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Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XV. Interference of AOA and AOPP with the establishment of accessibility
Physiological and Molecular Plant Pathology (1997) 51, 227–241
Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XV. Interference of AOA and AOPP with the establishment of accessibility* M. A, S. S and H. K† Laboratory of Plant Pathology, Faculty of Bioresources, Mie Uniersity, Tsu-city, 514, Japan (Accepted for publication September 1997)
Several researchers have reported that the level of phenylalanine ammonia-lyase (PAL) is elevated in barley and wheat leaves within 3–4 h after inoculation with either a compatible or an incompatible race of Erysiphe graminis, and that the susceptibility of oat leaves to the powdery mildew fungus increases when leaves are treated with the PAL inhibitors, α-aminooxyacetic acid (AOA) or α-aminooxy-β-phenylpropionic acid (AOPP). In this paper we consider induction of accessibility in barley cells by inoculation with E. graminis f. sp. hordei, a fungal pathogen of barley, in relation to PAL activity in coleoptile cells. We studied events at the single spore – single host cell level, and examined influences exerted by the fungus at the prepenetration stage of development using AOA and AOPP to inhibit PAL activity in coleoptile cells. The degree of accessibility induced in cells successfully penetrated by E. graminis (first inoculum) was evaluated in terms of the penetration efficiency into those cells that was achieved by a subsequent challenge inoculum of either E. graminis or a non-pathogen of barley E. pisi. Treatment of lower surfaces of coleoptiles with an appropriate concentration of either PAL inhibitor did not affect the penetration capability of either fungus inoculated onto the upper surfaces of the same coleoptiles. Sequential H O-AOA or H O-AOPP treatment of inoculated coleoptiles revealed that the accessibility # # induced by the first inoculum was suppressed when AOA or AOPP treatment was initiated before 6 h after inoculation. The first inoculum attempted penetration from appressoria at 9–10 h after inoculation. The present results suggest that components released from E. graminis germlings could affect the physiological condition of coleoptile cells, possibly through phenylpropanoid metabolism, which ultimately affects accessibility. # 1997 Academic Press Limited
INTRODUCTION
In this paper the terms ‘ accessibility ’ and ‘ accessible state ’ are used to describe a plant cell condition which allows successful penetration by an attacking fungal germling, and the terms ‘ inaccessibility ’ and ‘ inaccessible state ’ are used to describe the condition of plant cells which the fungus fails to penetrate. These definitions are consistent with the concepts of accessibility and inaccessibility originally proposed by Ouchi et al. [24 ] and Kunoh et al. [20 ]. As described previously, accessibility is induced in barley coleoptile *Contribution No. 133 from the Laboratory of Plant Pathology, Mie University. †Author to whom correspondence should be addressed. Abbreviations used in the text : AOA, α-aminooxyacetic acid ; AOPP, α-aminooxy-β-phenylpropionic acid ; DMSO, dimethylsulfoxide ; PAL, phenylalanine ammonia-lyase ; PE, penetration efficiency. 0885–5765}97}10022715 $25.00}0}pp970125
# 1997 Academic Press Limited
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cells when Erysiphe graminis f. sp. hordei is used as the primary inoculum, regardless of whether the secondary inoculum is the same (pathogenic) fungus [22 ] or a nonpathogen such as E. pisi [16, 17 ]. This paper examines effects of the PAL inhibitors, AOA and AOPP, on the induction of accessibility in barley coleoptile cells in relation to a pathogen and a non-pathogen. In previous work [16–18, 20, 22 ], we studied accessibility induced by prior attack by a pathogen (E. graminis f. sp. hordei ) and inaccessibility enhanced by prior attack by a non-pathogen (E. pisi ) at the single cell – single spore level in barley coleoptiles. We found that when penetration failure occurred in a cell attacked by a non-pathogen, the inaccessible state of the host cell was enhanced leading to failure of subsequent penetration attempts by a compatible race of the pathogen [16–18 ]. In contrast, the accessible state of the host cell, which would allow E. pisi to form an haustorium, was induced by successful penetration by E. graminis. These results led us to assume that penetration attempts by either fungus could trigger physiological changes in host cells towards either the inaccessible or the accessible state. Later work demonstrated that E. pisi released elicitor components at the pre-penetration stage which conditioned host cells toward the inaccessible state [21, 30 ]. Similarly, E. graminis released a suppressor, prior to the time of penetration, and the suppressor blocked the effect of the E. pisi elicitor [15 ]. Thus penetration by E. graminis was not necessary as the suppressor induced the condition of accessibility. These studies suggest that some components released from conidia of both fungi and}or their germlings might affect the condition of the host plant cell before the fungus attempts penetration. In 1975, Green et al. [9 ] reported that E. graminis f. sp. tritici elicits an increase in the level of the activity of phenylalanine ammonia-lyase (PAL) in wheat leaves, the first enzyme in phenylpropanoid biosynthesis, as early as 4 h after inoculation. Thereafter, Shiraishi et al. [26, 27 ] and Clark et al. [7 ] confirmed this phenomenon by showing that within 3–4 h after inoculation of barley leaves with E. graminis f. sp. hordei there was an increase in the synthesis of cinnamic acid, the immediate product of the PAL reaction, and that extractable activity of PAL had increased by 6 h and again between 12–15 h after inoculation. PAL activity began to increase during the prepenetration stage, before fungal appressoria matured. A recent report by Carver et al. [6 ] also addressed the significance of PAL to infection by E. graminis in oats. They demonstrated that the PAL inhibitors, α-aminooxyacetic acid (AOA) and α-aminooxy-β-phenylpropionic acid (AOPP), increased the susceptibility of oat leaf tissue to the fungus and significantly reduced the occurrence of an autofluorescence response by the host’s cells. This was presumed to indicate that the accumulation of phenolic compounds that limited development of the pathogen had been blocked [6 ]. Each of these studies suggest that treatment of host cells with either AOA or AOPP during the early stages of host–pathogen interaction could interfere with the induction of accessibility by successful penetration of a pathogen into the host cell. In this paper we examine the effects of treatment of the host with PAL inhibitors on accessibility.
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MATERIALS AND METHODS
Plant and fungal materials Barley, Hordeum ulgare L., cv. Kobinkatagi, was grown from seed in vermiculite in a growth chamber under fluorescent lights (c. 11±8 Wm−#) and a 12 h photoperiod at 20³2 °C and 70 % relative humidity. The coleoptiles were excised from seedlings 8 days after sowing, and layers of the epidermal cells of partially dissected coleoptiles were prepared as described previously [28 ]. Erysiphe graminis de Candolle f. sp. hordei Em. Marchal, race I, was maintained on the compatible barley cv. Kobinkatagi and E. pisi de Candolle, race I, on the compatible pea cv. Midoriusui in separate growth chambers with the same environmental conditions. Inoculation Partially dissected coleoptiles were inoculated with conidia of test fungi by two methods : (i) by paint brush, (ii) by individual transfer onto target coleoptile cells by manipulation [15–18 ] using a 200¬light microscope, as described in detail below. Preparation of test inhibitors and determination of their test concentrations Two competitive inhibitors of PAL [1, 2, 4, 5, 10, 25 ] were used : α-aminooxy acetic acid (AOA : Nacalai Tesque Co., Japan) and α-aminooxy-β-phenylpropionic acid (AOPP : gift from Dr. T. L. W. Carver). AOA was dissolved in distilled water to give a 1 m stock solution. AOPP was moderately water soluble and was dissolved by adding it to water gradually while continuously stirring to give a 1 m stock solution. Both stock solutions were kept in the dark at 4 °C until used. Cytochalasin A (Sigma Co.) was used as an actin polymerization inhibitor [29, 31, 32 ]. It was dissolved in dimethylsulfoxide (DMSO) to give a stock solution of 2 mg ml−" which was stored at ®20 °C until used. All stock solutions were diluted with distilled water to required concentrations immediately before use. The final concentration of DMSO in the test solution of cytochalasin A was adjusted to less than 1 %. It was confirmed that 1 % DMSO did not affect the morphogenesis nor the penetration efficiency of test fungi. In control experiments, 1 % DMSO was added to distilled water. Determination of test concentrations of inhibitors The first experiment determined if AOA and AOPP applied to barley coleoptiles affected the accessibility induced by E. graminis attack in single cells. Coleoptiles treated with distilled water were used as controls and compared with coleoptiles treated with solutions of AOA or AOPP at 1, 10, 100, 1000 or 10 000 n. The resultant accessibility of coleoptile cells was evaluated based on the ability of E. pisi to penetrate the same cells using the procedure illustrated in Fig. 1. The experiment involved micromanipulation to transfer fungal germlings at precise times, and this limited the number of germlings that could be transferred in a single test. Therefore, data were accumulated from repeated tests until data from 100–200 individual coleoptile cells (from 10 coleoptiles) subjected to each treatment had been obtained.
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Determination of PE of E. pisi 37 h
F. 1. Time-course of inoculation of E. graminis, transfer of a challenger, E. pisi, and inoculation of both fungi. Erysiphe pisi was transferred onto the coleoptiles cells where E. graminis had formed an incipient haustorium. The penetration efficiency (PE) of E. pisi was determined 37 h after inoculation with E. graminis. ha ¯ haustorium.
