1.9.9
BIOTIC AND ABIOTIC ELICITATION OF CAMALEXIN IN PHYTOALEXIN-DEFICIENT (PAD) MUTANTS AND SEVERAL WILD-TYPE ACCESSIONS OF ARABIDOPSIS

F MERT-TURK1, MH BENNETT2, J GLAZEBROOK3, JW MANSFIELD2 and EB HOLUB1

1Horticulture Research International, Wellesbourne, Warwickshire CV35 9EQ, UK; 2Biological Sciences Department, Wye College, Kent TN25 5AH, UK; 3Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA

Background and objectives
Phytoalexins are small antimicrobial molecules which are synthesized by plants in response to pathogen infection. Although a number of observations support the hypothesis that phytoalexins play an important role in the defence response of plants to pathogens, previous experiments using phytoalexin-deficient (pad) mutants of Arabidopsis thaliana suggested that there was no correlation between pathogen avirulence and camalexin accumulation in response to isolates of Pseudomonas syringae [1]. It was suggested that the wild-type PAD genes are required for resistance to some but not all isolates of a biotrophic oomycete Peronospora parasitica (downy mildew). Our objective has been to investigate the role of camalexin in compatible and incompatible interactions between Peronospora and Arabidopsis, using the pad mutants and wild-type accessions of the host. We have also examined camalexin production in response to abiotic elicitors.

Material and methods
Mutants of the Arabidopsis accession Col-0 included four single pad mutants and three double mutants from combinations of Col-pad1, Col-pad2 and Col-pad3, as well as Col-ndr1 (non-specific disease resistance) and Col-npr1 (non-expressor of PR1 protein). Seedlings were inoculated with a Col-avirulent isolate (Emoy2) and phenotypic response was characterized according to the number of sporangiophores produced per cotyledon 7 days after inoculation (d.a.i.). Camalexin production was measured daily until 5 d.a.i. Ultraviolet B (UV-B) radiation and silver nitrate (AgNO3) were also used as abiotic elicitors to test the same Arabidopsis lines. Camalexin was quantified by high-performance liquid chromotography (HPLC) using a fluorescence detector (318 nm excitation and 385 nm emission wavelength).

Results and conclusions
Col-pad4 and Ws-eds1 exhibited a change to full susceptibility in response to Emoy2, whereas Col-pad3, Col-pad2, 3, Col-ndr1 and Col-npr1 exhibited moderate susceptibility; and Col-pad2 and Col-pad1, 3 exhibited only a weak enhancement of sporulation compared with wild type. Quantification of camalexin suggested that biosynthesis of this phytoalexin was not a specific response to avirulent isolates, but a more general response to pathogen attack. For example, Col-pad4 synthesized camalexin at a level similar to Col-0 at 1, 2 and 3 d.a.i. in response to Emoy2 and produced more than wild type at 4 and 5 d.a.i. A similar result was obtained with Ws-3 and the mutant Ws-eds1. Camalexin was not detectable in Col- pad3, and two double mutants (Col-pad1, 3 and Col-pad2, 3) in response to Emoy2 or either AgNO3 or UV-B radiation. Camalexin is clearly being induced during interaction with virulent pathogens, although this generally occurs more slowly than interactions with avirulent pathogens. The presence or absence of the phytoalexin does not appear to be closely correlated with the expression of resistance in Arabidopsis.

References
1. Glazebrook J, Zook M, Mert F et al., 1997. Genetics 146, 381-392.
2. Holub EB, Beynon JL, 1996. Advances in Botanical Research 24, 228-273.