Horizontal transfer of a conserved npc-2 like effector gene in rust fungi that suppresses cell death in plants

ML/MD-2 is a conserved lipid/sterol-binding protein family having a role in sterol transfer and innate immunity in lower and higher eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 25 fungal and five oomycetes plant pathogen, having a different nutrition mode. All the fungal species were found to have varied numbers of family members, a distinctively substantial expansion of the ML gene family was observed in Rhizophagus irregularis (RI) with 33 genes. Our analysis also showed that NPC2 like proteins, a subfamily of ML domain superfamily, were not only restricted to animals and insect species but also present in plant fungal pathogens, including members of Clavicipitaceae, Pucciniacease, and Tremellaceae family. The phylogenetic analysis showed that these NPC2 like fungal proteins are more closely related to animals/insects than other fungal species. The molecular docking studies of these proteins with cholesterol and other derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. Further, the full length CDS of one of the npc2 like genes from Puccinia triticina (Pt5643) was PCR amplified and further characterized using various studies such as qRT-PCR, expression in onion epidermal cells, Nicotiana benthamiana for subcellular localization studies, yeast functional complementation, and expression studies. The mRNA abundance of Pt5643 was observed to be increased along with the infection progression and exhibits the highest expression at 5thday post-infection (dpi), suggesting its important role in the P. triticina infection cycle in wheat. The fluorescent confocal microscopy of transiently expressed YFP tagged Pt5643 in onion epidermal cells and N. benthamiana shows its location in cytoplasm and nucleus, indicating its involvement in the manipulation of host genes. The functional complementation of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic npc2 gene. Further, the overexpression of Pt5643 also suppressed the BAX and H2O2 induced program cell death in N. benthamiana and yeast, respectively thus proving to be a novel horizontally transferred effector in rust fungal pathogens. Altogether the present study reports the novel function of fungal NPC2 like proteins playing a crucial role in host defense manipulation possibly through lipid binding/transport similar to animals.

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Apoptosis-like programmed cell death in the grey mould fungus Botrytis cinerea: genes and their role in pathogenicity

Biochemical Society Transactions ◽

10.1042/bst0391493 ◽

2011 ◽

Vol 39 (5) ◽

pp. 1493-1498 ◽

Cited By ~ 21

Author(s):

Neta Shlezinger ◽

Adi Doron ◽

Amir Sharon

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Botrytis Cinerea ◽

Fungal Pathogens ◽

Grey Mould ◽

Fungal Species ◽

Fungal Virulence ◽

Mould Fungus ◽

Apoptosis Protein ◽

Result Show

A considerable number of fungal homologues of human apoptotic genes have been identified in recent years. Nevertheless, we are far from being able to connect the different pieces and construct a primary structure of the fungal apoptotic regulatory network. To get a better picture of the available fungal components, we generated an automatic search protocol that is based on protein sequences together with a domain-centred approach. We used this protocol to search all the available fungal databases for domains and homologues of human apoptotic proteins. Among all known apoptotic domains, only the BIR [baculovirus IAP (inhibitor of apoptosis protein) repeat] domain was found in fungi. A single protein with one or two BIR domains is present in most (but not all) fungal species. We isolated the BIR-containing protein from the grey mould fungus Botrytis cinerea and determined its role in apoptosis and pathogenicity. We also isolated and analysed BcNMA, a homologue of the yeast NMA11 gene. Partial knockout or overexpression strains of BcBIR1 confirmed that BcBir1 is anti-apoptotic and this activity was assigned to the N′-terminal part of the protein. Plant infection assays showed that the fungus undergoes massive PCD (programmed cell death) during early stages of infection. Further studies showed that fungal virulence was fully correlated with the ability of the fungus to cope with plant-induced PCD. Together, our result show that BcBir1 is a major regulator of PCD in B. cinerea and that proper regulation of the host-induced PCD is essential for pathogenesis in this and other similar fungal pathogens.

