Utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by the fungus Metarhizium robertsii.

Plant-derived sugars and lipids are key nutritional sources for plant associated fungi. However, the relationship between utilization of host-derived sugars and lipids during development of the symbiotic association remains unknown. Here we show that the fungus Metarhizium robertsii also needs plant-derived lipids to develop symbiotic relationship with plants. The fatty acid binding proteins FABP1 and FABP2 are important for utilization of plant-derived lipids as the deletion of Fabp1 and Fabp2 significantly reduced the ability of M. robertsii to colonize rhizoplane and rhizosphere of maize and Arabidopsis thaliana. Deleting Fabp1 and Fabp2 increased sugar utilization by upregulating six sugar transporters, and this explains why deleting the monosaccharide transporter gene Mst1, which plays an important role in utilization of plant-derived sugars, had no impact on the ability of the double-gene deletion mutant ΔFabp1::ΔFabp2 to colonize plant roots. FABP1 and FABP2 were also found in other plant-associated Metarhizium species, and they were highly expressed in the medium using the tomato root exudate as the sole carbon and nitrogen source, suggesting that they could be also important for these species to develop symbiotic relationship with plants. In conclusion, we discovered that utilization of plant-derived sugars and lipids are coupled during colonization of rhizoplane and rhizosphere by M. robertsii.

A recombinant fungal photolyase autonomously enters human cell nuclei to fix UV-induced DNA lesions.

Solar ultraviolet radiations induced DNA damages in human skin cells with cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) as the most frequent lesions. CPDs are repaired much slower than 6-4PPs by the nucleotide excision repair pathway, which are thus the major lesions that interfere with key cellular processes and give rise to gene mutations, possibly resulting in skin cancer. In prokaryotes and multicellular eukaryotes other than placental mammals, CPDs can be rapidly repaired by CPD photolyases in one simple enzymatic reaction using the energy of blue light. In this study, we aim to construct recombinant CPD photolyases that can autonomously enter human cell nuclei to fix UV-induced CPDs. A fly cell penetration peptide and a viral nucleus localization signal peptide were recombined with a fungal CPD photolyase to construct a recombinant protein. This engineered CPD photolyase autonomously crosses cytoplasm and nuclear membrane of human cell nuclei, which then efficiently photo-repairs UV-induced CPD lesions in the genomic DNA. This further protects the cells by increasing SOD activity, and decreasing cellular ROSs, malondialdehyde and apoptosis.

Air Combat Intention Recognition with Incomplete Information Based on Decision Tree and GRU Network.

Battlefield information is generally incomplete, uncertain, or deceptive. To realize enemy intention recognition in an uncertain and incomplete air combat information environment, a novel intention recognition method is proposed. After repairing the missing state data of an enemy fighter, the gated recurrent unit (GRU) network, supplemented by the highest frequency method (HFM), is used to predict the future state of enemy fighter. An intention decision tree is constructed to extract the intention classification rules from the incomplete a priori knowledge, where the decision support degree of attributes is introduced to determine the node-splitting sequence according to the information entropy of partitioning (IEP). Subsequently, the enemy fighter intention is recognized based on the established intention decision tree and the predicted state data. Furthermore, a target maneuver tendency function is proposed to screen out the possible deceptive attack intention. The one-to-one air combat simulation shows that the proposed method has advantages in both accuracy and efficiency of state prediction and intention recognition, and is suitable for enemy fighter intention recognition in small air combat situations.

Bioremediation of mercury-polluted soil and water by the plant symbiotic fungus Metarhizium robertsii.

Fungi are central to every terrestrial and many aquatic ecosystems, but the mechanisms underlying fungal tolerance to mercury, a global pollutant, remain unknown. Here, we show that the plant symbiotic fungus Metarhizium robertsii degrades methylmercury and reduces divalent mercury, decreasing mercury accumulation in plants and greatly increasing their growth in contaminated soils. M. robertsii does this by demethylating methylmercury via a methylmercury demethylase (MMD) and using a mercury ion reductase (MIR) to reduce divalent mercury to volatile elemental mercury. M. robertsii can also remove methylmercury and divalent mercury from fresh and sea water even in the absence of added nutrients. Overexpression of MMD and MIR significantly improved the ability of M. robertsii to bioremediate soil and water contaminated with methylmercury and divalent mercury. MIR homologs, and thereby divalent mercury tolerance, are widespread in fungi. In contrast, MMD homologs were patchily distributed among the few plant associates and soil fungi that were also able to demethylate methylmercury. Phylogenetic analysis suggests that fungi could have acquired methylmercury demethylase genes from bacteria via two independent horizontal gene transfer events. Heterologous expression of MMD in fungi that lack MMD homologs enabled them to demethylate methylmercury. Our work reveals the mechanisms underlying mercury tolerance in fungi, and may provide a cheap and environmentally friendly means of cleaning up mercury pollution.

