Anti-apoptotic machinery protects the necrotrophic fungus Botrytis cinerea from host-induced apoptotic-like cell death during plant infection.

Abstract

Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity. Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal drugs.

Full text

Page 1

Anti-Apoptotic Machinery Protects the Necrotrophic
Fungus Botrytis cinerea from Host-Induced Apoptotic-
Like Cell Death during Plant Infection
Neta Shlezinger1, Anna Minz1, Yonatan Gur1, Ido Hatam1, Yasin F. Dagdas2, Nicholas J. Talbot2,
Amir Sharon1*
1Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv, Israel, 2School of Biosciences, University of Exeter, Exeter, United Kingdom
Abstract
Necrotrophic fungi are unable to occupy living plant cells. How such pathogens survive first contact with living host tissue
and initiate infection is therefore unclear. Here, we show that the necrotrophic grey mold fungus Botrytis cinerea undergoes
massive apoptotic-like programmed cell death (PCD) following germination on the host plant. Manipulation of an anti-
apoptotic gene BcBIR1 modified fungal response to PCD-inducing conditions. As a consequence, strains with reduced
sensitivity to PCD were hyper virulent, while strains in which PCD was over-stimulated showed reduced pathogenicity.
Similarly, reduced levels of PCD in the fungus were recorded following infection of Arabidopsis mutants that show enhanced
susceptibility to B. cinerea. When considered together, these results suggest that Botrytis PCD machinery is targeted by plant
defense molecules, and that the fungal anti-apoptotic machinery is essential for overcoming this host-induced PCD and
hence, for establishment of infection. As such, fungal PCD machinery represents a novel target for fungicides and antifungal
drugs.
Citation: Shlezinger N, Minz A, Gur Y, Hatam I, Dagdas YF, et al. (2011) Anti-Apoptotic Machinery Protects the Necrotrophic Fungus Botrytis cinerea from Host-
Induced Apoptotic-Like Cell Death during Plant Infection. PLoS Pathog 7(8): e1002185. doi:10.1371/journal.ppat.1002185
Editor: Brett Tyler, Virginia Polytechnic Institute and State University, United States of America
Received March 9, 2011; Accepted June 14, 2011; Published August 18, 2011
Copyright: ? 2011 Shlezinger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant #206/09 from the ISF to AS. ISF web page: http://www.isf.org.il/english/default.asp. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: amirsh@ex.tau.ac.il
Introduction
Plant pathogenic fungi have evolved two prominent infection
strategies: biotrophic pathogens proliferate within the living plant
tissue deriving nutrition from living plant cells, and necrotrophic
pathogens that do not occupy living plant cells and instead, kill
host cells before tissue colonization. In order to infect plants,
biotrophic pathogens suppress plant defenses [1], a strategy that is
commonly used by pathogens to overcome host immunity [2,3]. In
contrast, necrotrophic pathogens secrete enzymes and toxins that
kill the host tissue ahead of pathogen invasion, thus avoiding direct
contact with defense molecules in living plant cells. It is unclear
how this group of pathogens overcomes the host defenses during
the early stages of infection, when the fungus is in contact with
living host cells.
The grey mold fungus Botrytis cinerea has become an important
model for the study of interactions between plants and
necrotrophic pathogens [4]. B. cinerea infects over 200 cultivated
plant species and causes significant economic damage to crops
worldwide. Host-specific resistance is ineffective against B. cinerea
and, conversely, the fungus utilizes the hypersensitive resistance
response (HR) and the associated programmed cell death of the
host to advance infection [5,6]. The plant defense responses
activated by B. cinerea are regulated in several ways, including the
jasmonate and ethylene signaling pathways, as well as by
additional signaling cascades that have not yet been identified
[7,8,9]. Collectively, these defense responses can slow B. cinerea
infection, but they do not completely block disease development
[10,11,12].
Production of anti-microbial secondary metabolites represents
an important component of plant defense responses against
pathogens [13,14]. In case of B. cinerea, the most significant
secondary metabolites are glucosinolate, indolic, and phenylpro-
panoid compounds [15,16]. Specifically, the indolic phytoalexin
camalexin has been shown to play important role in defense of A.
thaliana against a range of pathogens, including B. cinerea
[10,11,15]. Production of camalexin is induced in plants following
infection with B. cinerea, and A. thaliana mutant plants affected in
camalexin biosynthesis show variable levels of enhanced sensitivity
to pathogens [17]. The final step of camalexin production is
catalyzed by a cytochrome P-450 monooxygenase encoded by the
locus PAD3 [18]; pad3 mutant plants do not produce camalexin
and are hypersensitive to a wide range of pathogens including B.
cinerea. It has been proposed that PAD3 is required for basal
resistance to B. cinerea strains sensitive to camalexin [7,15].
Although it has been shown that camalexin is toxic to a wide
range of fungi, the mode of action remained unclear.
At least some plant antimicrobial compounds have the potential
to induce apoptotic-like programmed cell death (PCD) in fungi
[19,20,21]. Because necrotrophic fungi are unable to evade the
plant defenses, we reasoned that plant defense molecules might
target the fungal PCD machinery as a way to induce cell death in
necrotrophic pathogens. Here we show that B. cinerea is exposed to
massive, plant-induced PCD during the early phase of infection.
PLoS Pathogens | www.plospathogens.org1 August 2011 | Volume 7 | Issue 8 | e1002185

