Apoptosis-like programmed cell death in the grey mould fungus Botrytis cinerea: genes and their role in pathogenicity

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 domain-centred 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.     

Licensed to kill: the lifestyle of a necrotrophic plant pathogen

Necrotrophic plant pathogens have received an increasing amount of attention over the past decade. Initially considered to invade their hosts in a rather unsophisticated manner, necrotrophs are now known to use subtle mechanisms to subdue host plants. The gray mould pathogen Botrytis cinerea is one of the most comprehensively studied necrotrophic fungal plant pathogens. The genome sequences of two strains have been determined. Targeted mutagenesis studies are unraveling the roles played in the infection process by a variety of B. cinerea genes that are required for penetration, host cell killing, plant tissue decomposition or signaling. Our increasing understanding of the tools used by a necrotrophic fungal pathogen to invade plants will be instrumental to designing rational strategies for disease control.     

Antisense expression of the Arabidopsis thaliana AtPGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea

Polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall pectin and are important virulence factors of several phytopathogenic fungi. In response to abiotic and biotic stress, plants accumulate PG-inhibiting proteins (PGIPs) that reduce the activity of fungal PGs. In Arabidopsis thaliana, PGIPs with comparable activity against BcPG1, an important pathogenicity factor of the necrotrophic fungus Botrytis cinerea, are encoded by two genes, AtPGIP1 and AtPGIP2. Both genes are induced by fungal infection through different signaling pathways. We show here that transgenic Arabidopsis plants expressing an antisense AtPGIP1 gene have reduced AtPGIP1 inhibitory activity and are more susceptible to B. cinerea infection. These results indicate that PGIP contributes to basal resistance to this pathogen and strongly support the vision that this protein plays a role in Arabidopsis innate immunity.     

Functional analysis of the cytochrome P450 monooxygenase gene bcbot1 of Botrytis cinerea indicates that botrydial is a strain-specific virulence factor

The micrographic phytopathogen Botrytis cinerea causes gray mold diseases in a large number of dicotyledonous crop plants and ornamentals. Colonization of host tissue is accompanied by rapid killing of plant cells ahead of the growing hyphen, probably caused by secretion of nonspecific phytotoxins, e.g., the sesquiterpene botrydial. Although all pathogenic strains tested so far had been shown to secrete botrydial and although the toxin causes comparable necrotic lesions as infection by the fungus, the role of botrydial in the infection process has not been elucidated so far. Here, we describe the functional characterization of bcbot1, encoding a P450 monooxygenase and provide evidence that it is involved in the botrydial pathway, i.e., it represents the first botrydial biosynthetic gene identified. We show that bcbot1 is expressed in planta and that expression in vitro and in planta is controlled by an alpha-subunit of a heterotrimeric GTP-binding protein, BCG1. Deletion of bcbot1 in three standard strains of B. cinerea shows that the effect on virulence (on several host plants) is strain-dependent; only deletion in one of the strains (T4) led to reduced virulence.     

Flux of nitric oxide between the necrotrophic pathogen Botrytis cinerea and the host plant

Nitric oxide (NO) production by Botrytis cinerea and the effect of externally supplied NO were studied during saprophytic growth and plant infection. Fluorescence analysis with 4,5-diaminofluorescein diacetate and electrochemical studies were conducted in vitro between 4 and 20 h of incubation and in planta between 15 and 75 h post-inoculation. The production of NO by B. cinerea in vitro was detected inside the germinating spores and mycelium and in the surrounding medium. In planta production of NO showed a large variation that was dependent on the host plant and developmental stage of the infection. The induced production of NO was detected from 16 h of in vitro incubation in response to externally added NO. The production of NO by B. cinerea is probably modulated to promote fungal colonization of the plant tissue. The production of NO which diffuses outside the fungal cells and the induction of NO production by exogenous NO open up the possibility of NO cross-talk between the fungus and the plant. Finally, the existence of an NO concentration threshold is proposed, which may increase or reduce the plant defence against necrotrophic fungal pathogens.     

Arabidopsis thaliana: a model host plant to study plant-pathogen interaction using Chilean field isolates of Botrytis cinerea

One of the fungal pathogens that causes more agriculture damage is Botrytis cinerea. Botrytis is a constant threat to crops because the fungus infects a wide range of host species, both native and cultivated. Furthermore, Botrytis persists on plant debris in and on the soil. Some of the most serious diseases caused by Botrytis include gray mold on vegetables and fruits, such as grapes and strawberries. Botrytis also causes secondary soft rot of fruits and vegetables during storage, transit and at the market. In many plant-pathogen interactions, resistance often is associated with the deposition of callose, accumulation of autofluorescent compounds, the synthesis and accumulation of salicylic acid as well as pathogenesis-related proteins. Arabidopsis thaliana has been used as a plant model to study plant-pathogen interaction. The genome of Arabidopsis has been completely sequenced and this plant serves as a good genetic and molecular model. In this study, we demonstrate that Chilean field isolates infect Arabidopsis thaliana and that Arabidopsis subsequently activates several defense response mechanisms associated with a hypersensitive response. Furthermore, we propose that Arabidopsis may be used as a model host species to analyze the diversity associated with infectivity among populations of Botrytis cinerea field isolates.     

