Apoptosis-like programmed cell death in the grey mould fungus ...

8th International Meeting on Yeast Apoptosis

Apoptosis-like programmed cell death in the grey mould fungus Botrytis cinerea: genes and their role in pathogenicity Neta Shlezinger, Adi Doron and Amir Sharon1 Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv 69978, Israel

Abstract A considerable number of fungal homologues of human apoptotic genes have been identified in recent years. Nevertheless, we are far from being able to connect the different pieces and construct a primary structure of the fungal apoptotic regulatory network. To get a better picture of the available fungal components, we generated an automatic search protocol that is based on protein sequences together with a domaincentred approach. We used this protocol to search all the available fungal databases for domains and homologues of human apoptotic proteins. Among all known apoptotic domains, only the BIR [baculovirus IAP (inhibitor of apoptosis protein) repeat] domain was found in fungi. A single protein with one or two BIR domains is present in most (but not all) fungal species. We isolated the BIR-containing protein from the grey mould fungus Botrytis cinerea and determined its role in apoptosis and pathogenicity. We also isolated and analysed BcNMA, a homologue of the yeast NMA11 gene. Partial knockout or overexpression strains of BcBIR1 confirmed that BcBir1 is anti-apoptotic and this activity was assigned to the N -terminal part of the protein. Plant infection assays showed that the fungus undergoes massive PCD (programmed cell death) during early stages of infection. Further studies showed that fungal virulence was fully correlated with the ability of the fungus to cope with plant-induced PCD. Together, our result show that BcBir1 is a major regulator of PCD in B. cinerea and that proper regulation of the host-induced PCD is essential for pathogenesis in this and other similar fungal pathogens.

Introduction Apoptosis was initially discovered and studied in animals [1], in which it is essential for development and homoeostasis [2,3]. Studies in the last two decades have shown that apoptosis-like PCD (programmed cell death) also occurs in most other living systems, including plants, fungi and bacteria. Moreover, molecular-evolution studies suggest that the eukaryotic apoptotic machinery has originated from acquisition of bacterial genes of a mitochondrial endosymbiont [4] and that the evolution of apoptotic networks was associated with various events including horizontal gene transfer, recruitment, invention and proliferation [5]. Considerable differences exist in the molecular components, regulation and the role of the apoptotic machinery in different living systems [6–10]. Such variability is expected in light of the ancient origin of the core apoptotic machinery and the extensive modifications that took place during the evolution of apoptotic networks.

Key words: baculovirus inhibitor of apoptosis protein (IAP) repeat (Bir1), Botrytis cinerea, cell death, fungus, Nma111, pathogenesis, programmed cell death (PCD). Abbreviations used: DAPI, 4 ,6-diamidino-2-phenylindole; GFP, green fluorescent protein; HMM, hidden Markov model; IAP, inhibitor of apoptosis protein; BIR, baculovirus IAP repeat; IBM, IAPbinding motif; PCD, programmed cell death; PI, post-inoculation; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. 1 To whom correspondence should be addressed (email [email protected]).

Biochem. Soc. Trans. (2011) 39, 1493–1498; doi:10.1042/BST0391493

In Saccharomyces cerevisiae, PCD was first described over 10 years ago (see [11] for a recent review of apoptosis in yeasts). The recognition of the importance of PCD in yeast life cycle stimulated research on PCD in additional species, including a growing number of economically and medically important filamentous fungi. These studies showed that PCD is important for proper fungal development and that it is associated with aging and stress responses [11–14]. However, research on apoptotic cell death in fungi is still limited in scope, and information on apoptosis regulatory elements and networks is very partial. Much more research is necessary in order to unravel the molecular components and roles of PCD in species with different life styles. The study of PCD in pathogenic species is of special interest, since it could lead to a better understanding of fungal pathogenesis and to the identification of novel targets for antifungal cures. We have been studying PCD in the grey mould fungus Botrytis cinerea. This fungus is pathogenic on a wide range of important cultivated plants and causes significant crop losses worldwide. B. cinerea is a necrotrophic pathogen and is used as a model to study pathogenicity in this class of plant pathogens [15]. Importantly, B. cinerea and possibly other necrotrophic fungi induce PCD in plants, which is essential for successful infection. Conversely, some plant antimicrobial compounds have the potential to induce PCD in fungi [16– 18]. As necrotrophic fungi are unable to evade the plant C The

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defences, we reasoned that anti-apoptotic machinery might be necessary for the protection of the pathogen during the early infection phase, when the fungus might be exposed to PCDinducing plant defence molecules. Indeed, we found that B. cinerea undergoes massive PCD during the early infection phase. However, the fungus quickly recovers on transition to the second phase of infection, when it is no longer in contact with living host cells [19]. In the present paper, we report on recent progress in molecular-genomic analyses of the apoptotic network in B. cinerea and on the role of the anti-apoptotic machinery in fungal pathogenicity.

