Victorin Triggers Programmed Cell Death and the Defense Response via Interaction with a Cell ...

12 Pages·2005·0.85 MB·English

Plant Cell Physiol. 46(11): 1787–1798 (2005)
doi:10.1093/pcp/pci193, available online at www.pcp.oupjournals.org
JSPP © 2005
Victorin Triggers Programmed Cell Death and the Defense Response via
Interaction with a Cell Surface Mediator
Yasuomi Tada 1, 2, Koh Kusaka 1, 3, Shigeyuki Betsuyaku 1, 3, Takeshi Shinogi 4, Masaru Sakamoto,
Yasuko Ohura, Shingo Hata, Tomoyo Mori, Yukio Tosa and Shigeyuki Mayama *
Graduate School of Natural Science and Technology, Kobe University, Kobe, 657-8501 Japan
;
The host-selective toxin victorin is produced by Coch- Introduction
liobolus victoriae, the causal agent of victoria blight of oats.
Victorin has been shown to bind to the P protein of the In many plant–pathogen interactions, plants recognize
glycine decarboxylase complex (GDC) in mitochondria, and pathogen-produced avirulence gene products by matching
induce defense-related responses such as phytoalexin syn- resistance gene products and subsequently evoke a battery of
thesis, extracellular alkalization and programmed cell defense responses, including a local and rapid cell death termed
death. However, evidence demonstrating that the GDC hypersensitive response (HR) (Dangl and Jones 2001). In con-
plays a critical role in the onset of cell death is still lacking, trast, most pathogens have developed virulence factors that
and the role of defense-like responses in the pathogenicity interact with host molecules to cause a spectrum of disease in
has yet to be elucidated. Here, cytofluorimetric analyses, plants. Of such virulence factors, host-selective toxins (HSTs)
using the fluorescein (VicFluor) or bovine serum albumin– produced by certain pathogenic fungi are toxic only to suscep-
fluorescein derivative of victorin (VicBSA), demonstrated tible hosts and hence function as specificity determinants in
that victorin-induced cell death occurs before these conju- plant–fungus interactions (Walton 1996). Although approxi-
gates traverse the plasma membrane. As with native vic- mately 20 HSTs have been identified, the molecular and bio-
torin, VicBSA clearly elicits apoptosis-like cell death, chemical basis of disease susceptibility has remained to be
production of phytoalexin, extracellular alkalization, and elucidated.
generation of nitric oxide and reactive oxygen intermedi-
The fungus Cochliobolus victoriae, a causal agent of vic-
ates. These results suggest that the initial recognition of vic-
toria blight of oat (Avena sativa L.), produces an HST, victorin
torin takes place on the cell surface, not in mitochondria,
(Wolpert et al. 1985). Victorin is a peptide toxin that is required
and leads to the activation of a battery of victorin-induced
for the pathogenicity on oat cultivars carrying the dominant Vb
responses. Pharmacological studies showed that extra-
allele (Wolpert et al. 1985). The Vb gene has not been sepa-
cellular alkalization is the essential regulator for both vic-
rated genetically from Pc-2 (Rines and Luke 1985, Mayama et
torin- and VicBSA-induced cellular responses. We propose
al. 1995), a resistance gene against certain races of crown rust
a model where victorin may kill the host cell by activating
fungi, Puccinia coronata f. sp. avenae (Meehan and Murphy
an HR-like response, independent of the binding to the
1946, Meehan and Murphy 1947), suggesting that both genes
GDC, through ion fluxes across the plasma membrane.
may be identical. Therefore, characterization of the Vb gene
and the underlying mechanisms of victorin sensitivity may lead
Keywords: Apoptosis — Avena sativa — Cochliobolus victo-
to understanding of both disease susceptibility and resistance.
riae — Programmed cell death — Vb — Victorin.
In susceptible oat leaves, a radiolabeled victorin derivative
was shown to bind specifically to the P protein component of
Abbreviations: DAF, 4,5-diaminofluorescein; DCF, 2′,7′,-dichlo-
the glycine decarboxylase complex (GDC) located in the mito-
rofluorescein; DPI, diphenyleneiodonium chloride; EDC, 1-ethyl-3-(3-
chondrial matrix (Wolpert et al. 1994). Victorin strongly inhib-
dimethylaminopropyl)carbodiimide hydrochloride; FDA, fluorescein
diacetate; GDA, glycine decarboxylase activity; GDC, glycine its glycine decarboxylase activity (GDA), which plays a pivotal
decarboxylase complex; HST, host-selective toxin; MPT, mito- role in the photorespiration cycle, in victorin-sensitive leaf
chondrial permeability transition; NAC, N-acetylcysteine; NHS, N- tissues (Navarre and Wolpert 1995). The in vitro binding of
hydroxysuccinimide; PCD, programmed cell death; ROI, reactive oxy- victorin to the P protein occurs in both victorin-sensitive and
gen intermediate; SOD, superoxide dismutase; VicBSA, bovine serum
-insensitive oats (Wolpert and Macko 1989), and it requires
albumin–fluorescein derivative of victorin; VicFluor, fluorescein
derivative of victorin. a mitochondrial permeability transition (MPT) (Curtis and
Wolpert 2002), which is elicited by opening of permeability
1These authors contributed equally to this work.
2Present address: Duke University, Department of Biology, LSRC Building, Research Drive, Box 91000, Durham, NC 27708-1000, USA.
3Present address: Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, D-50829 Köln, Germany.
4Present address: Hiroshima University, Department of Applied Bioscience, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8528
Japan.
