The PKR regulatory subunit of protein kinase A (PKA) is involved in the regulation of growth, sexual and asexual development, and pathogenesis in Fusarium graminearum

Abstract

Fusarium graminearum is a causal agent of wheat scab disease and a producer of deoxynivalenol (DON) mycotoxins. Treatment with exogenous cyclic adenosine monophosphate (cAMP) increases its DON production. In this study, to better understand the role of the cAMP-protein kinase A (PKA) pathway in F. graminearum, we functionally characterized the PKR gene encoding the regulatory subunit of PKA. Mutants deleted of PKR were viable, but showed severe defects in growth, conidiation and plant infection. The pkr mutant produced compact colonies with shorter aerial hyphae with an increased number of nuclei in hyphal compartments. Mutant conidia were morphologically abnormal and appeared to undergo rapid autophagy-related cell death. The pkr mutant showed blocked perithecium development, but increased DON production. It had a disease index of less than unity and failed to spread to neighbouring spikelets. The mutant was unstable and spontaneous suppressors with a faster growth rate were often produced on older cultures. A total of 67 suppressor strains that grew faster than the original mutant were isolated. Three showed a similar growth rate and colony morphology to the wild-type, but were still defective in conidiation. Sequencing analysis with 18 candidate PKA-related genes in three representative suppressor strains identified mutations only in the CPK1 catalytic subunit gene. Further characterization showed that 10 of the other 64 suppressor strains also had mutations in CPK1. Overall, these results showed that PKR is important for the regulation of hyphal growth, reproduction, pathogenesis and DON production, and mutations in CPK1 are partially suppressive to the deletion of PKR in F. graminearum.

Keywords: DON; autophagy; conidiation; fungal pathogenicity; suppressor.

Figure 1

The pkr mutant was defective in growth and nuclear distribution. (A) Three day‐old potato dextrose agar (PDA) cultures of the wild‐type PH‐1 (WT), pkr mutant P1 (pkr) and pkr/PKR complemented strain (comp). (B) 12‐h germlings of PH‐1 and pkr mutant were examined by epifluorescence microscopy after staining with 4′,6‐diamidino‐2‐phenylindole (DAPI) and calcofluor white (CFW). DIC, differential interference contrast. Bar, 10 µm.

Figure 2

Defects of the pkr mutant in conidiation, cell viability and glycogen accumulation. (A) Conidia of wild‐type PH‐1 (WT), pkr mutant (pkr) and pkr/PKR complemented strain (comp) harvested from 5‐day‐old carboxymethyl cellulose (CMC) cultures. The pkr conidia appeared to be highly vacuolated or empty. Arrows indicate the foot cell. Bar, 10 µm. (B) Five‐day‐old highly vacuolated pkr conidia were stained with 5 μg/mL propidium iodide (PI). Bar, 10 μm. DIC, differential interference contrast. (C) Conidia of PH‐1 and pkr mutant harvested from 3‐day‐old CMC cultures were stained with KI/I₂ solution. Bar, 10 μm.

Figure 3

Assays for the localization of green fluorescent protein (GFP)‐FgAtg8 fusion proteins. (A) Conidia harvested from 3‐day‐old carboxymethyl cellulose (CMC) cultures of transformants of the wild‐type PH‐1 and pkr mutant expressing the GFP‐FgATG8 construct were examined by differential interference contrast (DIC) and epifluorescence microscopy. (B) 24‐h germlings of the same GFP‐FgATG8 transformants of the wild‐type and pkr mutant were examined for the localization of GFP‐FgAtg8 fusion proteins. Bar, 10 µm.

Figure 4

Defects of pkr in sexual reproduction and plant infection. (A) Carrot agar cultures of the wild‐type PH‐1 (WT), pkr mutant (pkr) and complemented pkr/PPKR transformant (comp) were examined at 14 days post‐fertilization (dpf). Fertile perithecia with cirrhi were observed in PH‐1 and the complemented transformant. (B) Flowering wheat heads were drop inoculated with conidia of the same set of strains and photographed at 14 days post‐inoculation (dpi). Black dots mark the inoculated spikelets. (C) Corn silks inoculated with culture blocks were photographed at 6 dpi.

Figure 5

Defects of the pkr mutant in response to exogenous cyclic adenosine monophosphate (cAMP). Deoxynivalenol (DON) production in 5‐day‐old liquid trichothecene biosynthesis (LTB) cultures of wild‐type PH‐1 (WT) and the pkr mutant treated with or without 4 mm cAMP.

Figure 6

Subcellular localization of PKR‐GFP fusion proteins. Conidia (A) and 12‐h germlings (B) of the pkr/PKR‐GFP transformant were examined by differential interference contrast (DIC) and epifluorescence microscopy. Green fluorescent protein (GFP) signals were observed in the nucleus and cytoplasm in conidia and 12‐h germlings. Bar, 10 µm. DAPI, 4′,6‐diamidino‐2‐phenylindole.

Figure 7

Spontaneous suppressors of the pkr mutant. (A) Six‐day‐old potato dextrose agar (PDA) cultures of the pkr mutant with fast‐growing sectors (marked with arrows). (B) Three‐day‐old PDA cultures of four representative spontaneous suppressor strains with different growth rates. WT, wild‐type.

Figure 8

Schematic drawing of mutation sites identified in CPK1. Amino acid changes in CPK1 identified in 12 suppressor strains are labelled above the schematic drawing of Cpk1 or sequence alignments of Cpk1 and its orthologues from Fusarium oxysporum (Fo), Fusarium verticillioides (Fv), Magnaporthe oryzae (Mo), Neurospora crassa (Nc) and Saccharomyces cerevisiae (Sc). The conserved amino acid residues with suppressor mutation in Cpk1 are labelled with blue boxes. The predicted ATP‐binding site and the serine/threonine (S/T) kinase active site are labelled underneath the schematic drawing of Cpk1. I–XI, 11 protein kinase subdomains.