Inductive Production of the Iron-Chelating 2-Pyridones Benefits the Producing Fungus To Compete for Diverse Niches.

Diverse 2-pyridone alkaloids have been identified with an array of biological and pharmaceutical activities, including the development of drugs. However, the biosynthetic regulation and chemical ecology of 2-pyridones remain largely elusive. Here, we report the inductive activation of the silent polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) (tenS) gene cluster for the biosynthesis of the tenellin-type 2-pyridones in the insect-pathogenic fungus Beauveria bassiana when cocultured with its natural competitor fungus Metarhizium robertsii. A pathway-specific transcription factor, tenR, was identified, and the overexpression of tenR well expanded the biosynthetic mechanism of 15-hydroxytenellin (15-HT) and its derivatives. In particular, a tandemly linked glycosyltransferase-methyltransferase gene pair located outside the tenS gene cluster was verified to mediate the rare and site-specific methylglucosylation of 15-HT at its N-OH residue. It was evident that both tenellin and 15-HT can chelate iron, which could benefit B. bassiana to outcompete M. robertsii in cocultures and to adapt to iron-replete and -depleted conditions. Relative to the wild-type strain, the deletion of tenS had no obvious negative effect on fungal virulence, but the overexpression of tenR could substantially increase fungal pathogenicity toward insect hosts. The results of this study well advance the understanding of the biosynthetic machinery and chemical ecology of 2-pyridones. IMPORTANCE Different 2-pyridones have been identified, with multiple biological activities but unclear chemical ecology. We found that the silent tenS gene cluster was activated in the insect pathogen Beauveria bassiana when the fungus was cocultured with its natural competitor Metarhizium robertsii. It was established that the gene cluster is regulated by a pathway-specific regulator, tenR, and the overexpression of this transcription factor expanded the biosynthetic machinery of the tenellin 2-pyridones. It was also found that the paired genes located outside the tenS cluster contribute to the site-specific methylglucosylation of the main compound 15-hydroxytenellin. Both tenellin and 15-hydroxytenellin can chelate and sequester iron to benefit the producing fungus to compete for different niches. This study well advances the biosynthetic mechanism and chemical ecology of 2-pyridones.

Dipeptidase PEPDA Is Required for the Conidiation Pattern Shift in Metarhizium acridum.

Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involves a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum, which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in ΔpepdA mutant. The defect of MC in ΔpepdA can be rescued when nonpolar amino acids, α-alanine, ß-alanine, or proline, were added into sucrose yeast extract agar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum. IMPORTANCE Conidia, as the asexual propagules in many fungi, are the start and end of the fungal life cycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the ΔpepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.

A Baeyer-Villiger Monooxygenase Gene Involved in the Synthesis of Lysergic Acid Amides Affects the Interaction of the Fungus Metarhizium brunneum with Insects.

Several fungi, including the plant root symbiont and insect pathogen Metarhizium brunneum, produce lysergic acid amides via a branch of the ergot alkaloid pathway. Lysergic acid amides include important pharmaceuticals and pharmaceutical lead compounds and have potential ecological significance, making knowledge of their biosynthesis relevant. Many steps in the biosynthesis of lysergic acid amides have been determined, but terminal steps in the synthesis of lysergic acid α-hydroxyethylamide (LAH)-by far the most abundant lysergic acid amide in M. brunneum-are unknown. Ergot alkaloid synthesis (eas) genes are clustered in the genomes of fungi that produce these compounds, and the eas clusters of LAH producers contain two uncharacterized genes (easO and easP) not found in fungi that do not produce LAH. Knockout of easO via a CRISPR-Cas9 approach eliminated LAH and resulted in accumulation of the alternate lysergic acid amides lysergyl-alanine and ergonovine. Despite the elimination of LAH, the total concentration of lysergic acid derivatives was not affected significantly by the mutation. Complementation with a wild-type allele of easO restored the ability to synthesize LAH. Substrate feeding studies indicated that neither lysergyl-alanine nor ergonovine were substrates for the product of easO (EasO). EasO had structural similarity to Baeyer-Villiger monooxygenases (BVMOs), and labeling studies with deuterated alanine supported a role for a BVMO in LAH biosynthesis. The easO knockout had reduced virulence to larvae of the insect Galleria mellonella, indicating that LAH contributes to virulence of M. brunneum on insects and that LAH has biological activities different from ergonovine and lysergyl-alanine. IMPORTANCE Fungi in the genus Metarhizium are important plant root symbionts and insect pathogens. They are formulated commercially to protect plants from insect pests. Several Metarhizium species, including M. brunneum, were recently shown to produce ergot alkaloids, a class of specialized metabolites studied extensively in other fungi because of their importance in agriculture and medicine. A biological role for ergot alkaloids in Metarhizium species had not been demonstrated previously. Moreover, the types of ergot alkaloids produced by Metarhizium species are lysergic acid amides, which have served directly or indirectly as important pharmaceutical compounds. The terminal steps in the synthesis of the most abundant lysergic acid amide in Metarhizium species and several other fungi (LAH) have not been determined. The results of this study demonstrate the role of a previously unstudied gene in LAH synthesis and indicate that LAH contributes to virulence of M. brunneum on insects.