Enhanced Cd efflux capacity and physiological stress resistance: The beneficial modulations of Metarhizium robertsii on plants under cadmium stress.

Due to the high migration capacity in agricultural soil-crop systems, cadmium (Cd) is accumulated in various crops and severely inhibits plant growth. In this study, we showed that, under Cd stress, the plant-symbiotic fungus Metarhizium robertsii reduced Cd accumulation in Arabidopsis thaliana shoots and roots by 21.8 % and 23.8 %, respectively. This is achieved by M. robertsii colonization-induced elevation of Cd efflux capacity via upregulation of three PCR genes, which is confirmed by the fact that the extent to which M. robertsii reduced Cd accumulation in the WT plants was greater than the inactivating mutants of the PCR genes. M. robertsii also alleviated Cd-induced leaf etiolation in A. thaliana by increasing the chlorophyll amount and modified plant physiological status to increase Cd stress tolerance via increasing production of catalase, peroxidase and glutathione and upregulating multiple HIPP proteins involved in sequestration of Cd. Notably, consistent with that in A. thaliana, the colonization of M. robertsii also reduced the Cd accumulation in Oryza sativa seedlings by upregulating the PCR gene OsPCR1, and increased chlorophyll amount and alleviated oxidative stress. Therefore, M. robertsii colonization reduced Cd accumulation in plants, and promoted plant growth and health by elevating Cd efflux capacity and modifying physiological status.

Metarhizium robertsii as a promising microbial agent for rice in situ cadmium reduction and plant growth promotion.

The toxic chemical element cadmium (Cd) in paddy fields triggered increasing problems of growth inhibition and food security in rice consistently. In this study, we found Metarhizium robertsii, which is widely used as a bioinsecticide and biofertilizer in agriculture and recently found to be resistant to Cd, developed intraradical and extraradical symbiotic hyphae in rice seedlings, and successfully colonized in the rice rhizosphere soil to more than 103 CFUs g-1 soil at harvesting. M. robertsii colonization significantly reduced Cd accumulations in both hydroponically cultured seedlings and the matured rice cultured in Cd contaminated potting soil (2 ppm). Notably, Cd accumulation reduction of the roots, stems, leaves, husks and grains of the matured rice induced by the fungus were 44.3%, 32.1%, 35.3%, 31.9% and 24.7%, respectively. It was caused by the M. robertsii-induced suppression of Cd intake transporter gene osNramp5 in the rice roots, and the chemical stabilizing of Cd to the residual fraction in the rhizosphere soil. In addition, the colonization of M. robertsii significantly promoted the growth characters and the photosynthesis of the rice plants. This is achieved by the increase of endogenous hormone levels of indole-3-acetic, gibberellin A3 and brassinolide induced by M. robertsii. Furthermore, the fungus enhanced the antioxidative capacities via increasing enzyme activities of catalase, peroxidase and the production of glutathione, ascorbic acid, proline in the rice plants. Our work provides theoretical basis for expanding the use of M. robertsii as in situ Cd accumulation reduction and detoxification agents for rice in contaminated paddy fields.

An inactivating mutation in the vacuolar arginine exporter gene Vae results in culture degeneration in the fungus Metarhizium robertsii.