In each test, two sets of coleoptiles (coleoptiles A and B, Fig. 1) were prepared. Three A coleoptiles were inoculated, using a brush, with 15–20 freshly harvested conidia of E. graminis and immediately incubated by floating on 300 µl of either water or an inhibitor solution (1–10 000 n AOA or AOPP in watch glasses at 20³2 °C and 70 % RH for 15 h. Three B coleoptiles were inoculated similarly with about 50 freshly harvested conidia of E. pisi 13 h after inoculation of the A coleoptiles and incubated on water in Petri dishes under the same conditions as above for 2 h. The timing of inoculation on the B coleoptiles ensured that germlings of E. pisi reached a stage suitable for transfer at 15 h after inoculation of E. graminis on the A coleoptiles. Both coleoptiles were mounted in water on a glass slide 15 h after inoculation with E. graminis, and germlings of E. pisi were individually transferred by manipulation onto cells of the A coleoptiles where E. graminis had formed incipient haustoria. In this case, several germlings of E. graminis on the A coleoptiles were selected such that no other germlings were located within a distance of five host cells away. After a further 22 h incubation of the A coleoptiles they were mounted on glass slides with water, and haustorium formation by E. pisi in the target cells was observed with an Olympus BH-2 light microscope. Penetration efficiency (PE) of this fungus was evaluated as follows : PE ¯ No. of haustoria . Total No. of observed haustoria and}or papillae below lobed appressoria¬100 % The accessibility of cells subjected to the different treatments was statistically analysed by a t-test applied to PEs between treatments. The PE values given are means³standard error. Although this first experiment allowed identification of AOA and AOPP concentrations which affected accessibility induced by E. graminis, it was necessary to test the effect of the inhibitors on the base level of PE achieved by E. graminis inoculated alone. Therefore, a second experiment was conducted where coleoptiles inoculated with E. graminis were floated on water or 1–10% n AOA or AOPP in watch glasses and incubated under the same conditions as above for 24 h before PE was determined. A main objective of this study was to elucidate the effects of AOA and AOPP on the
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physiological condition of coleoptile cells in relation to induced accessibility. However, there was a possibility that E. pisi, used as the probe to determine accessibility of coleoptile cells, might be affected directly by AOA or AOPP. The fact that this fungus always fails to form haustoria in barley coleoptiles [12, 14 ] made it impossible to evaluate potential direct negative effects of the inhibitors on penetration capability of E. pisi on coleoptiles. However, as we demonstrated previously [14 ], E. pisi can penetrate to form haustoria in coleoptiles treated with cytochalasins (inhibitors of actin polymerization). Therefore, in the third experiment, coleoptiles inoculated with E. pisi were treated with mixtures of 10 n of AOA or AOPP with 1 µg ml−" cytochalasin. After 24 h incubation (incubation conditions described above) on test solutions, the PE of E. pisi was assessed. Timing of coleoptile treatment with AOA or AOPP in relation to induced accessibility to E. pisi The timing of inhibitor application to coleoptiles in relation to the effects of inhibitors on induced changes in cell accessibility to E. pisi was examined by the procedure illustrated in Fig. 2. Coleoptiles C were inoculated with E. graminis before they were
E. pisi Coleoptile D Incubation 2 h E. graminis Determination of PE of E. pisi
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3h 6h 9h 12 h 18 h 24 h Control F. 2. The H O-AOA or -AOPP sequential treatment of inoculated coleoptiles. Coleoptile C # was first inoculated with E. graminis, and E. pisi which had been incubated on coleoptile D for 2 h was transferred onto the cells of coleoptile C where E. graminis had formed incipient haustoria. Coleoptile C’s were floated on H O for the first period and transferred onto AOA or AOPP # solution, followed by incubation for the remaining period. PE of E. pisi in coleoptile C was determined 37 h after inoculation with E. graminis. ha ¯ haustorium. (*) Incubated with water ; (8) incubated with AOA or AOPP.
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floated on water for designated times and prior to transfer to 10 n AOA or AOPP for the remaining incubation period (incubation conditions described above). At 15 h after inoculation with E. graminis, a conidium of E. pisi which had germinated on a separate D coleoptile (floated on water) was transferred onto the coleoptile cell on which a conidium of E. graminis had formed an haustorial primordium. PE of E. pisi was then determined after a further 22 h incubation of the C coleoptile. Data were accumulated from 100–130 individual coleoptile cells (9–15 coleoptiles). Influence of AOA or AOPP on accessibility to challenging E. graminis In addition to inducing accessibility to the non-pathogen E. pisi, previous work [22 ] showed that accessibility induced by primary inoculation with E. graminis also effectively enhances the PE of E. graminis when applied as a second, challenge inoculum. Therefore, an experiment was conducted to test whether AOA or AOPP treatment of barley coleoptiles influenced induced accessibility of E. graminis conditioned by a previous inoculation with E. graminis using the procedure illustrated in Fig. 3. E. graminis 2 Coleoptile F Incubation 5.5 h E. graminis 1 ha
Coleoptile E 0h
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PE of E. graminis 2 was determined 37 h
F. 3. Sequential inoculation with E. graminis 1 (the first inoculum), transfer of a challenging E. graminis 2, and incubation of both. Erysiphe graminis 2 was transferred onto the coleoptile cells where E. graminis 1 had formed an incipient haustorium. PE of E. graminis 2 was determined 37 h after inoculation of E. graminis 1.
Two sets of coleoptiles (coleoptiles E and F : Fig. 3) were prepared. Three E coleoptiles were inoculated with 15–20 freshly harvested E. graminis conidia, floated on 300 µl of water or 10 n AOA or AOPP in watch glasses, and incubated at 20³2 °C and 70 % RH for 18±5 h. Three F coleoptiles were inoculated with about 50 freshly harvested E. graminis conidia 13 h after inoculation of the E coleoptiles and incubated on water in Petri dishes under the same conditions as above for 5±5 h. This timing of inoculation on the F coleoptiles ensured that germlings on these coleoptiles reached the stage suitable for transfer at 18±5 h after inoculation with E. graminis on the E coleoptiles. Both coleoptiles were mounted in water on a glass slide 18±5 h after inoculation of the E coleoptiles, and germlings of E. graminis on the F coleoptiles were individually transferred onto the cells of the E coleoptiles where previously inoculated E. graminis had formed incipient haustoria. PE of E. graminis was evaluated after a further 18±5 h incubation. For controls, germlings on the F coleoptiles were transferred onto cells of uninoculated coleoptiles floated on water, 10 n AOA or 10 n AOPP. Data were accumulated from 100–120 individual coleoptile cells (9–13 coleoptiles).
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RESULTS
Effects of AOA and AOPP on the induction of accessibility by E. graminis The protocol indicated in Fig. 1 was used to examine the influence of AOA and AOPP on induction of accessibility to E. pisi when conditioned by prior attack by E. graminis. As mentioned earlier, E. pisi normally fails completely to penetrate coleoptiles of barley (a non-host) when they are not previously infected by E. graminis. However, the PE of E. pisi transferred onto cells of coleoptile A in which E. graminis had previously formed incipient haustoria was increased to 43±2³8±3 % (Figs 4, 5). Thus, we judged that these
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F. 4. Effects of different concentrations of AOA and AOPP on the induction of accessibility by the first inoculum, E. graminis, in coleoptile cells of barley. The accessible cell condition was assessed by penetration efficiency of challenging E. pisi in the cells where the first inoculum had formed an haustorium. (*) Significantly different from PE in water control. (*) Water ; (8) AOA ; (+) AOPP.
cells had been directed towards an accessible state by successful penetration with E. graminis. In contrast, when the A coleoptile was treated with 1 n AOA or AOPP during incubation, the PE of E. pisi decreased to 28±5³5±5 % and 26±4³8±8 %, respectively. Furthermore, when the A coleoptile was treated with 10 or 100 n AOA and AOPP, the PE was drastically reduced (P ! 0±05) to 3±3³2±4–3±7³2±6 % and 2±8³3±0– 5±3³2±9 %, respectively. Thus, these concentrations of both inhibitors effectively
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F. 5. Series of interference contrast micrographs showing successive growth stages of E. graminis (g) and E. pisi (p) on single coleoptile cells. ap ¯ appressorium ; h ¯ haustorium ; sh ¯ secondary hypha. Scale in frame A ¯ 40 µm. Accessibility was induced in a coleoptile cell by successful penetration by the primary inoculum, E. graminis. (Frames A–D). By 15 h after inoculation, E. graminis succeeded in penetrating and forming a haustorium in a coleoptile cell (A). The secondary inoculum, E. pisi, was transferred onto the same cell around 15 h (B). The transferred E. pisi developed a lobed appressorium by 21 h (C). When both E. graminis and E. pisi germlings developed an haustorium in the same cell, secondary hyphae elongated from both by 37 h (D). Accessibility was also induced in a coleoptile cell by successful penetration by the primary inoculum, E. graminis g1 (E–G). By 18±5 h after inoculation, g1 succeeded in penetrating and forming an haustorium in a coleoptile cell (E). The secondary inoculum, E. graminis g2, was transferred onto the same cell around 18±5 h (F). Secondary hyphae elongated from both g1 and g2 by 37 h (G).
suppressed the induction of accessibility brought on by the previous inoculation with E. graminis. Induction of accessibility never occurred in coleoptiles treated with inhibitor concentrations higher than 100 n. Therefore, we decided to use 10 n AOA and AOPP, the lowest concentration to interfere with induction of accessibility, as a standard concentration for subsequent experiments to examine the effects of the inhibitors on induced accessibility. Effects of AOA and AOPP on the penetration efficiency (PE) of E. graminis and E. pisi Figure 6 shows PE values obtained for E. graminis attacking coleoptiles treated with distilled water (controls) or with various concentrations of AOA or AOPP. The PE of E. graminis in coleoptiles floated on water was 57±3³6±2 %. When coleoptiles were
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F. 6. Effects of different concentrations of AOA and AOPP on the penetration efficiency of E. graminis in coleoptile cells of barley. (*) Significantly different from PE in water control. (*) Water ; (8) AOA ; (+) AOPP.