The deletion of chiMaD1, a horizontally acquired chitinase of Metarhizium anisopliae, led to higher virulence towards the cattle tick (Rhipicephalus microplus).

The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus.

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC50 by evaluating conidia germination (38µM). To ensure high cell integrity, a sublethal dose of MND (half of LC50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC50 and LT50, particularly at 108 conidia mL-1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.

Two white collar proteins protect fungal cells from solar UV damage by their interactions with two photolyases in Metarhizium robertsii.

The photolyases PHR1 and PHR2 enable photorepair of fungal DNA lesions in the forms of UV-induced cyclobutane pyrimidine dimer (CPD) and (6-4)-pyrimidine-pyrimidone (6-4PP) photoproducts, but their regulation remains mechanistically elusive. Here, we report that the white collar proteins WC1 and WC2 mutually interacting to form a light-responsive transcription factor regulate photolyase expression required for fungal UV resistance in the insect-pathogenic fungus Metharhizum robertsii. Conidial UVB resistance decreased by 54% in Δwc1 and 67% in Δwc2. Five-hour exposure of UVB-inactivated conidia to visible light resulted in photoreactivation rates of 30% and 9% for the Δwc1 and Δwc2 mutants, contrasting to 79%-82% for wild-type and complemented strains. Importantly, abolished transcription of phr1 in Δwc-2 and of phr2 in Δwc1 resulted in incapable photorepair of CDP and 6-4PP DNA lesions in UVB-impaired Δwc2 and Δwc1 cells respectively. Yeast two-hybrid assays revealed interactions of either WC protein with both PHR1 and PHR2. Therefore, the essential roles for WC1 and WC2 in both photorepair of UVB-induced DNA lesions and photoreactivation of UVB-inactivated conidia rely upon their interactions with, and hence transcriptional activation of, PHR1 and PHR2. These findings uncover a novel WC-cored pathway that mediates filamentous fungal response and adaptation to solar UV irradiation.

The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii.

Metarhizium spp. are well-known biocontrol agents used worldwide to control different insect pests. Keto-acid reductoisomerase (ILVC) is a key enzyme for branched-chain amino acid (BCAA) biosynthesis, and it regulates many physiological activities. However, its functions in insect-pathogenic fungi are poorly understood. In this work, we identified MrilvC in M. robertsii and dissected its roles in fungal growth, conidiation, germination, destruxin biosynthesis, environmental stress response, and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidial germination of an MrilvC-deficient strain. Further feeding assays with intermediates showed that some conidia of the ΔMrilvC mutant start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in the ΔMrilvC mutant. In conclusion, we found intermediates in BCAA biosynthesis are indispensable for Metarhizium robertsii conidial germination. This study will advance our understanding of the fungal germination mechanism.IMPORTANCE Branched-chain amino acid (BCAA) metabolism plays a significant role in many biological activities beyond protein synthesis. Spore germination initiates the first stage of vegetative growth, which is critical for the virulence of pathogenic fungi. In this study, we demonstrated that the keto-acid reductoisomerase MrILVC, a key enzyme for BCAA biosynthesis, from the insect-pathogenic fungus Metarhizium robertsii is associated with conidial germination and fungal pathogenicity. Surprisingly, the germination of the ΔMrilvC mutant was restored when supplemented with the intermediates of BCAA metabolism rather than three BCAAs. The result was significantly different from that of plant-pathogenic fungi. Therefore, this report highlights that the intermediates in BCAA biosynthesis are indispensable for conidial germination of M. robertsii.