*Corresponding author: E-mail, [email protected]; Fax, [phone].
1787
1788 Victorin elicits an HR-like response
transition pores in the mitochondrial inner membrane, lead- victorin do not traverse the plasma membrane before they
ing to mitochondrial swelling and membrane depolarization induce cell death. As with native victorin, VicBSA rapidly
(Zamzami et al. 1996, Kroemer et al. 1998). These data suggest stimulates the generation of reactive oxygen intermediates
that the interaction of victorin with the product of the Vb gene (ROIs) and nitric oxide (NO), phytoalexin synthesis, extra-
precedes the occurrence of an MPT and the binding of victorin cellular alkalization and cell death only in victorin-sensitive
to the GDC. Akimitsu et al. (1993b) have clearly shown, using leaves. These results suggest that victorin is recognized on the
anti-victorin anti-idiotypic antibodies, that victorin is recog- cell surface and may elicit an HR-like response independent of
nized on the cell surface in victorin-sensitive oats. Therefore, it the binding of victorin to the GDC. We propose that the vic-
is unlikely that the Vb gene encodes a subunit of GDC, and it is torin-induced defense-like response may be the primary cause
possible that the inhibition of GDA is not the direct cause of of cell death.
cell death. Further study is required to determine the primary
binding site of victorin for the induction of cell death. Results
Victorin has been shown unambiguously to induce apop-
tosis-like responses such as DNA laddering and heterochroma-
VicFluor and VicBSA elicit cell death before their entry into
tin condensation (Navarre and Wolpert 1999, Tada et al. 2001,
cells
Yao et al. 2001). Recently, Curtis and Wolpert (2004) demon-
To determine the causative relationship between the tim-
strated that victorin elicits the reduction of mitochondrial
ing of victorin-induced cell death and the dynamic distribution
membrane potential, followed by cell shrinkage without loss of
of victorin in the cell, we performed cytological analyses using
plasma membrane and tonoplast integrity. As reported in
a fluorescein conjugate of victorin (VicFluor) (Curtis and
animals, victorin-induced apoptosis-like characteristics are
Wolpert 2002), in resistant (Iowa X424) and susceptible plants
mediated by Ca2+ ions, oxidative stress, serine and cysteine
(Iowa X469). As shown in Fig. 1A, VicFluor induced cell death
proteases and nuclease activities (Navarre and Wolpert 1999,
in a dose- and time-dependent manner in victorin-sensitive
Tada et al. 2001, Yao et al. 2001). Thus, victorin-induced cell
leaves. As with native victorin, a high concentration of 0.6µg
death is probably caused by transcriptional and/or metabolic
ml–1 VicFluor did not affect cell viability by 1h after treat-
reprogramming of host cells, leading to the activation of a cel-
ment, then 95% of the cells died after another 1h incubation,
lular suicide machinery, rather than a necrotic disorganization
while a low concentration of 0.05µg ml–1 VicFluor elicited the
of the cell.
production of avenanthramide A without severe cell death (data
The most intriguing characteristic of victorin is that it can
not shown). These data demonstrate that VicFluor retains the
also serve as a specific elicitor for the induction of phytoalex-
ability to activate the same signaling pathways that are stimu-
ins, avenanthramides, in oat cultivars carrying the Pc-2 gene
lated by native victorin, although VicFluor is less effective than
(Mayama et al. 1986, Mayama et al. 1995). There are gene dos-
native victorin (Curtis and Wolpert 2002).
age effects of Pc-2 on the accumulation of avenanthramides
We performed quantitative and time course analyses of the
and cell death induced by victorin, suggesting that the Pc-2
fluorescence intensity of VicFluor in mesophyll cells. The mor-
gene might control both victorin sensitivity and rust resistance
phological changes in the cell were confirmed by detecting the
(Mayama et al. 1995). Despite the interest in the toxic nature of
red autofluorescence of chloroplasts, which exhibited an abnor-
victorin, few studies expressed concern about a possible link
between its elicitor activity and toxicity. Pharmacological stud- mal distribution after cell death (Fig. 1B, arrowhead). While
ies indicated that the production of avenanthramide A is treatment of Iowa X424 leaves with 0.6µg ml–1 VicFluor did
mediated by Ca2+ influx, protein kinase and de novo protein not induce cell death, a small percentage of dead cells (<2%,
synthesis (Tada et al. 2001). Also, victorin has been shown to Fig. 1A, C, asterisk), which might be caused by peeling stress,
induce other defense-related responses such as extracellular had about 2-fold higher VicFluor fluorescence compared with
alkalization (Ullrich and Novacky 1991), K+ efflux (Wheeler that in living cells at 4h after treatment (Fig. 1C, striped bars).
and Black 1962) and callose synthesis (Walton and Earle There was no significant increase in intracellular VicFluor fluo-
1985). Taken together with the toxic action of victorin, these rescence in living cells during the time course, indicating that
results give rise to questions: whether victorin can trigger a VicFluor may not permeate resistant cells before dying or dead
general plant defense response, whether the onset of the cells may lose their membrane integrity. In victorin-sensitive
defense response and cell death is due to side effects of the tox- cells, the VicFluor signal increased 2.3-fold at 4h after treat-
icity caused by the binding of victorin to the GDC, and whether ment compared with the control treatment (30s) (Fig. 1B, C),
the activation of defense signaling is essential for the virulence demonstrating that dead cells in the two lines displayed equiva-
function of victorin. lent intensity of VicFluor fluorescence. Interestingly, although
Here we demonstrate that a virulence factor of the fila- treatment with VicFluor for 2h resulted in massive cell death
mentous fungus induces a set of defense responses presumably (Fig. 1A), no significant increase in the intracellular VicFluor
to subvert the host cell. We show that a fluorescein (VicFluor) signal was observed in victorin-sensitive cells by this time (Fig.
and bovine serum albumin–fluorescein (VicBSA) conjugate of 1B, C). These data suggest that VicFluor may activate the cell
Victorin elicits an HR-like response 1789
Fig. 1 VicFluor permeates victorin-sensitive cells after cell death. (A) The kinetics of VicFluor-induced cell death. Epidermis-peeled victorin-
sensitive (Iowa X469, Vb/Vb) and insensitive leaves (Iowa X424, vb/vb) were incubated with VicFluor at the indicated concentrations for 1–4h.