treated with 1 n AOA, the PE of E. graminis was similar to the control (PE ¯ 63±7³8±9 % ; P " 0±05). However, treatment with either 10 or 100 n AOA significantly increased (P ! 0±05) the PE of E. graminis compared to the control (PE ¯ 76±6³4±2 % and 76±7³5±1 % for 10 and 100 n, respectively). However, AOPP had no detectable effect on the PE of E. graminis, as PE values were statistically indistinguishable from the control (Fig. 6). It is possible that the inability of AOPP to increase accessibility to E. graminis was a result of the compound’s relative insolubility as compared to that of AOA. Treatment of coleoptiles with 1 µg ml−" cytochalasin A allowed E. pisi to form haustoria with a PE of 37±2³7±8 %, a condition previously shown to occur only after inoculation first with E. graminis (Fig. 4). When coleoptiles were treated with a mixture of 1 µg ml−" cytochalasin A and 10 n of AOA or AOPP, PEs of 32±0³2±5 % and 36±3³7±5 %, respectively, were obtained. These were not significantly different from treatment with cytochalasin alone, demonstrating that at this concentration neither PAL inhibitor interfered with the penetration capability of E. pisi. These results confirmed that suppression of induced accessibility brought about by treatment of coleoptiles with 10 n of AOA or AOPP could not be explained by direct, negative effects on the morphogenesis of test fungi inoculated on these coleoptiles, but was likely to be dependent on the physiological condition of the coleoptile cells.
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F. 7. Effects of the timing and duration of sequential H O-AOA or H O-AOPP treatment on # # the accessibility induced by the first inoculum, E. graminis. The accessibility induced was assessed by measuring the penetration efficiency of challenging E. pisi in the cells where the first inoculum had formed a haustorium. (*) Significantly different from PE in water control. (*) Water ; (8) AOA ; (+) AOPP.
Effects of timing and duration of AOA or AOPP treatment on induction of accessibility to E. pisi The procedure illustrated in Fig. 2 was used to determine if there was a critical period during the interaction between coleoptile cells and E. graminis germlings applied as primary inoculum that determined the induction of coleoptile cell accessibility to E. pisi. Figure 7 shows that the PE of E. pisi on coleoptile cells in which E. graminis had formed incipient haustoria was 43±2³8±3 %, when the coleoptiles were floated on water during the entire incubation period. However, when coleoptiles were treated with AOA or AOPP immediately after inoculation, induction of accessibility to E. pisi was almost completely suppressed (PE ¯ 3±3³2±5 % and 5±3³2±9 %, for AOA and AOPP, respectively) giving PE values that were significantly lower (P ! 0±05) than in watertreated cells. When the AOA or AOPP treatment was initiated 3 h after the onset of incubation, PEs again were significantly (P ! 0±05) lower than in water-treated cells. (PE ¯ 6±7³5±0 % and 14±1³4±5 % for AOA and AOPP, respectively). In contrast, when coleoptiles were initially incubated for 6 h or more on water before transfer to AOA or AOPP, the PEs were not significantly different (P " 0±05) from those obtained
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where coleoptiles were held continuously on water (Fig. 7). Therefore, we concluded that coleoptile cell—E. graminis interaction(s) occurring within 6 h after inoculation are important in inducing physiological changes leading to accessibility of the cell to E. pisi. Effects of AOA and AOPP on the induction of accessibility to E. graminis In the above experiments, E. pisi was used to test for induction of accessibility. As mentioned above, accessibility using E. graminis as the primary inoculum also increases the PE of E. graminis when applied as a secondary inoculum [22 ]. The experiments reported in this section examined whether AOA and AOPP interfered with induction of accessibility of coleoptile cells to E. graminis. In a preliminary experiment, we tested whether AOA or AOPP treatment affected the PE of transferred E. graminis germlings using a modification of the procedure illustrated in Fig. 3. Erysiphe graminis germlings which had been incubated on the F coleoptile floating on water and where no previous inoculation had been made were transferred onto cells of a separate E coleoptile, followed by a further incubation for 18±5 h. In this control, PE was 55±7³8±3 % (Fig. 8). When similar germlings were transferred onto the E coleoptiles floating on 10 n AOA or AOPP where no previous inoculation had been made, PEs were 66±7³12±3 % and 68±3³11±9 %, respectively. 100
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F. 8. Effects of AOA or AOPP on the induction of accessibility which is effective to promote successful penetration by challenging E. graminis in the same cells where the first inoculum, E. graminis, has formed a haustorium. (*) Significantly different from PE in water control. (+) Transferred control ; (8) challenging.
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These values were not significantly ( P " 0±05) higher than that of the water control and they demonstrate that 10 n of either inhibitor did not directly affect the penetration capability of transferred E. graminis. When germlings of challenging E. graminis were transferred onto cells of a waterfloated E coleoptile (Fig. 3) where the primary inoculum of E. graminis previously had formed incipient haustoria, PE of the former was 88±8³4±9 % after an additional 18±5 h incubation (Fig. 8). However, when similar transfers were made to the E coleoptiles which had been floated on 10 n AOA or AOPP solutions, PEs of challenging E. graminis were reduced to 72±1³5±0 % and 67±4³4±5 %, respectively. Both values were significantly (P ! 0±05) lower than that of the water control, demonstrating that both inhibitors also interfered with the induction of the accessible cell condition for the challenging E. graminis conidia. DISCUSSION
AOPP and AOA are effective competitive inhibitors of PAL in most plant species [1, 2, 4, 10, 25 ]. Both inhibited oat PAL in itro, although like the results with cowpea PAL [25 ], AOPP was a more effective inhibitor than AOA. AOPP is the more specific inhibitor of PAL [2 ], whereas AOA is a general transamination inhibitor affecting various other metabolic processes [3, 11 ]. Thus, care must be taken when interpreting AOA results [4 ]. The present results showed that treatment of dissected coleoptiles with AOA or AOPP within 6 h after inoculation interfered with the induction of accessibility, which is normally the consequence of an initial attack by E. graminis. Kobayashi et al. [13 ] demonstrated that conidia of E. graminis produce primary germ tubes between 0±5 and 1±0 h after inoculation of coleoptiles and subsequently form appressorial germ tubes after about 4±2 h. The latter germ tubes stop elongation by 6±5 h, form a lobe at their swollen tip, and become mature appressoria by 6±9 h on average. In previous papers [13, 15 ] we demonstrated that both elicitor and suppressor factors were released from germlings of E. graminis with an immature appressorium. This earlier work led us to assume that some components released from E. graminis germlings could affect the physiological condition of the underlying coleoptile cells [13, 15 ]. Because of the effects of the PAL inhibitors, the present results suggest that accessibility may be linked to phenolic compound biosynthesis, especially phenylpropanoid biosynthesis. Green et al. [9 ] reported that inoculation of wheat leaves with E. graminis f. sp. tritici enhanced the level of PAL in leaves within 4 h. Shiraishi et al. [26 ] then demonstrated that within 3–4 h after inoculation of barley leaves with E. graminis f sp. hordei, synthesis of cinnamic acid was significantly enhanced and PAL activity increased by as early as 2 h. Furthermore, Shiraishi et al. [27 ] reported that inoculation of barley leaves with the same fungus induced a two phase increase in PAL activity in leaves, the first at 6 h and the second between 12–15 h after inoculation. Similar results were obtained by Clark et al. [7 ], who showed biphasic patterns of PAL transcript accumulation at 2 h and 12 h in seedling leaves of four near-isogenic barley lines inoculated with the same fungus. They also showed that temporal patterns of PAL enzyme activity were roughly similar to those of PAL transcript accumulation. Based on these results, Clark et al. [7 ] concluded that PAL induction was associated with a general defence to infection.
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However, a strong correlation between PAL induction and major gene resistance was not found. As indicated in Fig. 7, the induction of accessibility was suppressed significantly when coleoptiles inoculated with E. graminis were treated with AOA and AOPP within 0–3 h of inoculation. When such treatments were initiated later, there was no significant effect. However, if AOA or AOPP were applied 9 h after the primary inoculation, the PEs of E. pisi were 24±4³5±7 % and 25±2³6±5 %, respectively (Fig. 7). These values were not significantly lower than the PE of the control at the 5 % level, but were lower at the 10 % level of probability. Although a more detailed examination is needed, the marginally lower PEs given by these treatments might be related to the biphasic PAL-associated metabolic changes noted by others [7, 27 ].It is possible that biphasic elevation of PAL activity might be dependent on different isomers of PAL being produced in the different phases. At present there is no evidence for this, but different isomers have been demonstrated in pea and tobacco [8, 33 ]. At present it is uncertain whether the first phase between 0–3 h is associated with the penetration attempt by primary germ tubes as was discussed by Shiraishi et al. [27 ], Clark et al. [7 ] and Carver et al. [5 ], or whether it is related to other factors such as a response to exudates released by conidia [19, 23 ]. As described previously [16, 17, 22 ], accessibility induced in coleoptile cells by E. graminis applied as the primary inoculum effectively enhanced successful penetration by both E. graminis and E. pisi applied as secondary inoculum to the same cells. However, when coleoptiles first inoculated with E. graminis were floated on either 10 n AOA or AOPP immediately after inoculation, the accessible cell condition for secondary E. pisi or E. graminis was not induced. These results suggest that the accessible cell condition for these two fungi may be related to a common metabolic feature which is interfered with by AOA or AOPP. Although the present results suggested that effects on host cell metabolism as early as 0–3 h after primary inoculation was possibly associated with the host cell condition at the post-penetration stage, further studies are required to elucidate how this physiological connection might be regulated at the biochemical level. It is also necessary to determine whether the concentrations of AOA and AOPP applied in this study were high enough to inhibit PAL activity in barley coleoptiles totally. The biologically effective concentrations used here (10 n for both inhibitors), were much lower than the concentrations reported [5 ] to give 50 % inhibition of PAL activity in itro (200 n AOPP and 500 µ AOA). The authors thank Dr. T. L. W. Carver, Institute of Grassland & Environmental Research, UK and Dr. R. L. Nicholson, Purdue University, USA for their critical reading of the manuscript and correcting the English in the text. This work was partially supported by Grant-in-Aid for Scientific Research (B) (No. 08456025) from the Ministry of Education and Culture of Japan. REFERENCES 1. Amrhein N, Go$ decke KH, Keffeli VI. 1976. The estimation of relative intracellular phenylalanine ammonia-lyase (PAL)-activities and their modulation in io and in itro by competitive inhibitors. Brichte der Deutschen Gesellschaft 89 : 247–259.