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An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

Indian Journal of Genetics and Plant Breeding (The) ◽

10.31742/ijgpb.80.3.17 ◽

2020 ◽

Vol 80 (03) ◽

Author(s):

Yu-Miao Zhang ◽

Jun Wang ◽

Tao Wu

Keyword(s):

Subcellular Localization ◽

Protein Interaction ◽

Protein Interactions ◽

Epidermal Cells ◽

Cyclin Dependent Kinase ◽

Protein Subcellular Localization ◽

Protein Protein Interactions ◽

Efficient System ◽

Protein Protein Interaction ◽

Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

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Substituted 6,7-dimethoxy-5-oxo-2,3,5,9b-tetrahydrothiazolo[2,3-a]isoindole- 3-1,1-dioxide Derivatives with Antimicrobial Activity and Docking Assisted Prediction of the Mechanism of their Antibacterial and Antifungal Properties

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200922114735 ◽

2020 ◽

Vol 20 (29) ◽

pp. 2681-2691

Author(s):

Athina Geronikaki ◽

Victor Kartsev ◽

Phaedra Eleftheriou ◽

Anthi Petrou ◽

Jasmina Glamočlija ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antifungal Activity ◽

Drug Targets ◽

Bacterial Species ◽

Pharmaceutical Research ◽

Docking Studies ◽

Fungal Species ◽

Docking Analysis ◽

E Coli ◽

Antibacterial And Antifungal

Background: Although a great number of the targets of antimicrobial therapy have been achieved, it remains among the first fields of pharmaceutical research, mainly because of the development of resistant strains. Docking analysis may be an important tool in the research for the development of more effective agents against specific drug targets or multi-target agents 1-3. Methods: In the present study, based on docking analysis, ten tetrahydrothiazolo[2,3-a]isoindole derivatives were chosen for the evaluation of the antimicrobial activity. Results: All compounds showed antibacterial activity against eight Gram-positive and Gram-negative bacterial species being, in some cases, more potent than ampicillin and streptomycin against all species. The most sensitive bacteria appeared to be S. aureus and En. Cloacae, while M. flavus, E. coli and P. aeruginosa were the most resistant ones. The compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited good antifungal activity better than reference drugs bifonazole (1.4 – 41 folds) and ketoconazole (1.1 – 406 folds) against all fungal species. In order to elucidate the mechanism of action, docking studies on different antimicrobial targets were performed. Conclusion: According to docking analysis, the antifungal activity can be explained by the inhibition of the CYP51 enzyme for most compounds with a better correlation of the results obtained for the P.v.c. strain (linear regression between estimated binding Energy and log(1/MIC) with R 2 =0.867 and p=0.000091 or R 2 = 0.924, p= 0.000036, when compound 3 is excluded.

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Post-translational regulation of autophagy is involved in intra-microbiome suppression of fungal pathogens

Microbiome ◽

10.1186/s40168-021-01077-y ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Jing Wang ◽

Chaoyun Xu ◽

Qiming Sun ◽

Jinrong Xu ◽

Yunrong Chai ◽

...

Keyword(s):