Living and dead cells were observed by FDA staining at the indicated time points, and dead cells were expressed as a percentage of the total cells
counted. Data are means (n = 2). (B) Cytofluorimetric analyses of VicFluor distribution. The localization of VicFluor fluorescence (upper panels)
and chloroplasts (lower panels) in victorin-sensitive and -insensitive leaves was monitored in the same field by a fluorescence microscope coupled
with a CCD camera. Epidermis-peeled leaf segments were treated with 0.6µg ml–1 VicFluor for the indicated time and viewed with excitation of
488nm. The images are representative of at least four experiments. Note that dead cells demonstrated by arrowheads show the abnormal distribu-
tion of the chloroplasts. (C) Changes in the intensity of intracellular VicFluor fluorescence. Epidermis-peeled leaf segments of Iowa X469 (black
bars) and Iowa X424 (striped bars) were treated with 0.6µg ml–1 VicFluor. The VicFluor signal in 20 independent mesophyll cells was monitored
with excitation of 488nm and evaluated by MetaMorph software. Data represent the mean value and SD of three independent experiments. Note
that an asterisk shows the fluorescence value obtained from dead cells (<2%) in toxin-insensitive leaves.
death program in the extracellular space, and enters the cell (data not shown). Furthermore, we confirmed that VicBSA
after the disruption of the plasma membrane integrity. does not release free victorin sufficient to induce cell death
Although the dramatic increase in the intracellular Vic- during the treatment period (see Materials and Methods, data
Fluor signal was observed after cell death, we could not rule not shown). VicBSA had an intense activity for cell death at the
out the possibility that VicFluor entered victorin-sensitive cells concentration of 50µg ml–1, equivalent to that of 5ng ml–1
below detectable levels before cell death. We produced a vic- native victorin, which killed >95% of sensitive cells 2h after
torin-conjugated BSA, which is larger than 66kDa and hence
incubation. Cytofluorimetric analyses showed that VicBSA
presumed not to traverse the plasma membrane, using 1-ethyl-
was excluded from the cell for at least 4h after treatment (Fig.
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
2B, C), and a significant increase in the intracellular VicBSA
and N-hydroxysuccinimide (NHS), and subsequently the conju-
signal was not detected until 6h after incubation, although
gate was coupled with fluorescein (VicBSA) to monitor the
localization of VicBSA. VicBSA clearly induced cell death almost 100% of cells treated with 50µg ml–1 VicBSA died 2h
only in victorin-sensitive leaves in a dose- and time-dependent after treatment (Fig. 2A, B, arrowheads). These results strongly
manner (Fig. 2A), whereas a fluorescein–BSA conjugate did support the hypothesis that victorin is recognized on the cell
not cause any visible cellular damage during the time course surface of victorin-sensitive leaves and may stimulate the cell
1790 Victorin elicits an HR-like response
Fig. 2 A bovine serum albumin–fluorescein conjugate of victorin (VicBSA) induces cell death in victorin-sensitive leaves. (A) The kinetics of
VicBSA-induced cell death. Epidermis-peeled leaf segments of victorin-sensitive (Iowa X469, Vb/Vb) and -insensitive cultivars (Iowa X424, vb/
vb) were incubated with VicBSA at the indicated concentrations for 1–3h. Living cells were observed by FDA staining, and dead cells were
expressed as percentages of the total cells counted. Data represent the mean value and SD of three independent experiments. (B) Distribution of
VicBSA in victorin-sensitive cells. The localization of VicBSA fluorescence (upper panels) and chloroplasts (lower panels) in Iowa X469 was
monitored in the same field by a fluorescence microscope coupled with a CCD camera. Epidermis-peeled leaves were treated with 50µg ml–1
VicBSA for the indicated time, and the fluorescence was detected at 488nm. The images are representative of at least three experiments. Note
that dead cells demonstrated by arrowheads show an abnormal distribution of the chloroplasts. (C) Changes in the intensity of the intracellular
VicBSA signal. Epidermis-peeled victorin-sensitive leaves were treated with 50µg ml–1 VicBSA for the indicated time. The intensity of VicBSA
fluorescence in 20 independent mesophyll cells was monitored with excitation of 488nm and evaluated by MetaMorph software. Data represent
the mean value and SD of three independent experiments.
