Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae

Fang, Weiguo; Pava‐Ripoll, Monica; Wang, Sibao; Leger, Raymond St.

doi:10.1016/j.fgb.2008.12.001

Cited by 111 publications

(112 citation statements)

References 45 publications

(45 reference statements)

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“…Using Agrobacterium tumefaciens-mediated transformation as described previously (Fang et al, 2004) with pFBARGFP (Fang et al, 2009b), we generated a set of 20,328 T-DNA-tagged strain ARSEF2575 mutants. In order to identify mutants putatively compromised in rhizosphere competence, transformants were individually inoculated on agar plates containing soybean (Glycine max) root exudate at 0.03 mg mL…”

Section: Characterization Of the Sugar Transporter Mrt In M Robertsiimentioning

confidence: 99%

“…To construct the Mrt disruption plasmid, the 5#-end and 3#-end of Mrt were cloned by PCR and inserted into the XbaI and SpeI sites, respectively, of the plasmid pFBARGFP (Fang et al, 2009b). The disruption mutant (DMrt) was obtained utilizing Agrobacterium tumefaciens.…”

Section: Gene Cloning and Disruptionmentioning

confidence: 99%

“…Mycelial inoculum from SDB cultures (Fang et al, 2009b) was incubated for 10 h in BS supplemented with different carbohydrates. RNA was isolated using the Plant RNeasy Kit (Qiagen), and qPCR analysis was conducted as described previously (Fang et al, 2009b).…”

Section: Expression Pattern Of Mrtmentioning

confidence: 99%

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Mrt, a Gene Unique to Fungi, Encodes an Oligosaccharide Transporter and Facilitates Rhizosphere Competency in Metarhizium robertsii

2010

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The symbiotic associations between rhizospheric fungi and plants have enormous environmental impact. Fungi are crucial to plant health as antagonists of pathogens and herbivores and facilitate the uptake of soil nutrients. However, little is known about the plant products obtained by fungi in exchange or how they are transported through the symbiotic interface. Here, we demonstrate that sucrose and raffinose family oligosaccharides in root exudates are important for rhizosphere competence in the insect pathogen Metarhizium robertsii (formerly known as Metarhizium anisopliae). We identified mutants in the Metarhizium raffinose transporter (Mrt) gene of M. robertsii that grew poorly in root exudate and were greatly reduced in rhizosphere competence on grass roots. Studies on sugar uptake, including competition assays, revealed that MRT was a sucrose and galactoside transporter. Disrupting MRT resulted in greatly reduced or no growth on sucrose and galactosides but did not affect growth on monosaccharides or oligosaccharides composed entirely of glucose subunits. Consistent with this, expression of Mrt is exclusively up-regulated by galactosides and sucrose. Expressing a green fluorescent protein gene under the control of the Mrt promoter confirmed that MRT was expressed by germlings in the vicinity of grass roots but not in surrounding bulk soil. Disrupting Mrt did not reduce virulence to insects, demonstrating that Mrt is exclusively involved in M. robertsii's interactions with plants. To our knowledge, MRT is the first oligosaccharide transporter identified and characterized in a fungus and is unique to filamentous fungi, but homologous genes in Magnaporthe, Ustilago, Aspergillus, Fusarium, Epichloe, and Penicillium species indicate that oligosaccharide transport is of widespread significance.

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Section: Characterization Of the Sugar Transporter Mrt In M Robertsiimentioning

confidence: 99%

Section: Gene Cloning and Disruptionmentioning

confidence: 99%

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Mrt, a Gene Unique to Fungi, Encodes an Oligosaccharide Transporter and Facilitates Rhizosphere Competency in Metarhizium robertsii

2010

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“…Under certain conditions, germlings can differentiate, producing infection structures known as appressoria that subsequently produce penetration pegs. Penetration of the insect cuticle occurs via a combination of mechanical pressure and cuticle-degrading enzymes, principally proteases and chitinases (4,9). Once inside the insect hemocoel, the penetrating hyphae differentiate into free-floating single cell propagules known as in vivo hyphal bodies that display distinct morphological and biochemical properties (22,41).…”