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2. Amrhein N, Go$ decke KH. 1977. α-Aminooxy-β-phenylpropionic acid – a potent inhibitor of Lphenylalanine ammonia-lyase in itro and in io. Plant Science Letters 8 : 313–317. 3. Amrhein N. 1979. Novel inhibition of phenylpropanoid metabolism in plants. In Regulation of Secondary Products and Plant Hormone Metabolism, Luckner M, Schreiber L, eds. Oxford : Pergamon Press, 173–182. 4. Carver TLW, Robbins MP, Zeyen RJ. 1991. Effects of two PAL inhibitors on the susceptibility and localized autofluorescent host cell responses of oat leaves attacked by Erysiphe graminis DC. Physiological and Molecular Plant Pathology 39 : 269–287. 5. Carver TLW, Zeyen RJ, Bushnell WR, Robbins MP. 1994. Inhibition of phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase increase quantitative susceptibility of barley to powdery mildew (Erysiphe graminis D.C.) Physiological and Molecular Plant Pathology 44 : 261–272. 6. Carver TLW, Zeyen RJ, Robbins MP, Vance CP, Boyles DA. 1994. Suppression of host cinnamyl alcohol dehydrogenase and phenylalanine ammonia lyase increases oat epidermal cell susceptibility to powdery mildew penetration. Physiological and Molecular Plant Pathology 44 : 243–259. 7. Clark TA, Zeyen RJ, Smith AG, Carver TLW, Vance CP. 1994. Phenylalanine ammonia lyase mRNA accumulation, enzyme activity and cytoplasmic responses in barley isolines, differing at Mla and Ml-o loci, attacked by Erysiphe graminis f. sp. hordei. Physiological and Molecular Plant Pathology 44 : 171–185. 8. Fukazawa-akada T, Kung SD, Watoson JC. 1996. Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana. Plant Molecular Biology 30 : 711–722. 9. Green N, Hadwiger LA, Graham SO. 1975. Phenylalanine ammonia lyase, tyrosine ammonia lyase, and lignin in wheat inoculated with Erysiphe graminis f. sp. tritici. Phytopathology 65 : 1071–1074. 10. Havir EA. 1981. Modification of L-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and L-2-aminooxy-3-phenylpropinate. Planta 152 : 124–130. 11. John RA, Charteris A, Fowler LJ. 1978. The reaction of amino-oxyacetate with pyridoxal phosphatedependent enzymes. Biochemical Journal 171 : 771–779. 12. Kobayashi I, Kobayashi Y, Yamaoka N, Kunoh H. 1992. Recognition of a pathogen and a nonpathogen by barley coleoptile cells. III. Responses of microtubules and actin filaments in barley coleoptile cells to penetration attempts. Canadian Journal of Botany 70 : 1815–1823. 13. Kobayashi I, Watanabe H, Kunoh H. 1995. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XIV. Evidence for elicitor(s) and suppressor(s) of host inaccessibility from Erysiphe graminis. Physiological and Molecular Plant Pathology 46 : 445–456. 14. Kobayashi I, Kobayashi Y, Yamada M, Kunoh H. 1996. The involvement of the cytoskeleton in the expression of nonhost resistance in plants. In : Mills D, Kunoh H, Keen NT, Mayama S, eds. Molecular Aspects of Pathogenicity and Resistance : Requirement for Signal Transduction. St. Paul, Minnesota : The American Phytopathological Press, 185–195. 15. Komura T, Kobayashi I, Yamaoka N, Kunoh H. 1990. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. VIII. Cytological evidence for suppressor(s) of host inaccessibility from Erysiphe graminis. Physiological and Molecular Plant Pathology 37 : 409–416. 16. Kunoh H, Hayashimoto A, Harui M, Ishizaki H. 1985. Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. I. The significance of timing of fungal invasion. Physiological Plant Pathology 27 : 43–54. 17. Kunoh H, Kuroda K, Hayashimoto A,Ishizaki H. 1986. Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. II. The timing and localization of induced susceptibility in a single coleoptile cell and its transfer to an adjacent cell. Canadian Journal of Botany 64 : 889–895. 18. Kunoh H, Katsuragawa N, Yamaoka N, Hayashimoto A. 1988. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. III. Timing and localization of enhanced inaccessibility in a single coleoptile cell and its transfer to an adjacent cell. Physiological and Molecular Plant Pathology 33 : 81–93. 19. Kunoh H, Yamaoka N, Yoshioka H, Nicholson RL. 1988. Preparation of the infection court by Erysiphe graminis. I. Contact-mediated changes in morphology of the conidium surface. Experimental Mycology 12 : 325–335. 20. Kunoh H, Toyoda K, Yamaoka N, Kobayashi I. 1989. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. V. Duration of stimulus by a non-pathogen in relation to enhanced inaccessibility. Physiological and Molecular Plant Pathology 35 : 507–518. 21. Kunoh H, Komura T, Kobayashi I, Yamaoka N. 1990. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. VII. Enhancement of inaccessibility at the prepenetration stage of a nonpathogen Physiological and Molecular Plant Pathology 37 : 399–407. 22. Kunoh H, Kuroda K, Toyoda K, Yamaoka N, Kobayashi I. 1991. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. IX. Accessibility induced by Erysiphe
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27. 28. 29. 30. 31. 32. 33.
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graminis which promotes the subsequent infection of challenging E. graminis. Annals of the Phytopathological Society of Japan 57 : 57–60. Nicholson RL, Yoshioka H, Yamaoka N, Kunoh H. 1988. Preparation of the infection court by Erysiphe graminis. II. Release of esterase enzyme from conidia in response to a contact stimulus. Experimental Mycology 12 : 336–349. Ouchi S, Oku H, Hibino C, Akiyama I. 1974. Induction of accessibility and resistance in leaves of barley by some races of Erysiphe graminis. Phytopathology Zeitschrift 79 : 24–34. Ralton JE, Howlett BJ, Clarke AE, Irwin JA, Imrie B. 1988. Interaction of cowpea with Phytophthora ignae : inheritance of resistance and production of phenylalanine ammonia-lyase as a resistance response. Physiological and Molecular Plant Pathology 32 : 89–103. Shiraishi T, Yamaoka N, Kunoh H. 1989. Association between increased phenylalanine ammonialyase activity and cinnamic acid synthesis and the induction of temporary inaccessibility caused by Erysiphe graminis primary germ tube penetration of the barley leaf. Physiological and Molecular Plant Pathology 34 : 75–83. Shiraishi T, Yamada T, Nicholson RL, Kunoh H. 1995. Phenylalanine ammonia-lyase in barley : activity enhancement in response to Erysiphe graminis f. sp. hordei (race 1) a pathogen, and Erysiphe pisi, a nonpathogen. Physiological and Molecular Plant Pathology 46 : 153–162. Takamatsu S, Ishizaki H, Kunoh H. 1978. Cytological studies of early stages of powdery mildew in barley and wheat. V. Effects of calcium on the infection of coleoptile of barley by Erysiphe graminis hordei. Canadian Journal of Botary 56 : 2544–2549. Thomas DD. 1978. Cytochalasin effects in plants and eukaryotic microbial systems. In : Tanenbaum SW, ed. Cytochalasins-Biochemical and Cell Biological Aspects. North-Holland : Biomedical Press, 257–275. Toyoda K, Shiraishi T, Kobayashi I, Yamaoka N, Kunoh H. 1993. A water-soluble extract from germlings of Erysiphe pisi suppresses infection by E. graminis in coleoptile cells of barley. Annals of the Phytopathological Society of Japan 59 : 135–142. Wieland T. 1977. Modification of actins by phallotoxins. Naturwissenschaften 64 : 303–309. Yahara I, Harada F, Sekita S, Yoshihira K, Natori S. 1982. Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro. Journal of Cell Biology 92 : 69–78. Yamada T, Sriprasertsak P, Kato H, Hashimoto T, Shimizu H, Shiraishi T. 1994. Functional analysis of the promoters of phenylalanine ammonia-lyase genes in pea. Plant and Cell Physiology 35 : 917–926.
Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XV. Interference of AOA and AOPP with the establishment of accessibility* M. A, S. S and H. K† Laboratory of Plant Pathology, Faculty of Bioresources, Mie Uniersity, Tsu-city, 514, Japan (Accepted for publication September 1997)
Several researchers have reported that the level of phenylalanine ammonia-lyase (PAL) is elevated in barley and wheat leaves within 3–4 h after inoculation with either a compatible or an incompatible race of Erysiphe graminis, and that the susceptibility of oat leaves to the powdery mildew fungus increases when leaves are treated with the PAL inhibitors, α-aminooxyacetic acid (AOA) or α-aminooxy-β-phenylpropionic acid (AOPP). In this paper we consider induction of accessibility in barley cells by inoculation with E. graminis f. sp. hordei, a fungal pathogen of barley, in relation to PAL activity in coleoptile cells. We studied events at the single spore – single host cell level, and examined influences exerted by the fungus at the prepenetration stage of development using AOA and AOPP to inhibit PAL activity in coleoptile cells. The degree of accessibility induced in cells successfully penetrated by E. graminis (first inoculum) was evaluated in terms of the penetration efficiency into those cells that was achieved by a subsequent challenge inoculum of either E. graminis or a non-pathogen of barley E. pisi. Treatment of lower surfaces of coleoptiles with an appropriate concentration of either PAL inhibitor did not affect the penetration capability of either fungus inoculated onto the upper surfaces of the same coleoptiles. Sequential H O-AOA or H O-AOPP treatment of inoculated coleoptiles revealed that the accessibility # # induced by the first inoculum was suppressed when AOA or AOPP treatment was initiated before 6 h after inoculation. The first inoculum attempted penetration from appressoria at 9–10 h after inoculation. The present results suggest that components released from E. graminis germlings could affect the physiological condition of coleoptile cells, possibly through phenylpropanoid metabolism, which ultimately affects accessibility. # 1997 Academic Press Limited
INTRODUCTION
In this paper the terms ‘ accessibility ’ and ‘ accessible state ’ are used to describe a plant cell condition which allows successful penetration by an attacking fungal germling, and the terms ‘ inaccessibility ’ and ‘ inaccessible state ’ are used to describe the condition of plant cells which the fungus fails to penetrate. These definitions are consistent with the concepts of accessibility and inaccessibility originally proposed by Ouchi et al. [24 ] and Kunoh et al. [20 ]. As described previously, accessibility is induced in barley coleoptile *Contribution No. 133 from the Laboratory of Plant Pathology, Mie University. †Author to whom correspondence should be addressed. Abbreviations used in the text : AOA, α-aminooxyacetic acid ; AOPP, α-aminooxy-β-phenylpropionic acid ; DMSO, dimethylsulfoxide ; PAL, phenylalanine ammonia-lyase ; PE, penetration efficiency. 0885–5765}97}10022715 $25.00}0}pp970125
# 1997 Academic Press Limited
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cells when Erysiphe graminis f. sp. hordei is used as the primary inoculum, regardless of whether the secondary inoculum is the same (pathogenic) fungus [22 ] or a nonpathogen such as E. pisi [16, 17 ]. This paper examines effects of the PAL inhibitors, AOA and AOPP, on the induction of accessibility in barley coleoptile cells in relation to a pathogen and a non-pathogen. In previous work [16–18, 20, 22 ], we studied accessibility induced by prior attack by a pathogen (E. graminis f. sp. hordei ) and inaccessibility enhanced by prior attack by a non-pathogen (E. pisi ) at the single cell – single spore level in barley coleoptiles. We found that when penetration failure occurred in a cell attacked by a non-pathogen, the inaccessible state of the host cell was enhanced leading to failure of subsequent penetration attempts by a compatible race of the pathogen [16–18 ]. In contrast, the accessible state of the host cell, which would allow E. pisi to form an haustorium, was induced by successful penetration by E. graminis. These results led us to assume that penetration attempts by either fungus could trigger physiological changes in host cells towards either the inaccessible or the accessible state. Later work demonstrated that E. pisi released elicitor components at the pre-penetration stage which conditioned host cells toward the inaccessible state [21, 30 ]. Similarly, E. graminis released a suppressor, prior to the time of penetration, and the suppressor blocked the effect of the E. pisi elicitor [15 ]. Thus penetration by E. graminis was not necessary as the suppressor induced the condition of accessibility. These studies suggest that some components released from conidia of both fungi and}or their germlings might affect the condition of the host plant cell before the fungus attempts penetration. In 1975, Green et al. [9 ] reported that E. graminis f. sp. tritici elicits an increase in the level of the activity of phenylalanine ammonia-lyase (PAL) in wheat leaves, the first enzyme in phenylpropanoid biosynthesis, as early as 4 h after inoculation. Thereafter, Shiraishi et al. [26, 27 ] and Clark et al. [7 ] confirmed this phenomenon by showing that within 3–4 h after inoculation of barley leaves with E. graminis f. sp. hordei there was an increase in the synthesis of cinnamic acid, the immediate product of the PAL reaction, and that extractable activity of PAL had increased by 6 h and again between 12–15 h after inoculation. PAL activity began to increase during the prepenetration stage, before fungal appressoria matured. A recent report by Carver et al. [6 ] also addressed the significance of PAL to infection by E. graminis in oats. They demonstrated that the PAL inhibitors, α-aminooxyacetic acid (AOA) and α-aminooxy-β-phenylpropionic acid (AOPP), increased the susceptibility of oat leaf tissue to the fungus and significantly reduced the occurrence of an autofluorescence response by the host’s cells. This was presumed to indicate that the accumulation of phenolic compounds that limited development of the pathogen had been blocked [6 ]. Each of these studies suggest that treatment of host cells with either AOA or AOPP during the early stages of host–pathogen interaction could interfere with the induction of accessibility by successful penetration of a pathogen into the host cell. In this paper we examine the effects of treatment of the host with PAL inhibitors on accessibility.
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MATERIALS AND METHODS
Plant and fungal materials Barley, Hordeum ulgare L., cv. Kobinkatagi, was grown from seed in vermiculite in a growth chamber under fluorescent lights (c. 11±8 Wm−#) and a 12 h photoperiod at 20³2 °C and 70 % relative humidity. The coleoptiles were excised from seedlings 8 days after sowing, and layers of the epidermal cells of partially dissected coleoptiles were prepared as described previously [28 ]. Erysiphe graminis de Candolle f. sp. hordei Em. Marchal, race I, was maintained on the compatible barley cv. Kobinkatagi and E. pisi de Candolle, race I, on the compatible pea cv. Midoriusui in separate growth chambers with the same environmental conditions. Inoculation Partially dissected coleoptiles were inoculated with conidia of test fungi by two methods : (i) by paint brush, (ii) by individual transfer onto target coleoptile cells by manipulation [15–18 ] using a 200¬light microscope, as described in detail below. Preparation of test inhibitors and determination of their test concentrations Two competitive inhibitors of PAL [1, 2, 4, 5, 10, 25 ] were used : α-aminooxy acetic acid (AOA : Nacalai Tesque Co., Japan) and α-aminooxy-β-phenylpropionic acid (AOPP : gift from Dr. T. L. W. Carver). AOA was dissolved in distilled water to give a 1 m stock solution. AOPP was moderately water soluble and was dissolved by adding it to water gradually while continuously stirring to give a 1 m stock solution. Both stock solutions were kept in the dark at 4 °C until used. Cytochalasin A (Sigma Co.) was used as an actin polymerization inhibitor [29, 31, 32 ]. It was dissolved in dimethylsulfoxide (DMSO) to give a stock solution of 2 mg ml−" which was stored at ®20 °C until used. All stock solutions were diluted with distilled water to required concentrations immediately before use. The final concentration of DMSO in the test solution of cytochalasin A was adjusted to less than 1 %. It was confirmed that 1 % DMSO did not affect the morphogenesis nor the penetration efficiency of test fungi. In control experiments, 1 % DMSO was added to distilled water. Determination of test concentrations of inhibitors The first experiment determined if AOA and AOPP applied to barley coleoptiles affected the accessibility induced by E. graminis attack in single cells. Coleoptiles treated with distilled water were used as controls and compared with coleoptiles treated with solutions of AOA or AOPP at 1, 10, 100, 1000 or 10 000 n. The resultant accessibility of coleoptile cells was evaluated based on the ability of E. pisi to penetrate the same cells using the procedure illustrated in Fig. 1. The experiment involved micromanipulation to transfer fungal germlings at precise times, and this limited the number of germlings that could be transferred in a single test. Therefore, data were accumulated from repeated tests until data from 100–200 individual coleoptile cells (from 10 coleoptiles) subjected to each treatment had been obtained.
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E. graminis Coleoptile A 0h
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Determination of PE of E. pisi 37 h
F. 1. Time-course of inoculation of E. graminis, transfer of a challenger, E. pisi, and inoculation of both fungi. Erysiphe pisi was transferred onto the coleoptiles cells where E. graminis had formed an incipient haustorium. The penetration efficiency (PE) of E. pisi was determined 37 h after inoculation with E. graminis. ha ¯ haustorium.
