The autophagy genes ChATG4 and ChATG8 are required for reproductive development, virulence, and septin assembly in Cochliobolus heterostrophus

2021 ◽  
Author(s):  
Huilin Yu ◽  
Wantong Jia ◽  
Kunqin Xiao ◽  
Wenli Jiao ◽  
Xianghui Zhang ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Fungal Growth ◽  
Reproductive Development ◽  
Gene Replacement ◽  
Cochliobolus Heterostrophus ◽  
Physical Interaction ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Growth Development ◽  
Two Hybrid

Autophagy is a highly conserved degrading process and is crucial for cell growth and development in eukaryotes, especially when they face starvation and stressful conditions. To evaluate the functions of Atg4 and Atg8 in mycelial growth, asexual and sexual development, and virulence in Cochliobolus heterostrophus, ΔChatg4 and ΔChatg8 mutants were generated by gene replacement. Strains deleted for ChATG4 and ChATG8 genes showed significant changes in vegetative growth and in development of conidia and ascospores compared with the wild-type strain. The autophagy process was blocked and the virulence was reduced dramatically in ΔChatg4 and ΔChatg8 mutants. In addition, deletion of ChATG4 and ChATG8 disordered Cdc10 subcellular localization and formation of septin rings. The direct physical interaction between ChAtg4 and ChAtg8 was detected by Yeast-two-hybrid, and ChAtg4-GFP was dispersed throughout the cytoplasm, although GFP-ChAtg8 appeared as punctate structures. All phenotypes were restored in complemented strains. Taken together, these findings indicated that ChATG4 and ChATG8 were crucial for autophagy to regulate fungal growth, development, virulence, and localization of septin in C. heterostrophus.

scholarly journals Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3535-3542 ◽  
Author(s):  
Zhihong Xie ◽  
Yuetang Dou ◽  
Shuzheng Ping ◽  
Ming Chen ◽  
Guoying Wang ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Pseudomonas Stutzeri ◽  
Physical Interaction ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Free Living ◽  
Yeast Two Hybrid System ◽  
Living State ◽  
Microaerobic Conditions ◽  
Two Hybrid

Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.

scholarly journals Subunit Interactions in the mariner Transposase

Genetics ◽  
1996 ◽  
Vol 144 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
Allan R Lohe ◽  
David T Sullivan ◽  
Daniel L Hartl
Keyword(s):  
Hybrid System ◽  
Catalytic Domain ◽  
Wild Type ◽  
Target Element ◽  
Yeast Two Hybrid ◽  
Subunit Interactions ◽  
Hsp70 Promoter ◽  
Yeast Two Hybrid System ◽  
Transposition Reaction ◽  
Two Hybrid

Abstract We have studied the Mos1 transposase encoded by the transposable element mariner. This transposase is a member of the “D,D(35)E” superfamily of proteins exhibiting the motif D,D(34)D. It is not known whether this transposase, or other eukaryote transposases manifesting the D,D(35)E domain, functions in a multimeric form. Evidence for oligomerization was found in the negative complementation of Mos1 by an EMS-induced transposase mutation in the catalytic domain. The transposase produced by this mutation has a glycine-to-arginine replacement at position 292. The G292R mutation strongly interferes with the ability of wild-type transposase to catalyze excision of a target element. Negative complementation was also observed for two other EMS mutations, although the effect was weaker than observed with G292R. Results from the yeast two-hybrid system also imply that Mos1 subunits interact, suggesting the possibility of subunit oligomerization in the transposition reaction. Overproduction of Mos1 subunits through an hsp70 promoter also inhibits excision of the target element, possibly through autoregulatory feedback on transcription or through formation of inactive or less active oligomers. The effects of both negative complementation and overproduction may contribute to the regulation of mariner transposition.

scholarly journals Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins in Fusarium graminearum

2008 ◽  
Vol 8 (2) ◽  
pp. 217-229 ◽  
Author(s):  
Vellaisamy Ramamoorthy ◽  
Edgar B. Cahoon ◽  
Mercy Thokala ◽  
Jagdeep Kaur ◽  
Jia Li ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fungal Growth ◽  
Wild Type ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Disease Symptoms ◽  
Plant Defensins ◽  
Antifungal Action

ABSTRACT The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The ΔFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The ΔFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the ΔFgmt2 mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1 and ΔFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.

scholarly journals ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

2008 ◽  
Vol 183 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Noveera T. Ahmed ◽  
Chunlei Gao ◽  
Ben F. Lucker ◽  
Douglas G. Cole ◽  
David R. Mitchell
Keyword(s):  
Binding Site ◽  
Intraflagellar Transport ◽  
Site Formation ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
B Subunit ◽  
Dynein Arms ◽  
Two Hybrid

