Duplication and overexpression of the genes encoding a beta 1,3-glucan synthase confer the intrinsic resistance to echinocandin in Mucor circinelloides

Procedures such as solid organ transplants and cancer treatments can leave many patients in an immunocompromised state resulting in an increased susceptibility to opportunistic diseases including fungal infections. Mucormycosis infections are continually emerging and pose a serious threat to immunocompromised patients. Currently there has been a sharp increase in mucormycosis cases as a secondary infection in patients battling SARS-CoV-2 infections. Mucorales fungi are notorious for presenting resistance to most antifungal drugs. The absence of effective means to treat these infections results in mortality rates approaching 100% in cases of disseminated infection. One of the most effective antifungal drug classes currently available are echinocandins. Echinocandins seem to be efficacious in treatment of many other fungal infections. Unfortunately, susceptibility testing has found that echinocandins have no to little effect on Mucorales. In this study, we found that the model Mucorales Mucor circinelloides genome carries three copies of the genes encoding for the echinocandin target protein β-(1,3)-D-glucan synthase (fksA, fksB, and fksC). Interestingly, we revealed that exposing M. circinelloides to micafungin significantly increased the expression of the fksA and fksB genes when compared to an untreated control. We further uncovered that the serine/threonine phosphatase calcineurin is responsible for the overexpression of fksA and fksB as deletion of calcineurin results in a decrease in expression of all three fks genes and a lower minimal inhibitory concentration (MIC) to micafungin. Taken together, this study demonstrates that the fks gene duplication and overexpression by calcineurin contribute to the intrinsic resistance to echinocandins in Mucor.

Silencing of a Wheat Ortholog of Glucan Synthase-Like Gene Reduced Resistance to Blumeria graminis f. sp. tritici

Wheat powdery mildew, caused by the obligate biotrophic ascomycete fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a major threat to wheat production worldwide. It is known that Arabidopsis thaliana glucan synthase-like 5 (AtGSL5) improves the resistance of wheat to powdery mildew by increasing its anti-penetration abilities. However, the function of glucan synthase-like (GSL) orthologs in crop species remains largely unknown. In this study, TaGSL22, a novel functional ortholog of AtGSL5, was isolated as the only Bgt-induced GSL gene in wheat. Phylogenetic analysis indicated that TaGSL22 was conserved within the group of Gramineae and showed a closer relationship to GSL orthologs from monocots than to those from dicots. The TaGSL22 transcript was highest in the wheat leaves, followed by stems then roots. TaGSL22 was localized in the cell membrane and cytoplasm of wheat protoplasts, as predicted by transmembrane structure analysis. In addition, expression of TaGSL22 was induced by the plant hormones ethylene (ETH) and salicylic acid (SA), but down-regulated by jasmonate (JA) and abscisic acid (ABA). The transcript level of TaGSL22 was up-regulated in the incompatible interaction between Bgt and wheat, whereas it remained relatively unchanged in the compatible interaction. Knocking down of TaGSL22 by virus-induced gene silencing (VIGS) induced a higher infection type in the wheat–Bgt interaction. The TaGSL22-silenced plants exhibited reduced resistance to Bgt, accompanied by decreased callose accumulation. Our study shows a conserved function of GSL genes in plant immunity associated with penetration resistance, and it indicates that TaGSL22 can be used to improve papilla composition and enhance resistance to wheat powdery mildew.

Molecular Docking studies on the Anti-fungal activity of Allium sativum (Garlic) against Mucormycosis (black fungus) by BIOVIA discovery studio visualizer 21.1.0.0

Background: The COVID-19 pandemic is a major concern. However, its association and rising cases of mucormycosis, also known as black fungus make the scenario even more troublesome. In addition, no specific medication against mucormycosis/black fungus makes things even worse. Objective: Garlic phytoconstituents have shown remarkable antifungal properties against various fungal species in various studies. Thus, the objective of the study was to check the potency of garlic phytoconstituents against the 1,3-beta-glucan synthase fungal protein using in-silico methods. Method: Auto Dock was used to evaluate selected garlic phytochemical molecules against 1,3-beta-glucan synthase fungal protein, and Discovery studio visualizer was used to create 3D and 2D interaction photos. Results: Five out of 9 phytoconstituents were found to form conventional hydrogen bonds, and only alliin formed the highest number of hydrogen bonds. However, the binding energy and inhibition constant of all nine phytoconstituents were determined. Interestingly, Z-ajoene showed the lowest binding energy of -5.07 kcal/mol and inhibition constant of 192.57µM. Conclusion: The results of our investigation suggested that garlic phytochemicals can have a good impact against black fungi, pertaining to the significant binding energies of phytoconstituents during blind docking. Specifically, Z-ajoene could be a good alternate against black fungi. However, detailed research is required to explore the antifungal activity of garlic against mucormycosis.