Page 2

Mutant plants that are affected in defense responses and are
hypersensitive to B. cinerea, induce reduced levels of PCD in the
fungus. We also show that camalexin induces PCD in B. cinerea and
that it is important for the full scale host-driven PCD during the
early stages of infection. The fungal anti PCD machinery
attenuates camalexin and host-driven PCD and is therefore
necessary for infection.
Results
Generation of apoptosis-attenuated fungal strains
To investigate the importance of PCD in Botrytis pathogenesis,
we first generated B. cinerea strains showing both enhanced and
impaired levels of PCD. In order to generate strains showing
altered PCD, we isolated BcBIR1 (BC1G_14521.1), a B. cinerea
homolog of the Saccharomyces cerevisiae BIR1 gene (GenBank
P47134) (Figure S1). Bir1 is a component of the chromosomal
passenger complex, which regulates chromosome segregation, an
activity that is mediated by the carboxy-terminal domain of the
protein and essential for yeast survival [22]. Bir1p also has anti-
apoptotic activity, which is mediated by the two BIR domains at
the N’ terminal part of the protein and is not necessary for the cell
cycle regulating activities of the protein [23]. The B. cinerea BcBir1
protein also contains two BIR domains at the N’ part of the
protein, it shares 24% of amino acid sequence identity with Bir1,
but it is considerably shorter than Bir1 (601 amino acids and 954
amino acids, respectively).
Transgenic strains were first produced showing high level
expression of the entire BcBIR1 gene or an N’ terminal region of
the protein containing the two BIR domains (Figure S2).
Complete deletion of BcBIR1 was not possible because the gene
is essential, similar to the S. cerevisiae BIR1 gene. We therefore
used a partial knockout (heterokaryon) strain (Dbcbir1) in which
the expression of BcBIR1 was reduced, but not completely
eliminated.
PCD in BcBIR1 transgenic strains
Next, we tested the ability of these B. cinerea strains to undergo
PCD. When treated with apoptosis-inducing agents such as H2O2,
or under physiological conditions that induce PCD such as ageing
or in stationary phase [19,24]. BcBIR1 over-expression strains
(expressing either the entire protein or only the N’ part) retained
higher growth rates and showed reduced PCD markers (chromatin
condensation, DNA strand breaks, ROS accumulation), whereas
Dbcbir1 mutant strains exhibited reduced growth rates and showed
enhanced PCD compared with the isogenic wild type strain
(Figures 1, S3). These results show that BcBir1 has anti-apoptotic
activity, which is mediated by the BIR domains at the N’ terminal
part of the protein, similar to the S. cerevisiae homologue Bir1p.
The BcBIR1 mutant strains are affected in pathogenicity
To test the effect of BcBIR1 on pathogenicity, we inoculated
beans leaves and measured disease symptoms. The BcBIR1 over-
expression strains (in which PCD was attenuated) caused increased
disease symptoms, while Dbcbir1 mutant strains (in which PCD was
enhanced) caused restricted lesions (Figure 2A). Similar results
were observed on Arabidopsis thaliana (Figure 2B) and on a number
of additional plant species that are host for B. cinerea, such as peas,
tomato, tobacco and basil (data not shown). Thus, PCD and
pathogenicity in BcBIR1 transgenic strains are modified such that
enhanced or reduced PCD is associated with reduced or enhanced
pathogenicity, respectively. These results support the possibility
that the fungus undergoes apoptotic-like PCD during infection,
which might have an impact on disease establishment.
Use of H1-GFP tagged strains to follow fungal infection