death program independently of the binding to mitochondrial the interaction of native victorin with the host cell surface leads
GDC or the other unknown intracellular virulence targets. to cell death, the VicBSA-activated cell death pathway would
be blocked by the same inhibitors that prevent victorin-induced
Ca2+ and extracellular alkalization are essential signals for vic- cell death. Cell viability was assessed by the fluorescent dye
torin-induced cell death fluorescein diacetate (FDA), which determines membrane
Because victorin has been shown to trigger drastic defense integrity. Victorin- and VicBSA-induced cell death was
responses (Mayama et al. 1986), it is possible that victorin- strongly suppressed in the presence of Ca2+ metabolism inhibi-
induced cell death may occur as a result of immune responses. tors such as the Ca2+ chelator EGTA (10mM), the Ca2+ chan-
Thus, we performed pharmacological studies on cell death nel blocker LaCl (1.5mM) and the Ca2+ competitor ZnSO
3 4
induced by 5ng ml–1 victorin and 50µg ml–1 VicBSA using (1mM) (Fig. 3), suggesting that Ca2+ is a critical regulator of
various inhibitors that affect signal transduction pathways of cell death signaling. Changes in the cytosolic Ca2+ concentra-
defense responses and apoptotic cell death (Fig. 3). Given that tion are generally followed by the activation of Ca2+-sensing
Victorin elicits an HR-like response 1791
lular ATP content may play an important role in victorin- and
VicBSA-induced cell death. The inhibitor profiles of VicBSA
completely corresponded to those obtained with native victorin,
suggesting that VicBSA and victorin may stimulate the same
signal transduction pathway leading to the onset of cell death.
Furthermore, these findings led us to hypothesize that active
transport of ions (e.g. Ca2+ and H+), which requires ATP as an
energy source, might regulate cell death induced by victorin
and VicBSA, whereas the protein products of defense-related
genes may not be involved in the execution of cell death.
A previous study reported that change in the plasma mem-
brane permeability is the early toxic action of HSTs, and thus
electrolyte leakage is thought to be an indicator of cell death
(Walton 1996). However, the ion leakage is also a physiologi-
cal response associated with both the defense response
(Mackey et al. 2002) and toxin-induced cell death. Thus, to
confirm the assumption that stimulation of ion transport is the
critical checkpoint to victorin- and VicBSA-induced cell death,
we monitored the effects of various inhibitors on the kinetics of
extracellular alkalization induced by 5ng ml–1 victorin and
50µg ml–1 VicBSA (Fig. 4). As we expected, pre-treatment
with antimycin A for 2h strongly reduced extracellular
alkalization, which was evident by 30min after victorin or
Fig. 3 Effect of inhibitors of signal transduction pathways on vic- VicBSA treatment, while a shorter pre-treatment period of
torin- and VicBSA-induced cell death. Epidermis-peeled victorin-sen- 5min did not suppress the alkalization (data not shown). Addi-
sitive leaves were treated with 5ng ml–1 victorin C or 50µg ml–1 tion of LaCl also completely inhibited extracellular alkaliza-
3
VicBSA plus the indicated reagents for 3h. Living cells were esti-
tion during the time course. In contrast, the chemical agents
mated by FDA staining, and dead cells were expressed as a percent-
that did not reduce cell death, such as cycloheximide, K-252a
age of the total cells counted. Data represent the mean value and SD of
three independent experiments. and E-64, failed to block the rapid increase in extracellular pH.
It is important to note that both antimycin A and LaCl abro-
3
gated the alkalization induced by water treatment, while the
calmodulin, protein kinase and de novo protein synthesis (Chin other chemicals could not, demonstrating the potential ability
and Means 2000). However, the calmodulin inhibitor trifluop- of the two reagents to suppress the active ion movement. These
erazine (500µM), the protein kinase inhibitors K-252a (2µM) data indicate that the ion fluxes across the plasma membrane
and staurosporine (500nM), the protein phosphatase inhibitor may be a critical upstream signal in the cell death pathway.
okadaic acid (500nM) and the inhibitor of new protein synthe-
sis cycloheximide (2µM) did not have any effect on cell death, Victorin and VicBSA serve as specific elicitors
strongly suggesting that victorin- and VicBSA-induced cell Mayama et al. (1986) demonstrated that low concentra-
death is controlled by a constitutive machinery independent of tions of victorin, unlike some abiotic and biotic elicitors,
phosphorylation-based regulations. The NADPH oxidase inhib- stimulate the biosynthesis of phytoalexins, avenanthramides, in
itor diphenyleneiodonium chloride (DPI; 50µM), which is primary leaves of susceptible cultivar Iowa X469, suggesting
used to block the oxidative burst, did not abolish the induction that victorin functions as a specific elicitor, not as a general
of cell death. Although the addition of superoxide dismutase toxin which simply damages cells. To investigate the potential
(SOD; 150µg ml–1) and the radical scavenger N-acetyl- ability of VicBSA as a victorin derivative, we determined
cysteine (NAC; 2mM) partially reduced victorin-induced cell whether VicBSA treatment resulted in the induction of avenan-
death 3h after treatment, the treated cells consequently died by thramide A. Low concentrations of VicBSA (0.5–2µg ml–1)
5h after treatment (data not shown) as with VicBSA. The stimulated the accumulation of avenanthramide A in victorin-
cysteine (E-64, 500µM) and serine protease (aprotinin, 150µg sensitive leaves 24h after treatment in which >60% of the
ml–1) inhibitors did not suppress victorin- and VicBSA-induced treated cells were alive (data not shown), but had no signifi-
cell death. Interestingly, pre-treatment of victorin-sensitive cant effect in victorin-insensitive leaves (Fig. 5). At higher
leaves for 2h with antimycin A (10µM), which is known to concentrations (>5µg ml–1), the production of avenanthramide
deplete cellular ATP (Atkinson et al. 2004), resulted in signifi- A was dramatically decreased to the basal level, presumably
cant inhibition of cell death, while pre-treatment for 5min had because almost all the treated cells died within 12h, at which
no effect on cell death (data not shown), indicating that the cel- time little accumulation of avenanthramides was observed even
1792 Victorin elicits an HR-like response
Fig. 4 Victorin- and VicBSA-induced cell death is associated with extracellular alkalization. Epidermis-peeled victorin-sensitive leaves were
incubated with 5ng ml–1 victorin C (closed circle) or 50µg mL–1 VicBSA (open circle) plus the indicated chemicals or with inhibitors alone
(square). The kinetics of pH changes in test solutions were monitored by pH meter.
