Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro

Entomophthora thripidum is an obligate biotrophic insect pathogenic fungus that grows as protoplasts within the hemocoel of thrips. Prior to penetration through the insect cuticle and spore formation at the insect surface the protoplasts switch to hyphal growth. In vitro, the differentiation to hyphal growth was a prerequisite for the subsequent formation of infectious spores and was detected 10-20 days after inoculation. E. thripidum secreted a factor that autoinduced the differentiation to hyphal growth. The discovery of this activity inducing hyphal growth made possible the reliable production of spores, the infection of host insects and the consecutive re-isolation of the fungus from the infected insects.     

In vivo gene expression profiling of the entomopathogenic fungus Beauveria bassiana elucidates its infection stratagems in Anopheles mosquito

The use of entomopathogenic fungi to control mosquitoes is a promising tool for reducing vector-borne disease transmission.To better understand infection stratagems of insect pathogenic fungi,we analyzed the global gene expression profiling of Beauveria bassiana at 36,60,84 and 108 h after topical infection of Anopheles stephensi adult mosquitoes using RNA sequencing(RNA-Seq).A total of 5,354 differentially expressed genes(DEGs) are identified over the course of fungal infection.When the fungus grows on the mosquito cuticle,up-regulated DEGs include adhesion-related genes involved in cuticle attachment,Pthll-like GPCRs hypothesized to be involved in host recognition,and extracellular enzymes involved in the degradation and penetration of the mosquito cuticle.Once in the mosquito hemocoel,the fungus evades mosquito immune system probably through up-regulating expression of |3-l,3-glucan degrading enzymes and chitin synthesis enzymes for remodeling of cell walls.Moreover,six previous unknown SSCP(small secreted cysteine-rich proteins) are significantly up-regulated,which may serve as "effectors" to suppress host defense responses.B.bassiana also induces large amounts of antioxidant genes to mitigate host-generated exogenous oxidative stress.At late stage of infection,B.bassiana activates a broad spectrum of genes including nutrient degrading enzymes,some transporters and metabolism pathway components,to exploit mosquito tissues and hemolymph as a nutrient source for hyphal growth.These findings establish an important framework of knowledge for further comprehensive elucidation of fungal pathogenesis and molecular mechanism of Beauveria-mosqaito interactions.     

Immunocytochemical localization of a 32-kDa protease from the nematophagous fungusVerticillium suchlasporium in infected nematode eggs

Fluorescein-labeled anti-rabbit antiserum showed that a serine protease designated P32, produced by the nematophagous fungusVerticillium suchlasporium, is secreted during infection of nematode eggs. Increased fluorescence in appressoria of the fungus on eggshells of the plant parasitic nematodeHeterodera schachtii indicated the presence of P32 in these fungal structures. Appressoria are involved in host penetration and these results support a role for P32 in the pathogenicity of the fungus to nematode eggs. These findings agree with similar results of entomogenous fungi penetrating the insect cuticle.     

Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassiana as revealed by scanning electron microscopy

Conidia from highly pathogenic mutants of Beauveria bassiana germinate quickly (within 18 hr) on the surface of corn earworm larvae (Heliothis zea) and immediately begin penetration of the cuticle. Enzymes produced by the penetrating hyphae degrade the cuticle since holes are formed at the point of entry. Clustering of conidia around nodules of larvae is often seen, but penetration is not restricted to such areas. Direct evidence is presented to show that conidia can also germinate inside of spiracle openings and could invade larvae by this route. Once inside the hemocoel, the fungus multiplies extensively; however, larval death occurs with only minimal breakdown of internal tissues. During mummification, outgrowths of fungal hyphae occur first and most extensively in the intersegmental regions of larvae. Conidia from mutants exhibiting low levels of pathogenicity are either significantly delayed or do not germinate on larval surfaces. When germination does occur, hyphae from such mutants do not penetrate immediately; instead, extensive surface hyphal growth, with or without eventual penetration of the integument, is evident.     

Conservative production of galactosaminogalactan in Metarhizium is responsible for appressorium mucilage production and topical infection of insect hosts

The exopolysaccharide galactosaminogalactan (GAG) has been well characterized in Aspergilli, especially the human pathogen Aspergillus fumigatus. It has been found that a five-gene cluster is responsible for GAG biosynthesis in Aspergilli to mediate fungal adherence, biofilm formation, immunosuppression or induction of host immune defences. Herein, we report the presence of the conserved GAG biosynthetic gene cluster in the insect pathogenic fungus Metarhizium robertsii to mediate either similar or unique biological functions. Deletion of the gene cluster disabled fungal ability to produce GAG on germ tubes, mycelia and appressoria. Relative to the wild type strain, null mutant was impaired in topical infection but not injection of insect hosts. We found that GAG production by Metarhizium is partially acetylated and could mediate fungal adherence to hydrophobic insect cuticles, biofilm formation, and penetration of insect cuticles. In particular, it was first confirmed that this exopolymer is responsible for the formation of appressorium mucilage, the essential extracellular matrix formed along with the infection structure differentiation to mediate cell attachment and expression of cuticle degrading enzymes. In contrast to its production during A. fumigatus invasive growth, GAG is not produced on the Metarhizium cells harvested from insect hemocoels; however, the polymer can glue germ tubes into aggregates to form mycelium pellets in liquid culture. The results of this study unravel the biosynthesis and unique function of GAG in a fungal system apart from the aspergilli species.     

