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

A genomic approach highlights common and diverse effects and determinants of susceptibility on the yeast Saccharomyces cerevisiae exposed to distinct antimicrobial peptides

<p>Abstract</p> <p>Background</p> <p>The mechanism of action of antimicrobial peptides (AMP) was initially correlated with peptide membrane permeation properties. However, recent evidences indicate that action of a number of AMP is more complex and involves specific interactions at cell envelopes or with intracellular targets. In this study, a genomic approach was undertaken on the model yeast <it>Saccharomyces cerevisiae </it>to characterize the antifungal effect of two unrelated AMP.</p> <p>Results</p> <p>Two differentiated peptides were used: the synthetic cell-penetrating PAF26 and the natural cytolytic melittin. Transcriptomic analyses demonstrated distinctive gene expression changes for each peptide. Quantitative RT-PCR confirmed differential expression of selected genes. Gene Ontology (GO) annotation of differential gene lists showed that the unique significant terms shared by treatment with both peptides were related to the cell wall (CW). Assays with mutants lacking CW-related genes including those of MAPK signaling pathways revealed genes having influence on sensitivity to peptides. Fluorescence microscopy and flow cytometry demonstrated PAF26 interaction with cells and internalization that correlated with cell killing in sensitive CW-defective mutants such as Δ<it>ecm33 </it>or Δ<it>ssd1</it>. GO annotation also showed differential responses between peptides, which included ribosomal biogenesis, <it>ARG </it>genes from the metabolism of amino groups (specifically induced by PAF26), or the reaction to unfolded protein stress. Susceptibility of deletion mutants confirmed the involvement of these processes. Specifically, mutants lacking <it>ARG </it>genes from the metabolism of arginine pathway were markedly more resistant to PAF26 and had a functional CW. In the deletant in the arginosuccinate synthetase (<it>ARG1</it>) gene, PAF26 interaction occurred normally, thus uncoupling peptide interaction from cell killing. The previously described involvement of the glycosphingolipid gene <it>IPT1 </it>was extended to the peptides studied here.</p> <p>Conclusions</p> <p>Reinforcement of CW is a general response common after exposure to distinct AMP, and likely contributes to shield cells from peptide interaction. However, a weakened CW is not necessarily indicative of a higher sensitivity to AMP. Additional processes modulate susceptibility to specific peptides, exemplified in the involvement of the metabolism of amino groups in the case of PAF26. The relevance of the response to unfolded protein stress or the sphingolipid biosynthesis, previously reported for other unrelated AMP, was also independently confirmed.</p

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

Promotion of research motivation in Sustainable Food in undergraduate students

[EN] Research in Food Sciences is becoming more relevant every day. The world is facing unprecedented global problems affecting food sustainability and the link between food and health is scientifically proven. University degrees enable professional practice in various areas, but these are not equally known or contemplated by students. Bringing research into Sustainable Food to the classroom is the best way to inform and motivate people to choose this professional area. It is based on the hypothesis that the students of the Degree in Food Science and Technology of the University of Valencia do not know in depth the scientific career related to the area nor are they motivated to choose this professional option. The general objective is to verify this hypothesis through selfadministered questionnaires on knowledge and motivations in relation to research in this area to students of 2nd and 4th courses of this Degree. On the other hand, it is intended to provide training to 2nd-year students based on the experiences of researchers from universities, research centers, technology centers and companies, and to assess whether after such training there has been an improvement in knowledge and motivation towards a scientific career.[ES] La investigación en Ciencias de la Alimentación tiene cada día mayor relevancia. El mundo se enfrenta a problemas globales sin precedentes que afectan a la sostenibilidad alimentaria y el nexo entre alimentación y salud está científicamente demostrado. Los Grados Universitarios habilitan para la práctica profesional en diversas áreas, pero estas no son igualmente conocidas ni contempladas por los estudiantes. Acercar la investigación en Alimentación Sostenible a las aulas es la mejor manera de informar y motivar para la elección de esta área profesional. Se parte de la hipótesis de que los estudiantes del Grado en Ciencia y Tecnología de Alimentos de la Universitat de València no conocen en profundidad la carrera científica relacionada con el área ni están motivados para elegir esta opción profesional. El objetivo general es comprobar esta hipótesis mediante cuestionarios autoadministrados sobre conocimientos y motivaciones en relación a la investigación en esta área a estudiantes de 2º y 4º cursos de este Grado. Por otra parte, se pretende impartir una formación al alumnado de 2º basada en experiencias de investigadores de universidades, centros de investigación, centros tecnológicos y empresas, y evaluar si después de dicha formación se ha mejorado en conocimientos y motivación hacia la carrera científica.Gandía, M.; Roig, P.; Martínez-Culebras, P.; Gil, J.; Gamero, A. (2022). Fomento de la motivación investigadora en Alimentación Sostenible en estudiantes de Grado. Editorial Universitat Politècnica de València. 571-578. https://doi.org/10.4995/INRED2022.2022.1585057157

