Lipid-Binding Aegerolysin from Biocontrol Fungus Beauveria bassiana.
ABSTRACT: Fungi are the most common pathogens of insects and thus important regulators of their populations. Lipid-binding aegerolysin proteins, which are commonly found in the fungal kingdom, may be involved in several biologically relevant processes including attack and defense against other organisms. Aegerolysins act alone or together with membrane-attack-complex/perforin (MACPF)-like proteins to form transmembrane pores that lead to cell lysis. We performed an in-depth bioinformatics analysis of aegerolysins in entomopathogenic fungi and selected a candidate aegerolysin, beauveriolysin A (BlyA) from Beauveria bassiana. BlyA was expressed as a recombinant protein in Escherichia coli, and purified to further determine its functional and structural properties, including lipid-binding ability. Aegerolysins were found to be encoded in genomes of entomopathogenic fungi, such as Beauveria, Cordyceps, Metarhizium and Ophiocordyceps. Detailed bioinformatics analysis revealed that they are linked to MACPF-like genes in most genomes. We also show that BlyA interacts with an insect-specific membrane lipid. These results were placed in the context of other fungal and bacterial aegerolysins and their partner proteins. We believe that aegerolysins play a role in promoting the entomopathogenic and antagonistic activity of B. bassiana, which is an active ingredient of bioinsecticides.
Project description:Aegerolysins are small lipid-binding proteins particularly abundant in fungi. Aegerolysins from oyster mushrooms interact with an insect-specific membrane lipid and, together with MACPF proteins produced by the same organism, form pesticidal pore-forming complexes. The specific interaction with the same membrane lipid was recently demonstrated for nigerolysin A2 (NigA2), an aegerolysin from <i>Aspergillus niger</i>. In <i>Aspergillus</i> species, the aegerolysins were frequently found as secreted proteins, indicating their function in fungal defense. Using immunocytochemistry and live-cell imaging we investigated the subcellular localization of the nigerolysins A in <i>A. niger</i>, while their secretion was addressed by secretion prediction and Western blotting. We show that both nigerolysins A are leaderless proteins that reach the cell exterior by an unconventional protein secretion. NigA proteins are evenly distributed in the cytoplasm of fungal hyphae. A detailed bioinformatics analysis of <i>Aspergillus</i> aegerolysins suggests that the same function occurs only in a limited number of aegerolysins. From alignment, analysis of chromosomal loci, orthology, synteny, and phylogeny it follows that the same or a similar function described for pairs of pesticidal proteins of <i>Pleurotus</i> sp. can be expected in species of the subgenus <i>Circumdati</i>, section <i>Nigri</i>, series <i>Nigri</i>, and some other species with adjacent pairs of putative pesticidal proteins.
Project description:The long-term and excessive usage of pesticides is an enormous burden on the environment, which also increases pest resistance. To overcome this problem, research and application of entomopathogenic fungi, which are both environmentally friendly and cause lower resistance, have gained great momentum. Entomopathogenic fungi have a wide range of prospects. Apart from <i>Bacillus thuringiensis</i>, <i>Beauveria bassiana</i> is the most studied biopesticide. After invading insect hosts, <i>B. bassiana</i> produces a variety of toxins, which are secondary metabolites such as beauvericin, bassianin, bassianolide, beauverolides, tenellin, oosporein, and oxalic acid. These toxins help <i>B. bassiana</i> to parasitize and kill the hosts. This review unequivocally considers beauveria toxins highly promising and summarizes their attack mechanism(s) on the host insect immune system. Genetic engineering strategies to improve toxin principles, genes, or virulent molecules of <i>B. bassiana</i> have also been discussed. Lastly, we discuss the future perspective of <i>Beauveria</i> toxin research, including newly discovered toxins.