Cuticle degrading proteases from insect moulting fluid and culture filtrates of entomopathogenic fungi

Insects degrade their own cuticle during moulting, a process which is catalysed by a complex mixture of enzymes. Entomopathogenic fungi infect the insect host by penetration of the cuticle, utilizing enzymatic and/or physical mechanisms. Protein is a major component of insect cuticle and a major recyclable resource for the insect and, therefore, represents a significant barrier to the invading fungus. To this end, both insects and entomopathogenic fungi produce a variety of cuticle degrading proteases. The aim of this paper is to review these proteases and to highlight their similarities, with particular reference to the tobacco hornworm, Manduca sexta, and the entomopathogenic fungus, Metarhizium anisopliae     

Can Insects Develop Resistance to Insect Pathogenic Fungi?

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25^th generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses.     

TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi

Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.     

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.     

Fungal infection dynamics in response to temperature in the lepidopteran insect Galleria mellonella

This study examines how the dynamics of fungus–insect interactions can be modulated by temperature. The wax moth, Galleria mellonella, is a well‐studied and important model insect whose larvae in the wild develop optimally at around 34 °C in beehives. However, surprisingly little research on wax moths has been conducted at relevant temperatures. In this study, the entomopathogenic fungus Metarhizium robertsii inflicted rapid and substantial mortality on wax moth larvae maintained at a constant temperature of 24 °C, but at 34 °C a 10 fold higher dose was required to achieve an equivalent mortality. The cooler temperature favored fungal pathogenicity, with condial adhesion to the cuticle, germination and hemocoel invasion all significantly enhanced at 24 °C, compared with 34 °C. The wax moth larvae immune responses altered with the temperature, and with the infective dose of the fungus. Enzyme‐based immune defenses (lysozyme and phenoloxidase) exhibited enhanced activity at the warmer temperature. A dramatic upregulation in the basal expression of galiomicin and gallerimycin was triggered by cooling, and this was augmented in the presence of the fungus. Profiling of the predominant insect epicuticular fatty acids revealed a 4–7 fold increase in palmetic, oleic and linoleic acids in larvae maintained at 24 °C compared with those at 34 °C, but these failed to exert fungistatic effects on topically applied fungus. This study demonstrates the importance of choosing environmental conditions relevant to the habitat of the insect host when determining the dynamics and outcome of insect/fungus interactions, and has particular significance for the application of entomopathogens as biocontrol agents.     

Host-to-Pathogen Gene Transfer Facilitated Infection of Insects by a Pathogenic Fungus

Metarhizium robertsii is a plant root colonizing fungus that is also an insect pathogen. Its entomopathogenicity is a characteristic that was acquired during evolution from a plant endophyte ancestor. This transition provides a novel perspective on how new functional mechanisms important for host switching and virulence have evolved. From a random T-DNA insertion library, we obtained a pathogenicity defective mutant that resulted from the disruption of a sterol carrier gene (Mr-npc2a). Phylogenetic analysis revealed that Metarhizium acquired Mr-npc2a from an insect by horizontal gene transfer (HGT). Mr-NPC2a binds to cholesterol, an animal sterol, rather than the fungal sterol ergosterol, indicating it retains the specificity of insect NPC2 proteins. Mr-NPC2a is an intracellular protein and is exclusively expressed in the hemolymph of living insects. The disruption of Mr-npc2a reduced the amount of sterol in cell membranes of the yeast-like hyphal bodies that facilitate dispersal in the host body. These were consequently more susceptible to insect immune responses than the wild type. Transgenic expression of Mr-NPC2a increased the virulence of Beauveria bassiana, an endophytic insect-pathogenic fungus that lacks a Mr-NPC2a homolog.     

Process of infection of armored scale insects (Diaspididae) by an entomopathogenic Cosmospora sp

Several species in the fungal genus Cosmospora (synonym Nectria) (anamorph Fusarium) are specialist entomopathogens of armored scale insects (Diaspididae), known to cause periodic epizootics in host populations. Inconsistent mortality rates recorded under laboratory conditions prompted a study into the process of infection of armored scale insects by this fungus. Scale insect mortality following exposure to a Cosmospora sp. (Culture Collection Number: CC89) from New Zealand was related to insect age, with reproductively mature insects having a significantly higher infection rate than immature insects. Examination using scanning electron microscopy found no evidence that the fungus penetrated directly through the wax test (cap) of the scale insect or through the un-lifted interface between the test and the substrate on which the insect resided. However, fungal hyphae were observed growing beneath the test when the test of the reproductively mature insect lifted away from the substrate for the purpose of releasing crawlers, the mobile pre-settled juveniles. Once the hyphae of CC89 advanced under the test, germ-tubes readily penetrated the insect body through a number of natural openings (e.g. spiracles, vulva, stylet), with mycosis observed within seven days after inoculation. Direct penetration through the cuticle of the scale insect was not observed.     

