A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 106 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris.

The medicinal mushroom Cordyceps militaris is widely exploited in traditional medicine and nutraceuticals in Asian countries. However, fruiting body production in C. militaris is facing degeneration through cultivation batches, and the molecular mechanism of this phenomenon remains unclear. This study showed that fruiting body formation in three different C. militaris strains, namely G12, B12, and HQ1, severely declined after three successive culturing generations using the spore isolation method. PCR analyses revealed that these strains exist as heterokaryons and possess both the mating-type loci, MAT1-1 and MAT1-2. Further, monokaryotic isolates carrying MAT1-1 or MAT1-2 were successfully separated from the fruiting bodies of all three heterokaryotic strains. A spore combination of the MAT1-1 monokaryotic isolate and the MAT1-2 monokaryotic isolate promoted fruiting body formation, while the single monokaryotic isolates could not do that themselves. Notably, we found that changes in ratios of the MAT1-2 spores strongly influenced fruiting body formation in these strains. When the ratios of the MAT1-2 spores increased to more than 15 times compared to the MAT1-1 spores, the fruiting body formation decreased sharply. In contrast, when MAT1-1 spores were increased proportionally, fruiting body formation was only slightly reduced. Our study also proposes a new solution to mitigate the degeneration in the heterokaryotic C. militaris strains caused by successive culturing generations.

Penicillium digitatum as a Model Fungus for Detecting Antifungal Activity of Botanicals: An Evaluation on Vietnamese Medicinal Plant Extracts.

Medicinal plants play important roles in traditional medicine, and numerous compounds among them have been recognized for their antimicrobial activity. However, little is known about the potential of Vietnamese medicinal plants for antifungal activity. In this study, we examined the antagonistic activity of twelve medicinal plant species collected in Northern Vietnam against Penicillium digitatum, Aspergillus flavus, Aspergillus fumigatus, and Candida albicans. The results showed that the antifungal activities of the crude extracts from Mahonia bealei, Ficus semicordata, and Gnetum montanum were clearly detected with the citrus postharvest pathogen P. digitatum. These extracts could fully inhibit the growth of P. digitatum on the agar medium, and on the infected citrus fruits at concentrations of 300-1000 µg/mL. Meanwhile, the other tested fungi were less sensitive to the antagonistic activity of the plant extracts. In particular, we found that the ethanolic extract of M. bealei displayed a broad-spectrum antifungal activity against all four pathogenic fungi. Analysis of this crude extract by enrichment coupled with high-performance liquid chromatography revealed that berberine and palmatine are major metabolites. Additional inspections indicated berberine as the key compound responsible for the antifungal activity of the M. bealei ethanolic extract. Our study provides a better understanding of the potential of Vietnamese medicinal plant resources for combating fungal pathogens. This work also highlights that the citrus pathogen P. digitatum can be employed as a model fungus for screening the antifungal activity of botanicals.

In vitro antiviral activities of ethanol and aqueous extracts of Vietnamese traditional medicinal plants against Porcine Epidemic Diarrhea virus: a coronavirus family member.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea in pigs leading to severe illnesses and high mortality rates. The development of medicinal agents to treat PEDV infection is therefore crucial. In this study, antiviral activities against PEDV of ethanol and aqueous extracts of 17 Vietnamese traditional medicinal plants were evaluated using the cytopathic effect-based assay. The results showed that 14 out of 17 medicinal plants could inhibit the cytopathic effect of PEDV. The ethanol extract of Stixis scandens was identified as the most active extract with its MIC (minimum inhibitory concentration) being 0.15 µg/mL. Other plant extracts also displayed strong antiviral activity against PEDV, including Anisomeles indica, Pericampylus glaucus and Croton kongensis. The results demonstrate that certain medicinal plants have a high antiviral potential and may serve as a lead to develop novel pharmaceutical agents to cure PED as well as the diseases caused by other coronaviruses.

A newly constructed Agrobacterium-mediated transformation system revealed the influence of nitrogen sources on the function of the LaeA regulator in Penicillium chrysogenum.

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 µM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 106 spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.

A highly efficient Agrobacterium tumefaciens-mediated transformation system for the postharvest pathogen Penicillium digitatum using DsRed and GFP to visualize citrus host colonization.

Penicillium digitatum is a major postharvest pathogen of citrus crops. This fungus broadly spreads worldwide and causes green mold disease, which results in severe losses for citrus production. Understanding of the citrus infection by P. digitatum may help develop effective strategies for controlling this pathogen. In this study, we have characterized a virulent strain of P. digitatum isolated in Vietnam and established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for this fungal strain with two newly constructed binary vectors. These binary vectors harbor dominant selectable markers for hygromycin or nourseothricin resistance, and expression cassettes for the red fluorescent protein (DsRed) or the green fluorescent protein (GFP), respectively. Using the established ATMT system, the transformation efficiency of the Vietnamese strain could reach a very high yield of 1240±165 transformants per 106 spores. Interestingly, we found that GFP is much better than DsRed for in situ visualization of citrus fruit colonization by the fungus. Additionally, we showed that the transformation system can also be used to generate T-DNA insertion mutants for screening non-pathogenic or less virulent strains. Our work provides a new platform including a virulent tropical strain of P. digitatum, an optimized ATMT method and two newly constructed binary vectors for investigation of the postharvest pathogen. This platform will help develop strategies to dissect molecular mechanisms of host-pathogen interactions in more detail as well as to identify potential genes of pathogenicity by either insertional mutagenesis or gene disruption in this important pathogenic fungus.