The <i>paf</i> gene product modulates asexual development in <i>Penicillium chrysogenum</i>

Hegedűs, Nikoletta; Sigl, Claudia; Zadra, Ivo; Pócsi, István; Marx, Florentine

doi:10.1002/jobm.201000321

Cited by 40 publications

(50 citation statements)

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“…The production of PAF[Pc], PAF F31N , PAF Y48Q and NFAP[Pc] required the use of the P. chrysogenum ∆ paf mutant as cell factory, where the paf coding sequence was deleted by replacement with the nourseothricin ( nat1 ) resistance gene [29]. …”

Section: Resultsmentioning

confidence: 99%

“…S3). In all P. chrysogenum transformants the nat1 resistant gene was still present that originated from the deletion of the paf gene in the P. chrysogenum Δ paf strain [29] (Additional file 1: Fig. S3).…”

Section: Resultsmentioning

confidence: 99%

“…Fungal strains used in this study are listed in Table 3. For transformation , P. chrysogenum ∆paf [29] shaking cultures were grown in complete medium (CM; Additional file 1: Table S1) for 36 h at 25 °C. Conidia were harvested with spore buffer (0.9% NaCl (w/v), 0.01% Tween 80 (v/v)).…”

Section: Methodsmentioning

confidence: 99%

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A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

et al. 2016

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BackgroundSmall, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.ResultsThe APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ∆paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays.ConclusionThis study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0586-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

et al. 2016

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“…Tsitsigiannis and Keller (24) proposed that oxylipins could bind to and sensitize self G protein-coupled receptors, activating downstream signaling cascades. Furthermore, oxylipins regulate conidiation of A. nidulans and Penicillium chrysogenum through the transcription factor Bristle A (BrlA) (28), leading us to suggest that, as in plants and animals, oxylipins might play an important role in the response to injury of T. atroviride through the formation of lipoperoxides along the injured sites and oxylipins could serve as signaling molecules (Fig. S4).…”

Section: Discussionmentioning

confidence: 99%

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

Hernández‐Oñate¹,

Esquivel-Naranjo²,

Mendoza‐Mendoza

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

101

139

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A conserved injury-defense mechanism is present in plants and animals, in which the production of reactive oxygen species (ROS) and lipid metabolism are essential to the response. Here, we describe that in the filamentous fungus Trichoderma atroviride, injury results in the formation of asexual reproduction structures restricted to regenerating cells. High-throughput RNA-seq analyses of the response to injury in T. atroviride suggested an oxidative response and activation of calcium-signaling pathways, as well as the participation of lipid metabolism, in this phenomenon. Gene-replacement experiments demonstrated that injury triggers NADPH oxidase (Nox)-dependent ROS production and that Nox1 and NoxR are essential for asexual development in response to damage. We further provide evidence of H 2 O 2 and oxylipin production that, as in plants and animals, may act as signal molecules in response to injury in fungi, suggesting that the three kingdoms share a conserved defense-response mechanism.conidiation | stress | transcriptome

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“…Interestingly, transcriptome analysis of a T. atroviride ΔnoxR mutant, which fails to develop conidiophores in response to injury, showed that all genes related to oxylipin biosynthesis were either repressed or not responsive to injury, suggesting that oxylipin biosynthesis is compromised in a ΔnoxR mutant background. In this regard, as mentioned above, oxylipins are known to regulate the conidiation of A. nidulans and Penicillium chrysogenum (433,434) and might serve as signaling molecules in the response of T. atroviride to injury.…”

Section: Injury Induces Developmentmentioning

confidence: 97%

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures

Riquelme

Aguirre

Bartnicki-García

et al. 2018

Microbiol Mol Biol Rev

288

246

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SUMMARYFilamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established.

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The paf gene product modulates asexual development in Penicillium chrysogenum

Cited by 40 publications

References 57 publications

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures

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