oat plants (Tada et al. 2004). We determined the kinetics of
ROI and NO generation by monitoring the fluorescence of
2′,7′,-dichlorofluorescein (DCF) and 4,5-diaminofluorescein
(DAF), respectively. The lower epidermis of primary leaves
was peeled off and floated on a test solution containing DCF or
DAF, with the peeled surfaces in contact with the liquid. After
treatment with 5ng ml–1 victorin (closed symbols) and 50µg
ml–1 VicBSA (open symbols), significant production of ROIs
and NO was detected by 90min (Fig. 6), when VicBSA was
restricted to the extracellular space of susceptible cells (Fig. 2).
At 3h after victorin treatment, the fluorescence intensity of
DCF and DAF in toxin-sensitive leaves (circles) reached 4.0-
fold and 3.3-fold higher than that detected in the toxin-insensi-
tive cultivar Iowa X424 (squares), respectively. VicBSA elic-
ited changes in the kinetics of ROI and NO production similar
to those induced by native victorin. The increase in the fluores-
cence of DCF and DAF in a test solution was continuously
Fig. 5 VicBSA elicits the production of avenanthramide (A) Peeled
leaf segments of victorin-sensitive (circle) and insensitive cultivars observed even after the 2h time point at which most of the
(square) were treated with VicBSA at the indicated concentrations. exposed cells died (Fig. 2A, 6). These results are consistent
Avenanthramide A in test solutions was estimated 24h after treatment
with our previous observation that although the cells most
by HPLC. Data represent the mean ± SD from three experiments.
proximal to the peeled side died 2h after victorin treatment, the
other layers remained intact (Yao et al. 2001), suggesting that
in leaves treated with a low concentration of VicBSA (data not the generation of ROIs and NO could be elicited in the remain-
shown). ing viable cells. These results indicate that victorin and
The oxidative burst and the generation of NO are the most VicBSA stimulate a set of defense responses typically incited
prominent defense responses highly associated with the HR in during a gene-for-gene type resistance.
Victorin elicits an HR-like response 1793
Fig. 7 VicBSA induces apoptotic responses in victorin-sensitive
leaves. (A) Time course of VicBSA-induced DNA laddering. Peeled
leaf segments of victorin-sensitive (Iowa X469) and -insensitive culti-
vars (Iowa X424) were treated with 50µg ml–1 VicBSA for the indi-
cated time. DNA was analyzed by ethidium bromide staining on a 2%
(w/v) agarose gel. (B–D) Ultrastructural changes in VicBSA-treated
cells. Peeled victorin-sensitive leaves were incubated with 50µg ml–1
VicBSA for the indicated time. Open arrowheads show localized con-
densation of heterochromatin, and closed arrowheads indicate plasmo-
lysis. N, nucleus; Ch, chloroplast. Bar in (F) to (H) = 5µm.
evenly scattered within the nucleus (Fig. 7B). Although the
plasma membrane shrunk away from the cell wall at 2h after
Fig. 6 Victorin and VicBSA induce the generation of reactive oxygen
treatment (Fig. 7C, arrowheads), other organelles remained
intermediates (ROIs, A) and nitric oxide (NO, B) in victorin-sensitive
morphologically intact as observed in the control cells. As
leaves. Primary leaves of sensitive cultivar Iowa X469 (circle) and
insensitive cultivar Iowa X424 (square) were treated with 5ng ml–1 reported for native victorin (Yao et al. 2001), further con-
victorin C (closed symbols) or 50µg ml–1 VicBSA (open symbols) for densed chromatin was observed in the cells adjacent to the
the indicated time. The time course of ROI and NO accumulation was VicBSA-exposed layer in which almost all cells collapsed 4h
monitored by the fluorescence of dichlorofluorescein (DCF) and
after treatment (Fig. 7D). We conclude that VicBSA also stimu-
diaminofluorescein (DAF), respectively. Points and bars represent the
lates apoptosis-like cell death in victorin-sensitive leaves via a
mean value and SD of three independent experiments.
cell surface mediator.
VicBSA induces apoptosis-like cell death in susceptible cells Discussion
In previous studies, we showed that victorin causes the
cell to undergo apoptosis-like cell death characterized by DNA Although the primary function of phytotoxic compounds
laddering and heterochromatin condensation in the nuclei (Tada is thought to be to induce cell death and/or to suppress defense
et al. 2001, Yao et al. 2001, Yao et al. 2002). Thus we assessed response in host plants, we propose here an alternative func-
whether VicBSA activates the same signal cascade that is elic- tion of phytotoxic compound to trigger the plant defense
ited by victorin. The genomic DNA extracted from victorin- machinery for pathogenicity. Our data have demonstrated that
sensitive leaves treated with 50µg ml–1 VicBSA exhibited victorin serves as a specific elicitor in oat plants carrying the
clear DNA laddering from 4h after treatment, at which time Vb/Pc-2 gene, as initially suggested by Mayama et al. (1986).