Translational Regulation ◽

Fungal Pathogens ◽

Fungal Growth ◽

Fungal Species ◽

26S Proteasome ◽

Healthy Plant ◽

Autophagy Induction ◽

Histone Acetyltransferase Gcn5 ◽

Plant Microbiota ◽

Autophagic Process

Abstract Background Microbiome interactions are important determinants for ecosystem functioning, stability, and health. In previous studies, it was often observed that bacteria suppress potentially pathogenic fungal species that are part of the same plant microbiota; however, the underlying microbe-microbe interplay remains mostly elusive. Here, we explored antagonistic interactions of the fungus Fusarium graminearum and bacterium Streptomyces hygroscopicus at the molecular level. Both are ubiquitous members of the healthy wheat microbiota; under dysbiosis, the fungus causes devastating diseases. Results In co-cultures, we found that Streptomyces alters the fungal acetylome leading to substantial induction of fungal autophagy. The bacterium secrets rapamycin to inactivate the target of rapamycin (TOR), which subsequently promotes the degradation of the fungal histone acetyltransferase Gcn5 through the 26S proteasome. Gcn5 negatively regulates fungal autophagy by acetylating the autophagy-related protein Atg8 at the lysine site K13 and blocking cellular relocalization of Atg8. Thus, degradation of Gcn5 triggered by rapamycin was found to reduce Atg8 acetylation, resulting in autophagy induction in F. graminearum. Conclusions Autophagy homeostasis plays an essential role in fungal growth and competition, as well as for virulence. Our work reveals a novel post-translational regulation of autophagy initiated by a bacterial antibiotic. Rapamycin was shown to be a powerful modulator of bacteria–fungi interactions with potential importance in explaining microbial homeostasis in healthy plant microbiomes. The autophagic process provides novel possibilities and targets to biologically control pathogens.

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Mitotic Recombination and Adaptive Genomic Changes in Human Pathogenic Fungi

Genes ◽

10.3390/genes10110901 ◽

2019 ◽

Vol 10 (11) ◽

pp. 901 ◽

Cited By ~ 10

Author(s):

Asiya Gusa ◽

Sue Jinks-Robertson

Keyword(s):

Fungal Pathogens ◽

Repetitive Sequences ◽

Pathogenic Fungi ◽

Genetic Alterations ◽

Fungal Species ◽

Chronic Infections ◽

Yeast Saccharomyces Cerevisiae ◽

Genomic Changes ◽

Break Site ◽

Repair Mechanisms

Genome rearrangements and ploidy alterations are important for adaptive change in the pathogenic fungal species Candida and Cryptococcus, which propagate primarily through clonal, asexual reproduction. These changes can occur during mitotic growth and lead to enhanced virulence, drug resistance, and persistence in chronic infections. Examples of microevolution during the course of infection were described in both human infections and mouse models. Recent discoveries defining the role of sexual, parasexual, and unisexual cycles in the evolution of these pathogenic fungi further expanded our understanding of the diversity found in and between species. During mitotic growth, damage to DNA in the form of double-strand breaks (DSBs) is repaired, and genome integrity is restored by the homologous recombination and non-homologous end-joining pathways. In addition to faithful repair, these pathways can introduce minor sequence alterations at the break site or lead to more extensive genetic alterations that include loss of heterozygosity, inversions, duplications, deletions, and translocations. In particular, the prevalence of repetitive sequences in fungal genomes provides opportunities for structural rearrangements to be generated by non-allelic (ectopic) recombination. In this review, we describe DSB repair mechanisms and the types of resulting genome alterations that were documented in the model yeast Saccharomyces cerevisiae. The relevance of similar recombination events to stress- and drug-related adaptations and in generating species diversity are discussed for the human fungal pathogens Candida albicans and Cryptococcus neoformans.

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The lysosomal Rag-Ragulator complex licenses RIPK1– and caspase-8–mediated pyroptosis by Yersinia

Science ◽

10.1126/science.abg0269 ◽

2021 ◽

Vol 372 (6549) ◽

pp. eabg0269

Author(s):

Zengzhang Zheng ◽

Wanyan Deng ◽

Yang Bai ◽

Rui Miao ◽

Shenglin Mei ◽

...

Keyword(s):

Cell Death ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Host Cells ◽

Caspase 8 ◽

Death Domain ◽

Interacting Protein ◽

A Genome ◽

Infection Inhibition ◽

Guanosine Triphosphatase

Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor–β–activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide CRISPR screen to uncover mediators of caspase-8–dependent pyroptosis identified an unexpected role of the lysosomal folliculin (FLCN)–folliculin-interacting protein 2 (FNIP2)–Rag-Ragulator supercomplex, which regulates metabolic signaling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, Fas-associated death domain (FADD), RIPK1, and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag guanosine triphosphatase activity and lysosomal tethering of Rag-Ragulator but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.