In each test, two sets of coleoptiles (coleoptiles A and B, Fig. 1) were prepared. Three A coleoptiles were inoculated, using a brush, with 15–20 freshly harvested conidia of E. graminis and immediately incubated by floating on 300 µl of either water or an inhibitor solution (1–10 000 n AOA or AOPP in watch glasses at 20³2 °C and 70 % RH for 15 h. Three B coleoptiles were inoculated similarly with about 50 freshly harvested conidia of E. pisi 13 h after inoculation of the A coleoptiles and incubated on water in Petri dishes under the same conditions as above for 2 h. The timing of inoculation on the B coleoptiles ensured that germlings of E. pisi reached a stage suitable for transfer at 15 h after inoculation of E. graminis on the A coleoptiles. Both coleoptiles were mounted in water on a glass slide 15 h after inoculation with E. graminis, and germlings of E. pisi were individually transferred by manipulation onto cells of the A coleoptiles where E. graminis had formed incipient haustoria. In this case, several germlings of E. graminis on the A coleoptiles were selected such that no other germlings were located within a distance of five host cells away. After a further 22 h incubation of the A coleoptiles they were mounted on glass slides with water, and haustorium formation by E. pisi in the target cells was observed with an Olympus BH-2 light microscope. Penetration efficiency (PE) of this fungus was evaluated as follows : PE ¯ No. of haustoria . Total No. of observed haustoria and}or papillae below lobed appressoria¬100 % The accessibility of cells subjected to the different treatments was statistically analysed by a t-test applied to PEs between treatments. The PE values given are means³standard error. Although this first experiment allowed identification of AOA and AOPP concentrations which affected accessibility induced by E. graminis, it was necessary to test the effect of the inhibitors on the base level of PE achieved by E. graminis inoculated alone. Therefore, a second experiment was conducted where coleoptiles inoculated with E. graminis were floated on water or 1–10% n AOA or AOPP in watch glasses and incubated under the same conditions as above for 24 h before PE was determined. A main objective of this study was to elucidate the effects of AOA and AOPP on the
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physiological condition of coleoptile cells in relation to induced accessibility. However, there was a possibility that E. pisi, used as the probe to determine accessibility of coleoptile cells, might be affected directly by AOA or AOPP. The fact that this fungus always fails to form haustoria in barley coleoptiles [12, 14 ] made it impossible to evaluate potential direct negative effects of the inhibitors on penetration capability of E. pisi on coleoptiles. However, as we demonstrated previously [14 ], E. pisi can penetrate to form haustoria in coleoptiles treated with cytochalasins (inhibitors of actin polymerization). Therefore, in the third experiment, coleoptiles inoculated with E. pisi were treated with mixtures of 10 n of AOA or AOPP with 1 µg ml−" cytochalasin. After 24 h incubation (incubation conditions described above) on test solutions, the PE of E. pisi was assessed. Timing of coleoptile treatment with AOA or AOPP in relation to induced accessibility to E. pisi The timing of inhibitor application to coleoptiles in relation to the effects of inhibitors on induced changes in cell accessibility to E. pisi was examined by the procedure illustrated in Fig. 2. Coleoptiles C were inoculated with E. graminis before they were
E. pisi Coleoptile D Incubation 2 h E. graminis Determination of PE of E. pisi
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3h 6h 9h 12 h 18 h 24 h Control F. 2. The H O-AOA or -AOPP sequential treatment of inoculated coleoptiles. Coleoptile C # was first inoculated with E. graminis, and E. pisi which had been incubated on coleoptile D for 2 h was transferred onto the cells of coleoptile C where E. graminis had formed incipient haustoria. Coleoptile C’s were floated on H O for the first period and transferred onto AOA or AOPP # solution, followed by incubation for the remaining period. PE of E. pisi in coleoptile C was determined 37 h after inoculation with E. graminis. ha ¯ haustorium. (*) Incubated with water ; (8) incubated with AOA or AOPP.
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floated on water for designated times and prior to transfer to 10 n AOA or AOPP for the remaining incubation period (incubation conditions described above). At 15 h after inoculation with E. graminis, a conidium of E. pisi which had germinated on a separate D coleoptile (floated on water) was transferred onto the coleoptile cell on which a conidium of E. graminis had formed an haustorial primordium. PE of E. pisi was then determined after a further 22 h incubation of the C coleoptile. Data were accumulated from 100–130 individual coleoptile cells (9–15 coleoptiles). Influence of AOA or AOPP on accessibility to challenging E. graminis In addition to inducing accessibility to the non-pathogen E. pisi, previous work [22 ] showed that accessibility induced by primary inoculation with E. graminis also effectively enhances the PE of E. graminis when applied as a second, challenge inoculum. Therefore, an experiment was conducted to test whether AOA or AOPP treatment of barley coleoptiles influenced induced accessibility of E. graminis conditioned by a previous inoculation with E. graminis using the procedure illustrated in Fig. 3. E. graminis 2 Coleoptile F Incubation 5.5 h E. graminis 1 ha
Coleoptile E 0h
18.5 h
PE of E. graminis 2 was determined 37 h
F. 3. Sequential inoculation with E. graminis 1 (the first inoculum), transfer of a challenging E. graminis 2, and incubation of both. Erysiphe graminis 2 was transferred onto the coleoptile cells where E. graminis 1 had formed an incipient haustorium. PE of E. graminis 2 was determined 37 h after inoculation of E. graminis 1.
Two sets of coleoptiles (coleoptiles E and F : Fig. 3) were prepared. Three E coleoptiles were inoculated with 15–20 freshly harvested E. graminis conidia, floated on 300 µl of water or 10 n AOA or AOPP in watch glasses, and incubated at 20³2 °C and 70 % RH for 18±5 h. Three F coleoptiles were inoculated with about 50 freshly harvested E. graminis conidia 13 h after inoculation of the E coleoptiles and incubated on water in Petri dishes under the same conditions as above for 5±5 h. This timing of inoculation on the F coleoptiles ensured that germlings on these coleoptiles reached the stage suitable for transfer at 18±5 h after inoculation with E. graminis on the E coleoptiles. Both coleoptiles were mounted in water on a glass slide 18±5 h after inoculation of the E coleoptiles, and germlings of E. graminis on the F coleoptiles were individually transferred onto the cells of the E coleoptiles where previously inoculated E. graminis had formed incipient haustoria. PE of E. graminis was evaluated after a further 18±5 h incubation. For controls, germlings on the F coleoptiles were transferred onto cells of uninoculated coleoptiles floated on water, 10 n AOA or 10 n AOPP. Data were accumulated from 100–120 individual coleoptile cells (9–13 coleoptiles).
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RESULTS
Effects of AOA and AOPP on the induction of accessibility by E. graminis The protocol indicated in Fig. 1 was used to examine the influence of AOA and AOPP on induction of accessibility to E. pisi when conditioned by prior attack by E. graminis. As mentioned earlier, E. pisi normally fails completely to penetrate coleoptiles of barley (a non-host) when they are not previously infected by E. graminis. However, the PE of E. pisi transferred onto cells of coleoptile A in which E. graminis had previously formed incipient haustoria was increased to 43±2³8±3 % (Figs 4, 5). Thus, we judged that these
Penetration efficiency of challenging E. pisi (%)
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50
40
30
20
*
10
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*
0
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*
*
10 100 Concentration (nM)
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* *
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10 000
F. 4. Effects of different concentrations of AOA and AOPP on the induction of accessibility by the first inoculum, E. graminis, in coleoptile cells of barley. The accessible cell condition was assessed by penetration efficiency of challenging E. pisi in the cells where the first inoculum had formed an haustorium. (*) Significantly different from PE in water control. (*) Water ; (8) AOA ; (+) AOPP.
cells had been directed towards an accessible state by successful penetration with E. graminis. In contrast, when the A coleoptile was treated with 1 n AOA or AOPP during incubation, the PE of E. pisi decreased to 28±5³5±5 % and 26±4³8±8 %, respectively. Furthermore, when the A coleoptile was treated with 10 or 100 n AOA and AOPP, the PE was drastically reduced (P ! 0±05) to 3±3³2±4–3±7³2±6 % and 2±8³3±0– 5±3³2±9 %, respectively. Thus, these concentrations of both inhibitors effectively
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F. 5. Series of interference contrast micrographs showing successive growth stages of E. graminis (g) and E. pisi (p) on single coleoptile cells. ap ¯ appressorium ; h ¯ haustorium ; sh ¯ secondary hypha. Scale in frame A ¯ 40 µm. Accessibility was induced in a coleoptile cell by successful penetration by the primary inoculum, E. graminis. (Frames A–D). By 15 h after inoculation, E. graminis succeeded in penetrating and forming a haustorium in a coleoptile cell (A). The secondary inoculum, E. pisi, was transferred onto the same cell around 15 h (B). The transferred E. pisi developed a lobed appressorium by 21 h (C). When both E. graminis and E. pisi germlings developed an haustorium in the same cell, secondary hyphae elongated from both by 37 h (D). Accessibility was also induced in a coleoptile cell by successful penetration by the primary inoculum, E. graminis g1 (E–G). By 18±5 h after inoculation, g1 succeeded in penetrating and forming an haustorium in a coleoptile cell (E). The secondary inoculum, E. graminis g2, was transferred onto the same cell around 18±5 h (F). Secondary hyphae elongated from both g1 and g2 by 37 h (G).
suppressed the induction of accessibility brought on by the previous inoculation with E. graminis. Induction of accessibility never occurred in coleoptiles treated with inhibitor concentrations higher than 100 n. Therefore, we decided to use 10 n AOA and AOPP, the lowest concentration to interfere with induction of accessibility, as a standard concentration for subsequent experiments to examine the effects of the inhibitors on induced accessibility. Effects of AOA and AOPP on the penetration efficiency (PE) of E. graminis and E. pisi Figure 6 shows PE values obtained for E. graminis attacking coleoptiles treated with distilled water (controls) or with various concentrations of AOA or AOPP. The PE of E. graminis in coleoptiles floated on water was 57±3³6±2 %. When coleoptiles were
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F. 6. Effects of different concentrations of AOA and AOPP on the penetration efficiency of E. graminis in coleoptile cells of barley. (*) Significantly different from PE in water control. (*) Water ; (8) AOA ; (+) AOPP.
