The exocyst regulates hydrolytic enzyme secretion at hyphal tips and septa in banana Fusarium wilt fungus, Fusarium odoratissimum

Hyphal polarized growth in filamentous fungi requires tip-directed secretion, while additional evidence suggests that fungal exocytosis for the hydrolytic enzyme secretion can occur at other sites in hyphae, including the septum. In this study, we analyzed the role of the exocyst complex involved in the secretion in banana wilt fungal pathogen Fusarium odoratissimum . All eight exocyst components in F. odoratissimum not only localized to the tips ahead of the Spitzenkörper in growing hyphae, but also localized to the outer edges of septa in mature hyphae. To further analyze the exocyst in F. odoratissimum , we tried to do single gene deletion for all the genes encoding the eight exocyst components and only succeed to construct the gene deletion mutants for exo70 and sec5 , we suspect that the other 6 exocyst components are encoded by essential genes. Deletion of exo70 or sec5 led to defects in vegetative growth, conidiation and pathogenicity in F. odoratissimum . Notably, the deletion of exo70 resulted in decreased activities for endoglucosidase, filter paper enzymes and amylase, while the loss of sec5 only led to a slight reduction in amylase activity. Septa-localized α-amylase (AmyB) was identified as the marker for septum-directed secretion, and we found that Exo70 is essential for the localization of AmyB to septa. Meanwhile the loss of Sec5 did not affect AmyB localization to septa but led to a higher accumulation of AmyB in cytoplasm. This suggested while Exo70 and Sec5 both take part in the septum-directed secretion, the two conduct different roles in this process. IMPORTANCE The exocyst complex is a multisubunit tethering complex (MTC) for secretory vesicles at the plasma membrane and contains eight subunits, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. While the exocyst complex is well defined in eukaryotes from yeast to humans, the exocyst components in filamentous fungi show different localization patterns in the apical tips of hyphae that suggests filamentous fungi have evolved divergent strategies to regulate endomembrane trafficking. In this study, we demonstrated that the exocyst components in Fusarium odoratissimum are not only localized to the tips of growing hyphae but also to the outer edge of the septa in mature hyphae, suggesting that the exocyst complex plays a role in the regulation of septum-directed protein secretion in F. odoratissimum . We further found that Exo70 and Sec5 are required for the septum-directed secretion of α-amylase in F. odoratissimum but with different influence.

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Mutability of demographic noise in microbial range expansions

The ISME Journal ◽

10.1038/s41396-021-00951-9 ◽

2021 ◽

Author(s):

QinQin Yu ◽

Matti Gralka ◽

Marie-Cécilia Duvernoy ◽

Megan Sousa ◽

Arbel Harpak ◽

...

Keyword(s):

Gene Deletion ◽

Genetic Manipulation ◽

Single Gene ◽

Population Level ◽

Growth Conditions ◽

Establishment Probability ◽

In The Wild ◽

Order Of Magnitude ◽

Demographic Noise ◽

Single Gene Deletion

AbstractDemographic noise, the change in the composition of a population due to random birth and death events, is an important driving force in evolution because it reduces the efficacy of natural selection. Demographic noise is typically thought to be set by the population size and the environment, but recent experiments with microbial range expansions have revealed substantial strain-level differences in demographic noise under the same growth conditions. Many genetic and phenotypic differences exist between strains; to what extent do single mutations change the strength of demographic noise? To investigate this question, we developed a high-throughput method for measuring demographic noise in colonies without the need for genetic manipulation. By applying this method to 191 randomly-selected single gene deletion strains from the E. coli Keio collection, we find that a typical single gene deletion mutation decreases demographic noise by 8% (maximal decrease: 81%). We find that the strength of demographic noise is an emergent trait at the population level that can be predicted by colony-level traits but not cell-level traits. The observed differences in demographic noise from single gene deletions can increase the establishment probability of beneficial mutations by almost an order of magnitude (compared to in the wild type). Our results show that single mutations can substantially alter adaptation through their effects on demographic noise and suggest that demographic noise can be an evolvable trait of a population.

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Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63)

Molecular Microbiology ◽

10.1046/j.1365-2958.1998.00689.x ◽

1998 ◽

Vol 27 (3) ◽

pp. 519-530 ◽

Cited By ~ 84

Author(s):

Phalgun B. Joshi ◽

David L. Sacks ◽

Govind Modi ◽

W. Robert McMaster

Keyword(s):

Developmental Stage ◽

Gene Deletion ◽

Leishmania Major ◽

Major Genes ◽

Targeted Gene Deletion ◽

Genes Encoding

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Effect of garlic-derived allyl sulphides on morphogenesis and hydrolytic enzyme secretion inCandida albicans

Medical Mycology ◽

10.3109/13693786.2010.539629 ◽

2011 ◽

Vol 49 (4) ◽

pp. 444-448 ◽

Cited By ~ 16

Author(s):