Culture degeneration usually results in great commercial losses in the economically important filamentous fungi, but the genetic causes of the degeneration remain elusive. In the fungus Metarhizium robertsii, we found that deletion of the vacuolar arginine exporter gene Vae caused culture degeneration. Compared to the WT strain, the mutant showed increased apoptosis, reactive oxygen species (ROS) level and mitochondrial membrane potential collapse, reduced conidial yield and abnormal lipid droplet formation. The extent of the degeneration in the mutant gradually increased over the successive subculturing, which eventually became irreversible; compared to the third subculture of the mutant, the seventh subculture showed a lower conidial yield and pathogenicity to insects, stronger apoptosis, higher ROS level and a smaller number of conidial lipid droplets. Incorporation of the genomic clone of Vae could not restore the WT phenotypes in the seventh subculture, but could in the third one. Loss-of-function in Vae resulted in vacuolar arginine accumulation and reduction in the cytosolic arginine. This downregulated the expression of the regulator CAG9 of G protein signalling pathway, which accounted for most of the phenotypic changes associated with the degeneration of the mutant. We identified a deleterious mutation that causes culture degeneration in a filamentous fugus.

New Downstream Signaling Branches of the Mitogen-Activated Protein Kinase Cascades Identified in the Insect Pathogenic and Plant Symbiotic Fungus Metarhizium robertsii.

Fungi rely on major signaling pathways such as the MAPK (Mitogen-Activated Protein Kinase) signaling pathways to regulate their responses to fluctuating environmental conditions, which is vital for fungi to persist in the environment. The cosmopolitan Metarhizium fungi have multiple lifestyles and remarkable stress tolerance. Some species, especially M. robertsii, are emerging models for investigating the mechanisms underlying ecological adaptation in fungi. Here we review recently identified new downstream branches of the MAPK cascades in M. robertsii, which controls asexual production (conidiation), insect infection and selection of carbon and nitrogen nutrients. The Myb transcription factor RNS1 appears to be a central regulator that channels information from the Fus3- and Slt2-MAPK cascade to activate insect infection and conidiation, respectively. Another hub regulator is the transcription factor AFTF1 that transduces signals from the Fus3-MAPK and the membrane protein Mr-OPY2 for optimal formation of the infection structures on the host cuticle. Homologs of these newly identified regulators are found in other Metarhizium species and many non-Metarhizium fungi, indicating that these new downstream signaling branches of the MAPK cascades could be widespread.

Transcriptional Differences Guided Discovery and Genetic Identification of Coprogen and Dimerumic Acid Siderophores in Metarhizium robertsii.

Siderophores are small molecular iron chelators and participate in the multiple cellular processes in fungi. In this study, biosynthesis gene clusters of coprogens and dimerumic acids were identified by transcriptional level differences of genes related to iron deficiency conditions in Metarhizium robertsii. This leads to the characterization of new coprogen metachelin C (1) and five known siderophores metachelin A (2), metachelin A-CE (3), metachelin B (4), dimerumic acid 11-mannoside (5), and dimerumic acid (6). The structure of metachelin C (1) was elucidated by NMR spectroscopy and HR-ESI-MS analysis. Genetic deletions of mrsidA, and mrsidD abolished the production of compounds 1-6 that implied their involvement in the biosynthesis of coprogen and dimerumic acid. Interestingly, NRPS gene mrsidD is responsible for biosynthesis of both coprogen and dimerumic acid, thus we proposed plausible biosynthetic pathways for the synthesis of coprogen and dimerumic acid siderophores. Therefore, our study provides the genetic basis for understanding the biosynthetic pathway of coprogen and dimerumic acid in Metarhizium robertsii.

The Sugar Transporter MST1 Is Involved in Colonization of Rhizosphere and Rhizoplane by Metarhizium robertsii.

It is widely recognized that plant-symbiotic fungi are supported by photosynthates; however, little is known about the molecular mechanisms underlying the utilization of plant-derived sugars by rhizospheric fungi. In the insect-pathogenic and plant-symbiotic fungus Metarhizium robertsii, we previously showed that the utilization of oligosaccharides by the transporter MRT (Metarhizium raffinose transporter) is important for rhizosphere competency. In this study, we identified a novel monosaccharide transporter (MST1) that is involved in the colonization of the rhizoplane and acts additively with MRT to colonize the rhizosphere. MST1 is not involved in infection of insects by M. robertsii. MST1 is an H+ symporter and is able to transport a broad spectrum of monosaccharides, including glucose, sorbose, mannose, rhamnose, and fructose. Deletion of the Mst1 gene impaired germination and mycelial growth in medium containing the sugars that it can transport. Homologs of MST1 were widely found in many fungi, including plant symbionts such as Trichoderma spp. and mycorrhizal fungi and plant pathogens such as Fusarium spp. This work significantly advances insights into the development of symbiotic relationships between plants and fungi. IMPORTANCE Over 90% of all vascular plant species develop an intimate symbiosis with fungi, which has an enormous impact on terrestrial ecosystems. It is widely recognized that plant-symbiotic fungi are supported by photosynthates, but little is known about the mechanisms for fungi to utilize plant-derived carbon sources. In the fungus Metarhizium robertsii, we identified a novel monosaccharide transporter (MST1) that is an H+ symporter and can transport a broad spectrum of monosaccharides, including glucose, sorbose, mannose, rhamnose, and fructose. MST1 is involved in the colonization of the rhizoplane and acts additively with the previously characterized oligosaccharide transporter MRT to colonize the rhizosphere. Homologs of MST1 were found in many fungi, including plant symbionts and plant pathogens, suggesting that the utilization of plant-derived sugars by MST1 homologs could also be important for other fungi to develop a symbiotic or parasitic relationship with their respective plant hosts.

