Gα-cAMP/PKA pathway positively regulates pigmentation, chaetoglobosin A biosynthesis and sexual development in Chaetomium globosum

Abstract

Sensing the environmental signals, the canonical Gα-cAMP/PKA pathway modulates mycelial growth and development, and negatively regulates some secondary metabolism in filamentous fungi, e.g. aflatoxin in Aspergillus nidulans. Here we report the characterization of this signaling pathway in Chaetomium globosum, a widely spread fungus known for synthesizing abundant secondary metabolites, e.g. chaetoglobosin A (ChA). RNAi-mediated knockdown of a putative Gα-encoding gene gna-1, led to plural changes in phenotype, e.g. albino mycelium, significant restriction on perithecium development and decreased production of ChA. RNA-seq profiling and qRT-PCR verified significantly fall in expression of corresponding genes, e.g. pks-1 and CgcheA. These defects could be restored by simultaneous knock-down of the pkaR gene encoding a regulatory subunit of cAMP-dependent protein kinase A (PKA), suggesting that pkaR had a negative effect on the above mentioned traits. Confirmatively, the intracellular level of cAMP in wild-type strain was about 3.4-fold to that in gna-1 silenced mutant pG14, and addition of a cAMP analog, 8-Br-cAMP, restored the same defects, e.g., the expression of CgcheA. Furthermore, the intracellular cAMP in gna-1 and pkaR double silenced mutant was approaching the normal level. The following activity inhibition experiment proved that the expression of CgcheA was indeed regulated by PKA. Down-regulation of LaeA/VeA/SptJ expression in gna-1 mutant was also observed, implying that Gα signaling may crosstalk to other regulatory pathways. Taken together, this study proposes that the heterotrimeric Gα protein-cAMP/PKA signaling pathway positively mediates the sexual development, melanin biosynthesis, and secondary metabolism in C. globosum.

Regulation of the Gα-cAMP/PKA signaling pathway in cellulose utilization of Chaetomium globosum

“…DEGs between the wild-type strain and pG14 were selected based on FDR < 0.001 and |log 2 Ratio| ≥ 1. In pG14, among a total of 3326 DEGs, 688 were upregulated and 2638 were downregulated [ 34 ]. The result of the transcriptome analysis done by RNA-seq as a complementary data in our previous work is online (10.1371/journal.pone.0195553.s004).…”

“…One of the targets of the heterotrimeric G protein is the enzyme adenylyl cyclase that converts ATP to cAMP for use as a secondary messenger in filamentous fungi, as in other eukaryotic organisms [ 24 – 27 ]. Previous work revealed that intracellular levels of cAMP were modulated in a Gna1-dependent manner in C. globosum [ 34 ]. The cAMP level in the pG14 mutant was clearly reduced relative to that in the wild-type strain, while pGP6 increased cAMP accumulation nearly to the wild-type level [ 34 ].…”

“…pGP6 was obtained by transforming the protoplasts of C. globosum NK102 with the RNAi cassette pGNA-PKAR, which can simultaneously knockdown gna1 and pkaR . Southern blot results proved that the RNAi cassettes were inserted into all the transformants [ 34 ]. Quantitative reverse transcription (qRT)-PCR results verified that gna1 was knocked down in pG14, and gna1 and pkaR were simultaneously knocked down in pGP6 [ 34 ].…”

“…The culture conditions of the wild-type and mutant strains have been described in previous work [ 34 ]. For DNA or RNA isolation, 5-mm agar plaques containing the fungal hypha inoculated in potato dextrose broth were incubated for 4 or 8 days in a rotary shaker at 28 °C and 180 rpm.…”

“…The results showed that the canonical G protein-cAMP/PKA pathway plays a pivotal role in the production of ChA in C. globosum NK102. We generated a knockdown mutant of the gene gna1 (the pG14 mutant) and a knockdown mutant of both gna1 and pkaR (the pGP6 mutant) [ 34 ]. In this study, we further demonstrated that the transcriptional regulation of cellulase genes involves the G protein-cAMP/PKA pathway.…”