DNA methyltransferase implicated in the recovery of conidiation, through successive plant passages, in phenotypically degenerated Metarhizium.

Metarhizium robertsii is a fungus with two lifestyles; it is a plant root symbiont and an insect pathogen. A spontaneously phenotypically degenerated strain of M. robertsii strain ARSEF 2575 (M. robertsii lc-2575; lc = low conidiation) showed a reduction in conidiation and fungal virulence after successive subculturing on agar medium. In order to recover conidiation, we experimentally passaged M. robertsii lc-2575 through plant (soldier bean and switchgrass) root or insect (Galleria mellonella) larvae. After five passages, the resultant strains had significantly increased conidial yields on agar and increased virulence in insect bioassays. Concomitantly, DNA methyltransferase, MrDIM-2 expression was downregulated in BR5 (a strain after 5 bean root passages) and isolates after switchgrass and insect passages. Bisulfite sequencing showed little difference in overall genomic DNA methylation levels (~ 0.37%) between M. robertsii lc-2575 and BR5. However, a finer comparison of the different methylated regions (DMRs) showed that DMRs of BR5 were more abundant in the intergenic regions (69.32%) compared with that of M. robertsii lc-2575 (33.33%). The addition of DNA methyltransferase inhibitor, 5-azacytidine, to agar supported the role of DNA methyltransferases and resulted in an increase in conidiation of M. robertsii lc-2575. Differential gene expression was observed in selected DMRs in BR5 when compared with M. robertsii lc-2575. Here we implicated epigenetic regulation in the recovery of conidiation through the effects of DNA methyltransferase and that plant passage could be used as a method to recover fungal conidiation and virulence in a phenotypically degenerated M. robertsii. KEY POINTS: • Passage of Metarhizium through plant root or insect results in increased conidiation. • DNA methyltransferase is downregulated after host passage. • Bisulfite sequencing identified potentially methylated genes involved in conidiation.

Members of the nitronate monooxygenase gene family from Metarhizium brunneum are induced during the process of infection to Plutella xylostella.

Metarhizium species are the most abundant fungi that can be isolated from soil, with a well-known biopesticide capacity. Metarhizium recognizes their hosts when the conidium interacts with insects, where the fungi are in contact with the hydrocarbons of the outermost lipid layer cuticle. These cuticular hydrocarbons comprise a mixture of n-alkanes, n-alkenes, and methyl-branched chains. Metarhizium can degrade insect hydrocarbons and use these hydrocarbons for energy production and the biosynthesis of cellular components. The metabolism of nitroalkanes involves nitronate monooxygenase activity. In this work, we isolated a family of six genes with potential nitronate monooxygenase activity from Metarhizium brunneum. The six genes were expressed in Escherichia coli, and the nitronate monooxygenase activity was verified in the recombinant proteins. Additionally, when the conidia of M. brunneum were grown in medium with nitroalkanes, virulence against Plutella xylostella increased. Furthermore, we analyzed the expression of the six Npd genes during the infection to this insect, which showed differential expression of the six Npd genes during infection.

A M35 family metalloprotease is required for fungal virulence against insects by inactivating host prophenoloxidases and beyond.

