Comparative gene expression and genomics reflect geographical divergence in the plant symbiotic and entomopathogenic fungal genus Metarhizium

Several species within the fungal genus Metarhizium can both infect insects and colonize plant roots. In Brazil, a specific subgroup within Metarhizium anisopliae s.str. named “subclade Mani 2” is frequently observed infecting above-ground insects, whereas sympatric M. robertsii and M. brunneum predominantly occur in the soil environment. Genotypic variability within the genus may be linked to adaptations to these different habitats. We present a comparative analysis of the complete genomes and the adhesin genes Mad1 and Mad2 of 14 Metarhizium isolates representing M. anisopliae Mani 2 (n = 6), M. robertsii (n = 5) and M. brunneum (n = 3). In addition, the relative gene expression of six selected target genes was compared in root exudate solution and insect cuticle suspension. We hypothesized that M. anisopliae Mani 2 is adapted to insect-pathogenicity in the above-ground environment, reflected by higher relative expression of pathogenicity-related genes. In contrast, M. robertsii and M. brunneum are adapted to the soil environment, hence hypothesized to have a higher expression of genes related to plant associations. Phylogenomic and adhesin phylogenetic trees revealed species differences but also intraspecific variability associated with the geographic origin of isolates. Differences in relative gene expression were observed, with one pathogenicity-related gene (Pr1) being higher expressed in M. anisopliae. The insect adhesion Mad1 gene was more conserved than the plant adhesion Mad2 and similarly expressed in exudate solution, while Mad2 was highly expressed by all Brazilian isolates in both exudate and cuticle conditions. The variabilities observed correlated with different habitats and lifestyles, demonstrating the importance of selecting a diverse collection of isolates in genomic and gene expression studies.     

Differential expression of insect and plant specific adhesin genes, Mad1 and Mad2, in Metarhizium robertsii

Metarhizium robertsii is an entomopathogenic fungus that is also plant rhizosphere competent. Two adhesin-encoding genes, Metarhizium adhesin-like protein 1 (Mad1) and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. Here we examined the differential expression of the Mad genes when grown on a variety of soluble (carbohydrates and plant root exudate) and insoluble substrates (locust, tobacco hornworm, and cockroach cuticle, chitin, tomato stems, cellulose, and starch) and during insect, Plutella xylostella, infection. On insect cuticles Mad1 was up regulated, whereas bean root exudate and tomato stems resulted in the up regulation of Mad2. During the early stages of insect infection Mad1 was expressed while Mad2 was not expressed until fungal hyphae emerged and conidiated on the insect cadaver. The regulation of Mad2 was compared to that of other stress-related genes (heat shock protein (Hsp)30, Hsp70, and starvation stress gene A (ssgA)). Mad2 was generally up regulated by nutrient starvation (similar to ssgA) but not by pH, temperature, oxidative or osmotic stresses. Whereas Hsp30 and Hsp70 were generally up regulated at 37 °C or by oxidative stress even under nutrient enriched conditions. We fused the promoter of the Mad2 gene to a marker gene (green fluorescent protein (GFP)) and confirmed that Mad2 was up regulated when M. robertsii was grown in the presence of nutrient starvation. Examination of the promoter region of Mad2 revealed that it possessed two copies of a stress-response element (STRE) known to be regulated under the general stress-response pathway.     

Mad1’s ability to interact with Mad2 is essential to regulate and monitor meiotic synapsis in C. elegans