treated with 1 n AOA, the PE of E. graminis was similar to the control (PE ¯ 63±7³8±9 % ; P " 0±05). However, treatment with either 10 or 100 n AOA significantly increased (P ! 0±05) the PE of E. graminis compared to the control (PE ¯ 76±6³4±2 % and 76±7³5±1 % for 10 and 100 n, respectively). However, AOPP had no detectable effect on the PE of E. graminis, as PE values were statistically indistinguishable from the control (Fig. 6). It is possible that the inability of AOPP to increase accessibility to E. graminis was a result of the compound’s relative insolubility as compared to that of AOA. Treatment of coleoptiles with 1 µg ml−" cytochalasin A allowed E. pisi to form haustoria with a PE of 37±2³7±8 %, a condition previously shown to occur only after inoculation first with E. graminis (Fig. 4). When coleoptiles were treated with a mixture of 1 µg ml−" cytochalasin A and 10 n of AOA or AOPP, PEs of 32±0³2±5 % and 36±3³7±5 %, respectively, were obtained. These were not significantly different from treatment with cytochalasin alone, demonstrating that at this concentration neither PAL inhibitor interfered with the penetration capability of E. pisi. These results confirmed that suppression of induced accessibility brought about by treatment of coleoptiles with 10 n of AOA or AOPP could not be explained by direct, negative effects on the morphogenesis of test fungi inoculated on these coleoptiles, but was likely to be dependent on the physiological condition of the coleoptile cells.
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F. 7. Effects of the timing and duration of sequential H O-AOA or H O-AOPP treatment on # # the accessibility induced by the first inoculum, E. graminis. The accessibility induced was assessed by measuring the penetration efficiency of challenging E. pisi in the cells where the first inoculum had formed a haustorium. (*) Significantly different from PE in water control. (*) Water ; (8) AOA ; (+) AOPP.
Effects of timing and duration of AOA or AOPP treatment on induction of accessibility to E. pisi The procedure illustrated in Fig. 2 was used to determine if there was a critical period during the interaction between coleoptile cells and E. graminis germlings applied as primary inoculum that determined the induction of coleoptile cell accessibility to E. pisi. Figure 7 shows that the PE of E. pisi on coleoptile cells in which E. graminis had formed incipient haustoria was 43±2³8±3 %, when the coleoptiles were floated on water during the entire incubation period. However, when coleoptiles were treated with AOA or AOPP immediately after inoculation, induction of accessibility to E. pisi was almost completely suppressed (PE ¯ 3±3³2±5 % and 5±3³2±9 %, for AOA and AOPP, respectively) giving PE values that were significantly lower (P ! 0±05) than in watertreated cells. When the AOA or AOPP treatment was initiated 3 h after the onset of incubation, PEs again were significantly (P ! 0±05) lower than in water-treated cells. (PE ¯ 6±7³5±0 % and 14±1³4±5 % for AOA and AOPP, respectively). In contrast, when coleoptiles were initially incubated for 6 h or more on water before transfer to AOA or AOPP, the PEs were not significantly different (P " 0±05) from those obtained
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where coleoptiles were held continuously on water (Fig. 7). Therefore, we concluded that coleoptile cell—E. graminis interaction(s) occurring within 6 h after inoculation are important in inducing physiological changes leading to accessibility of the cell to E. pisi. Effects of AOA and AOPP on the induction of accessibility to E. graminis In the above experiments, E. pisi was used to test for induction of accessibility. As mentioned above, accessibility using E. graminis as the primary inoculum also increases the PE of E. graminis when applied as a secondary inoculum [22 ]. The experiments reported in this section examined whether AOA and AOPP interfered with induction of accessibility of coleoptile cells to E. graminis. In a preliminary experiment, we tested whether AOA or AOPP treatment affected the PE of transferred E. graminis germlings using a modification of the procedure illustrated in Fig. 3. Erysiphe graminis germlings which had been incubated on the F coleoptile floating on water and where no previous inoculation had been made were transferred onto cells of a separate E coleoptile, followed by a further incubation for 18±5 h. In this control, PE was 55±7³8±3 % (Fig. 8). When similar germlings were transferred onto the E coleoptiles floating on 10 n AOA or AOPP where no previous inoculation had been made, PEs were 66±7³12±3 % and 68±3³11±9 %, respectively. 100
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F. 8. Effects of AOA or AOPP on the induction of accessibility which is effective to promote successful penetration by challenging E. graminis in the same cells where the first inoculum, E. graminis, has formed a haustorium. (*) Significantly different from PE in water control. (+) Transferred control ; (8) challenging.
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These values were not significantly ( P " 0±05) higher than that of the water control and they demonstrate that 10 n of either inhibitor did not directly affect the penetration capability of transferred E. graminis. When germlings of challenging E. graminis were transferred onto cells of a waterfloated E coleoptile (Fig. 3) where the primary inoculum of E. graminis previously had formed incipient haustoria, PE of the former was 88±8³4±9 % after an additional 18±5 h incubation (Fig. 8). However, when similar transfers were made to the E coleoptiles which had been floated on 10 n AOA or AOPP solutions, PEs of challenging E. graminis were reduced to 72±1³5±0 % and 67±4³4±5 %, respectively. Both values were significantly (P ! 0±05) lower than that of the water control, demonstrating that both inhibitors also interfered with the induction of the accessible cell condition for the challenging E. graminis conidia. DISCUSSION
AOPP and AOA are effective competitive inhibitors of PAL in most plant species [1, 2, 4, 10, 25 ]. Both inhibited oat PAL in itro, although like the results with cowpea PAL [25 ], AOPP was a more effective inhibitor than AOA. AOPP is the more specific inhibitor of PAL [2 ], whereas AOA is a general transamination inhibitor affecting various other metabolic processes [3, 11 ]. Thus, care must be taken when interpreting AOA results [4 ]. The present results showed that treatment of dissected coleoptiles with AOA or AOPP within 6 h after inoculation interfered with the induction of accessibility, which is normally the consequence of an initial attack by E. graminis. Kobayashi et al. [13 ] demonstrated that conidia of E. graminis produce primary germ tubes between 0±5 and 1±0 h after inoculation of coleoptiles and subsequently form appressorial germ tubes after about 4±2 h. The latter germ tubes stop elongation by 6±5 h, form a lobe at their swollen tip, and become mature appressoria by 6±9 h on average. In previous papers [13, 15 ] we demonstrated that both elicitor and suppressor factors were released from germlings of E. graminis with an immature appressorium. This earlier work led us to assume that some components released from E. graminis germlings could affect the physiological condition of the underlying coleoptile cells [13, 15 ]. Because of the effects of the PAL inhibitors, the present results suggest that accessibility may be linked to phenolic compound biosynthesis, especially phenylpropanoid biosynthesis. Green et al. [9 ] reported that inoculation of wheat leaves with E. graminis f. sp. tritici enhanced the level of PAL in leaves within 4 h. Shiraishi et al. [26 ] then demonstrated that within 3–4 h after inoculation of barley leaves with E. graminis f sp. hordei, synthesis of cinnamic acid was significantly enhanced and PAL activity increased by as early as 2 h. Furthermore, Shiraishi et al. [27 ] reported that inoculation of barley leaves with the same fungus induced a two phase increase in PAL activity in leaves, the first at 6 h and the second between 12–15 h after inoculation. Similar results were obtained by Clark et al. [7 ], who showed biphasic patterns of PAL transcript accumulation at 2 h and 12 h in seedling leaves of four near-isogenic barley lines inoculated with the same fungus. They also showed that temporal patterns of PAL enzyme activity were roughly similar to those of PAL transcript accumulation. Based on these results, Clark et al. [7 ] concluded that PAL induction was associated with a general defence to infection.
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However, a strong correlation between PAL induction and major gene resistance was not found. As indicated in Fig. 7, the induction of accessibility was suppressed significantly when coleoptiles inoculated with E. graminis were treated with AOA and AOPP within 0–3 h of inoculation. When such treatments were initiated later, there was no significant effect. However, if AOA or AOPP were applied 9 h after the primary inoculation, the PEs of E. pisi were 24±4³5±7 % and 25±2³6±5 %, respectively (Fig. 7). These values were not significantly lower than the PE of the control at the 5 % level, but were lower at the 10 % level of probability. Although a more detailed examination is needed, the marginally lower PEs given by these treatments might be related to the biphasic PAL-associated metabolic changes noted by others [7, 27 ].It is possible that biphasic elevation of PAL activity might be dependent on different isomers of PAL being produced in the different phases. At present there is no evidence for this, but different isomers have been demonstrated in pea and tobacco [8, 33 ]. At present it is uncertain whether the first phase between 0–3 h is associated with the penetration attempt by primary germ tubes as was discussed by Shiraishi et al. [27 ], Clark et al. [7 ] and Carver et al. [5 ], or whether it is related to other factors such as a response to exudates released by conidia [19, 23 ]. As described previously [16, 17, 22 ], accessibility induced in coleoptile cells by E. graminis applied as the primary inoculum effectively enhanced successful penetration by both E. graminis and E. pisi applied as secondary inoculum to the same cells. However, when coleoptiles first inoculated with E. graminis were floated on either 10 n AOA or AOPP immediately after inoculation, the accessible cell condition for secondary E. pisi or E. graminis was not induced. These results suggest that the accessible cell condition for these two fungi may be related to a common metabolic feature which is interfered with by AOA or AOPP. Although the present results suggested that effects on host cell metabolism as early as 0–3 h after primary inoculation was possibly associated with the host cell condition at the post-penetration stage, further studies are required to elucidate how this physiological connection might be regulated at the biochemical level. It is also necessary to determine whether the concentrations of AOA and AOPP applied in this study were high enough to inhibit PAL activity in barley coleoptiles totally. The biologically effective concentrations used here (10 n for both inhibitors), were much lower than the concentrations reported [5 ] to give 50 % inhibition of PAL activity in itro (200 n AOPP and 500 µ AOA). The authors thank Dr. T. L. W. Carver, Institute of Grassland & Environmental Research, UK and Dr. R. L. Nicholson, Purdue University, USA for their critical reading of the manuscript and correcting the English in the text. This work was partially supported by Grant-in-Aid for Scientific Research (B) (No. 08456025) from the Ministry of Education and Culture of Japan. REFERENCES 1. Amrhein N, Go$ decke KH, Keffeli VI. 1976. The estimation of relative intracellular phenylalanine ammonia-lyase (PAL)-activities and their modulation in io and in itro by competitive inhibitors. Brichte der Deutschen Gesellschaft 89 : 247–259.
