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

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Abstract

Metarhizium anisopliae is a model system for studying insect fungal pathogenesis. The role of cAMP signal transduction in virulence was studied by disrupting the class I PKA catalytic subunit gene (MaPKA1). The PKA mutant (ΔMaPKA1) showed reduced growth and greatly reduced virulence. PKA was dispensable for differentiation of infection structures (appressoria), but differentiation was delayed and the appressoria were defective because of reduced turgor pressure. ΔMaPKA1 germinated at similar rates as the wild type in glucose and glycerol, but germination was delayed on alanine. Conidial adhesion and appressorium formation by ΔMaPKA1 against a plastic surface was fully inhibited with glucose as sole nutrient source. Adhesion to plastic was not inhibited with glycerol or alanine, but appressorium formation was delayed. ΔMaPKA1 showed reduced tolerance to the oxidative agent diamide, but not to H2O2 and methyl-viologen. Comparative transcriptome analysis of ΔMaPKA1 and the wild type strain showed that PKA is responsible for up-regulating approximately one-third of the genes induced by insect cuticle, including subsets of those responsible for differentiation of appressoria and penetration pegs, cuticle degradation, nutrient acquisition, pH regulation, lipid synthesis, cell cycle control and the cytoskeleton. PKA was not however required for expression of toxin-producing genes. We conclude therefore that MaPKA1 is required for sensing host-related stimuli and transduction of these signals to regulate many infection processes.

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.

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