Snowber Yousuf ◽

Aijaz Ahmad ◽

Amber Khan ◽

Nikhat Manzoor ◽

Luqman A. Khan

Keyword(s):

Hydrolytic Enzyme ◽

Enzyme Secretion ◽

Incandida Albicans

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For want of a nail. ramifications of a single gene deletion, dystrophin, in the brain of the mouse

Frontiers in Bioscience ◽

10.2741/1209 ◽

2004 ◽

Vol 9 (1-3) ◽

pp. 74 ◽

Cited By ~ 9

Author(s):

Trent Wallis

Keyword(s):

Gene Deletion ◽

Single Gene ◽

The Brain ◽

Single Gene Deletion

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Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

Nucleic Acids Research ◽

10.1093/nar/gkaa204 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4585-4600

Author(s):

Gabriel A Suárez ◽

Kyle R Dugan ◽

Brian A Renda ◽

Sean P Leonard ◽

Lakshmi Suryateja Gangavarapu ◽

...

Keyword(s):

Gene Deletion ◽

Single Gene ◽

Acinetobacter Baylyi ◽

Gene Deletions ◽

Multiple Gene ◽

Transposon Insertion ◽

A Genome ◽

Acinetobacter Baylyi Adp1 ◽

Transposon Insertion Sequencing ◽

Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

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Characterization of Aminoacyl-tRNA Synthetases in Chromerids

Genes ◽

10.3390/genes10080582 ◽

2019 ◽

Vol 10 (8) ◽

pp. 582 ◽

Cited By ~ 1

Author(s):

Sharaf ◽

Gruber ◽

Jiroutová ◽

Oborník

Keyword(s):

Single Gene ◽

Single Copy ◽

Duplicated Genes ◽

Endosymbiotic Gene Transfer ◽

Cellular Compartment ◽

Trna Synthetases ◽

Aminoacyl Trna Synthetases ◽

Genes Encoding ◽

Eukaryotic Origin

Aminoacyl-tRNA synthetases (AaRSs) are enzymes that catalyze the ligation of tRNAs to amino acids. There are AaRSs specific for each amino acid in the cell. Each cellular compartment in which translation takes place (the cytosol, mitochondria, and plastids in most cases), needs the full set of AaRSs; however, individual AaRSs can function in multiple compartments due to dual (or even multiple) targeting of nuclear-encoded proteins to various destinations in the cell. We searched the genomes of the chromerids, Chromera velia and Vitrella brassicaformis, for AaRS genes: 48 genes encoding AaRSs were identified in C. velia, while only 39 AaRS genes were found in V. brassicaformis. In the latter alga, ArgRS and GluRS were each encoded by a single gene occurring in a single copy; only PheRS was found in three genes, while the remaining AaRSs were encoded by two genes. In contrast, there were nine cases for which C. velia contained three genes of a given AaRS (45% of the AaRSs), all of them representing duplicated genes, except AsnRS and PheRS, which are more likely pseudoparalogs (acquired via horizontal or endosymbiotic gene transfer). Targeting predictions indicated that AaRSs are not (or not exclusively), in most cases, used in the cellular compartment from which their gene originates. The molecular phylogenies of the AaRSs are variable between the specific types, and similar between the two investigated chromerids. While genes with eukaryotic origin are more frequently retained, there is no clear pattern of orthologous pairs between C. velia and V. brassicaformis.

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Mls is not a single gene, allelic system. Different stimulatory Mls determinants are the products of at least two nonallelic, unlinked genes.

Journal of Experimental Medicine ◽

10.1084/jem.166.4.1150 ◽

1987 ◽

Vol 166 (4) ◽

pp. 1150-1155 ◽

Cited By ~ 27

Author(s):

R Abe ◽

J J Ryan ◽

R J Hodes

Keyword(s):

T Cells ◽

Single Gene ◽

Surface Structures ◽

Chromosome 1 ◽

Gene Products ◽

Spleen Cells ◽

Gene Encoding ◽

Cell Surface Structures ◽

Genes Encoding ◽

Polymorphic Cell

Mls determinants share with MHC products the unique property of stimulating T cells at extraordinarily high precursor frequencies. The Mls system was originally described as a single locus on chromosome 1, with four alleles, Mlsa, Mlsb, Mlsc, and Mlsd, that encode polymorphic cell surface structures. However, the fundamental issues of polymorphism and allelism in the Mls system remain controversial. To clarify these questions, a formal segregation analysis of the genes encoding Mlsa and Mlsc determinants was carried out by testing the capacity of spleen cells from progeny of (Mlsa X Mlsc)F1 X Mlsb breedings to stimulate responses by unprimed T cells and by Mlsa- and Mlsc-specific cloned T cells. The results of this analysis indicated that the gene encoding Mlsa determinants is neither allelic to nor linked to the gene encoding Mlsc determinants. Together with previous findings, these results also suggest that another strongly stimulatory type, Mlsd, in fact results from the independent expression of unlinked Mlsa and Mlsc gene products. Based on these observations, it is concluded that, contrary to conventional concepts, the stimulatory phenotypes designated as Mlsa, Mlsc, and Mlsd can be accounted for by the independent expression of the products of at least two unlinked gene loci.

