Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae
Introduction
Biological control agents, such as insect pathogenic fungi, offer an environmentally friendly alternative to chemical pesticides. However, their use has been limited by poor efficacy (St. Leger et al., 1996). Detailed knowledge of the mechanisms of fungal pathogenesis is needed for mycoinsecticide improvement. The ascomycete Metarhizium anisopliae has been used as model to study insect fungal pathogenesis. Its conidia adhere to the insect cuticle, germinate and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via a combination of mechanical pressure and cuticle degrading enzymes, principally proteases and chitinases. Hyphae proliferate as a yeast like phase (blastospores) within the insect which is killed by a combination of fungal growth and toxins. Hyphae then emerge from and conidiate on the cadaver. Several key genes involved in these processes have been identified including an adhesin (MAD1) and hydrophobins that are responsible for adherence to the cuticle (Wang and St. Leger, 2007a, St. Leger et al., 1992). The cuticle degrading enzymes and their genes have also been characterized (Bagga et al., 2004). A regulator of the G protein signaling pathway is involved in conidiation and hydrophobin synthesis (Fang et al., 2007). An osmosensor signals to penetrant hyphae that they have reached the hemocoel (Wang et al., 2008) and a perilipin (the first characterized in fungi) regulates the turgor pressure of infection structures (Wang and St. Leger, 2007b). The production of MCL1 is required for evading insect immune responses. It contains a collagen domain, and is so far unique to Metarhizium (Wang and St. Leger, 2006).
We have not yet identified the signal transduction pathways that are the master regulators of these virulence determinants. However, in previous studies we found that specific inhibitors of protein kinase A (PKA) delayed both appressorium formation and expression of cuticle degrading enzymes by M. anisopliae (St. Leger et al., 1990). Sensing of environmental stimuli and transduction of the corresponding signal via the cAMP–PKA signal pathway plays an essential role in the virulence of a variety of human and plant pathogenic fungi. PKA is required for the production of functional appressoria and pathogenicity in the rice blast pathogen Magnaporthe grisea (Mitchell and Dean, 1995). The disruption of PKA reduced capsule size and attenuated virulence in the human pathogen Cryptococcus neoformans (D’Souza et al., 2001). A PKA deficient mutant of Ustilago maydis has a constitutively filamentous phenotype and is nonpathogenic (Gold et al., 1994, Larraya et al., 2005). Using microarray analysis, SAGE (serial analysis of gene expression) and proteomic approaches to analyze mutants of the cAMP cascade has shown that cAMP–PKA signaling regulates various genes involved in cell wall synthesis, translation, transport functions, the tricarboxylic acid cycle and glycolysis in C. neoformans (Hu et al., 2007), Candida albicans (Harcus et al., 2004) and Aspergillus fumigatus (Grosse et al., 2008).
In this study, a PKA catalytic subunit gene Mapka1 (Metarhizium anisopliae protein kinase A 1) was cloned and disrupted in M. anisopliae. The mutant had impaired appressorium development and was almost avirulent. Transcriptomes of WT and the mutant were profiled by microarray analysis to identify genes regulated by PKA. We identified 244 down-regulated genes and one up-regulated gene (Pr1D) in ΔMaPKA1, and found that links between PKA, components of the translation machinery, transport, stress response and metabolic functions were conserved in M. anisopliae. However, we also found that down-regulated genes included those involved in appressorium and penetration peg formation, cuticle degradation and pH regulation (Fig. 5). M. anisopliae also differed from other fungi in that sterol metabolism, rather than phospholipid synthesis was controlled by PKA.
Section snippets
Gene disruption
We constructed a master Ti vector, pFBARGFP, for gene disruption in M. anisopliae. The Hygromycin resistance gene cassette in pPk2 (Covert et al., 2001) was replaced by the herbicide resistance bar gene cassette from pBARGPE1 (Pall and Bruhelli, 1993) to form pBAR. The bar cassette was inserted into EcoR I and Xba I sites. The egfp cassette was excised from SK–GFP (Fang et al., 2006) by digestion with EcoRI and SpeI and inserted into the corresponding sites in pBAR to form pFBARGFP. Using this
Gene cloning and characterization
A full length PKA catalytic subunit gene MaPKA1 and its upstream and downstream regulatory sequences were cloned. The ORF (open reading frame), which is interrupted by four introns, is 1569 bp long and predicts a protein with 522 amino acid residues. MaPKA1 has a typical catalytic domain (S_TKc) of Serine/Threonine protein kinases. MaPKA1 showed 59.1%, 56.9%, 60% and 66% amino acid identity to the PkaA from Aspergillus nidulans (Fillinger et al., 2002), adr1 from Ustilago maydis (U23730) (
Discussion
PKA is a central enzyme of cAMP signaling. Previously, we used exogenously applied cAMP and PKA inhibitors to implicate cAMP–PKA signaling in the differentiation of appressoria by germinating conidia (St. Leger et al., 1990). In this study, we further characterized the role of the cAMP–PKA pathway by disruption of a catalytic subunit of PKA. ΔMaPKA1 is almost avirulent and this presumably results from the pleiotropic effect of the many growth functions controlled by PKA in its role as a master
Acknowledgment
This work was supported by NSF Grant MCB-0542904.
References (42)
- et al.
Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae
Gene
(2004) - et al.
Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum
Mycol. Res.
(2001) - et al.
Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR
Mycol. Res.
(2006) - et al.
Cuticle-degrading enzymes of entomopathogenic fungi: cuticle degradation in vitro
J. Invertebr. Pathol.
(1986) - et al.
The effect of Melanization of Manduca Sexta cuticle on growth and infection by Metarhizium anisopliae
J. Invertebr. Pathol.
(1988) - et al.
Cloning and regulatory analysis of starvation–stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Metarhizium anisopliae
Gene
(1992) - et al.
Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase
Cell
(1987) - et al.
Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae
FEMS. Yeast Res.
(2001) - et al.
Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation
Fungal. Genet. Biol.
(2005) - et al.
The Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure, and virulence
J. Biol. Chem.
(2007)
Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea
The Plant Cell
PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea
Proc. Natl. Acad. Sci. USA
Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans
Mol. Cell Biol.
Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis
Proc. Natl. Acad. Sci. USA
Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens
Can. J. Microbiol.
A regulator of a G protein signalling (RGS) gene, cag8, from the insect-pathogenic fungus Metarhizium anisopliae is involved in conidiation, virulence and hydrophobin synthesis
Microbiology
Implication of a regulator of G protein signalling (BbRGS1) in conidiation and conidial thermotolerance of the insect pathogenic fungus Beauveria bassiana
FEMS. Microbiol. Lett.
cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans
Mol. Microbiol.
Variation in gene expression patterns as the insect pathogen Metarhizium anisopliae adapts to different host cuticles or nutrient deprivation in vitro
Microbiology
Protein kinase A regulates growth, sporulation, and pigment formation in Aspergillus fumigatus
Appl. Environ. Microbiol.
Culture age, temperature, and pH affect the polyol and trehalose contents of fungal propagules
Appl. Environ. Microbiol.
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