Molecular Docking Studies on the Anti-Fungal Activity of Allium Sativum (Garlic) Against Mucormycosis (Black Fungus) by BIOVIA Discovery Studio Visualizer 21.1.0.0

Background: The COVID-19 pandemic is a major concern. However, its association and rising cases of mucormycosis, also known as black fungus make the scenario even more troublesome. In addition, no specific medication against mucormycosis/black fungus makes things even worse.Objective: Garlic phytoconstituents have shown remarkable antifungal properties against various fungal species in various studies. Thus, the objective of the study was to check the potency of garlic phytoconstituents against the 1,3-beta-glucan synthase fungal protein using in-silico methods.Method: Auto Dock was used to evaluate selected garlic phytochemical molecules against 1,3-beta-glucan synthase fungal protein, and Discovery studio visualizer was used to create 3D and 2D interaction photos.Results: Five out of 9 phytoconstituents were found to form conventional hydrogen bonds, and only alliin formed the highest number of hydrogen bonds. However, the binding energy and inhibition constant of all nine phytoconstituents were determined. Interestingly, Z-ajoene showed the lowest binding energy of -5.07 kcal/mol and inhibition constant of 192.57µM.Conclusion: The results of our investigation suggested that garlic phytochemicals can have a good impact against black fungi, pertaining to the significant binding energies of phytoconstituents during blind docking. Specifically, Z-ajoene could be a good alternate against black fungi. However, detailed research is required to explore the antifungal activity of garlic against mucormycosis.

A Review on Microorganisms-Derived Products as Potential Antimicrobial Agents

Microorganisms including actinomycetes, archaea, bacteria, fungi, yeast, and micro algae are the auspicious source of vital bioactive compounds. In this review, the existing state of the art re-garding antimicrobial molecules from microorganisms has been summarized. The potential an-timicrobial compounds from actinomycetes, particularly Streptomyces sp.; archaea; fungi including endophytic and marine-derived fungi, mushroom; yeast, and microalgae were briefly described. Furthermore, this review briefly summarized the activity and mode of action of bacteriocins, a ribosomally synthesized antimicrobial peptides product of Eurotium sp., Streptomyces parvulus, S. thermophiles, Lactococcus lactis, etc. Bacteriocins have inherent properties such as targeting multi-ple-drug resistant pathogens, which allows them to be considered next-generation antibiotics. Similarly, Glarea lozoyensis derived antifungal lipohexpeptides i.e., pneumocandins, inhibits 1,3-β-glucan synthase of the fungal cell wall and acts as a precursor for the synthesis of caspo-fungin, is also elaborated. In conclusion, this review highlights the possibility of using microor-ganisms as an antimicrobial resource for biotechnological, nutraceutical, and pharmaceutical ap-plications. However, more investigations are still required to separate, purify, and characterize these bioactive compounds and transfer these primary drugs into clinically approved antibiotics.

Application of Double-Strand RNAs Targeting Chitin Synthase, Glucan Synthase, and Protein Kinase Reduces Fusarium graminearum Spreading in Wheat

Controlling the devastating fungal pathogen Fusarium graminearum (Fg) is a challenge due to inadequate resistance in nature. Here, we report on the identification of RNAi molecules and their applications for controlling Fg in wheat through silencing chitin synthase 7 (Chs7), glucan synthase (Gls) and protein kinase C (Pkc). From transgenic Fg strains four RNAi constructs from Chs7 (Chs7RNAi−1, −2, −3, and −4), three RNAi constructs from Gls (GlsRNAi−2, −3, and −6), and one RNAi construct from Pkc (PkcRNAi−5) were identified that displayed effective silencing effects on mycelium growth in medium and pathogenicity in wheat spikes. Transcript levels of Chs7, Gls and Pkc were markedly reduced in those strains. Double-strand RNAs (dsRNAs) of three selected RNAi constructs (Chs7RNAi-4, GlsRNAi-6 and PkcRNA-5) strongly inhibited mycelium growth in vitro. Spray of those dsRNAs on detached wheat leaves significantly reduced lesion sizes; the independent dsRNAs showed comparable effects on lesions with combination of two or three dsRNAs. Expression of three targets Chs7, Gls, and Pkc was substantially down-regulated in Fg-infected wheat leaves. Further application of dsRNAs on wheat spikes in greenhouse significantly reduced infected spikelets. The identified RNAi constructs may be directly used for spray-induced gene silencing and stable expression in plants to control Fusarium pathogens in agriculture.

CHARACTERISATION OF HORDEUM VULGARE CELLULOSE SYNTHASE-LIKE F6 PROMOTER VIA TRANSGENE EXPRESSION IN RICE

Beta-glucan in cereal crops is known as a functional food, which can reduce cardiovascular diseases by lowering blood cholesterol levels. However, beta-glucan content is relatively low in rice grains, despite being relatively abundant in barley and oat grains. Taking advantage of rice as the staple food for Asians, increasing beta-glucan content in rice for their consumption may help to reduce cardiovascular-related diseases among them. Previous attempts in increasing beta-glucan content in rice via transgene expression of beta-glucan synthase genes from barley into rice were unsuccessful due to the use of non-tissue specific as well as constitutively expressing promoter. The current transgenic expression study was performed to characterise the promoter of beta-glucan synthase gene in barley using beta-glucuronidase (GUS) reporter gene. Two fragments of HvCslF6 promoter (2771 bp and 1257 bp) were successfully fused with GUS reporter gene and integrated into rice plants, demonstrated that the promoter was functional in the heterologous plant system. The presence of blue GUS staining was observed on the leaf, root, stem, and grain of the transgenic rice regardless of the promoter length used and stayed functional up to the next generation. GUS qualitative analysis confirmed that the shorter promoter length generated a stronger GUS activity in comparison to the longer one. This indicated that the presence of repressor elements in between the -2771 bp and -1257 bp regions. The preliminary results shed light on the strong promoter activity in the rice endosperm tissue. It can become an alternative to the collection of plant promoters that can be used for grain quality improvement and biofortification.