To test whether the fungus undergoes apoptotic-like PCD
during infection, we generated a transgenic B. cinerea strain (H1-
GFP), in which nuclei were tagged with GFP, and followed
changes in the nuclear GFP signal in fungal hyphae during
progression of plant infection. Cytoplasmic localization of the
histone H1 was shown to play an important role during apoptosis
in mammals [25,26]. Furthermore, the disappearance of the
nuclear signal in H1-GFP tagged strains has previously been used
to indicate autophagic programmed cell death in Magnaporthe oryzae
during appressorium formation [27], and apoptotic-like pro-
grammed cell death in Colletotrichum gloeosporioides following
treatment with apoptosis-inducing agents [28].
First, we tested the effect of H2O2on the GFP signal in the B.
cinerea H1-GFP strain and compared it with accumulation of
apoptotic markers, DNA strand breaks (determined by TUNEL)
and chromatin condensation and fragmentation (determined by
DAPI staining). During the first hours after treatment with H2O2,
the nuclear GFP signal faded out and it could no longer be
detected 12 h after treatment (Figure S4A). At this time point, as
well as at 6 h post treatment, green fluorescence could be
visualized in the entire hyphae, which might be attributed to
dislocation of the GFP-tagged histone to cytoplasm during
apoptosis, as was shown for mammalian cells [25,26]. Alterna-
tively, the green fluorescence in damaged hyphae at this stage
might also result from disintegration of the nuclei, or due to auto-
fluorescence of destroyed hyphae. DAPI staining showed en-
hanced staining of nuclei 6 and 12 h post H2O2treatment, which
is indicative of chromatin condensation (Figure S4A). Since it is
impossible to distinguish between the TUNEL and GFP signals (in
both cases nuclei fluoresce in green), we performed TUNEL assay
on B. cinerea H1-GFP hyphae 12 h after treatment with H2O2,
when the nuclear H1-GFP signal completely disappeared (Figure
S4B). At this time point there was intense TUNEL staining,
further demonstrating the correlation between loss of the nuclear
Author Summary
Our work provides a fundamental new insight into the
biology and infection strategy of Botrytis cinerea, an
economically important plant pathogen, which is used as
a model for studying pathogenicity of necrotrophic plant
pathogens. A fundamental question that we sought to
address in this study is how B. cinerea, and possibly other
necrotrophic pathogens, overcome the plant defenses in
living host cells. For the first time we show evidence that
help explain the basic events behind early stages of
necrotrophic interaction. Our results show that plant
defense molecules target the fungal PCD machinery and
trigger massive cell death in the pathogen. Conversely, the
fungal anti-apoptotic machinery, mediated by the IAP-like
protein BcBir1, protects the fungus from host-induced
PCD, thereby allowing establishment of small infection
zones, from which spreading lesions will be produced at
the second infection stage. Weakening or strengthening of
B. cinerea anti-apoptotic machinery results in reduced or
enhanced disease symptoms, respectively, whereas weak-
ening of the plant defense system is associated with
reduced fungal PCD and enhanced disease. While the
current results are restricted to B. cinerea, we believe that
our findings have important implications for understand-
ing of fungal-host interactions, including the interplay
between pathogenic fungi and animal hosts, particularly
human pathogens.
PCD-Mediated Fungal Pathogenesis
PLoS Pathogens | www.plospathogens.org2August 2011 | Volume 7 | Issue 8 | e1002185

