Amino Acid Sensor Kinase Gcn2 Is Required for Conidiation, Secondary Metabolism, and Cell Wall Integrity in the Taxol-Producer Pestalotiopsis microspora

The canonical Gcn2/Cpc1 kinase in fungi coordinates the expression of target genes in response to amino acid starvation. To investigate its possible role in secondary metabolism, we characterized a gcn2 homolog in the taxol-producing fungus Pestalotiopsis microspora. Deletion of the gene led to severe physiological defects under amino acid starvation, suggesting a conserved function of gcn2 in amino acid sensing. The mutant strain Δgcn2 displayed retardation in vegetative growth. It generated dramatically fewer conidia, suggesting a connection between amino acid metabolism and conidiation in this fungus. Importantly, disruption of the gene altered the production of secondary metabolites by HPLC profiling. For instance, under amino acid starvation, the deletion strain Δgcn2 barely produced secondary metabolites including the known natural product pestalotiollide B. Even more, we showed that gcn2 played critical roles in the tolerance to several stress conditions. Δgcn2 exhibited a hypersensitivity to Calcofluor white and Congo red, implying a role of Gcn2 in maintaining the integrity of the cell wall. This study suggests that Gcn2 kinase is an important global regulator in the growth and development of filamentous fungi and will provide knowledge for the manipulation of secondary metabolism in P. microspora

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

Abstract Background The canonical heterotrimeric G protein-cAMP/PKA pathway regulates numerous cellular processes in filamentous fungi. Chaetomium globosum, a saprophytic fungus, is known for producing many secondary metabolites, including cytotoxic chaetoglobosin A (ChA), as well as abundant cellulase and xylanase. Results Here we report on the functional characterization of this signaling pathway in C. globosum. We blocked the pathway by knocking down the putative Gα-encoding gene gna1 (in the pG14 mutant). This led to impaired cellulase production and significantly decreased transcription of the major cellulase and xylanase genes. Almost all the glycohydrolase family genes involved in cellulose degradation were downregulated, including the major cellulase genes, cel7a, cel6a, egl1, and egl2. Importantly, the expression of transcription factors was also found to be regulated by gna1, especially Ace1, Clr1/2 and Hap2/3/5 complex. Additionally, carbon metabolic processes including the starch and sucrose metabolism pathway were substantially diminished, as evidenced by RNA-Seq profiling and quantitative reverse transcription (qRT)-PCR. Interestingly, these defects could be restored by simultaneous knockdown of the pkaR gene encoding the regulatory subunit of cAMP-dependent PKA (in the pGP6 mutant) or supplement of the cAMP analog, 8-Br-cAMP. Moreover, the Gα-cAMP/PKA pathway regulating cellulase production is modulated by environmental signals including carbon sources and light, in which VelB/VeA/LaeA complex and ENVOY probably work as downstream effectors. Conclusion These results revealed, for the first time, the positive role of the heterotrimeric Gα-cAMP/PKA pathway in the regulation of cellulase and xylanase utilization in C. globosum

Distinct Roles of Velvet Complex in the Development, Stress Tolerance, and Secondary Metabolism in Pestalotiopsis microspora, a Taxol Producer

The velvet family proteins have been shown to play critical roles in fungal secondary metabolism and development. However, variations of the roles have been observed in different fungi. We report here the observation on the role of three velvet complex components VeA, VelB, and LaeA in Pestalotiopsis microspora, a formerly reported taxol-producing fungus. Deletion of individual members led to the retardation of vegetative growth and sporulation and pigmentation, suggesting critical roles in these processes. The mutant strain △velB appeared hypersensitive to osmotic stress and the dye Congo red, whereas △veA and △laeA were little affected by the pressures, suggesting only velB was required for the integrity of the cell wall. Importantly, we found that the genes played distinct roles in the biosynthesis of secondary metabolites in P. microspora. For instance, the production of pestalotiollide B, a previously characterized polyketide, required velB and laeA. In contrast, the veA gene appeared to inhibit the pestalotiollide B (PB) role in its biosynthesis. This study suggests that the three components of the velvet complex are important global regulators, but with distinct roles in hyphal growth, asexual production, and secondary metabolism in P. microspora. This work provides information for further understanding the biosynthesis of secondary metabolism in the fungus

The AMP-Activated Protein Kinase Homolog Snf1 Concerts Carbon Utilization, Conidia Production and the Biosynthesis of Secondary Metabolites in the Taxol-Producer Pestalotiopsis microspora

