1.9.31
LOCALIZED DEFENCE RESPONSES IN ONION BULB SCALE EPIDERMIS CHALLENGED BY BOTRYTIS ALLII

S McLUSKY1, MH BENNETT1, M BEALE2 and J MANSFIELD1

1Department of Biological Sciences, Wye College, University of London, Wye, Kent TN25 5AH, UK; 2IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BS18 9AF, UK

Background and objectives
When onion bulb scale epidermis is inoculated with Botrytis allii spore suspension, granular deposits, termed reaction material (RM), accumulate in very localized regions around the sites of attempted fungal penetration. As the fungus generally fails to penetrate at these sites, the RM deposits are believed to have a role in disease resistance [1]. These deposits autofluoresce blue in UV light, and yellow in blue light indicative of phenolic compounds. The aim of this work was to identify and quantify the accumulated phenolics and try to ascertain their role in planta. The role of the cytoskeleton in delivery of the RM granules to the challenge site has also been investigated.

Results and conclusions
Reversed-phase HPLC of methanolic extracts of onion epidermis revealed four major phenolic compounds which were present in the infected, but not in the control tissue. GCMS, LCMS and HPLC identified these compounds as feruloyl 3-methoxytyramine (FMT), feruloyl tyramine (FT), p-coumaryl tyramine (CT) and 2(4-hydroxyphenyl) ethyl ferulate. These compounds accumulate rapidly in challenged tissues up to a maximum 72 h post-inoculation. FMT is the most abundant. As these components autofluoresce blue under UV light they are believed to be components of the RM granules. Germination and TLC plate bioassays have shown that these compounds are not significantly fungitoxic.

After fungal challenge, onion cell walls accumulate autofluorescence and become more resistant to enzymic degradation [1]. Drastic alkaline hydrolysis of infected onion epidermal cell walls 72 h post inoculation released ferulic acid, p-coumaric acid, vanillin and p-hydroxybenzaldehyde. These are breakdown products of FMT, FT and CT, and imply that these amides are ether-linked into the cell walls, where their role is probably to strengthen them to resist fungal penetration.

Confocal microscopy with rhodamine-phalloidin labelling (specific for F-actin) revealed that, in uninfected onion epidermal cells, the actin microfilaments (MFs) are fine and randomly distributed; however, within a few hours of inoculation they thicken and become directed towards the challenge site. Thus the localization of RM granules to the site of attempted penetration appears to be directed along actin MFs.

References
1. Stewart A, Mansfield JW, 1985. Plant Pathology 34, 25-37.