Subcellular compartmentation, interdependency and dynamics of the cyclic AMP‐dependent PKA subunits during pathogenic differentiation in rice blast

Abstract

The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast.

Cpk2, a Catalytic Subunit of Cyclic AMP-PKA, Regulates Growth and Pathogenesis in Rice Blast

“…A major challenge in deciphering the overall cAMP-PKA signaling is to fully understand the contribution(s) of both the catalytic subunits, CpkA and Cpk2, in M. oryzae . Previous studies revealed the importance of CPKA in appressorium formation and pathogenicity in M. oryzae ( Mitchell and Dean, 1995 ; Xu et al, 1997 ; Selvaraj et al, 2017 ). 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 .…”

“…To get cpk AΔ cpk 2Δ, the cpk 2Δ construct was introduced into cpk AΔ strain. The CPK2 -GFP in pFGL820 (encoding sulfonyl urea resistance gene cassette) was constructed by sequential cloning of the eGFP ORF, the last 1kb and the downstream fragment of CPK2 ORF to yield the final construct pCPK2-GFP-Trpc construct ( Selvaraj et al, 2017 ). The GFP-CPK2 overexpression construct was created by fusing the Moh3 promoter with the cpk2 ORF, sequentially cloned in to pFGL1010G which encodes sulfonyl urea resistance with an ilV locus which facilitate ectopic single copy integration ( Yang and Naqvi, 2014 ).…”

“…For plant infection assays, freshly harvested conidia at a concentration of 1 × 10 6 /ml in 0.2% gelatin were used. Plant inoculation, incubation and lesion examination were conducted as mentioned previously ( Ramanujam and Naqvi, 2010 ; Bosch et al, 2012 ; Selvaraj et al, 2017 ). Rice leaf sheath infection assay was performed as described ( Kankanala et al, 2007 ).…”

“…PKA assay was performed using a non-radioactive cAMP-dependent protein kinase assay system fluorescent using the PKA model substrate, Kemptide (Promega, Madison, WI, United States), following the manufacturer’s instructions and the sample preparation, determination of protein concentration were carried out were prepared as mentioned previously ( Kang et al, 1999 ; Selvaraj et al, 2017 ). The samples for cAMP estimation were prepared as described previously ( Liu et al, 2007 ; Ramanujam and Naqvi, 2010 ), and the assays were carried out using the cAMP Biotrak Immuno-assay system (Amersham Biosciences, Piscataway, NJ, United States) according to the manufacturer’s protocol.…”

“…Mutants disrupted in the catalytic subunit gene CPKA exhibit normal growth and conidiation, but show delayed appressorium formation and loss of pathogenicity, which results from the defects in appressorial function ( Mitchell and Dean, 1995 ; Xu et al, 1997 ). We showed that loss of the regulatory subunit of PKA ( RPKA ) results in complete loss of pathogenicity; and a suppressor mutant that partially restores the pathogenicity in rpk AΔ represents a point mutation in the CPKA locus ( Selvaraj et al, 2017 ). Recently, the second catalytic subunit of PKA, Cpk2, has been characterized in M. oryzae ( Li Y. et al, 2017 ).…”