Page 3

GFP signal in the H1-GFP strain and progression of PCD. These
analyses confirmed that loss of the nuclear GFP signal in the H1-
GFP strain is correlated with early stages of apoptotic-like cell
death.
We then used the H1-GFP strain to determine the fate of the
fungal cells during plant infection. A. thaliana Col-0 plants were
inoculated with conidia of the H1-GFP strain and GFP signal in
fungal hyphae was monitored using live cell imaging during the
first 72 h of plant infection (PI). The nuclear GFP signal was
retained for the first 24 h PI, but during the next 24 h (24–48 h PI)
it dissociated; some GFP fluorescence was first observed in entire
hyphae (as was also seen following H2O2treatment) and later
faded out, consistent with progression of PCD within proliferating
hyphae (Figure 3A). In accordance with results of the H2O2
treatment, significant reduction in the number of DAPI stained
nuclei, which represents a final stage of cell death, lagged behind
the disappearance of the H1-GFP signal and could only be
observed at 48 h PI (Figure 3A, 48 h, Figure S4D).
Very few GFP-labeled nuclei could be detected at the 48 h time
point in isolated fungal cells within the mass of mycelium in the
necrotic zone (Figure 3A, 48 h arrowhead). However, despite
massive cell death at 48 h, the fungus quickly recovered as
indicated by reappearance of the nuclear signal at 72 h PI. Similar
results were obtained on additional host plants, such as beans and
peas (Figure S4C). This sequence of events only occurred on
plants; when germinated on a glass slide, a stable nuclear signal
Figure 1. BcBIR1 encodes a protein with anti-apoptotic activity. (A, B) PCD induced by H2O2(see also Figure S3 for additional conditions). (A)
Fungi were grown in PDB for 24 h, 8 mM H2O2was added and relative biomass was determined after 48 h by measuring the OD of the cultures.
Percent of growth inhibition was calculated by dividing the OD of treated and untreated cultures. Data shown represent mean 6SEM for four
independent experiments performed in triplicates. (B) Accumulation of apoptotic markers. Chromatin condensation (DAPI) and DNA strand breaks
(TUNEL) were recorded 4 h after treatment with H2O2. Data shown represent mean 6SEM for triplicate samples of 200 nuclei per sample (n=3).
Percentage of apoptotic nuclei in untreated cultures were less than 0.5% for WT, BcBir124 and nBir15, and lower than 7% (DAPI) and 4% (TUNEL) for
Dbir14. (C) Ageing. Cultures were maintained under normal growth conditions, spores were collected after 7 and 14 days, stained with DAPI, and
percent of apoptotic nuclei was determined. Data shown represent mean 6SEM for triplicate samples of 100 spores per sample (n=4). Columns and
lines not connected with the same letter are statistically different (p,0.05) as determined by one-way ANOVA (p,0.001) followed by a post-hoc
Tukey HSD analysis. Pictures show increased accumulation of black pigments, which indicates senescence of the cultures in the Dbcbir1 mutant. The
strains were cultured on PDA plates as described above. Pictures were taken after 14 days of culturing.
doi:10.1371/journal.ppat.1002185.g001
PCD-Mediated Fungal Pathogenesis
PLoS Pathogens | www.plospathogens.org3August 2011 | Volume 7 | Issue 8 | e1002185

Page 4

was retained at all time points (Figure 3A, control). Thus, following
germination and establishment of contact with the host, nuclear
signal in H1-GFP strain starts to disappear between 24–36 h PI,
and almost no nuclei can be detected by 48 h PI. Since
disappearance of the H1-GFP signal is correlated with accumu-
lation of apoptotic markers, these results suggest that the majority
of fungal cells are exposed to plant-induced PCD during the early
infection phase, when the fungus is in direct contact with living
plant tissue.
We observed a similar pattern (though on a different time scale)
of disappearance and reappearance of the H1-GFP nuclear signal
during infection of corn plants by the necrotrophic fungus
Cochliobolus heterostrophus (Figure S5, Cc-Zm). In contrast, in the
hemibiotrophic pathogen C. gloeosporioides the H1-GFP signal was
stable throughout the infection process on pea leaves (Figure S5,
Cg-Ps). Significantly, inoculation of pea leaves with B. cinerea
produced similar results to inoculation of beans (Figure S4C),
indicating that the differences in H1-GFP signal did not result
from different host responses and are therefore specific to the
fungus. Further, similar to C. gloeosporioides, the nuclear signal was
stable throughout infection also in M. oryzae, a hemibiotrophic
fungus that infects rice and barley (Figure S5, Mo-Os). Thus,
retention or loss of the H1-GFP signal is not a universal
phenomenon of fungal pathogenesis, but rather it is specific to
B. cinerea, and possibly to other necrotrophic pathogens.
PCD is induced in Botrytis during the early infection
phase
To verify that the results obtained with the H1-GFP strain are
indicative of PCD, we measured DNA strand breaks (TUNEL
positive nuclei) and chromatin condensation (condensed nuclei
following DAPI staining) during infection of A. thaliana plants by
the wild type B05.10 strain. In accordance with the results
obtained with the H1-GFP marker, the level of PCD markers
increased between 24 h and 48 h PI, and then reduced to near
baseline levels at 72 h (Figure 3B–C). These results support the
Figure 2. BcBIR1 mutant strains are affected in pathogenicity. Leaves of P. vulgaris (A) and A. thaliana (B) were inoculated with B. cinerea
B05.10 wild type strain, Dbir14 mutant strain and BIR124 and nBIR15 strains that over-express the entire BcBIR1 or the N’ part of the protein,
respectively. Percent of primary lesions on beans (P. vulgaris) leaves was recorded 48 h PI by counting the number of droplets that produced lesions.
Size of expending lesions (on beans and Arabidopsis) was recorded 72 h PI. Data represent mean 6SEM of 32 droplets from four different plants per
each fungal genotype (n=4). Columns not connected with the same letter are statistically different (p,0.05) as determined by one-way ANOVA
(p,0.001) followed by a post-hoc Tukey HSD analysis.
doi:10.1371/journal.ppat.1002185.g002
PCD-Mediated Fungal Pathogenesis
PLoS Pathogens | www.plospathogens.org4August 2011 | Volume 7 | Issue 8 | e1002185