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Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

10.1101/2021.08.02.454728 ◽

2021 ◽

Author(s):

Zachary D Stolp ◽

Madhura Kulkarni ◽

Yining Liu ◽

Chengzhang Zhu ◽

Alizay Jalisi ◽

...

Keyword(s):

Cell Death ◽

Yeast Cell ◽

Vesicle Trafficking ◽

Membrane Integrity ◽

Time Lapse ◽

Membrane Permeabilization ◽

Cell Death Pathway ◽

Sequence Of Events ◽

A Genome ◽

Lysosome Membrane

Unicellular eukaryotes are suggested to undergo self-inflicted destruction. However, molecular details are sparse by comparison to the mechanisms of cell death known for human cells and animal models. Here we report a molecular pathway in Saccharomyces cerevisiae leading to vacuole/lysosome membrane permeabilization and cell death. Following exposure to heat-ramp conditions, a model of environmental stress, we observed that yeast cell death occurs over several hours, suggesting an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 adaptor complex. AP-3 promotes stress-induced cell death through its Arf1-GTPase-dependent vesicle trafficking function, which is required to transport and install proteins on the vacuole/lysosome membrane, including a death-promoting protein kinase Yck3. Time-lapse microscopy revealed a sequence of events where AP-3-dependent vacuole permeability occurs hours before the loss of plasma membrane integrity. An AP-3-dependent cell death pathway appears to be conserved in the human pathogen Cryptococcus neoformans.

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Epidemiology, Clinical Profile and Treatment Outcomes of Bacterial and Fungal Keratitis

10.21203/rs.3.rs-573494/v1 ◽

2021 ◽

Author(s):

Nabila Mabrouk ◽

Mohamed Abdelkader ◽

Mohamed Abdelhakeem ◽

Khaled Mourad ◽

Ahmed Abdelghany

Keyword(s):

Treatment Outcomes ◽

Fungal Pathogens ◽

Fungal Growth ◽

Fungal Species ◽

Clinical Findings ◽

Fungal Keratitis ◽

Clinical Profile ◽

Clinical Series ◽

Vegetative Matter ◽

Department Faculty

Abstract Purpose: The purpose of the study is to determine the microbiological aetiology, epidemiological factors, and clinical profile and treatment outcomes of infective keratitis in Ophthalmology department, Minia University. EgyptMethods:Prospective, non-randomized, observational clinical series of cases, including 150 patients with mean age 30 (range 12 to 85 years), 90 patients (60 %) were males and 60 (40%) were females, clinically diagnosed as infective corneal ulcer, attending the Ophthalmology Department – Faculty of Medicine. Minia University, Minia, Egypt. From 2018 to 2020.Detailed history taking and all clinical findings were collected. Corneal scrapings were obtained from patients and subjected to staining and culture for bacterial and fungal pathogens; Bacterial and fungal growth were identified by standard laboratory procedures.Results:Corneal trauma by a vegetative matter was the commesnest risk factor associated with infective keratitis in 92 cases (61.3%). Smear and Culture was positive in 83 cases (58.4%) of 142 corneal scrapings obtained, of which 60 cases were fungal (72.3%), 21 cases were bacterial (25.3%) and 2 cases were mixed bacterial and fungal (2.4%), Aspergillus species was the commonest fungal species isolated in fungal keratitis. 142 cases (94.67 %) healed completely with scar. Only 6 cases (4%) required evisceration due to aggressive presentation from the start and keratoplasty was performed for 2 cases (1.33%).Conclusions:Fungal keratitis was the commonest type in cases attending to our department. Adequate diagnosis, management and follow up helped in achieving high successful curative outcomes. Clinical Trials.gov ID: NCT04894630. Time of registration 1 December 2018

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