Caracterización funcional de genes del tipo APF en el hongo fitopatógeno de frutos cítricos Penicillium digitatum

[ES] Penicillium digitatum es el principal patógeno postcosecha de frutos cítricos. Es un hongo ascomiceto necrótrofo que penetra a través de heridas preexistentes e infecta el fruto, afectando su calidad comercial, produciendo su podredumbre y causando graves pérdidas económicas a nivel mundial. Los péptidos antimicrobianos (AMPs), tanto naturales como sintéticos, se consideran una alternativa prometedora frente a la utilización de fungicidas químicos para la protección de los cultivos y la conservación postcosecha de productos vegetales. Entre los AMPs naturales se encuentran las denominadas proteínas antifúngicas (AFPs), pequeñas proteínas catiónicas y ricas en cisteína de origen fúngico. Nuestro grupo de laboratorio ha trabajado en la identificación y caracterización de péptidos antifúngicos sintéticos (PAF) para su aplicación en el ámbito de la protección postcosecha de frutos cítricos. La secuencia del genoma de P.digitatum revela la existencia de genes que podrían codificar proteínas tipo AFP. En este trabajo se han elegido tres de ellos (PdigORF_17907, PDIG_23520, y PDIG_31210) para su caracterización funcional a través de la generación de mutantes nulos mediante el mecanismo de transformación génica mediado por A.tumefaciens (ATMT) y recombinación homóloga. Paralelamente se ha determinado la sensibilidad a péptidos antimicrobianos en diferentes cepas mutantes de P.digitatum, y se ha estudiado la sinergia entre el péptido sintético PAF26 y el péptido PAF112 derivado de la secuencia AFP de P.digitatum PDIG_68840. Los resultados revelan la necesidad de la secuencia completa de PAF112 para ejercer una actividad antimicrobiana sinérgica con PAF26.[EN] Penicillium digitatum is the main citrus postharvest pathogen. It is an ascomycete necrotrophic fungus that penetrates orange fruits through pre-existent wounds, affecting their commercial quality, producing fruit rot and causing economic losses worldwide. Natural and synthetic antimicrobial peptides (AMPs) are considered a potential alternative to chemical fungicides for crop protection. Among natural AMPs, we can find small cationic cysteine-rich proteins called antifungal proteins (AFPs) of fungal origin. In our laboratory we have worked in the identification and characterization of synthetic antifungal peptides (PAF) for their application in the protection of citrus fruits. The P.digitatum recently sequenced genome shows existing genes that could probably codify AFP-like proteins. In this work, we have chosen three of these genes for their functional characterization using the A.tumefaciens mediated transformation mechanism (ATMT) and homologous recombination. On the other hand, the sensitivity to antifungal peptides has been determined in different P.digitatum strains, and the synergy between PAF26 and PAF112, derived from the P.digitatum AFP sequence PDIG_68840, has been identified. The results show the need for the complete sequence of PAF112 to establish an antimicrobial synergetic interaction with PAF26.[CA] Penicillium digitatum és el principal patogen postcollita de fruits cítrics. És un fong ascomicet necròtrof que penetra a través de ferides preexistents i infecta el fruit, afectant a la seua qualitat comercial, produint la seua podridura i causant greus pèrdues econòmiques a nivell mundial. Els pèptids antimicrobians (AMPs), tant naturals com sintètics, es consideren una alternativa prometedora enfront la utilització de fungicides químics per a la protecció dels cultius i la conservació postcollita de productes vegetals. Entre els AMPs naturals es troben les denominades proteïnes antifúngiques (AFPs), unes xicotetes proteïnes catiòniques i riques en cisteïna d'origen fúngic. El nostre grup de laboratori ha treballat en la identificació i caracterització de pèptids antifúngics sintètics (PAF) per a la seua aplicació en l'àmbit de la protecció de fruits cítrics. La seqüència del genoma de P.digitatum revela l'existència de gens que podrien codificar proteïnes tipus AFP. En este treball s'han triat tres d'ells (PdigORF_17907, PDIG23520 i PDIG31210) per a la seua caracterització funcional a través de la generació de mutants nuls per mitjà del mecanisme de transformació gènica mediat per A.tumefaciens (ATMT) i recombinació homòloga. Paral•lelament s'ha determinat la sensibilitat a pèptids antimicrobians en diferents ceps mutants de P.digitatum, i s'ha caracteritzat la sinergia entre l'hexapèptid sintètic PAF26 i el pèptid PAF112 derivat de la seqüència AFP de P.digitatum PDIG_68840. Els resultats revelen la necessitat de la seqüència completa de PAF112 per a exercir una activitat inhibitòria sinèrgica amb PAF26.Garrigues Cubells, SM. (2014). Caracterización funcional de genes del tipo APF en el hongo fitopatógeno de frutos cítricos Penicillium digitatum .http://hdl.handle.net/10251/57212.Archivo delegad