Figure 1 Apoptotic markers in B. cinerea B05.10 in aged cultures Fungi were grown for 120 h, and samples of mycelium were stained with the various dyes and visualized under the microscope. Similar results were obtained following treatment with 250 mM lovastatin, 8 mM H2 O2 and 1.5 mM hexanoic acid (not shown). (A) ROS were detected after staining with DHR-123 using the rhodamine filter. Scale bar, 100 μm. (B) Chromatin condensation was detected following nuclei staining with DAPI or Hoechst 3342 using the DAPI filter. Scale bar, 5 μm. (C) DNA strand breaks were detected following TUNEL assay using the GFP filter. Scale bar, 5 μm.

Analysis of apoptosis in B. cinerea In order to investigate PCD in B. cinerea, we first determined the conditions under which apoptotic cell death can be detected and quantified. Apoptotic cell death was determined by standard methods after proper modifications. The main methods that we used included TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay for the determination of DNA strand breaks, chromatin condensation and fragmentation as determined by staining of nuclei with DAPI (4 ,6-diamidino-2-phenylindole) or Hoechst 3342, and production of ROS (reactive oxygen species), which was determined by staining with DHR-123 (Figure 1). First, we tested the appearance of markers of apoptotic cell death after treatment with compounds that are known to induce PCD in other organisms, including fungi. Conditions that induce PCD were determined after treatments with a number of such compounds including hydrogen peroxide, lovastatin or high salt [16,19]. Apoptotic cell death was also detected in aged cultures and on transition of cultures to stationary phase [19]. In addition to accumulation of biochemical markers, at the microscopic level, hyphae and conidia exhibit apoptosis-associated morphological changes, particularly increased vacuolization and cell lysis. For the detection of PCD in living cells, we used a transgenic strain of B. cinerea that expresses a histone– GFP (green fluorescent protein) fusion protein and hence the nuclei can be visualized by fluorescence microscopy (Figure 2). Transgenic strains expressing similar cassettes have been widely used to monitor nuclear division and cell cycle [20–24]. Histone H1–GFP-tagged strains have also been used as indicators of apoptosis-like PCD in Colletotrichum gloeosporioides [22], and autophagic PCD in Magnaporthe oryzae [24]. Furthermore, cytoplasmic localization of histone H1 was shown to play an important role during apoptosis in mammals [25,26]. Thus the live imaging of the GFP signal in the histone H1–GFP-expressing strain is a useful tool to follow cell fate, both in axenic growth and in infected plants. Indeed, after treatment with hydrogen peroxide, the nuclear GFP signal in the B. cinerea H1–GFP strain faded out within 6 h post-treatment and in opposite correlation to increased TUNEL staining, confirming that disappearance of the nuclear GFP signal is indicative of early PCD (Figure 2). C The


Antifungal plant metabolites induce apoptotic cell death in B. cinerea Production of antimicrobial secondary metabolites represents an important component of plant defence responses against pathogens [27,28]. In the case of B. cinerea, the most significant secondary metabolites are glucosinolate, indolic and phenylpropanoid compounds [29,30]. Specifically, the indolic phytoalexin camalexin has been shown to play an important role in the defence of Arabidopsis thaliana against a range of pathogens, including B. cinerea [29,31,32]. A number of reports showed that certain antifungal plant compounds target the fungal apoptosis machinery, thereby inducing fungal cell death [17,33]. Therefore we tested the induction of apoptotic cell death by two plant compounds with known


Figure 2 Detection of PCD in B. cinerea using the H1-GFP tagged strains (A) Microscopic visualization of nuclei in the histone H1-GFP tagged strain following H2 O2 treatment. Fungi were grown for 24 h in PDB (potato dextrose broth) medium, H2 O2 was added to a final concentration of 10 mM, and the cultures were incubated for additional 12 h and stained with Hoechst 3342. Images were captured using the GFP and DAPI filter sets. (B) Culture of the histone H1–GFP strain was treated with H2 O2 (as in A), and a TUNEL assay was performed on mycelium collected 12 h after H2 O2 treatment, when the histone H1 nuclear signal had completely disappeared. Note that in this assay, the nuclear GFP signal indicates apoptotic nuclei, just opposite the histone H1–GFP marker. Con., control; DIC, differential interference contrast.