A diverse family of metalloproteases (MPs) is distributed in eukaryotes. However, the functions of MPs are still understudied. We report that seven MPs belonging to the M35 family are encoded in the genome of the insect pathogenic fungus Metarhizium robertsii. By gene deletions and insect bioassays, we found that one of the M35-family MPs, i.e. MrM35-4, is required for fungal virulence against insect hosts. MrM35-4 is a secretable enzyme and shows a proteolytic activity implicated in facilitating fungal penetration of insect cuticles. After gene rescue and overexpression, insect bioassays indicated that MrM35-4 contributes to inhibiting insect cuticular and hemocyte melanization activities. Enzymatic cleavage assays revealed that the recombinant prophenoloxidases PPO1 and PPO2 of Drosophila melanogaster could be clipped by MrM35-4 in a manner differing from a serine protease that can activate PPO activities. In addition, it was found that MrM35-4 is involved in suppressing antifungal gene expression in insects. Consistent with the evident apoptogenic effect of MrM35-4 on host cells, we found that the PPO mutant flies differentially succumbed to the infections of the wild-type and mutant strains of M. robertsii. Thus, MrM35-4 plays a multifaceted role beyond targeting PPOs during fungus-insect interactions, which represents a previously unsuspected strategy employed by Metarhizium to outmaneuver insect immune defenses.

Biocontrol potential and genetic diversity of Metarhizium anisopliae lineage in agricultural habitats.

AIMS: To assess phylogenetic and genotypic diversity of Metarhizium anisopliae lineage within diverse agroecosystems in the Karnataka State of India and to compare their chitinase activity and pathogenicity against insect pest of field crops subterranean termite, Odontotermes obesus. METHODS AND RESULTS: Three phylogenetic and 27 microsatellite markers were used to assess the genetic diversity of M. anisopliae lineage within multiple agroecosystems. Multilocus phylogeny of the Metarhizium isolates identified two species: Metarhizium pingshaense and Metarhizium guizhouense. Multilocus phylogeny and microsatellite markers resolved two phylogenetic species of M. pingshaense, Mp_1 and Mp_2, and one phylogenetic species of M. guizhouense, Mg_1. Phylogenetic species, Mp_2 and Mg_1, were detected with one genotype each and Mp_1 with eleven genotypes. Metarhizium pingshaense GKVK 02_16 isolate caused significantly high mortality of O. obesus in bioassays and detected with high chitinase activity. CONCLUSIONS: The study revealed phylogenetic and genotypic diversity of M. anisopliae lineage in agroecosystems of Karnataka State. Findings of pathogenicity and chitinase activity suggest that M. pingshaense GKVK 02_16 isolate provides effective control of O. obesus. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation provided an understanding of the genetic diversity and biocontrol efficiency of M. anisopliae lineage in agroecosystem. These data will serve as a resource in the future pest management strategies at a regional scale.

Molecular evolution of Pr1 proteases depicts ongoing diversification in Metarhizium spp.

The Pr1 family of serine endopeptidases plays an important role in pathogenicity and virulence of entomopathogens such as Metarhizium anisopliae (Ascomycota: Hypocreales). These virulence factors allow for the penetration of the host cuticle, a vital step in the infective process of this fungus, which possesses 11 Pr1 isoforms (Pr1A through Pr1K). The family is divided into two classes with Class II (proteinase K-like) comprising 10 isoforms further split into three subfamilies. It is believed that these isoforms act synergistically and with other virulence factors, allowing pathogenicity to multiple hosts. As virulence coevolves through reciprocal selection with hosts, positive selection may lead to the evolution of new protease families or isoforms of extant ones that can withstand host defenses. This work tests this hypothesis in Class II Pr1 proteins, focusing on M. anisopliae, employing different methods for phylogenetic inference in amino acid and nucleotide datasets in multiple arrangements for Metarhizium spp. and related species. Phylogenies depict groups that match the taxonomy of their respective organisms with high statistical support, with minor discrepancies. Positively selected sites were identified in six out of ten Pr1 isoforms, most of them located in the proteolytic domain and spatially close to the catalytic residues. Moreover, there was evidence of functional divergence in the majority of pairwise comparisons. These results imply the existence of differential selective pressure acting on Pr1 proteins and a potential new isoform, likely affecting host specificities, virulence, or even adapting the organism to different host-independent lifestyles.

Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease.