Leaf hairs influence phytopathogenic fungus infection and confer an increased resistance when expressing a Trichoderma alpha-1,3-glucanase

The leaf surface of a very large number of plant species are covered by trichomes. Non-glandular trichomes are specialized unicellular or multicellular structures that occur in many different plant species and function in xenobiotic detoxification and protecting the plant against pest attack. By analysing the susceptibility of trichome mutants, evidence is provided that indicates the influence of leaf trichomes on foliar fungal infections in Arabidopsis thaliana, probably by facilitating the adhesion of the fungal spores/hyphae to the leaf surface. A decreased trichome number in the hairless Arabidopsis mutant gl1 enhances tolerance against the necrotrophic fungus Botrytis cinerea. By contrast, the try mutant shows an increased susceptibility to both fungal infection and accumulation. Trichome density does not influence infection by the soil-borne pathogen Rhizoctonia solani. In addition, the influence of trichomes on foliar infection is supported by targeting the high-level expression of the Trichoderma harzianum alpha-1,3-glucanase protein to the specialized cell structures. Trichome expression of this anti-fungal hydrolase shows a significant resistance to infection by the foliar pathogen Botrytis cinerea. Resistance to this fungus is not dependent on the constitutive induction of the salicylic or jasmonic defence signalling pathways, but the presence of the alpha-1,3-glucanase protein in trichomes.     

The BMP1 gene is essential for pathogenicity in the gray mold fungus Botrytis cinerea

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.     

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.     

Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea

Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola results in systemic induction of genes encoding a plant defensin (PDF1.2), a basic chitinase (PR-3), and an acidic hevein-like protein (PR-4). Pathogen-induced induction of these three genes is almost completely abolished in the ethylene-insensitive Arabidopsis mutant ein2-1. This indicates that a functional ethylene signal transduction component (EIN2) is required in this response. The ein2-1 mutants were found to be markedly more susceptible than wild-type plants to infection by two different strains of the gray mold fungus Botrytis cinerea. In contrast, no increased fungal colonization of ein2-1 mutants was observed after challenge with avirulent strains of either Peronospora parasitica or A. brassicicola. Our data support the conclusion that ethylene-controlled responses play a role in resistance of Arabidopsis to some but not all types of pathogens.     

Root inoculation with a forest soil streptomycete leads to locally and systemically increased resistance against phytopathogens in Norway spruce

Soil streptomycetes are commonly antagonistic against plant pathogens. However, interactions involving increased defense responses in the host plant, leading to suppression of plant disease development, have not yet been detailed. Here, the mechanisms were studied of disease suppression by Streptomyces sp. GB 4-2 against Heterobasidion root and butt rot in Norway spruce (Picea abies) seedlings. GB 4-2 promoted mycelial growth of the phytopathogenic fungus, germination rate of fungal spores, extension of germ tubes and early colonization of outer cortical layers of the plant root. Reduced colonization of the inner cortical cell layers was accompanied by the induction of cell wall appositions, and increased xylem formation in the vascular cylinder emerged after bacterium-fungus coinoculation. Bacterial treatment led to decreased water content in roots and needles and increased photosynthetic yield (F(v)/F(m)) and peroxidase activities in needles. The infection of needles by Botrytis cinerea was reduced by bacterial pretreatment. Complex interactions of GB 4-2 with Norway spruce and Heterobasidion abietinum were discovered. The bacterium promoted the growth of the phytopathogenic fungus but induced plant defense responses. Host responses indicate that GB 4-2 induces both local and systemic defense responses in Norway spruce.     

A tomato metacaspase gene is upregulated during programmed cell death in<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>-infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.     

Botrytis cinerea BcNma is involved in apoptotic cell death but not in stress adaptation

Apoptotic-like programmed cell death (PCD) occurs naturally in fungi during development and might also be induced by external conditions. Candidate apoptotic genes have been characterized in several model fungal species but not in plant pathogenic fungi. Here we report on the isolation and characterization of BcNMA, an orthologue of the human pro-apoptotic gene HtrA2 from the plant pathogen Botrytis cinerea. The predicted BcNma protein shows high homology to the previously characterized Nma111p from Saccharomyces cerevisiae and despite some structural differences it complemented the function of Nma111p in Δnma111 mutant strains. BcNMA-over-expression and mutant strains had enhanced or reduced appearance of apoptotic markers, respectively. However there was no difference in growth response of the wild type and BcNMA-transgenic strains to application of various stresses, and the effect on pathogenicity was marginal in both the over-expression and mutant strains. When considered together these results suggest that although BcNma has a pro-apoptotic activity, it is not a major regulator of apoptosis. The protein probably has additional roles that are unrelated to apoptosis, which lead to the pleotrophic phenotype of the transgenic strains and lack of a clear effect on stress adaptation and pathogenicity.     

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.