scholarly journals Ultrastructural Characterization of Infection and Colonization of Maize Leaves by Colletotrichum graminicola, and by a C. graminicola Pathogenicity Mutant

2002 ◽  
Vol 92 (7) ◽  
pp. 803-812 ◽  
Author(s):  
C. W. Mims ◽  
L. J. Vaillancourt
Keyword(s):  
Host Cells ◽  
Signal Peptidase ◽  
Colletotrichum Graminicola ◽  
Wild Type ◽  
Mesophyll Cells ◽  
Ultrastructural Characterization ◽  
Significant Difference ◽  
Maize Leaves ◽  
Short Time

Observations were made of the ultrastructure of infection and colonization of leaves of a susceptible maize inbred by Colletotrichum graminicola and by a C. graminicola pathogenicity mutant. The mutant causes no symptoms on either maize leaves or stalks. Prior evidence suggested that it is deficient in production of signal peptidase, responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum. There was no significant difference in the process of infection or colonization by the mutant and wild-type strains up to 48 h after inoculation. Both the mutant and the wild type produced globose, melanized appressoria within 24 h after inoculation on the host surface. By 36 h, both strains had penetrated the host epidermal cells directly. The host cells frequently formed papillae in response to appressoria, but these were not usually successful in preventing fungal ingress in either case. Penetration was followed by formation of irregularly shaped, swollen infection hyphae. Infection hyphae of both strains grew biotrophically for a relatively short time (less than 12 h). One or more hyphal branches was produced from each infection hypha, and these invaded adjacent mesophyll cells. Both strains of the fungus grew cell-to-cell, setting up new biotrophic interactions in each cell, between 36 and 48 h after inoculation. Papillae were frequently formed by the mesophyll cells, but these were not successful in preventing fungal ingress. The first noticeable difference between the mutant and the wild type was related to their interaction with mesophyll cells. Cells invaded by the wild type died relatively quickly, whereas those infected by the mutant appeared to survive longer. The most dramatic difference between the mutant and wild type occurred when the mutant completely failed to make a transition to necrotrophic growth, while the wild type made that switch at 48 to 72 h after inoculation. The mutant may be unable to secrete sufficient quantities of one or more proteins that are necessary to support the switch between biotrophy and necrotrophy.

scholarly journals CPR1: A Gene Encoding a Putative Signal Peptidase That Functions in Pathogenicity of Colletotrichum graminicola to Maize

2002 ◽  
Vol 15 (2) ◽  
pp. 120-128 ◽  
Author(s):  
M. R. Thon ◽  
E. M. Nuckles ◽  
J. E. Takach ◽  
L. J. Vaillancourt
Keyword(s):  
Protein Transport ◽  
Stop Codon ◽  
Host Cells ◽  
Signal Peptidase ◽  
Colletotrichum Graminicola ◽  
Wild Type ◽  
Gene Encoding ◽  
Knock Out ◽  
Stalk Rot ◽  
Rot Disease

Colletotrichum graminicola causes anthracnose leaf blight and stalk rot of maize. We used restriction-enzyme mediated insertional (REMI) mutagenesis to identify a gene in this fungus that is required for pathogenicity to both stalks and leaves. The predicted polypeptide encoded by this gene, which we have named CPR1, is similar to a family of proteins that comprise one subunit of the eukaryotic micro-somal signal peptidase. The nonpathogenic CPR1 REMI mutant contains a plasmid integration in the 3′ untranslated region of the gene, 19 bp downstream from the stop codon. The result is a significant reduction in transcript levels in comparison to the wild type, perhaps as a result of increased transcript instability. We were unable to knock out the CPR1 gene, and it may be essential for viability. Microscopic examination of the REMI mutant on maize leaves revealed that it is fully capable of penetrating and colonizing host cells during the initial, biotrophic phases of the disease interaction but, unlike the wild type, it appears to be unable to switch to a necrotrophic mode of growth. We suggest that the CPR1 REMI mutant may be unable to secrete sufficient quantities of degradative enzymes to support that transition. The CPR1 REMI mutant provides us with a useful tool for future studies of the role of fungal protein transport in this important stalk rot disease of maize.