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout, Sequence Analysis, and Comparison to Other C-5(6) Sterol Desaturases

2009 ◽  
Vol 8 (8) ◽  
pp. 1287-1297 ◽  
Author(s):  
Alejandro D. Nusblat ◽  
Sebastián R. Najle ◽  
Mariela L. Tomazic ◽  
Antonio D. Uttaro ◽  
Clara B. Nudel
Keyword(s):  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Tetrahymena Thermophila ◽  
Gene Replacement ◽  
Gene Identification ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Gene Coding ◽  
Transmembrane Regions

ABSTRACT The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.

scholarly journals Interactions between QnrB, QnrB Mutants, and DNA Gyrase

2015 ◽  
Vol 59 (9) ◽  
pp. 5413-5419 ◽  
Author(s):  
Eu Suk Kim ◽  
Chunhui Chen ◽  
Molly Braun ◽  
Hyo Youl Kim ◽  
Ryo Okumura ◽  
...  
Keyword(s):  
No Reduction ◽  
Substantial Reduction ◽  
Dna Gyrase ◽  
Single Amino Acid ◽  
Physical Interaction ◽  
Wild Type ◽  
Physical Interactions ◽  
Substitution Mutant ◽  
Two Hybrid ◽  
Intermediate Effect

ABSTRACTPlasmid-encoded protein QnrB1 protects DNA gyrase from ciprofloxacin inhibition. Using a bacterial two-hybrid system, we evaluated the physical interactions between wild-type and mutant QnrB1, the GyrA and GyrB gyrase subunits, and a GyrBA fusion protein. The interaction of QnrB1 with GyrB and GyrBA was approximately 10-fold higher than that with GyrA, suggesting that domains of GyrB are important for stabilizing QnrB1 interaction with the holoenzyme. Sub-MICs of ciprofloxacin or nalidixic acid reduced the interactions between QnrB1 and GyrA or GyrBA but produced no reduction in the interaction with GyrB or a quinolone-resistant GyrA:S83L (GyrA with S83L substitution) mutant, suggesting that quinolones and QnrB1 compete for binding to gyrase. Of QnrB1 mutants that reduced quinolone resistance, deletions in the C or N terminus of QnrB1 resulted in a marked decrease in interactions with GyrA but limited or no effect on interactions with GyrB and an intermediate effect on interactions with GyrBA. While deletion of loop B and both loops moderately reduced the interaction signal with GyrA, deletion of loop A resulted in only a small reduction in the interaction with GyrB. The loop A deletion also caused a substantial reduction in interaction with GyrBA, with little effect of loop B and dual-loop deletions. Single-amino-acid loop mutations had little effect on physical interactions except for a Δ105I mutant. Therefore, loops A and B may play key roles in the proper positioning of QnrB1 rather than as determinants of the physical interaction of QnrB1 with gyrase.

scholarly journals Functional interactions between the hBRM/hBRG1 transcriptional activators and the pRB family of proteins.

1996 ◽  
Vol 16 (4) ◽  
pp. 1576-1583 ◽  
Author(s):  
B E Strober ◽  
J L Dunaief ◽  
Guha ◽  
S P Goff
Keyword(s):  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Adenocarcinoma Cell Line ◽  
Cycle Progression ◽  
Yeast Two Hybrid System ◽  
Flat Cell ◽  
Two Hybrid

hBRG1 and hBRM are mammalian homologs of the SNF2/SW12 yeast transcriptional activator. These proteins exist in a large multisubunit complex that likely serves to remodel chromatin and, in so doing, facilitates the function of specific transcription factors. The retinoblastoma protein (pRB) inhibits cell cycle progression by repressing transcription of specific growth-related genes. Using the yeast two-hybrid system, we demonstrate that the members of the hBRG1/hBRM family of proteins interact with the pRB family of proteins, which includes pRB, p107, and p130. Interaction between the hBRG1/hBRM family with the pRB family likely influences cellular proliferation, as both hBRG1 and hBRM, but not mutants of these proteins unable to bind to pRB family members, inhibit the formation of drug-resistant colonies when transfected into the SW13 human adenocarcinoma cell line, which lacks endogenous hBRG1 or hBRM. Further, hBRM and two isoforms of hBRG1 induce the formation of flat, growth-arrested cells in a pRB family-dependent manner when introduced into SW13 cells. This flat-cell inducing activity is severely reduced by cotransfection of the wild-type E1A protein and variably reduced by the cotransfection of mutants of E1A that lack the ability to bind to some or all members of the pRB family.

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