Meiotic homolog synapsis is essential to ensure accurate segregation of chromosomes during meiosis. In C. elegans, proper regulation of synapsis and a checkpoint that monitors synapsis relies on the spindle checkpoint components, Mad1 and Mad2, and Pairing Centers (PCs), cis-acting loci that interact with the nuclear envelope to mobilize chromosomes within the nucleus. Here, we test what specific functions of Mad1 and Mad2 are required to regulate and monitor synapsis. We find that a mutation that prevents Mad1’s localization to the nuclear periphery abolishes the synapsis checkpoint but has no effect on Mad2’s localization to the nuclear periphery or synapsis. By contrast, a mutation that prevents Mad1’s interaction with Mad2 abolishes the synapsis checkpoint, delays synapsis and fails to localize Mad2 to the nuclear periphery. These data indicate that Mad1’s primary role in regulating synapsis is through control of Mad2 and that Mad2 can bind other factors at the nuclear periphery. We also tested whether Mad2’s ability to adopt a specific conformation associated with its activity during spindle checkpoint function is required for its role in meiosis. A mutation that prevents Mad2 from adopting its active conformer fails to localize to the nuclear periphery, abolishes the synapsis checkpoint and exhibits substantial defects in meiotic synapsis. Thus, Mad2, and its regulation by Mad1, is an important regulator of meiotic synapsis in C. elegans.     

Plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and Beauveria

Endophytic fungi may display preferential tissue colonization within their plant hosts. Here we tested if the endophytic, insect pathogenic fungi (EIPF) Metarhizium and Beauveria showed preferential localization within plant tissues, in the field and under laboratory conditions. In the field, plants were sampled from three separate sites (Brock University, St. Catharines, Ontario; Pelham, Ontario; and Torngat Mountains National Park, Newfoundland, Canada) and EIPF were isolated from plant roots, the hypocotyl, and stem and leaves. Two genera of EIPF, Metarhizium spp. and Beauveria bassiana, were isolated from plants sampled, as well as the nematophagous fungus, Pochonia chlamydosporium. Metarhizium spp. were almost exclusively found in roots, whereas B. bassiana and P. chlamydosporium were found throughout the plant. The Metarhizium species were identified by RFLP and 95 % were Metarhizium robertsii, 4.3 % were M. brunneum, and 0.7 % were M. guizhouense. Lab studies with M. robertsii and B. bassiana reflected observations found in the field, that is, Metarhizium was restricted to the roots of plants while B. bassiana was found throughout the plant. Insect infection by these EIPF is preferential with respect to above and below ground insects, and the present study correlates above and below ground insect infections with endophytic colonization by these EIPF.     

Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301–1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242–246; Li, Y., and R. Benezra. Science. 274: 246–248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK₁ cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929–939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK₁ cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after ~12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.     

p31comet-Induced Cell Death Is Mediated by Binding and Inactivation of Mad2

Mad2, a key component of the spindle checkpoint, is closely associated with chromosomal instability and poor prognosis in cancer. p31^comet is a Mad2-interacting protein that serves as a spindle checkpoint silencer at mitosis. In this study, we showed that p31^comet-induced apoptosis and senescence occur via counteraction of Mad2 activity. Upon retroviral transduction of p31^comet, the majority of human cancer cell lines tested lost the ability to form colonies in a low-density seeding assay. Cancer cells with p31^comet overexpression underwent distinct apoptosis and/or senescence, irrespective of p53 status, confirming the cytotoxicity of p31^comet. Interestingly, both cytotoxic and Mad2 binding activities were eliminated upon deletion of the C-terminal 30 amino acids of p31^comet. Point mutation or deletion of the region affecting Mad2 binding additionally abolished cytotoxic activity. Consistently, wild-type Mad2 interacting with p31^comet, but not its non-binding mutant, inhibited cell death, indicating that the mechanism of p31^comet-induced cell death involves Mad2 inactivation. Our results clearly suggest that the regions of p31^comet affecting interactions with Mad2, including the C-terminus, are essential for induction of cell death. The finding that p31^comet-induced cell death is mediated by interactions with Mad2 that lead to its inactivation is potentially applicable in anticancer therapy.     

Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.     