Autophagy vitalizes the pathogenicity of pathogenic fungi

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.     

转录组分析罗伯茨绿僵菌精胺合成酶MrSPS介导真菌血腔定殖功能

【背景】精胺在植物应对逆境胁迫、动物抵抗疲劳和衰老、真菌生长代谢等过程中发挥重要作用,但目前在昆虫病原真菌中的研究未见报道。【目的】在分子水平上探究罗伯茨绿僵菌精胺合成关键酶——精胺合成酶在昆虫血腔定殖中的作用机制。【方法】显微注射法测定Mrsps敲除株ΔMrsps的致病力变化,并观察血腔中ΔMrsps生长状态;收集ΔMrsps和野生型WT注射侵染30 h后的大蜡螟血淋巴进行转录组测序,分别与罗伯茨绿僵菌和大蜡螟参考基因组进行比对分析,并结合定量PCR进行验证。【结果】与WT和回补株ΔMrsps-cp相比较,ΔMrsps致病力显著下降,而且随着注射浓度的降低,ΔMrsps致病力下降越显著。侵染36 h后WT和ΔMrsps孢子都能正常萌发且开始以类酵母状态生长,60 h后,相较于WT,ΔMrsps的生长繁殖数量较少。转录组共检测到3 202个罗伯茨绿僵菌基因,其中1 769个基因在ΔMrsps中表达上调,922个基因表达下调;差异表达基因涉及碳水化合物代谢、运输、分解代谢、翻译和氨基酸代谢等多条途径;筛选出28个血腔致病相关基因全部在ΔMrsps中表达下调;定量PCR检测发现在整个血腔定殖阶段免疫逃避蛋白Mcl1基因和血腔定殖Colonization of hemocoel 1基因在WT和ΔMrsps-cp中的表达量高于ΔMrsps。共检测到13 249个大蜡螟基因,其中4 026个差异表达基因;KEGG注释分析显示大量差异表达基因富集到内分泌系统和免疫系统等途径;深入分析发现22个差异表达基因归属于Toll和Imd信号通路,其中18个基因在ΔMrsps侵染的大蜡螟中表达上调,表明ΔMrsps侵染大蜡螟过程中更易引起免疫系统的激活。【结论】揭示了Mrsps在罗伯茨绿僵菌血腔定殖阶段作用的分子机制,为进一步揭示精胺在真菌中的作用机理提供了理论基础。     

虫生真菌分子致病机理及基因工程改造研究进展

虫生真菌侵染寄主昆虫的复杂过程可分为体表附着、体壁穿透及体内定殖和致死等不同阶段.近年来,以金龟子绿僵茵(Metarhizium anisopliae)和球孢白僵菌(Beauveria bassiana)为代表的基因功能研究取得了长足的进展,从不同角度阐明了虫生真菌的分子致病机理;同时,基因工程技术的应用为昆虫病原真菌的遗传改良和选育高毒力杀虫菌株开辟了新的途径.对近年来昆虫病原真菌侵染寄主的分子对策及基因工程改造的研究进展进行了综述,并就进一步研究虫生真菌的毒力基因及功能进行了探讨.     

虫生真菌分子致病机理及基因工程改造研究进展

虫生真菌侵染寄主昆虫的复杂过程可分为体表附着、体壁穿透及体内定殖和致死等不同阶段。近年来, 以金龟子绿僵菌(Metarhizium anisopliae)和球孢白僵菌(Beauveria bassiana)为代表的基因功能研究取得了长足的进展, 从不同角度阐明了虫生真菌的分子致病机理; 同时, 基因工程技术的应用为昆虫病原真菌的遗传改良和选育高毒力杀虫菌株开辟了新的途径。对近年来昆虫病原真菌侵染寄主的分子对策及基因工程改造的研究进展进行了综述, 并就进一步研究虫生真菌的毒力基因及功能进行了探讨。     

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.     

The Magas1 gene is involved in pathogenesis by affecting penetration in Metarhizium acridum

Appressorium is a specialized infection structure of filamentous pathogenic fungi and plays an important role in establishing a pathogenic relationship with the host. The Egh16/Egh16H family members are involved in appressorium formation and pathogenesis in pathogenic filamentous fungi. In this study, a homolog of Egh16H, Magas1, was identified from an entomopathogenic fungus, Metarhizium acridum. The Magas1 protein shared a number of conserved motifs with other Egh16/Egh16H family members and specifically expressed during the appressorium development period. Magas1-EGFP fusion expression showed that Magas1 protein was not localized inside the cell. Deletion of the Magas1 gene had no impact on vegetative growth, conidiation and appressorium formation, but resulted in a decreased mortality of host insect when topically inoculated. However, the mortality was not significant between the Magas1 deletion mutant and wild-type treatment when the cuticle was bypassed by injecting conidia directly into the hemocoel. Our results suggested that Magas1 may influence virulence by affecting the penetration of the insects' cuticle.