VicBSA is excluded from the susceptible cell, but was not Victorin triggers a wide range of defense responses such as
observed in toxin-insensitive leaves through the incubation programmed cell death (PCD) (Tada et al. 2001, Yao et al.
period (Fig. 7A). Ultrastructural observation showed that het- 2001), an oxidative burst, NO generation and the activation of
erochromatin had began to aggregate into large masses in vic- defense-related genes responsible for phytoalexin synthesis,
torin-sensitive leaves treated with 50µg ml–1 VicBSA for 2h PR protein accumulation and detoxification of ROI stress (S.
(Fig. 7C), while heterochromatin in the control leaves was Betsuyaku, H. Nakayashiki, Y. Tosa and S. Mayama, unpub-
1794 Victorin elicits an HR-like response
Fig. 8 The current model of victorin-induced cellular responses. Victorin-sensitive cells. (A) Victorin may interact with an extracellular media-
tor(s) in Vb/Pc-2 oats and stimulates ion fluxes across the plasma membrane, followed by the activation of defense responses and rapid cell death.
After cell death, victorin may permeate cells and is distributed in the mitochondria, inducing a senescence-like response (Navarre and Wolpert
1999). (B) Victorin-insensitive cells. Victorin neither induces cell death nor enters the resistant cells.
lished ). Recently, we demonstrated that two genes encoding K. Kusaka, Y. Tosa and S. Mayama, unpublished), and has
hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinn- been shown to accumulate in the mitochondria after the induc-
amoyltransferase and S-adenosyl-L-methionine:trans-caffeoyl- tion of cell shrinkage (Curtis and Wolpert 2004). Secondly,
CoA 3-O-methyltransferase, which catalyze the biosynthesis of VicBSA, a macromolecular conjugate in which victorin and
avenanthramides, are highly up-regulated in response to vic- fluorescein are covalently attached to BSA, clearly induces cell
torin (Yang et al. 2004). Considering that defense responses
death 2h after treatment without entering cells until 6h (Fig.
evoked by a pathogen-encoded avirulence gene product often
2). Interestingly, low concentrations of VicBSA elicit the pro-
accompany PCD (Lam et al. 2001), it is possible that victorin
duction of avenanthramide A in victorin-sensitive leaves (Fig.
carries out its virulence function by activating the innate
5). Furthermore, extracellular alkalization and the generation of
immune system.
ROIs and NO are rapidly induced in response to VicBSA,
We propose a model in which victorin is recognized on
before it accumulates in the cell, with similar kinetics to those
the cell surface triggering apoptosis-like cell death and defense
detected with native victorin. Thus, VicBSA and VicFluor most
responses independently of the binding to the GDC (Fig. 8) as
probably attack extracellular component(s) to incite nearly all,
originally demonstrated by Akimitsu et al. (Akimitsu et al.
or all, victorin-induced responses. These indicate that the pro-
1992, Akimitsu et al. 1993a, Akimitsu et al. 1993b). We pro-
tein product of the Vb gene could be involved in the recogni-
vide two lines of evidence for this proposal: first, VicFluor, a
fluorescein conjugate of victorin, does not accumulate in tion of victorin on the cell surface, including the plasma
victorin-sensitive cells until 4h after treatment, as shown by membrane and cell wall, and that although the binding of vic-
Curtis and Wolpert (2004), although most of the cells are dead torin to the mitochondrial GDC is initially suggested to play a
by 2h (Fig. 1). VicFluor also serves as a potent elicitor leading central role in victorin’s mode of action (Wolpert et al. 1994,
to the production of avenanthramide A, ROIs and NO (Y. Tada, Navarre and Wolpert 1995), the P protein might be the primary
Victorin elicits an HR-like response 1795
site of action neither for acute cell death nor for defense dering. The authors discussed that an MPT may be required for
responses as recently proposed by Wolpert et al. (2002). the access of victorin to the mitochondrial matrix. However,
Akimitsu et al. (Akimitsu et al. 1992, Akimitsu et al. there is an alternative possibility that FCCP may rescue cell
1993a) showed, using anti-victorin polyclonal antibodies, that death through the reduction of ATP levels as in leaves treated
victorin binds to 65 and 45kDa proteins in purified plasma with antimycin A. (ii) Among inhibitors of signal transduction
membrane of both susceptible and resistant cultivars. Further- pathways, only reagents, other than antimycin A, that affect
more, the authors demonstrated that anti-victorin anti-idiotypic divalent cation metabolism can strongly suppress victorin-
antibodies elicit callose synthesis in protoplasts from suscepti- induced cell death (Fig. 3). Administration of inhibitors that
ble leaves as induced by native victorin (Akimitsu et al. perturb the activity of protein kinase, phosphatase, calmodulin,
1993b). They suggested that the primary site of action of vic- proteases, ROI generation and protein synthesis has no sup-
torin exists on the surface of the plasma membrane, since the pressive effect on cell death in a long-term treatment. Consid-
antibodies are speculated to be too large to enter cells. The cur- ering our previous studies showing that cysteine and serine
rent study further supports these results and raises the possibil- proteases, protein kinases and ROIs play important roles in the
ity that this recognition can lead to a battery of victorin- induction of DNA laddering or heterochromatin condensation
induced responses. Actually, our unpublished data (R. Saito, S. (Tada et al. 2001, Yao et al. 2001, Yao et al. 2002), the current
Ryu, K. Kusaka, Y. Tada, H. Nakayashiki, Y. Tosa and S. data may suggest that the commitment to cell death precedes
Mayama) demonstrated that victorin does interact with a novel ROI generation, and activation of protein kinases, cysteine and
mediator(s) extracted from the extracellular space of both sus- serine proteases which lead to the late characteristics of apop-
ceptible and resistant leaves. tosis. These facts do not contradict the hypothesis that the ion
Despite the data showing that the cell surface molecule(s) fluxes across the plasma membrane could be the primary cause
may be a key mediator of victorin-induced responses, we can- of cell death. Supporting this assumption, (iii) all reagents
not rule out the possibility that the inactivation of GDA plays a including antimycin A that negate victorin-induced cell death
major role in the pathogenicity of C. victoriae. Navarre and remarkably inhibit the increase in extracellular pH, a hallmark
Wolpert (1999) demonstrated that victorin causes a senescence- of the activation of ion transport (Fig. 4). We suggest that extra-
like response in victorin-sensitive leaves characterized by a cellular alkalization is an upstream signal responsible for the
proteolytic cleavage of the Rubisco large subunit (LSU) as well cell death program and is not induced as a result of cell death
as chlorophyll loss. The binding of victorin to the GDC appears because only these chemicals can shut down the transient
to be prerequisite for LSU cleavage because pyridoxal phos- alkalization, which might be activated by peeling stress, in
phate, which competes with victorin for binding to the P- leaves treated with water, showing the predominant function as
protein, prevents Rubisco cleavage, indicating that the inhibi- inhibitors of ion transport systems.