The plant beneficial effects of Metarhizium species correlate with their association with roots

Metarhizium species have recently been found to be plant rhizosphere associates as well as insect pathogens. Because of their abundance, rhizospheric Metarhizium could have enormous environmental impact, with co-evolutionary implications. Here, we tested the hypothesis that some Metarhizium spp. are multifactorial plant growth promoters. In two consecutive years, corn seeds were treated with entomopathogenic Metarhizium spp. and field tested at the Beltsville Facility in Maryland. Seed treatments included application of green fluorescent protein (GFP)-tagged strains of Metarhizium brunneum, Metarhizium anisopliae, Metarhizium robertsii, and M. robertsii gene disruption mutants that were either avirulent (Δmcl1), unable to adhere to plant roots (Δmad2), or poorly utilized root exudates (Δmrt). Relative to seeds treated with heat-killed conidia, M. brunneum, M. anisopliae, and M. robertsii significantly increased leaf collar formation (by 15, 14, and 13 %), stalk length (by 16, 10, and 10 %), average ear biomass (by 61, 56, and 36 %), and average stalk and foliage biomass (by 46, 36, and 33 %). Their major impact on corn yield was during early vegetative growth by allowing the plants to establish earlier and thereby potentially outpacing ambient biotic and abiotic stressors. Δmcl1 colonized roots and promoted plant growth to a similar extent as the parent wild type, showing that Metarhizium populations are plant growth promoters irrespective of their role as insect pathogens. In contrast, rhizospheric populations and growth promotion by Δmrt were significantly reduced, and Δmad2 failed to colonize roots or impact plant growth, suggesting that colonization of the root is a prerequisite for most, if not all, of the beneficial effects of Metarhizium.     

The mitochondrial genome contribution to the phylogeny and identification of Metarhizium species and strains

The genus Metarhizium is composed of entomopathogenic fungal biological control agents (BCAs) used for invertebrate pest control. The phylogenetic relationships of species within this genus are still under scrutiny as several cryptic species can be found. In this work, the mitochondrial (mt) genome of Metarhizium brunneum ARSEF 4556 was fully sequenced and a comparative genome analysis was conducted with 7 other available mt genomes, belonging to 5 Metarhizium species: M. anisopliae, M. brunneum, M. robertsii, M. guizhouense and M. majus. Results showed that Metarhizium demonstrates greater conserved stability than other fungal mt genomes. Furthermore, this analysis located 7 diverse regions in both intergenic domains and gene fragments which were ideal for species/strain discrimination. The sequencing of these regions revealed several SNPs among 38 strains tested, 11 of which were uncharacterized. Single gene phylogenies presented variable results which may be used further for intra-species discrimination. Phylogenetic trees based on the concatenation of mt domains and the nuclear ITS1-5.8S-ITS2 region showed discrimination of the species studied and allowed the identification of uncharacterized strains. These were mostly placed within species M. anisopliae and M. brunneum. Five strains clustered together in a clade related to M. brunneum, suggesting that they comprise a cryptic species.     

Ubiquity of Insect-Derived Nitrogen Transfer to Plants by Endophytic Insect-Pathogenic Fungi: an Additional Branch of the Soil Nitrogen Cycle

The study of symbiotic nitrogen transfer in soil has largely focused on nitrogen-fixing bacteria. Vascular plants can lose a substantial amount of their nitrogen through insect herbivory. Previously, we showed that plants were able to reacquire nitrogen from insects through a partnership with the endophytic, insect-pathogenic fungus Metarhizium robertsii. That is, the endophytic capability and insect pathogenicity of M. robertsii are coupled so that the fungus acts as a conduit to provide insect-derived nitrogen to plant hosts. Here, we assess the ubiquity of this nitrogen transfer in five Metarhizium species representing those with broad (M. robertsii, M. brunneum, and M. guizhouense) and narrower insect host ranges (M. acridum and M. flavoviride), as well as the insect-pathogenic fungi Beauveria bassiana and Lecanicillium lecanii. Insects were injected with ¹⁵N-labeled nitrogen, and we tracked the incorporation of ¹⁵N into two dicots, haricot bean (Phaseolus vulgaris) and soybean (Glycine max), and two monocots, switchgrass (Panicum virgatum) and wheat (Triticum aestivum), in the presence of these fungi in soil microcosms. All Metarhizium species and B. bassiana but not L. lecanii showed the capacity to transfer nitrogen to plants, although to various degrees. Endophytic association by these fungi increased overall plant productivity. We also showed that in the field, where microbial competition is potentially high, M. robertsii was able to transfer insect-derived nitrogen to plants. Metarhizium spp. and B. bassiana have a worldwide distribution with high soil abundance and may play an important role in the ecological cycling of insect nitrogen back to plant communities.     

Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator

A putative spindle matrix has been hypothesized to mediate chromosome motion, but its existence and functionality remain controversial. In this report, we show that Megator (Mtor), the Drosophila melanogaster counterpart of the human nuclear pore complex protein translocated promoter region (Tpr), and the spindle assembly checkpoint (SAC) protein Mad2 form a conserved complex that localizes to a nuclear derived spindle matrix in living cells. Fluorescence recovery after photobleaching experiments supports that Mtor is retained around spindle microtubules, where it shows distinct dynamic properties. Mtor/Tpr promotes the recruitment of Mad2 and Mps1 but not Mad1 to unattached kinetochores (KTs), mediating normal mitotic duration and SAC response. At anaphase, Mtor plays a role in spindle elongation, thereby affecting normal chromosome movement. We propose that Mtor/Tpr functions as a spatial regulator of the SAC, which ensures the efficient recruitment of Mad2 to unattached KTs at the onset of mitosis and proper spindle maturation, whereas enrichment of Mad2 in a spindle matrix helps confine the action of a diffusible “wait anaphase” signal to the vicinity of the spindle.     

Spindle checkpoint requires Mad1-bound and Mad1-free Mad2

The spindle checkpoint prevents anaphase from occurring until all chromosomes have attached properly to the mitotic spindle. The checkpoint components Mad1 and Mad2 associate with unattached kinetochores and are probably involved in triggering the checkpoint. We now demonstrate that in Xenopus egg extracts Mad1 and Mad2 form a stable complex, whereas a fraction of Mad2 molecules is not bound to Mad1. The checkpoint establishment and maintenance are lost upon titrating out free Mad2 with an excess of Mad1 or a truncated Mad1 (amino acids 326-718, Mad1C) that contains the Mad2-binding region. Mad1N (amino acids 1-445) that binds kinetochores, but not Mad2, reduces Mad1 and Mad2 at kinetochores and abolishes checkpoint maintenance. Furthermore, the association between Mad2 and Cdc20, the activator for the anaphase-promoting complex, is enhanced under checkpoint-active condition compared with that at metaphase. Immunodepletion analysis shows that the Mad1-free Mad2 protein is unable to bind Cdc20, consistent with the model that kinetochore localization of Mad2 facilitates the formation of Mad2-Cdc20 complex. This study demonstrates that the ratio between Mad1 and Mad2 is critical for maintaining a pool of Mad1-free Mad2 that is necessary for the spindle checkpoint. We propose that Mad2 may become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2.     

A review on the genus Metarhizium as an entomopathogenic microbial biocontrol agent with emphasis on its use and utility in Mexico