of 66 fungal species spanning all main groups of fungi. Next, a second database was generated composed of sequences representing all major apoptotic proteins as well as all major apoptotic domains. A computer program was developed which includes an iterated blastp search [34], generation of an HMM (hidden Markov model) [35] for each sequence and then repeated search against the database using the HMM profile. Using this approach, we have been able to identify a large number of B. cinerea genes that are putative homologues of known apoptotic genes. However, wellconserved apoptotic domains such as the BH domains (Bcl2 homology domains) BH1–4, CARD (caspase recruitment domain), CAS (cellular apoptosis-susceptibility protein), DD (death domain), DED (death effector domain), CIDE [cell death-inducing DFFA (DNA fragmentation factor 45 kDa α)-like effector] or death receptors could not be found in fungi. The only classical apoptotic domain identified in fungi was BIR [baculovirus IAP (inhibitor of apoptosis protein) repeat]. This domain is found in proteins of the IAP family of proteins, which are important regulators of apoptosis in animals [36]. Proteins containing a single or two BIR domains were identified in most (but not all) fungal species, including in B. cinerea. The protein is homologous with S. cerevisiae Bir1p and was therefore designated BcBir1. Along with BcBir1, we also identified a homologue of S. cerevisiae Nma111p, an HtrA family serine protease that is a negative regulator of Bir1p. Because of the central role of IAPs in regulation of apoptosis, we decided to further analyse BcBir1 along with its putative regulator, BcNma.

Functional analysis of B. cinerea BcBir1 and BcNma

antifungal properties: hexanoic acid and camalexin. In both cases, treatment of B. cinerea mycelium with micromolar concentrations of the compound resulted in accelerated cell death, growth inhibition, accumulation of apoptotic markers and loss of the nuclear signal in the H1–GFP strain [16,19]. These results suggest that when exposed to plant defence molecules during the early phase of infection, B. cinerea might be subjected to host-induced PCD. In this event, fungal anti-apoptotic machinery might be necessary for survival and pathogenicity. In order to investigate this possibility, we tested potential apoptotic genes for their involvement in fungal PCD and pathogenicity.

The inventory of B. cinerea apoptotic genes We used a computer-guided approach in search of B. cinerea genes that might play an important role in regulation of apoptotic cell death. First, a database was generated which includes all the publicly available fungal genome sequences. Only complete sequences were included, representing a total

Bir1p belongs to the type-II (IAP-like) BIR-containing proteins, which also includes the mammalian survivin/BIRC5. As mentioned, IAPs are anti-apoptotic proteins and they play a central role in regulation of apoptosis in animals. All IAPs contain one to three BIR domains, which are essential for their anti-apoptotic activity [37]. Bir1p and survivin are components of the chromosomal passenger complex in yeast and humans respectively. This complex regulates chromosome segregation, an activity that is mediated by the C-terminal part of the proteins and is essential for survival. Bir1p and survivin also have anti-apoptotic activity, which is mediated by the BIR domains at the N -terminal part of the protein and is not necessary for the cell cycle regulating activities of these proteins [38]. Similar to Bir1p, the B. cinerea BcBir1 protein also contains two BIR domains at the N part, it shares 24% amino acid sequence identity with Bir1p, but it is considerably shorter than Bir1p (601 amino acids and 954 amino acids respectively). The S. cerevisiae Nma111p belongs to the hightemperature requirement (HtrA) family of serine proteases and is a homologue of the human HtrA2/Omi, a mitochondrial protein with pro-apoptotic activity [39]. Nma111p is also involved in the response to high temperatures and has a pro-apoptotic function which depends on its serine C The


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protease activity [40]. BcNma is highly homologous with Nma111p (51 % identity) and has moderate homology with HtrA2/Omi (27 % identity). Similar to HtrA2, the BcNma protein contains only a single PDZ domain and an IBM (IAPbinding motif), whereas Nma111p has two PDZ domains and lacks an IBM [16]. To study the possible role of BcBir1 and BcNma in apoptosis, the BcBIR1 and BcNMA genes were isolated and transgenic strains were produced in which the genes were either deleted (knockout strains) or expressed under the actin promoter (overexpression strains). 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 (bcbir1) in which the expression of BcBIR1 was reduced but not completely eliminated. To determine a possible role in regulation of PCD, we exposed the transgenic strains to apoptosis-promoting conditions (e.g. treatment with H2 O2 , aging, stationary phase) and measured the growth rate and apoptotic markers. Under all the apoptosis-promoting conditions, the BcBIR1 overexpression strains (expressing either the entire protein or only the N part with the two BIR domains) retained higher growth rates and showed reduced PCD markers. In contrast, the bcbir1 mutant strains exhibited reduced growth rates and enhanced PCD compared with the isogenic wild-type strain. Aged cultures of the bcbir1 mutant showed increased accumulation of black pigments, which is indicative of enhanced senescence. Conidia collected from these aged cultures had apoptotic morphology and reduced viability. 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. To test the effect of BcBIR1 on pathogenicity, we inoculated beans (Phaseolus vulgaris) or A. thaliana plants and measured disease symptoms. The BcBIR1 overexpression strains (in which apoptotic cell death was attenuated) caused increased disease symptoms, while bcbir1 mutant strains (in which PCD was enhanced) caused restricted lesions. Thus PCD and pathogenicity in BcBIR1 transgenic strains are modified such that enhanced or reduced PCD is associated with reduced or enhanced pathogenicity respectively. Unlike BcBir1, which has a structure similar to Bir1p, the BcNMA protein has several structural differences compared with Nma111, including the lack of an IBM and a single versus two PDZ domains [16]. In order to verify whether BcNma is a functional homologue of the yeast Nma111p, we transformed yeast cells with BcNMA and tested whether expression of BcNma could restore wild-type phenotype in nma111 mutant cells. The nma111 yeast strains expressing the BcNMA gene showed wild-type levels of apoptotic response to H2 O2 and exhibited wild-type sensitivity to high temperature. These results confirmed that BcNma is a functional homologue of Nma111p, and demonstrated that despite the structural differences between the two proteins it can complement both the pro-apoptotic activity and the high-temperature response that are mediated by Nma111p. C The


