solvent exposed and may confer specificity. DNA sequence comparisons in the coding region show that sequence novelty has been generated by sequence exchange either by crossovers or gene conversion. Analysis of the promoter sequences revealed extensive polymorphism due to a network of deletions and insertions which could reduce the rate of out-of-phase pairing and retard array homogenization by gene conversion [1].

Cf-gene function is being investigated at the whole plant and cell culture levels. Salicylic acid (SA) is dispensable for full resistance, though SA is induced late in the Cf-dependent response to Avr peptides, and may play a role in some aspects of the response. Cf-9 is active on transfer to tobacco, and transgenic tobacco suspension cultures respond to Avr9 by producing reactive oxygen species (ROS). This has permitted an investigation into the role of various signalling molecules, particularly protein kinases, in Cf-9-dependent ROS production. The availability of active suspension culture lines carrying a c-myc-tagged Cf-9 should facilitate the search for proteins or peptides that associate with Cf-9 during or before activation. ROS production is inhibited by diphenylene iodonium (DPI) which inhibits the mammalian neutrophil superoxide synthesizing NADPH oxidase. Arabidopsis carries six homologues of the mammalian NADPH oxidase subunit, and these genes may encode a component of the ROS-producing machinery.

References
1. Parniske M, Hammond-Kosack KE, Goistein C et al., 1997. Cell 91, 821-832.