Mapping and Identification of Antifungal Peptides in the Putative Antifungal Protein AfpB from the Filamentous Fungus Penicillium digitatum

Antifungal proteins (AFPs) from Ascomycetes are small cysteine-rich proteins that are abundantly secreted and show antifungal activity against non-producer fungi. A gene coding for a class B AFP (AfpB) was previously identified in the genome of the plant pathogen Penicillium digitatum. However, previous attempts to detect the AfpB protein were not successful despite the high expression of the corresponding afpB gene. In this work, the structure of the putative AfpB was modeled. Based on this model, four synthetic cysteine-containing peptides, PAF109, PAF112, PAF118, and PAF119, were designed and their antimicrobial activity was tested and characterized. PAF109 that corresponds to the gamma-core motif present in defensin-like antimicrobial proteins did not show antimicrobial activity. On the contrary, PAF112 and PAF118, which are cationic peptides derived from two surface-exposed loops in AfpB, showed moderate antifungal activity against P. digitatum and other filamentous fungi. It was also confirmed that cyclization through a disulfide bridge prevented peptide degradation. PAF116, which is a peptide analogous to PAF112 but derived from the Penicillium chrysogenum antifungal protein PAF, showed activity against P. digitatum similar to PAF112, but was less active than the native PAF protein. The two AfpB-derived antifungal peptides PAF112 and PAF118 showed positive synergistic interaction when combined against P. digitatum. Furthermore, the synthetic hexapeptide PAF26 previously described in our laboratory also exhibited synergistic interaction with the peptides PAF112, PAF118, and PAF116, as well as with the PAF protein. This study is an important contribution to the mapping of antifungal motifs within the AfpB and other AFPs, and opens up new strategies for the rational design and application of antifungal peptides and proteins

CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass

The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of D-xylose and L-arabinose from wheat bran, and D-galacturonic acid from sugar beet pulp.Peer reviewe

Efficient production and characterization of the novel and highly active antifungal protein AfpB from Penicillium digitatum

Filamentous fungi encode distinct antifungal proteins (AFPs) that offer great potential to develop new antifungals. Fungi are considered immune to their own AFPs as occurs in Penicillium chrysogenum, the producer of the well-known PAF. The Penicillium digitatum genome encodes only one afp gene (afpB), and the corresponding protein (AfpB) belongs to the class B phylogenetic cluster. Previous attempts to detect AfpB were not successful. In this work, immunodetection confirmed the absence of AfpB accumulation in wild type and previous recombinant constitutive P. digitatum strains. Biotechnological production and secretion of AfpB were achieved in P. digitatum with the use of a P. chrysogenum-based expression cassette and in the yeast Pichia pastoris with the α-factor signal peptide. Both strategies allowed proper protein folding, efficient production and single-step purification of AfpB from culture supernatants. AfpB showed antifungal activity higher than the P. chrysogenum PAF against the majority of the fungi tested, especially against Penicillium species and including P. digitatum, which was highly sensitive to the self-AfpB. Spectroscopic data suggest that native folding is not required for activity. AfpB also showed notable ability to withstand protease and thermal degradation and no haemolytic activity, making AfpB a promising candidate for the control of pathogenic fungi

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi

[EN] Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, readyto-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciensmediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA,P7760,Pef1¿, and PafpB constitutive promoters, and PglaA,PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.This work was supported by PROMETEO/2018/066 from "Conselleria d'Educacio" (Generalitat Valenciana, Comunitat Valenciana, Spain), grant PID2021-125858OB-100, and the Severo Ochoa Excellence Program CEX 2021-001189-S funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe." EM-G was the recipient of a predoctoral grant FPU18/02019 funded by MCIN/AEI/10.13039/501100011033 and by "ESF Investing in your future." SG holds a Juan de la Cierva Incorporacion grant (IJC 2020-042749-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR.Moreno-Giménez, E.; Gandía, M.; Sáez, Z.; Manzanares, P.; Yenush, L.; Orzáez Calatayud, DV.; Marcos, JF.... (2023). FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi. Frontiers in Bioengineering and Biotechnology. 11:1-17. https://doi.org/10.3389/fbioe.2023.12228121171