Project description:Aegerolysins ostreolysin A (OlyA) and pleurotolysin A (PlyA), and pleurotolysin B (PlyB) with the membrane-attack-complex/perforin domain are proteins from the mushroom genus Pleurotus. Upon binding to sphingomyelin/cholesterol-enriched membranes, OlyA and PlyA can recruit PlyB to form multimeric bi-component transmembrane pores. Recently, Pleurotus aegerolysins OlyA, PlyA2 and erylysin A (EryA) were demonstrated to preferentially bind to artificial lipid membranes containing 50?mol% ceramide phosphoethanolamine (CPE), the main sphingolipid in invertebrate cell membranes. In this study, we demonstrate that OlyA6, PlyA2 and EryA bind to insect cells and to artificial lipid membranes with physiologically relevant CPE concentrations. Moreover, these aegerolysins permeabilize these membranes when combined with PlyB. These aegerolysin/PlyB complexes show selective toxicity toward western corn rootworm larvae and adults and Colorado potato beetle larvae. These data strongly suggest that these aegerolysin/PlyB complexes recognize CPE as their receptor molecule in the insect midgut. This mode of binding is different from those described for similar aegerolysin-based bacterial complexes, or other Bacillus thuringiensis Cry toxins, which have protein receptors. Targeting of Pleurotus aegerolysins to CPE and formation of transmembrane pores in concert with PlyB suggest the use of aegerolysin/PlyB complexes as novel biopesticides for the control of western corn rootworm and Colorado potato beetle.
Project description:The European pepper moth, Duponchelia fovealis (Lepidoptera: Crambidae), is a key pest in strawberry production. Entomopathogenic fungi (EF) and parasitoids of the Trichogrammatidae family are effective biological control agents of this pest with the potential to be used jointly for improved efficacy. This study aims to evaluate the susceptibility of Trichogramma atopovirilia and Trichogramma pretiosum to two Beauveria bassiana strains (B2 and B3) and two commercial bioinsecticides (Bovemax® and Methamax®) by applying them to D. fovealis eggs in pre- and post-parasitism periods. Pre-parasitism application of B2 and B3 did not affect the percentage of D. fovealis eggs parasitized by either Trichogramma species, except in the case of T. atopovirilia when eggs were sprayed with B3 at 1.5 × 105 conidia mL-1 (16.7% less than the control). In contrast, eggs sprayed with 1.5 × 108 conidia mL-1 of the commercial bioinsecticides were not parasitized by any Trichogramma species. Overall, the EF tested reduced the parasitism rate, adult emergence, and longevity of Trichogramma adults by less than 30% in all cases. The adverse effects of the B. bassiana strains and commercial products on the biological traits of both Trichogramma species were minimal, meaning that these agents can be used jointly in D. fovealis control strategies.
Project description:BACKGROUND: The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt) genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic information to study the biogeography of the fungus. RESULTS: The complete mt genomes of B. bassiana (32,263 bp) and B. brongniartii (33,920 bp) were fully analysed. Apart from a typical gene content and organization, the Beauveria mt genomes contained several introns and had longer intergenic regions when compared with their close relatives. The phylogenetic diversity of a population of 84 Beauveria strains -mainly B. bassiana (n = 76) - isolated from temperate, sub-tropical and tropical habitats was examined by analyzing the nucleotide sequences of two mt intergenic regions (atp6-rns and nad3-atp9) and the nuclear ITS1-5.8S-ITS2 domain. Mt sequences allowed better differentiation of strains than the ITS region. Based on mt and the concatenated dataset of all genes, the B. bassiana strains were placed into two main clades: (a) the B. bassiana s. l. and (b) the "pseudobassiana". The combination of molecular phylogeny with criteria of geographic and climatic origin showed for the first time in entomopathogenic fungi, that the B. bassiana s. l. can be subdivided into seven clusters with common climate characteristics. CONCLUSIONS: This study indicates that mt genomes and in particular intergenic regions provide molecular phylogeny tools that combined with criteria of geographic and climatic origin can subdivide the B. bassiana s.l. entomopathogenic fungi into seven clusters with common climate characteristics.
