Abstract
The cAMP-Protein Kinase A signaling, anchored on CpkA, is necessary for appressorium development and host penetration, but indispensable for infectious growth in Magnaporthe oryzae. In this study, we identified and characterized the gene encoding the second catalytic subunit, CPK2, whose expression was found to be lower compared to CPKA at various stages of pathogenic growth in M. oryzae. Deletion of CPK2 caused no alterations in vegetative growth, conidiation, appressorium formation, or pathogenicity. Surprisingly, the cpkAΔcpk2Δ double deletion strain displayed significant reduction in growth rate and conidiation compared to the single deletion mutants. Interestingly, loss of CPKA and CPK2 resulted in morphogenetic defects in germ tubes (with curled/wavy and serpentine growth pattern) on hydrophobic surfaces, and a complete failure to produce appressoria therein, thus suggesting an important role for CPK2-mediated cAMP-PKA in surface sensing and response pathway. CPKA promoter-driven expression of CPK2 partially suppressed the defects in host penetration and pathogenicity in the cpkAΔ. Such ectopic CPK2 expressing strain successfully penetrated the rice leaves, but was unable to produce proper secondary invasive hyphae, thus underscoring the importance of CpkA in growth and differentiation in planta. The Cpk2-GFP localized to the nuclei and cytoplasmic vesicles in conidia and germ tubes. The Cpk2-GFP colocalized with CpkA-mCherry on vesicles in the cytosol, but such overlap was not evident in the nuclei. Our studies indicate that CpkA and Cpk2 share overlapping functions, but also play distinct roles during pathogenesis-associated signaling and morphogenesis in the rice blast fungus.
Highlights
The Protein kinase A (PKA) family of Ser/Thr kinases is highly conserved in eukaryotes, and serves important phosphorylation-dependent functions in signal transduction and development (Hanks and Hunter, 1995)
Analysis of the genome sequence of M. oryzae3 revealed an open reading frame (ORF) that encodes a catalytic subunit of PKA, CPK2 (MGG_02832; contig 6–2325: coordinates 2566–1350), which was distinct from the CPKA locus
Our study provides a detailed elucidation of the function, dynamics and organization of the individual signaling components, CpkA and Cpk2 of the cAMP-PKA signaling in M. oryzae
Summary
The Protein kinase A (PKA) family of Ser/Thr kinases is highly conserved in eukaryotes, and serves important phosphorylation-dependent functions in signal transduction and development (Hanks and Hunter, 1995). The PKA holoenzyme is an inactive heterotetramer composed of two regulatory (R) and two catalytic (C) subunits and the cooperative binding of two cAMP molecules to the R subunit frees and activates the C subunits to phosphorylate hundreds of targets and regulate a vast swath of biochemical and metabolic processes. The catalytic subunit of PKA (PKA-C) is a typical structure for protein kinases and PKA signaling plays a central role in vegetative growth, development, mating, stress response, and Functional Analyses of Cpk in M. oryzae pathogenicity in various fungi (Lengeler et al, 2000; D’Souza and Heitman, 2001). In Candida albicans, the catalytic isoforms Tpk1p and Tpk2p share positive roles in cell growth, while they have distinct roles in hyphal morphogenesis, stress response and regulation of glycogen metabolism (Sonneborn et al, 2000; Bockmühl et al, 2001; Cloutier et al, 2003). Only one PKA isoform plays a predominant role in other phytopathogens like Colletotrichum trifolii, C. lagenarium, Botrytis cinerea and Setosphaeria turcica that utilize appressoria to penetrate and infect the host; or in Mycosphaerella graminicola and Verticillium dahlia, that invade the host through stomata or other natural openings (Yang and Dickman, 1999; Takano et al, 2001; Yamauchi et al, 2004; Mehrabi and Kema, 2006; Schumacher et al, 2008; Tzima et al, 2010; Hao et al, 2015)
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