scholarly journals A Genetic Screen Identifies Two Novel Rice Cysteine-rich Receptor-like Kinases That Are Required for the Rice NH1-mediated Immune Response

2014 ◽  
Author(s):  
Mawsheng Chern ◽  
Rebecca Bart ◽  
Wei Bai ◽  
Deling Ruan ◽  
Wing Hoi Sze-To ◽  
...  
Keyword(s):  
Immune Response ◽  
Fungal Pathogens ◽  
Expression System ◽  
Tiling Array ◽  
Comparative Genome Hybridization ◽  
Over Expression ◽  
Lesion Mimic ◽  
Genes Encoding ◽  
Elevated Expression ◽  
Genome Tiling Array

Over-expression of rice NH1 (NH1ox), the ortholog of Arabidopsis NPR1, confers immunity to bacterial and fungal pathogens and induces the appearance of necrotic lesions due to activation of defense genes at the pre-flowering stage. This lesion-mimic phenotype can be enhanced by the application of benzothiadiazole (BTH). To identify genes regulating these responses, we screened a fast neutron-irradiated NH1ox rice population. We identified one mutant, called snl1 (suppressor of NH1-mediated lesion-mimic 1), which is impaired both in BTH-induced necrotic lesion formation and in the immune response. Using a comparative genome hybridization approach employing rice whole genome tiling array, we identified 11 genes associated with the snl1 phenotype. Transgenic analysis revealed that RNA interference of two of the genes, encoding previously uncharacterized cysteine-rich receptor-like kinases (CRK6 and CRK10), re-created the snl1 phenotype. Elevated expression of CRK10 using an inducible expression system resulted in enhanced immunity. Quantitative PCR revealed that BTH treatment and elevated levels of rice NH1 and its paralog NH3 induced expression of CRK10 and CRK6 RNA. These results indicate that CRK6 and CRK10 are required for the BTH-activated immune response mediated by NH1.

scholarly journals To have neighbour's fare: extending the molecular toolbox for Streptococcus pneumoniae

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 351-359 ◽  
Author(s):  
Tomas G. Kloosterman ◽  
Jetta J. E. Bijlsma ◽  
Jan Kok ◽  
Oscar P. Kuipers
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Expression System ◽  
Inducible Promoter ◽  
Defined Medium ◽  
Reporter System ◽  
Two Component System ◽  
Over Expression ◽  
Expression Studies ◽  
Genes Encoding

In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous β-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.

scholarly journals Camphor and Eucalyptol—Anticandidal Spectrum, Antivirulence Effect, Efflux Pumps Interference and Cytotoxicity

2021 ◽  
Vol 22 (2) ◽  
pp. 483
Author(s):  
Marija Ivanov ◽  
Abhilash Kannan ◽  
Dejan S. Stojković ◽  
Jasmina Glamočlija ◽  
Ricardo C. Calhelha ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Liver Cells ◽  
Ergosterol Biosynthesis ◽  
Antifungal Compound ◽  
Porcine Liver ◽  
Fungal Virulence ◽  
Genes Encoding ◽  
Plant Constituents

Candidaalbicans represents one of the most common fungal pathogens. Due to its increasing incidence and the poor efficacy of available antifungals, finding novel antifungal molecules is of great importance. Camphor and eucalyptol are bioactive terpenoid plant constituents and their antifungal properties have been explored previously. In this study, we examined their ability to inhibit the growth of different Candida species in suspension and biofilm, to block hyphal transition along with their impact on genes encoding for efflux pumps (CDR1 and CDR2), ergosterol biosynthesis (ERG11), and cytotoxicity to primary liver cells. Camphor showed excellent antifungal activity with a minimal inhibitory concentration of 0.125–0.35 mg/mL while eucalyptol was active in the range of 2–23 mg/mL. The results showed camphor’s potential to reduce fungal virulence traits, that is, biofilm establishment and hyphae formation. On the other hand, camphor and eucalyptol treatments upregulated CDR1;CDR2 was positively regulated after eucalyptol application while camphor downregulated it. Neither had an impact on ERG11 expression. The beneficial antifungal activities of camphor were achieved with an amount that was non-toxic to porcine liver cells, making it a promising antifungal compound for future development. The antifungal concentration of eucalyptol caused cytotoxic effects and increased expression of efflux pump genes, which suggests that it is an unsuitable antifungal candidate.

scholarly journals NtbHLH1, a JAF13-like bHLH, interacts with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yuxin Fan ◽  
Jiayu Peng ◽  
Jiacheng Wu ◽  
Ping Zhou ◽  
Ruijie He ◽  
...  
Keyword(s):  
Flavonoid Biosynthesis ◽  
Transcriptional Level ◽  
Flavonoid Pathway ◽  
Regulation Of Expression ◽  
Over Expression ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Regulatory Complex ◽  
Positive Regulators ◽  
R2r3 Myb

Abstract Background Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. Results In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. Conclusions Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

scholarly journals The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

2021 ◽  
Vol 7 (2) ◽  
pp. 86
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
Mating Partner ◽  
Fungal Virulence ◽  
Functional Studies ◽  
Infection Structures ◽  
Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

High content screening and computational prediction reveal viral genes that suppress innate immune response

2021 ◽  
Author(s):  
Tai L Ng ◽  
Erika J Olson ◽  
Tae Yeon Yoo ◽  
H. Sloane Weiss ◽  
Yukiye Koide ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Nuclear Transport ◽  
Computational Prediction ◽  
Viral Proteins ◽  
Innate Immune ◽  
Type I ◽  
Viral Genes ◽  
Genes Encoding

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-kB and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins.