Page 5

results obtained with the H1-GFP strain and confirm that massive
PCD occurs during the early infection phase, but that the fungus
manages to recover upon transition to the second phase.
Modified PCD in BcBIR1 mutant strains during plant
infection
Infection of A. thaliana wild type plants with BcBIR1 over-
expression strains led to larger necrotic lesions whereas the Dbcbir1
mutant strain caused attenuated disease symptoms (Figure 2B). If
PCD was a limiting factor in disease development, intensified and
reduced disease symptoms are expected to be paralleled by
reduced or increased levels of PCD. In support of this rational, the
amount of PCD in the BcBIR1 over-expression and Dbcbir1 mutant
strains on infected Col-0 plants was reduced or enhanced,
respectively (Figure 3C). These results further support the role of
BcBIR1-mediated anti-apoptotic response as a mechanism to cope
with plant-induced PCD during the direct contact of the fungus
with living host cells.
Figure 3. Disease symptoms are negatively correlated with PCD in Botrytis. (A) Plants were inoculated with the B. cinerea H1-GFP. Images
show gradual decrease in the number of fluorescent nuclei between 24–48 h, and then recovery of the signal at 72 h. Very few nuclei are visible at
48 h (arrowhead). Control panels (right) show hyphae that were developed following spore germination on PDA medium. Abundant nuclei signal are
observed at all times. (B) TUNEL and DAPI staining of B. cinerea on infected leave of A. thaliana. Leaves of Col-0 plants were inoculated with B. cinerea
B05.10 wild type strain and TUNEL assay was performed to determine PCD in the developing hyphae. The green fluoresce of nuclei in these images
indicate positive TUNEL staining of nuclei, i.e., apoptotic cells. Bar = 20 mm. (C) Levels of PCD during infection of A. thaliana Col-0 wild type plants
with the B. cinerea BcBIR1 mutant strains. Col-0 plants were inoculated with B05.10 wild type, Dbir14 mutant and BIR24 over expression strains. PCD
was recorded by DNA strand break (TUNEL assay) and chromatin condensation (DAPI staining). Data represent mean 6SEM for triplicate samples of
200 nuclei per sample (n=3). The effect of fungal genotype and infection phase on PCD level was determined using two-way ANOVA. Columns not
connected with the same letter are statistically different (p,0.05) as determined by two-way ANOVA (p,0.001) followed by a post-hoc Tukey HSD
analysis. (D) Typan blue staining of Botrytis-infected Arabidopsis leaves. Leaves of A. thaliana Col-0 plants were inoculated with a spore suspension of
B. cinerea B05.10. Leaves were stained with trypan blue 48 h and 72 h PI. Blue staining of plant cells surrounding the fungal mycelium indicates dead
plant cells. The right panel shows a higher magnification of the inset from the middle panel (both from 72 h PI). Degradation of the leaf veins (black
arrowheads) and accumulation of vesicles that are produced from remnants of plant cells (yellow arrowheads) show complete maceration of the
tissue in the center of the lesion. Bars in first two panels (48 h and 72 h) = 100 mm, bar in last panel (inset 72 h) = 20 mm.
doi:10.1371/journal.ppat.1002185.g003
PCD-Mediated Fungal Pathogenesis
PLoS Pathogens | www.plospathogens.org5August 2011 | Volume 7 | Issue 8 | e1002185