tion of GDA may facilitate plant destruction by abrogating the Several lines of evidence suggest that the stimulation
photorespiratory cycle. In addition, the application of the GDC of ion fluxes is tightly associated with the onset of cell
inhibitor aminoacetonitrile results in apoptosis-like cell death death and resistance response. The syringolide elicitors from
(Yao et al. 2002) and disease symptoms similar to those induced Pseudomonas syringae isolates elicit the HR (Keen and
by native victorin (Y. Tada, S. Hata, Y. Tosa and S. Mayama, Buzzell 1991) accompanied by extracellular alkalization, K+
unpublished). Therefore, although the inhibition of GDA by efflux and Ca2+ influx (Atkinson et al. 1996) in soybean culti-
victorin seems to play a partial role, if any, in the onset of cell vars that carry the complementary resistance gene Rpg4. As in
death and defense responses, it may contribute to symptom the case of victorin-treated leaves, the blockage of Ca2+ influx
development and provide subsequent benefit for the pathogen by LaCl significantly suppresses the increase in extracellular
3
in terms of feeding nutrients and colonizing host plants. pH and K+ efflux, demonstrating their important roles in a the
We propose that active transport of ions, which requires syringolide-activated signaling pathway (Atkinson et al. 1996).
ATP as an energy source, is the critical regulator of physiologi- Furthermore, the non-HR response of parsley elicited by an
cal and morphological changes induced by victorin. This oligopeptide elicitor Pep-13 of Phytophthora sojae also
hypothesis largely comes from the observations as follows. (i) requires a sustained increase in free Ca2+ and extracellular
ATP depletion by pre-treatment with antimycin A for 2h alkalization as components of the signal cascade (Nurnberger
results in the strong inhibition of cell death (Fig. 3), whereas a et al. 1994, Blume et al. 2000). Intriguingly, the expression of
shorter pre-treatment period of 5min with antimycin A does the proton pump bacterio-opsin (bO) from Halobacterium
not negate cell death. The significant reduction of cellular ATP halobium in various plants results in the induction of HR cell
levels occurs in oat suspension cells by 2h after antimycin A death, systemic resistance and genes encoding PR proteins and
treatment but not by 5min (M. Sakamoto, H. Nakayashiki, Y. PAL in the absence of pathogen (Mittler et al. 1995). These
Tosa and S. Mayama, unpublished). Curtis and Wolpert (2002) responses are caused by passive proton leakage through the bO
demonstrated that the mitochondrial uncoupler FCCP, which channel, indicating that the loss of ion homeostasis may be the
inhibits ATP synthesis and leads to the hydrolysis of cellular cause of the HR (Pontier et al. 2002). Here we show that the
ATP, prevents VicFluor binding to the P protein and DNA lad- continual exposure of toxin-sensitive cells to victorin leads to a
1796 Victorin elicits an HR-like response
sustained increase in extracellular pH until cell death occurs Materials and Methods
(Fig. 4), exhibiting the possible role of the abnormal ion bal-
ance across the plasma membrane in cell death. Plant materials
Unlike other HSTs, the current hypothesis is that victorin Seven-day-old primary leaves of oat (A. sativa L.) cultivars Iowa
induces disease susceptibility by activating host defense X424 and Iowa X469 were used in this study. Iowa X469 carrying the
Vb/Pc-2 gene is sensitive to the host-selective toxin victorin C pro-
responses. This proposal may seem to contradict a general con-
duced by C. victoriae. Iowa X424 is a near isogenic line of Iowa X469
cept that toxins abrogate resistance to pathogens. However,
that does not carry the Vb/Pc-2 gene and hence is insensitive to vic-
although high concentrations of victorin up-regulate a set of torin. Seeds were dipped in water for 12h, sown on vermiculite, and
defense-related genes (Yang et al. 2004, S. Betsuyaku, H. grown in a growth chamber at 20°C under illumination for 16h daily
Nakayashiki, Y. Tosa and S. Mayama, unpublished), the with 10,000lux fluorescent light.
production level of avenanthramides is very low (Mayama et
Victorin C and chemical treatments
al. 1995). Our unpublished data (Y. Takata, Y. Kimura, H.