MicroRNAs (miRNAs) have been recognized as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. However, the functions and working mechanisms of hundreds of fungal miRNA-like (miR-like) RNAs are obscure. Here, we report that a short tandem target mimic (STTM) triggered the degradation of several fungal miR-like RNAs in two different fungal species, Metarhizium robertsii and Aspergillus flavus, and that small-RNA-degrading nucleases (SDNs) were indispensable for such degradation. STTMs were most effective when the fungal polymerase II (Pol II) promoter was used for their expression, while the Pol III promoter was less effective. The length of the STTM spacer, approximately 48 to 96 nucleotides, and the number of miR-like RNA binding sites, from 2 to 4 copies, showed no significant difference in the degradation of miR-like RNAs. STTMs modulated the miR-like RNA expression levels in at least two different fungal species, which further impacted fungal asexual growth and sporulation. Further analysis showed that the degraded miR-like RNAs in STTM mutants led to the upregulation of potential target genes involved in fungal development and conidial production, which result in different phenotypes in these mutants. The STTM technology developed in this study is an effective and powerful tool for the functional dissection of fungal miR-like RNAs.IMPORTANCE The development and application of STTM technology to block miR-like RNAs in M. robertsii and A. flavus may allow for efficient generation of miR-like RNA mutants in various fungi, providing a powerful tool for functional genomics of small RNA molecules in fungi.

MaPmt4, a protein O-mannosyltransferase, contributes to cell wall integrity, stress tolerance and virulence in Metarhizium acridum.

In eukaryotic cells, protein O-glycosylation is an essential protein modification. Analysis of the Metarhizium acridum genome database revealed a total of three O-glycoside mannosyltransferase homologs (Pmt1, Pmt2 and Pmt4), closely related to Saccharomyces cerevisiae Pmt1, Pmt2, and Pmt4. In this study, the functions of MaPmt4, encoding a protein O-mannosyltransferase in M. acridum, were characterized using disruption and complementation strategies. Disruption of MaPmt4 delayed the conidial germination and reduced the fungal tolerances to heat shock and UV-B irradiation, but did not affect conidial yield. Inactivation of MaPmt4 displayed increased sensitivity to cell wall-perturbing agents, formed thinner cell walls, and changed composition of fungal cell wall, demonstrating that MaPmt4 was also important to maintain fungal cell wall integrity. Bioassays by topical inoculation and intrahemocoel injection showed that the MaPmt4 deletion mutant exhibited greatly reduced virulence. The subsequent examination revealed that the inactivation of MaPmt4 impaired appressorium formation, decreased fungal growth in locust hemolymph in vitro, and boosted insect immune responses, the latter in part potentially owing to the changes in conidial surface structures, and thus attenuated the virulence of MaPmt4 deletion mutant. Furthermore, the results of comparative proteomics showed that MaPmt4 played important roles in fungal cell wall integrity, stress tolerances, and virulence via broad genetic pathways.

[Transgenic Expression of chit42 gene from Metarhizium anisopliae in Trichoderma harzianum Enhances Antagonistic Activity against Botrytis cinerea].

Chitinases expressed by some beneficial fungi are crucial for the biocontrol of phytopathogens. The activity of chitinolytic strains of Trichoderma sp. may be enhanced by increasing the expression of chitinases. We describe the Trichoderma strain Mchit42 which expresses a transgenic chitinase chit42 from Metarhizium anisopliae. Inhibitory effects against plant pathogens were tested. Comparison of WT (T30) and OE (Mchit42) indicated that overexpression of M. anisopliae chit42 did not alter Trichoderma growth, while enhancing the expression of endogenous chitinase, ß-l,3-glucanases, and polygalacturonase and increasing the antagonistic activity of Trichoderma against Botrytis cinerea. This work confirmed that the expression of the entomopathogenic fungi-sourced chit42 genes in Trichoderma harzianum enhances the efficiency of Trichoderma biocontrol against targeted pathogens.

Pathogen-produced catalase affects immune priming: A potential pathogen strategy.

Immune priming in invertebrates occurs when the first contact with a pathogen/parasite enhances resistance after a second encounter with the same strain or species. Although the mechanisms are not well understood, there is evidence that priming the immune response of some hosts leads to greater pro-oxidant production. Parasites, in turn, might counteract the host attack with antioxidants. Virulent pathogen strains may therefore mask invertebrate immune priming. For example, different parasite species overexpress catalase as a virulence factor to resist host pro-oxidants, possibly impairing the immune priming response. The aim of this study was firstly to evaluate the specificity of immune priming in Tenebrio molitor when facing homologous and heterologous challenges. Secondly, homologous challenges were carried out with two Metarhizium anisopliae strains (Ma10 and CAT). The more virulent strain (CAT) overexpresses catalase, an antioxidant that perhaps impairs a host immune response mediated by reactive oxygen species (ROS). Indeed, T. molitor larvae exhibited better immune priming (survival) in response to the Ma10 than CAT homologous challenge. Moreover, the administration of paraquat, an ROS-promoting agent, favoured survival of the host upon exposure to each fungal strain. We propose that some pathogens likely overcome pro-oxidant-mediated immune priming defences by producing antioxidants such as catalase.