A novel cascade allows Metarhizium robertsii to distinguish cuticle and hemocoel microenvironments during infection of insects.

Pathogenic fungi precisely respond to dynamic microenvironments during infection, but the underlying mechanisms are not well understood. The insect pathogenic fungus Metarhizium robertsii is a representative fungus in which to study broad themes of fungal pathogenicity as it resembles some major plant and mammalian pathogenic fungi in its pathogenesis. Here we report on a novel cascade that regulates response of M. robertsii to 2 distinct microenvironments during its pathogenesis. On the insect cuticle, the transcription factor COH2 activates expression of cuticle penetration genes. In the hemocoel, the protein COH1 is expressed due to the reduction in epigenetic repression conferred by the histone deacetylase HDAC1 and the histone 3 acetyltransferase HAT1. COH1 interacts with COH2 to reduce COH2 stability, and this down-regulates cuticle penetration genes and up-regulates genes for hemocoel colonization. Our work significantly advances the insights into fungal pathogenicity in insects.

A Novel Nitrogen and Carbon Metabolism Regulatory Cascade Is Implicated in Entomopathogenicity of the Fungus Metarhizium robertsii.

The entomopathogenic fungus Metarhizium robertsii can switch among parasitic, saprophytic, and symbiotic lifestyles in response to changing nutritional conditions, which is attributed to its extremely versatile metabolism. Here, we found that the Fus3-mitogen-activated protein kinase (MAPK) and the transcription factor regulator of nutrient selection 1 (RNS1) constitute a novel fungal cascade that regulates the degradation of insect cuticular lipids, proteins, and chitin to obtain nutrients for hyphal growth and enter the insect hemocoel for subsequent colonization. On the insect cuticle, Fus3-MAPK physically contacts and phosphorylates RNS1, which facilitates the entry of RNS1 into nuclei. The phosphorylated RNS1 binds to the DNA motif BM2 (ACCAGAC) in its own promoter to self-induce expression, which then activates the expression of genes for degrading cuticular proteins, chitin, and lipids. We further found that the Fus3-MAPK/RNS1 cascade also activates genes for utilizing complex and less-favored nitrogen and carbon sources (casein, colloid chitin, and hydrocarbons) that were not derived from insects, which is repressed by favored organic carbon and nitrogen nutrients, including glucose and glutamine. In conclusion, we discovered a novel regulatory cascade that controls fungal nitrogen and carbon metabolism and is implicated in the entomopathogenicity of M. robertsii. IMPORTANCE Penetration of the cuticle, the first physical barrier to pathogenic fungi, is the prerequisite for fungal infection of insects. In the entomopathogenic fungus Metarhizium robertsii, we found that the Fus3-mitogen-activated protein kinase (MAPK) and the transcription factor regulator of nutrient selection 1 (RNS1) constitute a novel cascade that controls cuticle penetration by regulating degradation of cuticular lipids, proteins, and chitin to obtain nutrients for hyphal growth and entry into the insect hemocoel. In addition, during saprophytic growth, the Fus3-MAPK/RNS1 cascade also activates utilization of complex and less-favored carbon and nitrogen sources that are not derived from insects. The homologs of Fus3-MAPK and RNS1 are widely found in ascomycete filamentous fungi, including saprophytes and pathogens with diverse hosts, suggesting that the regulation of utilization of nitrogen and carbon sources by the Fus3-MAPK/RNS1 cascade could be widespread. Our work provides significant insights into regulation of carbon and nitrogen metabolism in fungi and fungal pathogenesis in insects.