The host-selective toxin victorin C was kindly provided by Dr.
Nakayashiki, Y. Tosa and S. Mayama) demonstrated that the
Wolpert (Oregon State University, Corvallis, OR, USA) and was
antifungal protein chitinase, which is highly induced by low applied at a concentration of 5ng ml–1. E-64d, aprotinin, cyclo-
concentrations of victorin, also does not accumulate in heximide, EGTA, LaCl, ZnSO, trifluoperazine, SOD and NAC were
3 4
response to toxic concentrations of victorin. It is plausible that obtained from Nacalai Tesque (Kyoto, Japan). Antimycin A, stau-
the effective production of such antimicrobial compounds rosporine, DPI and okadaic acid were obtained from Sigma (St Louis,
MO, USA). K-252a was purchased from Alomone Labs (Jerusalem,
would take longer than the execution of cell death and the
Israel). SOD and NAC were used as ROI scavengers at concentrations
expression of the defense-related genes. Furthermore, avenan- of 150µg ml–1 and 2mM, respectively. EGTA, a calcium chelator,
thramides have been shown not to inhibit the fungal growth ZnSO, a calcium antagonist, and LaCl, a calcium channel blocker,
4 3
effectively. Thus, C. victoriae may synchronously elicit HR- were used at 10, 1 and 1.5mM, respectively. K-252a and staurosporine
like cell death at and around the infection site and exploit the were used as protein kinase inhibitors at concentrations of 2µM and
500nM, respectively. Okadaic acid was used as a protein phosphatase
dead cells for the pathogenicity, avoiding the accumulation of
inhibitor at 500nM. E-64d was used as a cell-permeable cysteine
PR proteins and other antifungal compounds. The necrotrophic protease inhibitor at 500µM. Aprotinin was used as a serine protease
pathogen Botrytis cinerea has been shown to elicit an HR in inhibitor at 150µg ml–1. Cycloheximide was used as an inhibitor of de
Arabidopsis to utilize dead cells for successful colonization, novo protein synthesis at 2µM. DPI was used as an NADPH oxidase
while the Arabidopsis dnd1 (defense, no death) mutant, loss-of- inhibitor at a concentration of 50µM. Antimycin A was used as a
mitochondrial complex III inhibitor at 10µM. The effectiveness of
HR phenotype, significantly prevents fungal growth of B. cine-
inhibitors and an enzyme used in this experiment has already been
rea (Govrin and Levine 2000). In addition, the suppression of
demonstrated in our previous studies (Tada et al. 2001, Kusaka et al.
PCD by transgenic expression of the baculovirus p35 gene, a
2004, Tada et al. 2004). The lower epidermis of primary oat leaves
specific inhibitor of apoptogenic protease (caspase), protects was peeled off, and 5cm segments, taken at 1–6cm from the leaf tip,
tomato plants from disease symptoms induced by Alternaria were floated on 3ml of testing solutions with the exposed mesophyll
alternata and P. syringae pv. tomato (Lincoln et al. 2002). surface in contact with the liquid, and kept at 20°C under continuous
light for the indicated times.
Together with our data, these suggest that PCD during the HR
and pathogenicity does not necessarily retard fungal growth
Cytofluorimetric analysis
especially in plant–necrotroph interactions, and that certain
An IX70 (Olympus, Tokyo, Japan) fluorescent microscope
necrotrophic pathogens have evolved their tools to trigger cell equipped with a MicroMax 12-bit CCD camera system (Princeton
death by modification of plant intrinsic cell death signaling. Instruments, Inc., Trenton, NJ, USA) and an optical filter changer
In mammalian cells, effectors from pathogenic bacteria (Lambda 10–2, Sutter Instruments, Novato, CA, USA) was used. A
xenon lamp is coupled with a monochromator (Polychrome II, TILL
often mimic host molecules and interact with host cell surface
Photonics, Planegg, Germany) to provide the excitation light source.
receptors to reduce phagocytic activity and the development of
ROIs generated from oat leaves were measured by monitoring the flu-
immunity (Knodler et al. 2001). We demonstrated that victorin orescence of DCF (Molecular Probes, Eugene, OR, USA). NO accu-
elicits an HR-like response in Vb/Pc-2 oats, possibly for patho- mulation was measured by the fluorescence of DAF (Daiichi Kagaku,
logical cell death, suggesting that victorin might mimic a spe- Tokyo, Japan). The epidermis was peeled from 7-day-old leaves, and
cific elicitor against Pc-2 to stimulate cell death signaling, 1cm×5 segments were incubated with the test solutions containing
100µM DCF or 50µM DAF for the indicated times. The test solutions
which is normally triggered during defense against the crown
were observed using 488 and 515nm with excitation at 520 and
rust fungus. Although we cannot exclude the possibility that
495nm emission wavelengths, respectively. Images from the micro-
the Vb gene differs from the Pc-2 gene, our data suggest an alter- scope were acquired, and the fluorescence intensity values were
native strategy of the necrotrophic pathogen to invade plants. analyzed by MetaMorph software (Universal Imaging Corporation,
Downingtown, PA, USA). All fluorescence intensity values would be
in the range between 0 and 4,095, and experimental points represent
the mean ± SD of three replicates of the same experiment.
VicFluor was prepared as described by Curtis and Wolpert
(2002). A 100µg aliquot of victorin was incubated with 1mg of 5-
(and 6)-carboxyfluorescein, succinimidyl ester (NHS–fluorescein,