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A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging

Cell Metabolism ◽

10.1016/j.cmet.2015.09.008 ◽

2015 ◽

Vol 22 (5) ◽

pp. 895-906 ◽

Cited By ~ 129

Author(s):

Mark A. McCormick ◽

Joe R. Delaney ◽

Mitsuhiro Tsuchiya ◽

Scott Tsuchiyama ◽

Anna Shemorry ◽

...

Keyword(s):

Gene Deletion ◽

Single Gene ◽

Comprehensive Analysis ◽

Replicative Lifespan ◽

Single Gene Deletion

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Evaluation of Low Mean Corpuscular Volume in an Apheresis Donor Population.

Blood ◽

10.1182/blood.v108.11.4136.4136 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4136-4136

Author(s):

Barbara J. Bryant ◽

Julie A. Hopkins ◽

Susan F. Leitman

Keyword(s):

Iron Deficiency ◽

Mean Corpuscular Volume ◽

Gene Deletion ◽

Single Gene ◽

Transferrin Saturation ◽

Hemoglobin S ◽

Alpha Thalassemia ◽

Donor Population ◽

Thalassemia Trait ◽

Single Gene Deletion

Abstract Donors of apheresis blood components are routinely evaluated with a complete blood count (CBC) at the time of each donation. In otherwise healthy donors, recurrent low mean corpuscular volume (MCV) values (< 80 fL) in the presence of an acceptable hemoglobin (≥ 12.5 gm/dL) could be due to iron deficiency or to an hemoglobinopathy, such as alpha thalassemia trait or a beta chain variant trait. Iron deficiency in repeat blood donors may warrant treatment with oral iron supplementation, whereas donors with hemoglobinopathies in the absence of iron deficiency do not need treatment. Pre-donation samples for CBC (Cell-Dyn 4000, Abbott) were obtained from all apheresis donors donating platelets, plasma, granulocytes, lymphocytes, and monocytes. MCV values <80 fL were electronically flagged via a donor database module for review by medical staff. Donors with MCV ≤ 80 fL on two or more occasions were evaluated for iron deficiency and the presence of hemoglobinopathies. CBC, ferritin, serum iron, transferrin, percent transferrin saturation, and hemoglobin electrophoresis were performed at the time of a subsequent donation. Iron deficiency was defined as values below the reference range for ferritin or transferrin saturation. Alpha thalassemia trait was presumed if the red blood cell count was elevated, no variant hemoglobins were detected by electrophoresis, and the ferritin, percent transferrin saturation, serum iron, and transferrin levels were all within normal ranges. In a one-year period, 25 of 1333 healthy apheresis donors had a low MCV on more than one occasion. Donors with low MCV were more likely to be African American (AA) (12 of 25, 48%) or Asian (2 of 25, 8%) compared with donors without a low MCV (AA 193 of 1308, 15%; Asian 37 of 1308, 3%). Iron deficiency was present in 60% (15 of 25) of the low-MCV donors: 36% (9) had isolated iron deficiency, 20% (5) had iron deficiency with probable alpha thalassemia trait, and 4% (1) had hemoglobin C trait with coexistent iron deficiency. Hemoglobinopathy without concomitant iron deficiency was found in 40% (10 of 25) of the low-MCV donors and included 24% (6) with presumed alpha thalassemia trait, 4% (1) with hemoglobin S trait and single gene deletion alpha thalassemia trait (hemoglobin S concentration 34%), 4% (1) with hemoglobin S trait and double gene deletion alpha thalassemia trait (hemoglobin S concentration 28%), 4% (1) with hemoglobin Lepore trait, and 4% (1) with hemoglobin G-Philadelphia trait with at least a single gene deletion alpha thalassemia trait (hemoglobin G-Philadelphia concentration 36%). Although the combination of MCV, hemoglobin, and red cell count available from the routine CBC were often helpful in discriminating iron deficiency from hemoglobinopathy, the frequent coexistence of both processes resulted in a need for further laboratory evaluation, both before and after iron repletion, to confirm the diagnosis. In a sample of American repeat apheresis donors, iron deficiency is present in the majority with recurrent low MCV values and hemoglobin levels ≥ 12.5 gm/dL. Concurrent hemoglobinopathy is also commonly present but may not be easily recognized in the setting of iron deficiency. The MCV is a useful screening tool to detect iron deficiency in a repeat blood donor population, however low MCV values should be further investigated in the blood donor setting to determine if iron replacement therapy is indicated.

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