However, B. cinerea strains in which BcNMA was deleted or overexpressed did not show significant changes in growth when exposed to stress conditions such as high or low temperature, salt stress and nitrogen starvation. After exposure to apoptosis-inducing conditions, including aging and hexanoic acid, bcnma strains retained higher growth rates and showed reduced PCD markers, whereas BcNMAoverexpressing strains showed enhanced PCD levels but the effect on growth rate was similar to the effect on growth rate in the B05.10 wild-type strain. These results suggest that although BcNma has a pro-apoptotic activity, it is not a major regulator of apoptosis in B. cinerea, unlike BcBir1, which plays a central role in the apoptotic machinery. Pathogenicity assays showed that both the bcnma mutant and the BcNMA-overexpressing strains were slightly less virulent than the wild-type strain. This minor effect suggests that BcNma is not directly associated with pathogenicity. The similar effect in the mutant and overexpression strains is unclear, but it might be related to additional roles of BcNma. Thus, unlike BcBir1, which has a central role in apoptosis and is essential for pathogenicity, BcNma is not a major regulator of apoptosis and has only a minor effect on pathogenicity.

Role of BcBir1-mediated anti-apoptotic machinery in Botrytis infection To test whether the fungus undergoes apoptosis-like PCD during infection, we used the B. cinerea H1–GFP strain to determine the fate of the fungal cells during plant infection. A. thaliana Col-0 (wild-type) 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 PI (post-inoculation). The nuclear GFP signal was retained for the first 24 h PI, and then, during the next 24 h (24–48 h PI) it dissociated. However, despite the massive cell death at 48 h PI, the fungus quickly recovered as indicated by the reappearance of the nuclear signal at 72 h PI [19]. Similar results were obtained on additional host plants, such as beans and peas. Thus after germination and establishment of contact with the host, nuclear signal in H1–GFP strain starts to disappear between 24 and 36 h PI, and almost no nuclei can be detected at 48 h PI. The results obtained with the H1–GFP strain were reproduced by direct measurement of apoptotic markers: TUNEL assay and chromatin condensation. These results suggest that massive PCD occurs during the early infection phase, but the fungus manages to recover on transition to the second phase. In support of our hypothesis that fungal PCD is a limiting factor in disease development, the amount of PCD in BcBIR1 overexpression strains (causing intensified disease symptoms) and bcbir1 mutant strains (causing reduced disease symptoms) on infected A. thaliana plants was reduced or enhanced respectively. On A. thaliana mutant plants that are impaired in defence responses and hypersensitive to B. cinerea, the nuclear signal of the B. cinerea H1–GFP strain was retained also at 48 h


PI, confirming that the amount of fungal PCD negatively correlates with plant susceptibility to the fungus. One of the A. thaliana lines that we used was the phytoalexin-deficient pad3 mutant, which does not produce the phytoalexin camalexin. As mentioned, camalexin induced apoptotic cell death in B. cinerea wild-type strain in vitro. In accordance with this PCD-promoting effect of camalexin, the BcBIR1 overexpression and mutant strains showed reduced or enhanced sensitivity to camalexin respectively along with reduced PCD on the pad3 plants. In the present paper, we showed that the B. cinerea BcBir1 has a major function in the anti-apoptotic machinery of the fungus. During infection of plants, the B. cinerea is exposed to apoptosis-promoting compounds, which induce massive cell death in the initial hyphae. BcBir1-mediated antiapoptotic activity is essential at this stage to prevent complete elimination of the infecting hyphae. Similar results were obtained with Cochliobolus heterostrophus, a necrotrophic pathogen of corn. Thus our results propose that the antiapoptotic machinery has a major role in mediating disease in this class of pathogens.

Funding This research was supported by the Israel Science Foundation [grant number 206/09].

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Received 19 June 2011 doi:10.1042/BST0391493