1.3.25
IN SITU DETECTION OF AMINOPEPTIDASE ACTIVITY IN INFECTED PLANT CELLS

M ARAKAWA and H KUNOH

Faculty of Bioresources, Mie University, Tsu-city 514-8507, Japan

Background and objectives
Phenylalanine ammonia-lyase (PAL), a key enzyme of the phenylpropanoid pathway, plays a significant role in the expression of resistance of plant cells attacked by pathogens. In most host-pathogen interactions, enzyme activity has generally been analysed in whole-tissue preparations. However, as Graham and Graham [1] emphasized, events that determine host resistance and susceptibility often occur in very limited populations of cells or just in advance of the infection front, a very narrow zone of tissue defining the area of immediate contact between host and pathogen. Interactions taking place after infection progresses through tissue may be largely irrelevant to the outcome of the infection. Thus it is no longer sufficient to measure responses by gross tissue analyses, even in those cases where they represent discrete lesions, since these lesions may represent a composite of many cellular and molecular events. In most airborne fungal diseases, the interaction often initiates immediately after a single spore contacts a single host cell. Accordingly, the detection of activation of enzymes within a single cell is meaningful for understanding the initial events that lead to resistance or susceptibility. In this paper the activity of aminopeptidases, to which PAL belongs, was detected in single cells with a fluorescent probe.

Materials and methods
Aminopeptidases release fluorescent aminomethyl coumaril acetic acid (AMCA) of 445 nm emission wavelength from phenylalanine 4-methyl-coumaril 7-amide (Phe-MCA). Whether PAL, also an aminopeptidase, can release AMCA from MCA was examined. Phe-MCA and PAL from Rhodotorula glutinis were adsorbed to gel particles for chromatography (Tosoh, Toyopearl HW-50S) on glass slides and incubated at 30C, followed by heating in an oven at 100C to terminate the reaction. The intensity of fluorescence (445 nm) was measured after exposure to excitation light (345 nm). For comparison, the particles were incubated with Phe-MCA and PAL together with alpha-aminooxyacetic acid (AOA) or (x-aminooxy-r3-phenylpropionic acid (AOPP) to examine whether these inhibitors of PAL could suppress the reaction. Firstly, particles adsorbing Phe-MCA and PAL were heated at 100C before incubation to inactivate PAL. Secondly, partially dissected coleoptiles were prepared from 9-day old barley seedlings. They were then inoculated with conidia of Erysiphe graminis and incubated at 20C for 12 h. Specimens were fixed with 1% formaldehyde and washed with distilled water. Phe-MCA solution was dropped onto the fixed coleoptiles on glass slides, followed by incubation at 30C overnight. Specimens were observed and photographed with a fluorescence microscope. Partially dissected coleoptiles were also pretreated with AOA or AOPP and then inoculated and incubated with Phe-MCA to determine whether these inhibitors suppressed the intensity of fluorescence.

Results and conclusions
Intensity of fluorescence emitted from gel particles that had adsorbed Phe-MCA and PAL increased with the concentration of PAL applied. When the same particles were heated or treated with AOA or AOPP, the intensity of emitted fluorescence was markedly diminished. These results indicate that PAL was able to release AMCA from Phe-MCA. When the inoculated coleoptiles were treated with Phe-MCA, only the sites of cytoplasmic aggregation in coleoptile cells below appressoria exhibited intense fluorescence. The intensity of fluorescence in the cytoplasmic aggregation site was markedly reduced when coleoptiles were pretreated with AOA or AOPP. These results indicate that PAL could be activated locally in cytoplasmic aggregations at the initial infection site, and that Phe-MCA might be a suitable candidate to detect a local activity of PAL. However, the possibility remains that aminopeptidases other than PAL contribute to emission of fluorescence in cytoplasmic aggregates, since AOA and AOPP did not completely suppress fluorescence in these sites.

References
1. Graham TL, Graham MY, 1991. Molecular Plant-Microbe Interactions 4, 415-422.