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

Collaborative implementation of the Sustainable Development Goals in Food Science

[EN] In 2015, the United Nations approved the 2030 Agenda for Sustainable Development in which the 17 Sustainable Development Goals (SDGs) were proposed as a renewal and update of the Millennium Development Goals. The University must actively contribute to the fulfilment of the SDGs, providing students with the knowledge, tools and motivation to get involved. For this reason, a teaching innovation project (UV-SFPIE_PID-1641017) was proposed that aims to integrate the SDGs in the framework of different subjects of different courses of the degrees in Food Sciences of the University of Valencia (UVEG). The aim is to motivate students to use new ICT technologies to develop teaching materials that allow this integration. With the proposed activities, the students should try to establish relationships between sustainability and the teaching syllabus, in order to enhance skills, critical thinking, creativity, curiosity, teamwork and problem solving. The results obtained indicated an increase in knowledge related to sustainability, greater student involvement in the proposed activities and greater motivation. The response and opinion of the students was positive, pointing out the importance of the proposal and the need to extend this initiative to other subjects.[ES] Las Naciones Unidas aprobaron en 2015 la Agenda 2030 para el desarrollo sostenible en la que se propusieron los 17 Objetivos de Desarrollo Sostenible (ODS) como renovación y actualización de los Objetivos de Desarrollo del Milenio.La Universidad debe contribuir activamente al cumplimiento de los ODS, dotando al alumnado de los conocimientos, herramientas y motivaciones para que se involucre.Por ello se planteó un proyecto de innovación docente (UV-SFPIE_PID-1641017) que persigue encajar los ODS en el marco de varias asignaturas de diferentes cursos de los grados en Ciencias de la Alimentación de la Universitat de València (UVEG). El objetivo es motivar a los estudiantes a que, mediante las nuevas tecnologías TIC, elaboren material didáctico que permita esa integración. Con las actividades propuestas se busca que los alumnos establezcan relaciones entre la sostenibilidad y el temario docente, con el fin de potenciar habilidades, pensamiento crítico, creatividad, curiosidad, trabajo en equipo y resolución de problemas.Los resultados obtenidos señalaron un aumento de los conocimientos en sostenibilidad, mayor implicación del alumnado en las actividades propuestas y mayor motivación. La respuesta y opinión de los estudiantes fue positiva, señalando la importancia de la propuesta y la necesidad de extenderla a más asignaturas.Gamero, A.; Martínez-Culebras, P.; Soler, C.; Gil, J.; Roig, P.; Gandía, M. (2022). Implementación colaborativa de los Objetivos de Desarrollo Sostenible en Ciencias de la alimentación. Editorial Universitat Politècnica de València. 587-598. https://doi.org/10.4995/INRED2022.2022.1586358759

Interiorización del método científico en alimentación sostenible mediante la experimentación en el aula

