Selvaraj P, Shen Q, Yang F, Naqvi NI

Front Microbiol. 2017 Nov 21;8:2289. doi: 10.3389/fmicb.2017.02289. eCollection 2017.

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.

See: https://www.ncbi.nlm.nih.gov/pubmed/29209297

Figure 1: Cpk2-mediated cAMP-PKA signaling is necessary for proper vegetative and asexual development in M. oryzae (A) Radial and aerial hyphal growth of the wild type (WT) and the indicated PKA-C mutant strains. Mycelial plugs inoculated on PA medium was cultured in the dark at 28°C for 5 days. The left panel shows the comparative radial growth of individual CPK mutants cpkAΔ, cpk2Δ and cpkAΔcpk2Δ with the WT B157. The radial and cross-sectional view for the aerial hyphal growth of WT and the mutants are shown (right). (B) Bright field micrographs showing the conidiation at 48 h and 7 days post photoinduction. Individual cpkAΔ and cpk2Δ produced conidia normally as the WT at 48 h, while cpkAΔcpk2Δ showed very few conidia even at 7 dpi. Scale bar = 10 μm. (C) Bar graphs showing the difference in radial growth (left) and the quantification of conidiation and appressorium formation in WT and PKA-C mutants (right). Values represent mean ± SE of three independent replicates with approximately 200 conidia assessed per experiment; ^∗∗p < 0.001, ^∗∗∗p < 0.0001.