Plant volatile organic compound (E)-2-hexenal facilitates Botrytis cinerea infection of fruits by inducing sulfate assimilation.

Investigation into plant-fungal pathogen interactions is one of the most interesting fields in plant sciences. However, the roles of plant volatile organic compounds in the arms race are still largely unknown. Based on precise quantification of plant volatiles, we discovered that the plant volatile organic compound (E)-2-hexenal, at concentrations that were similar to or lower than those in tissues of strawberry and tomato fruits, upregulates sulfate assimilation in spores and hyphae of the phytopathogenic fungus Botrytis cinerea. This upregulation is independent of the types of sulfur sources in the plant and can be achieved in the presence of inorganic sulfate and organic sulfur sources. Using the fungal deletion mutants, we further found that sulfate assimilation is involved in the infection of tomato and strawberry fruits by B. cinerea, and that the severity of the disease is proportional to the sulfate content in the fruits. Both before and during the infection, (E)-2-hexenal induced utilisation of plant sulfate by B. cinerea facilitates its pathogenesis through enhancing its tolerance to oxidative stress. This work provides novel insights into the role of plant volatiles in plant-fungal pathogen interaction and highlights the importance of sulfur levels in the host in the prevention of grey mould disease.

Slt2-MAPK/RNS1 Controls Conidiation via Direct Regulation of the Central Regulatory Pathway in the Fungus Metarhizium robertsii.

Ascomycete fungi usually produce small hydrophobic asexual conidia that are easily dispersed and essential for long-term survival under a variety of environmental conditions. Several upstream signaling regulators have been documented to control conidiation via regulation of the central regulatory pathway that contains the transcription factors BrlA, AbaA and WetA. Here, we showed that the Slt2-MAPK signaling pathway and the transcription factor RNS1 constitute a novel upstream signaling cascade that activates the central regulatory pathway for conidiation in the Ascomycetes fungus M. robertsii. The BrlA gene has two overlapping transcripts BrlAα and BrlAß; they have the same major ORF, but the 5' UTR of BrlAß is 835 bp longer than the one of BrlAα. During conidiation, Slt2 phosphorylates the serine residue at the position 306 in RNS1, which self-induces. RNS1 binds to the BM2 motif in the promoter of the BrlA gene and induces the expression of the transcript BlrAα, which in turn activates the expression of the genes AbaA and WetA. In conclusion, the Slt2/RNS1 cascade represents a novel upstream signaling pathway that initiates conidiation via direct activation of the central regulatory pathway. This work provides significant mechanistic insights into the production of asexual conidia in an Ascomycete fungus.

Two-stage inventory management with financing under demand updates.

Considered is a retailer (she) facing non-stationary stochastic demand. Demand can be fully observed and backlogged, consequently the retailer can update the initial demand information using a Bayesian approach. To alleviate the demand risk, the retailer may use a secondary opportunity to replenish through an option contract. In addition, the retailer also has access to an immediate loan if she faces capital constraints and to a risk-free investment if she has surplus funds. The paper presents a recourse approach to solve the two-stage optimization problem and derive the optimal inventory/financing policies. The results show that the option procurement policy has a two-threshold base-stock structure depending on the first procurement, demand update and also the retailer's financial state. The initial procurement can be computed subsequently. A sufficiently large initial demand will induce the retailer to seize the secondary procurement opportunity. Finally, a series of numerical examples demonstrate the resulting policy under various inventory/financial situations. This research incorporates the financial and operational decisions into demand updates, and brings new managerial results and insights.

Metarhizium robertsii ammonium permeases (MepC and Mep2) contribute to rhizoplane colonization and modulates the transfer of insect derived nitrogen to plants.

The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis.

Unveiling the Mechanisms for the Plant Volatile Organic Compound Linalool To Control Gray Mold on Strawberry Fruits.