[ES] La alimentación sostenible hace referencia a aquella alimentación saludable que se adapta al entorno y la cultura, que disminuye el impacto ambiental, respeta los recursos naturales y la biodiversidad y es económicamente accesible. El alumnado que cursa alguno de los grados de Ciencias de la Alimentación debe ser consciente del concepto de sostenibilidad, integrarlo en su vida diaria, así como saber transmitirlo a la sociedad. Este alumnado busca adquirir conocimientos a través de metodologías diferentes y complementarias. La combinación de prácticas tradicionales y modernas son vitales para promover diferentes habilidades cognitivas en el estudiantado. Es importante aumentar su conocimiento y sus competencias en la resolución de problemas pero también lo es incrementar sus habilidades de razonamiento. Teniendo en cuenta que los y las estudiantes en Ciencias de la Alimentación deben trabajar desde la aplicación del método científico, una de las maneras más adecuadas de interiorizar este es llevándolo a la práctica. Por ello, en este proyecto se pretende que el alumnado sea capaz de plantear una hipótesis de trabajo en alimentación sostenible en consonancia con los ODS relacionados y llevar a cabo un taller práctico que potencie sus competencias comunicativas en el ámbito universitario.[EN] Sustainable food refers to healthy food that is adapted to the environment and culture, that reduces environmental impact, respects natural resources and biodiversity and is economically accessible. Students taking a degree in Food Science must be aware of the concept of sustainability, integrate it into their daily lives and know how to transmit it to society. These students want to acquire knowledge through different and complementary methodologies. The combination of traditional and modern practices are vital to promote different cognitive skills in students. It is important to increase their knowledge and problem solving competences but it is also important to increase their reasoning skills. Taking into account that students in Food Science must work from the application of the scientific method, one of the most appropriate ways to learn this method is by putting it into practice. Therefore, the aim of this project is to ensure that students are able to propose a working hypothesis on sustainable food in line with the related SDGs and to carry out a practical workshop that enhances their communicative skills in the university environment.Gandía Gómez, M.; Rodríguez-Carrasco, Y.; Cabrera-Pastor, A.; Pardo, E.; Gamero, A. (2023). Interiorización del método científico en alimentación sostenible mediante la experimentación en el aula. Editorial Universitat Politècnica de València. 581-589. https://doi.org/10.4995/INRED2023.2023.1653758158

The impact of childhood RSV infection on children’s and parents’ quality of life: a prospective multicenter study in Spain

Background: Several immunisation candidates against RSV are in late-stage clinical trials. To evaluate the benefts of a potential vaccination programme, both economic and health benefts will be needed. Health benefts are usually measured in Health-related Quality of Life (HRQoL) loss using standardised questionnaires. However, there are no RSV-specifc questionnaires validated for children under 2 years, in whom most RSV episodes occur. Therefore, HRQoL estimates are taken from literature or inadequate tools. We determined HRQoL loss and direct costs due to an RSV episode in children younger than 2 years and their caregivers during a month of follow up, using a new questionnaire administered online. Methods: An observational prospective multicentre surveillance study was conducted in children aged younger than two years. Children were recruited from 8 primary care centres and 1 hospital in the Valencia region and Cata‑ lonia (Spain). RSV-positive cases were obtained by immunochromatographic test. HRQoL was assessed using a new ad-hoc 38 item-questionnaire developed. Parents of infected children completed 4 questionnaires at four timepoints (day 0, 7, 14 and 30) after diagnosis. Results: 117 children were enrolled in the study and 86 (73.5%) were RSV+. Median (interquartile range; IQR) scores were 0.52 (0.42–0.68), 0.65 (0.49–0.79), 0.82 (0.68–0.97) and 0.94 (0.81–1), for days 0, 7, 14 and 30, respectively. Compared to total recovery (Q30), HRQoL loss was 37.5%, 31.5% and 8.9% on days 0, 7 and 14 since diagnosis of the disease. The total median cost per patient (including treatments) was €598.8 (IQR: 359.63–2425.85). Conclusions: RSV had almost 40% impact on HRQoL during the frst week since onset of symptoms and the median cost per episode and patient was about €600. These results represent a substantial input for health-economic evalua‑ tions of future RSV-related interventions such as vaccination.This study was funded by Conselleria d’Educació, Cultura i Esport of The Valencia Region of Spain.Medicin