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confidence: 99%

Requirement of a Mitogen-Activated Protein Kinase for Appressorium Formation and Penetration of Insect Cuticle by the Entomopathogenic Fungus Beauveria bassiana

Zhang

Jiang

et al. 2010

Appl Environ Microbiol

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Beauveria bassiana is an important insect-pathogenic fungus that invades insects by direct penetration of the host cuticle. To delineate the molecular mechanisms involved in fungal infection, a mitogen-activated protein kinase (MAPK) gene, Bbmpk1, which encodes a YERK1 family MAPK was isolated and characterized. Targeted gene disruption of Bbmpk1 resulted in a complete loss of virulence when applied topically to host insects but did not affect growth of the fungus when conidia were injected directly into the hemocoel. Hyphae of the mutant strain growing in the insect hemocoel were unable to penetrate the cuticle growing outwards and consequently failed to sporulate on the cadaver surface. These data suggest that BbMPK1 is essential for penetration of the insect cuticle both from the outside and from the inside-out in order to escape and disperse from the host. Inactivation of BbMPK1 also caused a significant decrease in fungal adhesion to insect cuticles and eliminated their ability to form appressoria. In order to identify downstream genes regulated by BbMPK1, a suppressive subtractive hybridization (SSH) library was generated comparing mutant and wild-type transcripts isolated during appressorium formation. Thirty-one genes screened from the SSH library were determined to be expressed in the wild-type strain but either significantly reduced or not expressed in the mutant. Ten genes showed high or medium similarity to known protein encoding genes, including proteins involved in cell surface hydrophobicity, lipid metabolism, microtubule dynamics, mitochondrial electron transport, chromatin remodeling, transcription, rRNA processing, small nucleolar RNA accumulation, oxidation of aldehydes, translation, and likely other cellular processes.

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“…For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13,14,18,20,29,49,50,51,52,53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle.…”

mentioning

confidence: 99%

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

Jin

Zhang

Fang

et al. 2010

Appl Environ Microbiol

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Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ⌬BbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.Mycoinsecticides are considered promising biological control agents and alternatives or supplements to chemical pesticides (15). However, the dearth of physiological, genetic, and molecular knowledge of entomopathogenic fungi has retarded their widespread application.For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13,14,18,20,29,49,50,51,52,53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle. M. anisopliae and B. bassiana produce hydrophobic spores which contact and adhere to the insect cuticle (12). Once attached, the conidium germinates and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via cuticle-degrading enzymes (11,19,46) as well as mechanical pressure (24, 53). Hyphae proliferate within the hemocoel, emerge from inside the insect, and subsequently conidiate on the cadaver (15). However, much remains to be elucidated regarding the mechanisms of insect fungal pathogenesis.To obtain detailed knowledge of the mechanisms of fungal pathogenesis, a pool of B. bassiana transfer DNA (T-DNA) insertional mutants had been generated through an Agrobacterium-mediated-transformation method (21). A mutant, designated T12, characterized by the presence of more conidia, was isolated, and its flanking sequence was obtained by T-DNA tagging. The flanking fragment contained an open reading frame (ORF), which corresponded to a gene termed JEN1, encoding a transporter of carboxylates (http://www.ncbi.nlm .nih.gov/Blast.cgi). Organic acid transportation is important for the metabolism of almost all cells ...

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Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae

Cited by 111 publications

References 45 publications

Mrt, a Gene Unique to Fungi, Encodes an Oligosaccharide Transporter and Facilitates Rhizosphere Competency in Metarhizium robertsii

Mrt, a Gene Unique to Fungi, Encodes an Oligosaccharide Transporter and Facilitates Rhizosphere Competency in Metarhizium robertsii

Requirement of a Mitogen-Activated Protein Kinase for Appressorium Formation and Penetration of Insect Cuticle by the Entomopathogenic Fungus Beauveria bassiana

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

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