Fungal infections significantly alter the emissions of volatile organic compounds (VOCs) by plants, but the mechanisms for VOCs affecting fungal infections of plants remain largely unknown. Here, we found that infection by Botrytis cinerea upregulated linalool production by strawberries and fumigation with linalool was able to inhibit the infection of fruits by the fungus. Linalool treatment downregulated the expression of rate-limiting enzymes in the ergosterol biosynthesis pathway, and this reduced the ergosterol content in the fungi cell membrane and impaired membrane integrity. Linalool treatment also caused damage to mitochondrial membranes by collapsing mitochondrial membrane potential and also downregulated genes involved in adenosine triphosphate (ATP) production, resulting in a significant decrease in the ATP content. Linalool treatment increased the levels of reactive oxygen species (ROS), in response to which the treated fungal cells produced more of the ROS scavenger pyruvate. RNA-Seq and proteomic analysis data showed that linalool treatment slowed the rates of transcription and translation.

MrSt12 implicated in the regulation of transcription factor AFTF1 by Fus3-MAPK during cuticle penetration by the entomopathogenic fungus Metarhizium robertsii.

Metarhizium robertsii is a versatile fungus with multifactorial lifestyles, and it is an emerging fungal model for investigating the mechanisms of multiple lifestyle transitions that involve trans-kingdom host jumping. Penetration of the insect cuticle is the necessary step for the transition from saprophytic or symbiotic to pathogenic lifestyle. Previously, we found the transcription factor AFTF1 plays an important role in cuticle penetration, which is precisely regulated by Fus3-MAPK, Slt2-MAPK, and the membrane protein Mr-OPY2. Here, we identified a transcription factor (MrSt12) that directly regulated the transcription of Aftf1 by physically interacting with the cis-acting element (ATGAAACA) in the promoter of Aftf1. The deletion mutant of MrSt12 failed to form the infection structure appressorium and was thus nonpathogenic. We further found that the regulation of Aftf1 by MrSt12 was directly controlled by the Fus3-MAPK. In conclusion, we found a new signaling cascade containing Fus3-MAPK, MrSt12, and AFTF1, which regulates cuticle penetration by M. robertsii.

Increasing Pyruvate Concentration Enhances Conidial Thermotolerance in the Entomopathogenic Fungus Metarhizium robertsii.

The fungal entomopathogens Metarhizium spp. have been developed as environmentally friendly mycoinsecticides. However, heat stress severely reduces the viability of Metarhizium conidia in the field, which is an important obstacle to the successful use of these mycoinsecticides. Heat treatment induces rapid accumulation of pyruvate, which timely scavenges heat-induced ROS (reactive oxygen species) in hyphal cells of M. robertsii. However, in heat-treated conidia, pyruvate accumulation occurs later than the rapid production of ROSs, which could harm the conidial cells. In the present study, a transgenic M. robertsii strain was constructed with the pyruvate kinases gene overexpressed during conidiation. Two independent transformants of the transgenic strain produced conidia under optimal conditions with elevated pyruvate concentration. This inhibits the rapid heat-induced ROS production and prevents the collapse of mitochondrial membrane potential, thereby increasing conidial tolerance to heat stress. In conclusion, the tolerance of M. robertsii conidia to heat stress was improved by increasing the conidial pyruvate concentration, which could be translated into a more effective pest control.

Horizontal gene transfer allowed the emergence of broad host range entomopathogens.

The emergence of new pathogenic fungi has profoundly impacted global biota, but the underlying mechanisms behind host shifts remain largely unknown. The endophytic insect pathogen Metarhizium robertsii evolved from fungi that were plant associates, and entomopathogenicity is a more recently acquired adaptation. Here we report that the broad host-range entomopathogen M. robertsii has 18 genes that are derived via horizontal gene transfer (HGT). The necessity of degrading insect cuticle served as a major selective pressure to retain these genes, as 12 are up-regulated during penetration; 6 were confirmed to have a role in penetration, and their collective actions are indispensable for infection. Two lipid-carrier genes are involved in utilizing epicuticular lipids, and a third (MrNPC2a) facilitates hemocoel colonization. Three proteases degraded the procuticular protein matrix, which facilitated up-regulation of other cuticle-degrading enzymes. The three lipid carriers and one of the proteases are present in all analyzed Metarhizium species and are essential for entomopathogenicity. Acquisition of another protease (MAA_01413) in an ancestor of broad host-range lineages contributed to their host-range expansion, as heterologous expression in the locust specialist Metarhizium acridum enabled it to kill caterpillars. Our work reveals that HGT was a key mechanism in the emergence of entomopathogenicity in Metarhizium from a plant-associated ancestor and in subsequent host-range expansion by some Metarhizium lineages.