Metarhizium is a genus of entomopathogenic fungi that was initially classified into three species and varieties. More recently, DNA sequencing has improved the phylogenetic resolution of Metarhizium which now includes 30 species. The insect host ranges vary within the genus and some species such as M. robertsii have broad host ranges, while others such as M. acridum show a narrow host range and are restricted to the order Orthoptera. Metarhizium spp. are ubiquitous naturally occurring soil inhabiting fungi, and some are rhizosphere colonisers and their diversity has been attributed to various selective factors (habitat type, climatic conditions, specific associations with plants and insect hosts). Metarhizium have been used for the biological control of insect pests that affect economically important agricultural crops and have been tested under laboratory and field conditions for the control of insect vectors of human disease, showing the effectiveness of the fungus against the target pest. In Mexico, Metarhizium species have been used for the control of insect pests such as the spittlebug (Hemiptera: Cercopidae), and locusts (Orthoptera) that affect crops such as corn, bean and sugarcane. Biosafety studies, such as dermal and intragastric tests in mammalian models have also been carried out to ensure safety to humans and other animals. Metarhizium shows great promise as an alternative to chemical insecticides that has relatively low impact on human health and the environment. Key features of Metarhizium for biocontrol of insects are outlined with special reference to their utility in Mexico.     

Molecular mechanism of Mad1 kinetochore targeting by phosphorylated Bub1

During metaphase, in response to improper kinetochore‐microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase‐promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation‐dependent signalling cascade. The Mad1‐Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C‐terminal domain of Mad1 (Mad1^CTD) bound to two phosphorylated Bub1^CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg‐Leu‐Lys) motif, but also directly acts as an N‐terminal cap to the CD1 α‐helix dipole. Surprisingly, only one Bub1^CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled‐coil of Mad1^CTD and has implications for how the Mad1‐Bub1 complex at kinetochores promotes efficient MCC assembly.     

Metarhizium: jack of all trades,master of many

The genus Metarhizium and Pochonia chlamydosporia comprise a monophyletic clade of highly abundant globally distributed fungi that can transition between long-term beneficial associations with plants to transitory pathogenic associations with frequently encountered protozoans, nematodes or insects. Some very common ‘specialist generalist’ species are adapted to particular soil and plant ecologies, but can overpower a wide spectrum of insects with numerous enzymes and toxins that result from extensive gene duplications made possible by loss of meiosis and associated genome defence mechanisms. These species use parasexuality instead of sex to combine beneficial mutations from separate clonal individuals into one genome (Vicar of Bray dynamics). More weakly endophytic species which kill a narrow range of insects retain sexuality to facilitate host–pathogen coevolution (Red Queen dynamics). Metarhizium species can fit into numerous environments because they are very flexible at the genetic, physiological and ecological levels, providing tractable models to address how new mechanisms for econutritional heterogeneity, host switching and virulence are acquired and relate to diverse sexual life histories and speciation. Many new molecules and functions have been discovered that underpin Metarhizium associations, and have furthered our understanding of the crucial ecology of these fungi in multiple habitats.     

Uncovering the Cultivable Microbial Diversity of Costa Rican Beetles and Its Ability to Break Down Plant Cell Wall Components

Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.     

Production of Destruxins from Metarhizium spp. Fungi in Artificial Medium and in Endophytically Colonized Cowpea Plants

Destruxins (DTXs) are cyclic depsipeptides produced by many Metarhizium isolates that have long been assumed to contribute to virulence of these entomopathogenic fungi. We evaluated the virulence of 20 Metarhizium isolates against insect larvae and measured the concentration of DTXs A, B, and E produced by these same isolates in submerged (shaken) cultures. Eight of the isolates (ARSEF 324, 724, 760, 1448, 1882, 1883, 3479, and 3918) did not produce DTXs A, B, or E during the five days of submerged culture. DTXs were first detected in culture medium at 2–3 days in submerged culture. Galleria mellonella and Tenebrio molitor showed considerable variation in their susceptibility to the Metarhizium isolates. The concentration of DTXs produced in vitro did not correlate with percent or speed of insect kill. We established endophytic associations of M. robertsii and M. acridum isolates in Vigna unguiculata (cowpeas) and Cucumis sativus (cucumber) plants. DTXs were detected in cowpeas colonized by M. robertsii ARSEF 2575 12 days after fungal inoculation, but DTXs were not detected in cucumber. This is the first instance of DTXs detected in plants endophytically colonized by M. robertsii. This finding has implications for new approaches to fungus-based biological control of pest arthropods.