Genetic regulation of nitrogen metabolism in the fungi

1997 ◽  
Vol 61 (1) ◽  
pp. 17-32
Author(s):  
G A Marzluf
Keyword(s):  
Nitrogen Metabolism ◽  
Protein Interactions ◽  
Fungal Pathogens ◽  
Metabolic Regulation ◽  
Specific Binding ◽  
Genetic Regulation ◽  
Nitrogen Sources ◽  
Specific Protein ◽  
Genes Encoding ◽  
Gata Family

In the fungi, nitrogen metabolism is controlled by a complex genetic regulatory circuit which ensures the preferential use of primary nitrogen sources and also confers the ability to use many different secondary nitrogen sources when appropriate. Most structural genes encoding nitrogen catabolic enzymes are subject to nitrogen catabolite repression, mediated by positive-acting transcription factors of the GATA family of proteins. However, certain GATA family members, such as the yeast DAL80 factor, act negatively to repress gene expression. Selective expression of the genes which encode enzymes for the metabolism of secondary nitrogen sources is often achieved by induction, mediated by pathway-specific factors, many of which have a GAL4-like C6/Zn2 DNA binding domain. Regulation within the nitrogen circuit also involves specific protein-protein interactions, as exemplified by the specific binding of the negative-acting NMR protein with the positive-acting NIT2 protein of Neurospora crassa. Nitrogen metabolic regulation appears to play a significant role in the pathogenicity of certain animal and plant fungal pathogens.

scholarly journals Streptomyces roseolus, A Promising Biocontrol Agent Against Aspergillus flavus, the Main Aflatoxin B1 Producer

Toxins ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 442 ◽  
Author(s):  
Isaura Caceres ◽  
Selma Snini ◽  
Olivier Puel ◽  
Florence Mathieu
Keyword(s):  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Biocontrol Agent ◽  
Cyclopiazonic Acid ◽  
Over Expression ◽  
Expression Of Genes ◽  
B1 Gene ◽  
Genes Encoding ◽  
Molecular Mechanism Of Action ◽  
Potential Use

Crop contamination by aflatoxin B1 is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute to the substitution of phytopharmaceutical products are thus needed. For this, a promising method resides in the use of biocontrol agents. Several actinobacteria strains have demonstrated to effectively reduce the aflatoxin B1 concentration. Nevertheless, the molecular mechanism of action by which these biological agents reduce the mycotoxin concentration has not been determined. The aim of the present study was to test the potential use of Streptomyces roseolus as a biocontrol agent against aflatoxin B1 contamination. Co-cultures with Aspergillus flavus were conducted, and the molecular fungal response was investigated through analyzing the q-PCR expression of 65 genes encoding relevant fungal functions. Moreover, kojic and cyclopiazonic acid concentrations, as well as morphological fungal changes were also analyzed. The results demonstrated that reduced concentrations of aflatoxin B1 and kojic acid were respectively correlated with the down-regulation of the aflatoxin B1 gene cluster and kojR gene expression. Moreover, a fungal hypersporulated phenotype and a general over-expression of genes involved in fungal development were observed in the co-culture condition.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel Yu ◽  
Megan A Outram ◽  
Emma Creen ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

Yeast Golgi SNARE interactions are promiscuous

2000 ◽  
Vol 113 (1) ◽  
pp. 145-152 ◽  
Author(s):  
M.M. Tsui ◽  
D.K. Banfield
Keyword(s):  
Protein Interactions ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Snare Proteins ◽  
Current View ◽  
Over Expression ◽  
Vesicular Traffic ◽  
Binding Data ◽  
Genes Encoding ◽  
Target Membrane

The transport of proteins between various compartments of the secretory pathway occurs by the budding of vesicles from one membrane and their fusion with another. A key event in this process is the selective recognition of the target membrane by the vesicle and the current view is that SNARE protein interactions likely play a central role in vesicle-target recognition and or membrane fusion. In yeast, only a single syntaxin (Sed5p) is required for Golgi transport and Sed5p is known to bind to at least 7 SNARE proteins. However, the number of Sed5p-containing SNARE complexes that exist in cells is not known. In this study we examined direct pair-wise interactions between full length soluble recombinant forms of SNAREs (Sed5p, Sft1p, Ykt6p, Vti1p, Gos1p, Sec22p, Bos1p, and Bet1p) involved in ER-Golgi and intra-Golgi membrane trafficking. In the binding assay that we describe here the majority of SNARE-binary interactions tested were positive, indicating that SNARE-SNARE interactions although promiscuous are not entirely non-selective. Interactions between a number of the genes encoding these SNAREs are consistent with our binding data and taken together our results suggest that functionally redundant Golgi SNARE-complexes exist in yeast. In particular, over-expression of Bet1p (a SNARE required for ER-Golgi and Golgi-ER traffic) and can bypass the requirement for the otherwise essential SNARE Sft1p (required for intra-Golgi traffic), suggesting that Bet1p either functions in a parallel pathway with Sft1p or can be incorporated into SNARE-complexes in place of Sftp1. None-the-less this result suggests that Bet1p can participate in two distinct trafficking steps, cycling between the ER and Golgi as well as in retrograde intra-Golgi traffic. In addition, suppressor genetics together with the analysis of the phenotypes of conditional mutations in Sft1p and Ykt6p, are consistent with a role for these SNAREs in more than one trafficking step. We propose that different combinations of SNAREs form complexes with Sed5p and are required for multiple steps in ER-Golgi and intra-Golgi vesicular traffic. And that the apparent promiscuity of SNARE-SNARE binding interactions, together with the requirement for some SNAREs in more than one trafficking step, supports the view that the specificity of vesicle fusion events cannot be explained solely on the basis of SNARE-SNARE interactions.

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