Duplication of a Pks gene cluster and subsequent functional diversification facilitate environmental adaptation in Metarhizium species.

The ecological importance of the duplication and diversification of gene clusters that synthesize secondary metabolites in fungi remains poorly understood. Here, we demonstrated that the duplication and subsequent diversification of a gene cluster produced two polyketide synthase gene clusters in the cosmopolitan fungal genus Metarhizium. Diversification occurred in the promoter regions and the exon-intron structures of the two Pks paralogs (Pks1 and Pks2). These two Pks genes have distinct expression patterns, with Pks1 highly expressed during conidiation and Pks2 highly expressed during infection. Different upstream signaling pathways were found to regulate the two Pks genes. Pks1 is positively regulated by Hog1-MAPK, Slt2-MAPK and Mr-OPY2, while Pks2 is positively regulated by Fus3-MAPK and negatively regulated by Mr-OPY2. Pks1 and Pks2 have been subjected to positive selection and synthesize different secondary metabolites. PKS1 is involved in synthesis of an anthraquinone derivative, and contributes to conidial pigmentation, which plays an important role in fungal tolerance to UV radiation and extreme temperatures. Disruption of the Pks2 gene delayed formation of infectious structures and increased the time taken to kill insects, indicating that Pks2 contributes to pathogenesis. Thus, the duplication of a Pks gene cluster and its subsequent functional diversification has increased the adaptive flexibility of Metarhizium species.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

The Rab GTPase activating protein Gyp2 contributes to UV stress tolerance in Metarhizium acridum.

GTPase activation protein (GAP) for Rab GTPases can accelerate GTP hydrolysis to alter the activity of Rab GTPases. To explore the function of GAP in entomopathogenic fungi, we constructed a deletion mutant of Gyp2 gene, a member of the Gyp (GAP for Ypt/Rab proteins) family in the locust-specific fungal pathogen, Metarhizium acridum. Results showed that the ∆MaGyp2 mutant had dramatically decreased tolerance to ultraviolet irradiation compared to wild type strain. Quantitative real-time PCR revealed that UV irradiation repair related genes Uve1 and WC1 were downregulated in ∆MaGyp2 mutant. Seven of other ten Gyp family members had significantly increased transcription in ∆MaGyp2 mutant compared with wild type, which may partly rescue the deficiency of MaGyp2.

Lipid biology in fungal stress and virulence: Entomopathogenic fungi.

Broad host range insect pathogenic fungi penetrate through the host cuticle, necessitating an ability to confront and overcome surface lipids and other molecules that often include antimicrobial compounds. In this context, induction of lipid assimilatory pathways by exogenous substrates is crucial for successful infection to occur, and lipid growth substrates can have significant effects on the virulence of fungal infectious propagules, e.g. conidia. The production of lipases is a critical part of the cuticle-degrading repertoire and pathways involved in triglyceride metabolism and phospholipid homeostasis have been shown to contribute to host invasion. Mobilization of endogenous lipid stores via the activities of the caleosin and perilipin lipid storage-turnover proteins, have been linked to diverse processes including formation of penetration structures, e.g. germ tubes and appressoria, spore properties and dispersal, and the ability to respond to lipid growth substrates and virulence. Here, we summarize recent advances in our understanding of lipid assimilation and mobilization pathways in the ability of entomogenous fungi to infect and use host substrates. Host surface and internal lipids can alternatively act as antifungal barriers, inducers of pathogenesis-related pathways, and/or as fungal growth substrates. Lipids and lipid assimilation can be considered as forming a co-evolutionary web between the insect host and entomogenous fungi.