Project description:The use of mycoviruses to manipulate the virulence of entomopathogenic fungi employed as biocontrol agents may lead to the development of novel methods to control attacks by insect pests. Such approaches are urgently required, as existing agrochemicals are being withdrawn from the market due to environmental and health concerns. The aim of this work is to investigate the presence and diversity of mycoviruses in large panels of entomopathogenic fungi, mostly from Spain and Denmark. In total, 151 isolates belonging to the genera <i>Beauveria</i>, <i>Metarhizium</i>, <i>Lecanicillium</i>, <i>Purpureocillium</i>, <i>Isaria</i>, and <i>Paecilomyces</i> were screened for the presence of dsRNA elements and 12 Spanish <i>B. bassiana</i> isolates were found to harbor mycoviruses. All identified mycoviruses belong to three previously characterised species, the officially recognised <i>Beauveria bassiana victorivirus 1</i> (BbVV-1) and the proposed Beauveria bassiana partitivirus 2 (BbPV-2) and Beauveria bassiana polymycovirus 1 (BbPmV-1); individual <i>B. bassiana</i> isolates may harbor up to three of these mycoviruses. Notably, these mycovirus species are under distinct selection pressures, while recombination of viral genomes increases population diversity. Phylogenetic analysis of the RNA-dependent RNA polymerase gene sequences revealed that the current population structure in Spain is potentially a result of both vertical and horizontal mycovirus transmission. Finally, pathogenicity experiments using the Mediterranean fruit fly <i>Ceratitis capitata</i> showed no direct correlation between the presence of any particular mycovirus and the virulence of the <i>B. bassiana</i> isolates, but illustrated potentially interesting isolates that exhibit relatively high virulence, which will be used in more detailed virulence experimentation in the future.
Project description:We investigated the transcriptional response of invasive Mediterranean (MED) species of the whitefly B. tabaci complex (commonly referred to as Q biotype) to entomopathogenic fungi Beauveria bassiana using Illumina sequencing technology.
Project description:Rhizoctonia potato disease is widespread in the world and causes substantial yield and quality losses in potato. This study aimed to evaluate the efficacy of entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana in the inhibition of potato Rhizoctonia complex disease. The efficacy of the entomopathogenic fungi M. robertsii and B. bassiana in the defense of potato against Rhizoctonia disease (stem cancer, black scrulf and other forms of manifestation on tubers) was estimated under field conditions in Western Siberia. Preplanting treatment of the tubers with B. bassiana decreased Rhizoctonia disease in the stems and stolons. At the same time, treatment with M. robertsii did not cause a decrease in Rhizoctonia disease in these organs. However, both fungi decreased the sclerotium index on the tubers of new crops. We demonstrated two mechanisms of inhibition of Rhizoctonia solani by M. robertsii and B. bassiana, including (1) direct effect, expressed as inhibition of R. solani sclerotium formation in cocultivation assays, and (2) indirect effect, which is associated with increased peroxidase activity in potato roots under the influence of colonization by entomopathogenic fungi. We suggest that the treatment of seed tubers with B. basiana can effectively manage Rhizoctonia disease during the plant vegetative season and that both fungi significantly improve the quality of the new tuber crop.
Project description:Since the discovery that entomopathogenic fungi can live inside plants as endophytes, researchers have been trying to understand how this affects mainly plants and herbivores. We studied how inoculation of Vicia faba L. (Fabales: Fabaceae) plants with Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) (strain GHA) either via the seeds or leaves influenced the nymph production of two successive generations of Aphis fabae Scopoli (Hemiptera: Aphididae). While we did not find any difference in nymph production for the first generation of aphids, second-generation aphids on both seed- and spray inoculated plants produced significantly higher numbers of nymphs than aphids on uninoculated plants. This emphasizes the importance of two (or multi-) generational experimentation. Beauveria bassiana was recovered from 26.0, 68.8 and 6.3% of respectively seed-, spray inoculated and control plants, thus, demonstrating its ability to live as an endophyte in V. faba. The confirmation that plants inoculated with entomopathogenic fungi can have a positive effect on pest insects makes careful consideration of these multi-trophic interactions imperative.
Project description:We investigated the transcriptional response of invasive Mediterranean (MED) species of the whitefly B. tabaci complex (commonly referred to as Q biotype) to entomopathogenic fungi Beauveria bassiana using Illumina sequencing technology. Nearly 1,000 of control whiteflies, 48h fungal-induced whiteflies and 72h fungal-induced whiteflies were collected, respectively.