Differential roles, crosstalk and response to the Antifungal Protein AfpB in the three Mitogen-Activated Protein Kinases (MAPK) pathways of the citrus postharvest pathogen Penicillium digitatum.

Fungi have three mitogen-activated protein kinases (MAPKs): Kss1/Fus3 involved in the invasive growth and virulence of pathogens, Hog1 in response to osmotic stress, and Slt2/Mpk1 in response to cell wall (CW) stress. We conducted comparative analyses of these MAPKs in the phytopathogen Penicillium digitatum and studied their role in the mode of action of the novel self-antifungal protein AfpB. The sensitivity to different stresses of Δhog1 and the reduced growth of Δkss1 coincided with previous reports. However, Δslt2 showed a strong reduction of growth and conidiation, abnormal morphology, and sensitivity to CW stress and temperature. The complementation of Δslt2 validated this mutant. Immunodetection of P-Hog1 and P-Slt2 confirmed the loss and gain of MAPKs in the mutant and complemented strains. Mutants Δslt2 and Δkss1 showed a strong reduction in virulence, whereas Δhog1 was the least affected, and none sporulated during infection. We studied the MAPK signalling induction in response to different treatments. Our data revealed a complex crosstalk involving the three MAPKs, the differential responses of Hog1 and Slt2 to various stresses and their induction by AfpB or the fungicide fludioxonil (FD). Δhog1 resistance to FD confirmed that Hog1 mediates the activity of FD, whereas Δkss1 sensitivity is probably due to the basal activation of Hog1 in Δkss1. None of the three MAPK mutants showed increased sensitivity to AfpB, contrary to previous reports of other antifungal proteins, which indicates that the observed AfpB-mediated activation of Hog1 and Slt2 would not have a defensive role.

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

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 HygR 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 pDGB3α/pDGB3Ω 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.