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

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond

This is the peer reviewed version of the following article: Vazquez-Vilar, M., Gandía, M., García-Carpintero, V., Marqués, E., Sarrion-Perdigones, A., Yenush, L., Polaina, J., Manzanares, P., Marcos, J. F., & Orzaez, D. (2020). Multigene engineering by goldenbraid cloning: from plants to filamentous fungi and beyond. Current Protocols in Molecular Biology, 130, e116, doi: 10.1002/cpmb.116, which has been published in final form at https://doi.org/10.1002/cpmb.116. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate¿based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host¿specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi¿a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal¿specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium¿mediated transformation. We also describe the use of a set of web¿based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms.This article is dedicated to the memory of our friend and colleague Dr. Alejandro Sarrion-Perdigones, an early developer of GoldenBraid. We acknowledge the excellent technical assistance provided by Marisol Gascón (IBMCP, Valencia, Spain) with the fluorescent images. This work was funded by Grant BIO2013- 42193 and Grant BIO2016-78601-R, Plan Nacional I+D, Spanish Ministry of Economy and Competitiveness, RTI2018-101115-B-C21 from the Ministerio de Ciencia, Innovación y Universidades (Spain) (MICINN/FEDER Funds), and PROMETEO/ 2018/066 from Conselleria d'Educació (Generalitat Valenciana, Comunitat Valenciana, Spain) and SUSPHIRE PCI2018- 092893-ERA CoBioTech (109) (MCIU/FEDER).Vázquez-Vilar, M.; Gandía, M.; García-Carpintero, V.; Marqués, E.; Sarrion-Perdigones, A.; Yenush, L.; Polaina, J.... (2020). Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond. Current Protocols in Molecular Biology. 130(1):1-31. https://doi.org/10.1002/cpmb.116S1311301Bernabé‐Orts, J. M., Casas‐Rodrigo, I., Minguet, E. G., Landolfi, V., Garcia‐Carpintero, V., Gianoglio, S., … Orzaez, D. (2019). Assessment of Cas12a‐mediated gene editing efficiency in plants. Plant Biotechnology Journal, 17(10), 1971-1984. doi:10.1111/pbi.13113Ballester, A.-R., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lázaro, C., Carmona, L., … Gabaldón, T. (2015). Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity. Molecular Plant-Microbe Interactions®, 28(3), 232-248. doi:10.1094/mpmi-09-14-0261-fiKhang, C. H., Park, S.-Y., Lee, Y.-H., & Kang, S. (2005). A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genetics and Biology, 42(6), 483-492. doi:10.1016/j.fgb.2005.03.004Chen, C., Liu, J., Duan, C., Pan, Y., & Liu, G. (2020). Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum. Fungal Genetics and Biology, 134, 103279. doi:10.1016/j.fgb.2019.103279Bai Flagfeldt, D., Siewers, V., Huang, L., & Nielsen, J. (2009). Characterization of chromosomal integration sites for heterologous gene expression inSaccharomyces cerevisiae. Yeast, 26(10), 545-551. doi:10.1002/yea.1705Fräbel, S., Wagner, B., Krischke, M., Schmidts, V., Thiele, C. M., Staniek, A., & Warzecha, H. (2018). Engineering of new-to-nature halogenated indigo precursors in plants. Metabolic Engineering, 46, 20-27. doi:10.1016/j.ymben.2018.02.003Fresquet-Corrales, S., Roque, E., Sarrión-Perdigones, A., Rochina, M., López-Gresa, M. P., Díaz-Mula, H. M., … Cañas, L. A. (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLOS ONE, 12(9), e0184839. doi:10.1371/journal.pone.0184839Garrigues, S., Gandía, M., & Marcos, J. F. (2015). Occurrence and function of fungal antifungal proteins: a case study of the citrus postharvest pathogen Penicillium digitatum. Applied Microbiology and Biotechnology, 100(5), 2243-2256. doi:10.1007/s00253-015-7110-3Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-345. doi:10.1038/nmeth.1318Gurgel, I. L. da S., Jorge, K. T. de O. S., Malacco, N. L. S. de O., Souza, J. A. M., Rocha, M. C., Fernandes, M. F., … Soriani, F. M. (2019). The Aspergillus fumigatus Mucin MsbA Regulates the Cell Wall Integrity Pathway and Controls Recognition of the Fungus by the Immune System. mSphere, 4(3). doi:10.1128/msphere.00350-19Hernanz-Koers, M., Gandía, M., Garrigues, S., Manzanares, P., Yenush, L., Orzaez, D., & Marcos, J. F. (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. doi:10.1016/j.fgb.2018.04.010Juarez, P., Huet-Trujillo, E., Sarrion-Perdigones, A., Falconi, E., Granell, A., & Orzaez, D. (2013). Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants. International Journal of Molecular Sciences, 14(3), 6205-6222. doi:10.3390/ijms14036205Kramer, M. F., & Coen, D. M. (2001). Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Molecular Biology. doi:10.1002/0471142727.mb1501s56Marcet-Houben, M., Ballester, A.-R., de la Fuente, B., Harries, E., Marcos, J. F., González-Candelas, L., & Gabaldón, T. (2012). Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus. BMC Genomics, 13(1). doi:10.1186/1471-2164-13-646Michielse, C. B., J Hooykaas, P. J., J J van den Hondel, C. A. M., & J Ram, A. F. (2008). Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nature Protocols, 3(10), 1671-1678. doi:10.1038/nprot.2008.154Müller, K. M., & Arndt, K. M. (2011). Standardization in Synthetic Biology. Synthetic Gene Networks, 23-43. doi:10.1007/978-1-61779-412-4_2Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., … Rogers, C. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. doi:10.1111/nph.13532Pérez-González, A., Kniewel, R., Veldhuizen, M., Verma, H. K., Navarro-Rodríguez, M., Rubio, L. M., & Caro, E. (2017). Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria. BMC Biotechnology, 17(1). doi:10.1186/s12896-017-0393-yPérez-Nadales, E., & Di Pietro, A. (2011). The Membrane Mucin Msb2 Regulates Invasive Growth and Plant Infection in Fusarium oxysporum  . The Plant Cell, 23(3), 1171-1185. doi:10.1105/tpc.110.075093Salazar-Cerezo, S., Kun, R. S., de Vries, R. P., & Garrigues, S. (2020). CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer. Enzyme and Microbial Technology, 133, 109463. doi:10.1016/j.enzmictec.2019.109463Sarrion-Perdigones, A., Chang, L., Gonzalez, Y., Gallego-Flores, T., Young, D. W., & Venken, K. J. T. (2019). Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying. Nature Communications, 10(1). doi:10.1038/s41467-019-13651-ySarrion-Perdigones, A., Falconi, E. E., Zandalinas, S. I., Juárez, P., Fernández-del-Carmen, A., Granell, A., & Orzaez, D. (2011). GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLoS ONE, 6(7), e21622. doi:10.1371/journal.pone.0021622Sarrion-Perdigones, A., Vazquez-Vilar, M., Palaci, J., Castelijns, B., Forment, J., Ziarsolo, P., … Orzaez, D. (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. PLANT PHYSIOLOGY, 162(3), 1618-1631. doi:10.1104/pp.113.217661Selma, S., Bernabé‐Orts, J. M., Vazquez‐Vilar, M., Diego‐Martin, B., Ajenjo, M., Garcia‐Carpintero, V., … Orzaez, D. (2019). Strong gene activation in plants with genome‐wide specificity using a new orthogonal CRISPR /Cas9‐based programmable transcriptional activator. Plant Biotechnology Journal, 17(9), 1703-1705. doi:10.1111/pbi.13138Shendure, J. A., Porreca, G. J., Church, G. M., Gardner, A. F., Hendrickson, C. L., Kieleczawa, J., & Slatko, B. E. (2011). Overview of DNA Sequencing Strategies. Current Protocols in Molecular Biology, 96(1). doi:10.1002/0471142727.mb0701s96Szewczyk, E., Nayak, T., Oakley, C. E., Edgerton, H., Xiong, Y., Taheri-Talesh, N., … Oakley, B. R. (2006). Fusion PCR and gene targeting in Aspergillus nidulans. Nature Protocols, 1(6), 3111-3120. doi:10.1038/nprot.2006.405Vafaee, Y., Staniek, A., Mancheno-Solano, M., & Warzecha, H. (2014). A Modular Cloning Toolbox for the Generation of Chloroplast Transformation Vectors. PLoS ONE, 9(10), e110222. doi:10.1371/journal.pone.0110222Vazquez-Vilar, M., Bernabé-Orts, J. M., Fernandez-del-Carmen, A., Ziarsolo, P., Blanca, J., Granell, A., & Orzaez, D. (2016). A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods, 12(1). doi:10.1186/s13007-016-0101-2Villiers, B. R. M., Stein, V., & Hollfelder, F. (2009). USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction. Protein Engineering, Design and Selection, 23(1), 1-8. doi:10.1093/protein/gzp063Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE, 6(2), e16765. doi:10.1371/journal.pone.0016765Sarrion‐Perdigones et al. (2013). See above.Hernanz‐Koersetal. (2018). See above.https://gbcloning.upv.es/https://benchling.co