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

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FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology

[EN] Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for Hyg(R) or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3 alpha/pDGB3 Omega destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.This work was funded by grants BIO2015-68790-C2-1-R and BIO2016-78601-R from the "Ministerio de Economia y Competitividad" (MINECO, Spain). SG was recipient of a predoctoral scholarship (FPU13/04584) within the FPU program from "Ministerio de Educacion, Cultura y Deporte" (MECD, Spain). We acknowledge the excellent technical assistance of Tania Campos and the help in the microscopy experiments of Jose M. Coll-Marques (IATA, Valencia, Spain). We also thank Dr. Pilar Moya (Universitat Politecnica de Valencia, Spain) for helpful discussions during the initial stages of this project.Hernanz-Koers, M.; Gandía-Gómez, M.; Garrigues-Cubells, SM.; Manzanares-Mir, PM.; Yenush, L.; Orzáez Calatayud, DV.; Marcos -Lopez, JF. (2018). FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology. Fungal Genetics and Biology. 116:51-61. https://doi.org/10.1016/j.fgb.2018.04.010S516111

Three Antifungal Proteins From Penicillium expansum: Different Patterns of Production and Antifungal Activity

Antifungal proteins of fungal origin (AFPs) are small, secreted, cationic, and cysteine-rich proteins. Filamentous fungi encode a wide repertoire of AFPs belonging to different phylogenetic classes, which offer a great potential to develop new antifungals for the control of pathogenic fungi. The fungus Penicillium expansum is one of the few reported to encode three AFPs each belonging to a different phylogenetic class (A, B, and C). In this work, the production of the putative AFPs from P. expansum was evaluated, but only the representative of class A, PeAfpA, was identified in culture supernatants of the native fungus. The biotechnological production of PeAfpB and PeAfpC was achieved in Penicillium chrysogenum with the P. chrysogenum-based expression cassette, which had been proved to work efficiently for the production of other related AFPs in filamentous fungi. Western blot analyses confirmed that P. expansum only produces PeAfpA naturally, whereas PeAfpB and PeAfpC could not be detected. From the three AFPs from P. expansum, PeAfpA showed the highest antifungal activity against all fungi tested, including plant and human pathogens. P. expansum was also sensitive to its self-AFPs PeAfpA and PeAfpB. PeAfpB showed moderate antifungal activity against filamentous fungi, whereas no activity could be attributed to PeAfpC at the conditions tested. Importantly, none of the PeAFPs showed hemolytic activity. Finally, PeAfpA was demonstrated to efficiently protect against fungal infections caused by Botrytis cinerea in tomato leaves and Penicillium digitatum in oranges. The strong antifungal potency of PeAfpA, together with the lack of cytotoxicity, and significant in vivo protection against phytopathogenic fungi that cause postharvest decay and plant diseases, make PeAfpA a promising alternative compound for application in agriculture, but also in medicine or food preservation

GalR, GalX and AraR co-regulate d-galactose and l-arabinose utilization in Aspergillus nidulans

Filamentous fungi produce a wide variety of enzymes in order to efficiently degrade plant cell wall polysaccharides. The production of these enzymes is controlled by transcriptional regulators, which also control the catabolic pathways that convert the released monosaccharides. Two transcriptional regulators, GalX and GalR, control d-galactose utilization in the model filamentous fungus Aspergillus nidulans, while the arabinanolytic regulator AraR regulates l-arabinose catabolism. d-Galactose and l-arabinose are commonly found together in polysaccharides, such as arabinogalactan, xylan and rhamnogalacturonan I. Therefore, the catabolic pathways that convert d-galactose and l-arabinose are often also likely to be active simultaneously. In this study, we investigated the interaction between GalX, GalR and AraR in d-galactose and l-arabinose catabolism. For this, we generated single, double and triple mutants of the three regulators, and analysed their growth and enzyme and gene expression profiles. Our results clearly demonstrated that GalX, GalR and AraR co-regulate d-galactose catabolism in A. nidulans. GalX has a prominent role on the regulation of genes of d-galactose oxido-reductive pathway, while AraR can compensate for the absence of GalR and/or GalX.Peer reviewe