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2. Amrhein N, Go$ decke KH. 1977. α-Aminooxy-β-phenylpropionic acid – a potent inhibitor of Lphenylalanine ammonia-lyase in itro and in io. Plant Science Letters 8 : 313–317. 3. Amrhein N. 1979. Novel inhibition of phenylpropanoid metabolism in plants. In Regulation of Secondary Products and Plant Hormone Metabolism, Luckner M, Schreiber L, eds. Oxford : Pergamon Press, 173–182. 4. Carver TLW, Robbins MP, Zeyen RJ. 1991. Effects of two PAL inhibitors on the susceptibility and localized autofluorescent host cell responses of oat leaves attacked by Erysiphe graminis DC. Physiological and Molecular Plant Pathology 39 : 269–287. 5. Carver TLW, Zeyen RJ, Bushnell WR, Robbins MP. 1994. Inhibition of phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase increase quantitative susceptibility of barley to powdery mildew (Erysiphe graminis D.C.) Physiological and Molecular Plant Pathology 44 : 261–272. 6. Carver TLW, Zeyen RJ, Robbins MP, Vance CP, Boyles DA. 1994. Suppression of host cinnamyl alcohol dehydrogenase and phenylalanine ammonia lyase increases oat epidermal cell susceptibility to powdery mildew penetration. Physiological and Molecular Plant Pathology 44 : 243–259. 7. Clark TA, Zeyen RJ, Smith AG, Carver TLW, Vance CP. 1994. Phenylalanine ammonia lyase mRNA accumulation, enzyme activity and cytoplasmic responses in barley isolines, differing at Mla and Ml-o loci, attacked by Erysiphe graminis f. sp. hordei. Physiological and Molecular Plant Pathology 44 : 171–185. 8. Fukazawa-akada T, Kung SD, Watoson JC. 1996. Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana. Plant Molecular Biology 30 : 711–722. 9. Green N, Hadwiger LA, Graham SO. 1975. Phenylalanine ammonia lyase, tyrosine ammonia lyase, and lignin in wheat inoculated with Erysiphe graminis f. sp. tritici. Phytopathology 65 : 1071–1074. 10. Havir EA. 1981. Modification of L-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and L-2-aminooxy-3-phenylpropinate. Planta 152 : 124–130. 11. John RA, Charteris A, Fowler LJ. 1978. The reaction of amino-oxyacetate with pyridoxal phosphatedependent enzymes. Biochemical Journal 171 : 771–779. 12. Kobayashi I, Kobayashi Y, Yamaoka N, Kunoh H. 1992. Recognition of a pathogen and a nonpathogen by barley coleoptile cells. III. Responses of microtubules and actin filaments in barley coleoptile cells to penetration attempts. Canadian Journal of Botany 70 : 1815–1823. 13. Kobayashi I, Watanabe H, Kunoh H. 1995. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XIV. Evidence for elicitor(s) and suppressor(s) of host inaccessibility from Erysiphe graminis. Physiological and Molecular Plant Pathology 46 : 445–456. 14. Kobayashi I, Kobayashi Y, Yamada M, Kunoh H. 1996. The involvement of the cytoskeleton in the expression of nonhost resistance in plants. In : Mills D, Kunoh H, Keen NT, Mayama S, eds. Molecular Aspects of Pathogenicity and Resistance : Requirement for Signal Transduction. St. Paul, Minnesota : The American Phytopathological Press, 185–195. 15. Komura T, Kobayashi I, Yamaoka N, Kunoh H. 1990. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. VIII. Cytological evidence for suppressor(s) of host inaccessibility from Erysiphe graminis. Physiological and Molecular Plant Pathology 37 : 409–416. 16. Kunoh H, Hayashimoto A, Harui M, Ishizaki H. 1985. Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. I. The significance of timing of fungal invasion. Physiological Plant Pathology 27 : 43–54. 17. Kunoh H, Kuroda K, Hayashimoto A,Ishizaki H. 1986. Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. II. The timing and localization of induced susceptibility in a single coleoptile cell and its transfer to an adjacent cell. Canadian Journal of Botany 64 : 889–895. 18. Kunoh H, Katsuragawa N, Yamaoka N, Hayashimoto A. 1988. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. III. Timing and localization of enhanced inaccessibility in a single coleoptile cell and its transfer to an adjacent cell. Physiological and Molecular Plant Pathology 33 : 81–93. 19. Kunoh H, Yamaoka N, Yoshioka H, Nicholson RL. 1988. Preparation of the infection court by Erysiphe graminis. I. Contact-mediated changes in morphology of the conidium surface. Experimental Mycology 12 : 325–335. 20. Kunoh H, Toyoda K, Yamaoka N, Kobayashi I. 1989. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. V. Duration of stimulus by a non-pathogen in relation to enhanced inaccessibility. Physiological and Molecular Plant Pathology 35 : 507–518. 21. Kunoh H, Komura T, Kobayashi I, Yamaoka N. 1990. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. VII. Enhancement of inaccessibility at the prepenetration stage of a nonpathogen Physiological and Molecular Plant Pathology 37 : 399–407. 22. Kunoh H, Kuroda K, Toyoda K, Yamaoka N, Kobayashi I. 1991. Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. IX. Accessibility induced by Erysiphe
Cellular accessibility in barley coleoptiles
23. 24. 25. 26.
27. 28. 29. 30. 31. 32. 33.
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graminis which promotes the subsequent infection of challenging E. graminis. Annals of the Phytopathological Society of Japan 57 : 57–60. Nicholson RL, Yoshioka H, Yamaoka N, Kunoh H. 1988. Preparation of the infection court by Erysiphe graminis. II. Release of esterase enzyme from conidia in response to a contact stimulus. Experimental Mycology 12 : 336–349. Ouchi S, Oku H, Hibino C, Akiyama I. 1974. Induction of accessibility and resistance in leaves of barley by some races of Erysiphe graminis. Phytopathology Zeitschrift 79 : 24–34. Ralton JE, Howlett BJ, Clarke AE, Irwin JA, Imrie B. 1988. Interaction of cowpea with Phytophthora ignae : inheritance of resistance and production of phenylalanine ammonia-lyase as a resistance response. Physiological and Molecular Plant Pathology 32 : 89–103. Shiraishi T, Yamaoka N, Kunoh H. 1989. Association between increased phenylalanine ammonialyase activity and cinnamic acid synthesis and the induction of temporary inaccessibility caused by Erysiphe graminis primary germ tube penetration of the barley leaf. Physiological and Molecular Plant Pathology 34 : 75–83. Shiraishi T, Yamada T, Nicholson RL, Kunoh H. 1995. Phenylalanine ammonia-lyase in barley : activity enhancement in response to Erysiphe graminis f. sp. hordei (race 1) a pathogen, and Erysiphe pisi, a nonpathogen. Physiological and Molecular Plant Pathology 46 : 153–162. Takamatsu S, Ishizaki H, Kunoh H. 1978. Cytological studies of early stages of powdery mildew in barley and wheat. V. Effects of calcium on the infection of coleoptile of barley by Erysiphe graminis hordei. Canadian Journal of Botary 56 : 2544–2549. Thomas DD. 1978. Cytochalasin effects in plants and eukaryotic microbial systems. In : Tanenbaum SW, ed. Cytochalasins-Biochemical and Cell Biological Aspects. North-Holland : Biomedical Press, 257–275. Toyoda K, Shiraishi T, Kobayashi I, Yamaoka N, Kunoh H. 1993. A water-soluble extract from germlings of Erysiphe pisi suppresses infection by E. graminis in coleoptile cells of barley. Annals of the Phytopathological Society of Japan 59 : 135–142. Wieland T. 1977. Modification of actins by phallotoxins. Naturwissenschaften 64 : 303–309. Yahara I, Harada F, Sekita S, Yoshihira K, Natori S. 1982. Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro. Journal of Cell Biology 92 : 69–78. Yamada T, Sriprasertsak P, Kato H, Hashimoto T, Shimizu H, Shiraishi T. 1994. Functional analysis of the promoters of phenylalanine ammonia-lyase genes in pea. Plant and Cell Physiology 35 : 917–926.