Abstract
In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. ‘Fuji’ leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes—important virulence factors in V. mali—are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.
Highlights
Signal transduction cascades are the primary means by which external stimuli are communicated to the nuclei of eukaryotic organisms
We found that Gvm2 has a role in regulating conidiation and virulence
We found that Gvm2 and Gvm3 play various roles in the modulation of vegetative growth, asexual development, and virulence possibly via the cAMP/protein kinase A (PKA) pathway in this pathogenic fungus
Summary
Signal transduction cascades are the primary means by which external stimuli are communicated to the nuclei of eukaryotic organisms. Heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, and virulence [1]. G-proteins are composed of three subunits: α, β and γ, which remain inactive in the heterotrimeric state with GDP bound to the Gα subunit [2]. Heterotrimeric G-proteins are activated by members of the seven-transmembrane-spanning family of receptors [3]. Binding of signal ligands to such receptors promotes an exchange of GDP to GTP on the Gα subunit, which triggers a conformational change and dissociation from the Gβγ heterodimer [4]. The guanine nucleotide state of the Gα subunit seems to play a crucial role in controlling G-protein signaling [4]
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