Highly conserved, the Snf1/AMPK is a central regulator of carbon metabolism and energy production in the eukaryotes. However, its function in filamentous fungi has not been well established. In this study, we reported functional characterization of Snf1/AMPK in the growth, development and secondary metabolism in the filamentous fungus Pestalotiopsis microspora. By deletion of the yeast SNF1 homolog, we found that it regulated the utilization of carbon sources, e.g., sucrose, demonstrating a conserved function of this kinase in filamentous fungus. Importantly, several novel functions of SNF1 were unraveled. For instance, the deletion strain displayed remarkable retardation in vegetative growth and pigmentation and produced a diminished number of conidia, even in the presence of the primary carbon source glucose. Deletion of the gene caused damages in the cell wall as shown by its hypersensitivities to Calcofluor white and Congo red, suggesting a critical role of Snf1 in maintaining cell wall integrity. Furthermore, the mutant strain Δsnf1 was hypersensitive to stress, e.g., osmotic pressure (1 M sorbitol), drug G418 and heat shock, though the mechanism remains to be illustrated. Significantly, disruption of the gene altered the production of secondary metabolites. By high-performance liquid chromatography (HPLC) profiling, we found that Δsnf1 barely produced secondary metabolites, e.g., the known product pestalotiollide B. This study suggests that Snf1 is a key regulator in filamentous fungus Pestalotiopsis microspora concerting carbon metabolism and the filamentous growth, conidiation, cell wall integrity, stress tolerance and the biosynthesis of secondary metabolites

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

<div><p>Sensing the environmental signals, the canonical Gα-cAMP/PKA pathway modulates mycelial growth and development, and negatively regulates some secondary metabolism in filamentous fungi, <i>e</i>.<i>g</i>. aflatoxin in <i>Aspergillus nidulans</i>. Here we report the characterization of this signaling pathway in <i>Chaetomium globosum</i>, a widely spread fungus known for synthesizing abundant secondary metabolites, <i>e</i>.<i>g</i>. chaetoglobosin A (ChA). RNAi-mediated knockdown of a putative Gα-encoding gene <i>gna-1</i>, led to plural changes in phenotype, <i>e</i>.<i>g</i>. 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, <i>e</i>.<i>g</i>. <i>pks-1</i> and <i>CgcheA</i>. These defects could be restored by simultaneous knock-down of the <i>pkaR</i> gene encoding a regulatory subunit of cAMP-dependent protein kinase A (PKA), suggesting that <i>pkaR</i> 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 <i>gna-1</i> silenced mutant pG14, and addition of a cAMP analog, 8-Br-cAMP, restored the same defects, <i>e</i>.<i>g</i>., the expression of <i>CgcheA</i>. Furthermore, the intracellular cAMP in <i>gna-1</i> and <i>pkaR</i> double silenced mutant was approaching the normal level. The following activity inhibition experiment proved that the expression of <i>CgcheA</i> was indeed regulated by PKA. Down-regulation of <i>LaeA/VeA/SptJ</i> expression in <i>gna-1</i> 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 <i>C</i>. <i>globosum</i>.</p></div

Expression variation of genes putatively related to ChA biosynthesis detected by RNA-seq profiling.

<p>Expression variation of genes putatively related to ChA biosynthesis detected by RNA-seq profiling.</p

Diminished biosynthesis of ChA.

<p><b>(A)</b> HPLC analysis on the production of ChA in the wild-type and the RNAi mutants. Arrow indicates the peak of ChA. <b>(B)</b> The cAMP assay for the silenced mutants. Relative expression of gene <i>CgcheA</i> from the wild-type (WT) and the transformant pG14 in the presence of 8-Br-cAMP<b>(C)</b> and H-89<b>(D)</b> at indicated concentrations.</p

Construction of RNA interference cassettes.

<p>RNA interference cassettes were constructed based on the pSilent-1 plasmid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195553#pone.0195553.ref025" target="_blank">25</a>]. The inserted fragments were obtained by PCR following restriction enzyme digestion. Primers are indicated in black boxes. IS, inserted fragment; IT, intron 2 of cutinase (CUT) gene from <i>Magnaporthe oryzae</i>; PtrpC, promotor of <i>trpC</i> from <i>A</i>. <i>nidulans</i>; TtrpC, <i>trpC</i> terminator of <i>A</i>. <i>nidulans</i>; <i>Hyg</i>^<i>r</i>, hygromycin resistance; <i>Amp</i>^<i>r</i>, ampicillin resistance. Restriction enzyme sites are also indicated.</p