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.

Molecular evolution and transcriptional profile of GH3 and GH20 ß-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae.

Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of ß-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study explored the transcriptional profile and evolution of putative GH20 NAGases (MaNAG1 and MaNAG2) and GH3 NAGases (MaNAG3 and MaNAG4) identified in M. anisopliae. While MaNAG2 orthologs are conserved in several ascomycetes, MaNAG1 clusters only with Aspergilllus sp. and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 were phylogenetically related with bacterial GH3 NAGases. The transcriptional profiles of M. anisopliae NAGase genes were evaluated in seven culture conditions showing no common regulatory patterns, suggesting that these enzymes may have specific roles during the Metarhizium life cycle. Moreover, the expression of MaNAG3 and MaNAG4 regulated by chitinous substrates is the first evidence of the involvement of putative GH3 NAGases in physiological cell processes in entomopathogens, indicating their potential influence on cell differentiation during the M. anisopliae life cycle.

Genome-Wide Analysis of Secondary Metabolite Gene Clusters in Ophiostoma ulmi and Ophiostoma novo-ulmi Reveals a Fujikurin-Like Gene Cluster with a Putative Role in Infection.

The emergence of new microbial pathogens can result in destructive outbreaks, since their hosts have limited resistance and pathogens may be excessively aggressive. Described as the major ecological incident of the twentieth century, Dutch elm disease, caused by ascomycete fungi from the Ophiostoma genus, has caused a significant decline in elm tree populations (Ulmus sp.) in North America and Europe. Genome sequencing of the two main causative agents of Dutch elm disease (Ophiostoma ulmi and Ophiostoma novo-ulmi), along with closely related species with different lifestyles, allows for unique comparisons to be made to identify how pathogens and virulence determinants have emerged. Among several established virulence determinants, secondary metabolites (SMs) have been suggested to play significant roles during phytopathogen infection. Interestingly, the secondary metabolism of Dutch elm pathogens remains almost unexplored, and little is known about how SM biosynthetic genes are organized in these species. To better understand the metabolic potential of O. ulmi and O. novo-ulmi, we performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in these species and assessed their conservation among eight species from the Ophiostomataceae family. Among 19 identified BGCs, a fujikurin-like gene cluster (OpPKS8) was unique to Dutch elm pathogens. Phylogenetic analysis revealed that orthologs for this gene cluster are widespread among phytopathogens and plant-associated fungi, suggesting that OpPKS8 may have been horizontally acquired by the Ophiostoma genus. Moreover, the detailed identification of several BGCs paves the way for future in-depth research and supports the potential impact of secondary metabolism on Ophiostoma genus' lifestyle.

Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae: genome identification and patterns of expression in a cuticle infection model.

BACKGROUND: The described species from the Metarhizium genus are cosmopolitan fungi that infect arthropod hosts. Interestingly, while some species infect a wide range of hosts (host-generalists), other species infect only a few arthropods (host-specialists). This singular evolutionary trait permits unique comparisons to determine how pathogens and virulence determinants emerge. Among the several virulence determinants that have been described, secondary metabolites (SMs) are suggested to play essential roles during fungal infection. Despite progress in the study of pathogen-host relationships, the majority of genes related to SM production in Metarhizium spp. are uncharacterized, and little is known about their genomic organization, expression and regulation. To better understand how infection conditions may affect SM production in Metarhizium anisopliae, we have performed a deep survey and description of SM biosynthetic gene clusters (BGCs) in M. anisopliae, analyzed RNA-seq data from fungi grown on cattle-tick cuticles, evaluated the differential expression of BGCs, and assessed conservation among the Metarhizium genus. Furthermore, our analysis extended to the construction of a phylogeny for the following three BGCs: a tropolone/citrinin-related compound (MaPKS1), a pseurotin-related compound (MaNRPS-PKS2), and a putative helvolic acid (MaTERP1). RESULTS: Among 73 BGCs identified in M. anisopliae, 20 % were up-regulated during initial tick cuticle infection and presumably possess virulence-related roles. These up-regulated BGCs include known clusters, such as destruxin, NG39x and ferricrocin, together with putative helvolic acid and, pseurotin and tropolone/citrinin-related compound clusters as well as uncharacterized clusters. Furthermore, several previously characterized and putative BGCs were silent or down-regulated in initial infection conditions, indicating minor participation over the course of infection. Interestingly, several up-regulated BGCs were not conserved in host-specialist species from the Metarhizium genus, indicating differences in the metabolic strategies employed by generalist and specialist species to overcome and kill their host. These differences in metabolic potential may have been partially shaped by horizontal gene transfer (HGT) events, as our phylogenetic analysis provided evidence that the putative helvolic acid cluster in Metarhizium spp. originated from an HGT event. CONCLUSIONS: Several unknown BGCs are described, and aspects of their organization, regulation and origin are discussed, providing further support for the impact of SM on the Metarhizium genus lifestyle and infection process.

Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins.

BACKGROUND: Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins. RESULTS: We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity. CONCLUSIONS: The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.

Genomic analyses and transcriptional profiles of the glycoside hydrolase family 18 genes of the entomopathogenic fungus Metarhizium anisopliae.

Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of ß-1,4-linkages in chitin. This linear homopolymer of N-acetyl-ß-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-ß-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes' roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus.

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.

Metarhizium anisopliae chitinase CHIT30 is involved in heat-shock stress and contributes to virulence against Dysdercus peruvianus.

Entomopathogenic fungi are able to produce several chitinases, which serve a variety of biological functions, such as fungal cell wall organization and the degradation of exogenous chitin for nutrition or insect infection processes. In this study, we analyzed the contribution of the CHIT30 chitinase from Metarhizium anisopliae in morphogenetic development and virulence as a model of chitinase function. The analysis of chi3 gene expression revealed transcript accumulation in response to heat-shock stress conditions as well as cultivation in medium supplemented with chitin. Null chi3 mutants were constructed to determine the biological role of CHIT30. No substantial differences in the secreted chitinase activity could be detected between the wild type and the Δchi3 mutant. However, both endochitinase and exochitinase activities were diminished in the mutant strain following heat-shock treatment, suggesting that CHIT30 is involved in heat-shock adaptation. Mutants lacking CHIT30 chitinase showed reduced virulence against the cotton stainer bug Dysdercus peruvianus, indicating that the CHIT30 chitinase plays a role in the infection process of M. anisopliae.

Evidence of alternative splicing of the chi2 chitinase gene from Metarhizium anisopliae.

Metarhizium anisopliae is a filamentous fungus used in the biological control of arthropods and produces several chitinases in order to break the host cuticle chitin fibers. Chitinase function during fungal cell development and/or infection processes is also an important aspect when analyzing the life cycle of entomopathogens. The expression profile analysis of the endochitinase chi2 gene acquired by RT-PCR experiments indicated the presence of two different transcripts, suggesting the occurrence of alternative splicing in the chi2 gene. The presence of two transcripts, characterized by the removal or retention of the second 72 bp intron, was further confirmed by DNA sequencing, Northern blot and qRT-PCR. Furthermore, we detected the synthesis of two different proteins from the transcripts by two-dimensional Western blot and mass spectrometry analyses. This is the first reported occurrence of alternative splicing in M. anisopliae.

Endochitinase CHI2 of the biocontrol fungus Metarhizium anisopliae affects its virulence toward the cotton stainer bug Dysdercus peruvianus.

Chitinases have been implicated in fungal cell wall remodeling and play a role in exogenous chitin degradation for nutrition and competition. Due to the diversity of these enzymes, assigning particular functions to each chitinase is still ongoing. The entomopathogenic fungus Metarhizium anisopliae produces several chitinases, and here, we evaluate whether endochitinase CHI2 is involved in the pathogenicity of this fungus. We constructed strains either overexpressing or lacking the CHI2 chitinase. These constructs were validated by Southern, Northern and Western blot analysis, and chitinase production. To access the effects of CHI2 chitinase in virulence, the cotton stainer bug Dysdercus peruvianus was used as a host. CHI2 overexpression constructs showed higher efficiency in host killing suggesting that the production of this chitinase by a constitutive promoter reduces the time necessary to kill the insect. More significantly, the knock out constructs showed decreased virulence to the insects as compared to the wild type strain. The lack of this single CHI2 chitinase diminished fungal infection efficiency, but not any other detectable trait, showing that the M. anisopliae family 18, subgroup B endochitinase CHI2 plays a role in insect infection.

Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae.

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild-type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.