Ultrastructural characterization of a pigment mutant of the red alga Palmaria palmata

1984 ◽  
Vol 62 (6) ◽  
pp. 1101-1107 ◽  
Author(s):  
C. M. Pueschel ◽  
J. P. van der Meer
Keyword(s):  
Red Alga ◽  
Palmaria Palmata ◽  
Wild Type ◽  
Ultrastructural Examination ◽  
Dark Treatment ◽  
Prolamellar Bodies ◽  
Ultrastructural Characterization ◽  
Wild Type Control

Ultrastructural examination of a green-pigmented mutant of the red alga Palmaria palmata (L.) O. Kuntze revealed unusual features of the chloroplasts. Encircling peripheral thylakoids, characteristic of the wild-type plastids and florideophyte plastids generally, were lacking. Parallel evenly spaced thylakoids occurred in groups, leaving large volumes of thylakoid-free stroma. Irregularly shaped, electron-dense inclusions with an amorphous substructure and diameters up to 3 μm occurred in some plastids. Cells of the sporeling holdfasts contained structures resembling prolamellar bodies. Attempts to induce formation of prolamellar bodies in blades by dark treatment for 5 weeks were unsuccessful. However, some plastids did develop highly corrugated thylakoids with the crests of one thylakoid apposed to the troughs of the adjacent thylakoid. Thylakoid morphology of the wild-type control was not altered by the absence of light.

OmpD but not OmpC is involved in adherence ofSalmonella entericaserovar Typhimurium to human cells

2004 ◽  
Vol 50 (9) ◽  
pp. 719-727 ◽  
Author(s):  
Bochiwe Hara-Kaonga ◽  
Thomas G Pistole
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Human Cells ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Human Macrophages ◽  
Significant Difference ◽  
Serovar Typhimurium

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 °C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.Key words: Salmonella, adherence, porins, intestinal epithelial cells, macrophage.

Ultrastructural characterization of male sterile33 (ms33) mutant in Arabidopsis affected in pollen desiccation and maturation

2001 ◽  
Vol 79 (1) ◽  
pp. 118-129 ◽  
Author(s):  
Houman Fei ◽  
Vipen K Sawhney
Keyword(s):  
Pollen Development ◽  
Ultrastructural Level ◽  
Mitotic Division ◽  
Wild Type ◽  
Pollen Maturation ◽  
Ultrastructural Characterization ◽  
In The Wild ◽  
Pollen Stage ◽  
Mutant Pollen

The MS33 gene in Arabidopsis is required for stamen filament growth and for pollen maturation. The objective of this study was to characterize the effects of ms33 mutation on pollen development at the ultrastructural level. There were no differences between the wild type and ms33 mutant pollen development before the first mitotic division of microspores. At the bicellular pollen stage, the first signs of abnormalities were observed in the ms33 tapetum, which started to degenerate early and released osmiophilic material in the anther locule. In ms33 pollen, the endintine was thicker, and exintine thinner, than in the wild type, and the mutant pollen had large vacuoles. Later in development, the mutant pollen underwent second mitosis and produced two normal-looking sperm cells; however, the intine was precociously formed, and there were abnormalities in tryphine deposition on the pollen wall, in the size of vacuoles, and in the formation of lipid bodies in the vegetative cell cytoplasm. Based on these observations it is suggested that mutation in the MS33 gene interferes with intine formation and tryphine deposition, both of which negatively affect pollen desiccation resulting in large, highly vacuolate pollen that are nonviable.Key words: Arabidopsis, male sterility, mutant, pollen, tapetum, ultrastructure.

Ultrastructural characterization of an extracellular fibrillar sheath on cells of Ascocalyx abietina, the scleroderris canker agent of conifers

1987 ◽  
Vol 65 (1) ◽  
pp. 154-167 ◽  
Author(s):  
Nicole Benhamou ◽  
G. B. Ouellete
Keyword(s):  
Rnase A ◽  
Host Cells ◽  
Biological Functions ◽  
Intact Cells ◽  
Physiological Conditions ◽  
Physical Conditions ◽  
Ultrastructural Characterization ◽  
Cationic Compounds ◽  
Rnase B

Morphology, ultrastructure, and some aspects of the chemical composition of a fibrillar sheath surrounding cells of the fungus Ascocalyx abietina (Lagerberg.) Schlaepfer-Bernhard were studied using electron microscopy and gold-labeled ligands. Although consistently present around all cells, the fibrillar matrix was found to vary greatly in morphology within the same isolate, depending apparently on age and (or) physiological conditions of the cells. Around cells considered younger, the sheath appeared always to be constituted of fibrillar masses that varied in size and shape but were delineated by a well-defined border. In contrast, cells expected to be older were generally bordered by a regular and uniform matrix composed of numerous intertwined fine fibrils, some being associated with small osmiophilic knobs. The presence of RNA in the denser layers of the sheath was revealed through gold complexes with either RNase A or RNase B. Continuity of portions of the sheath with similar material surrounding endocells or intact cells through gaps in the wall was frequently observed. This observation was considered as one of the possible explanations for the presence of RNA in the sheath. Association of sugars such as β-glucopyranosides and especially sialic acid with the extracellular matrix is, most probably, relevant to specific biological functions such as attachment to host cells, protection against unfavourable physical conditions and transport of cationic compounds. Peculiarities of this sheath produced by A. abietina contribute, therefore, to distinguish it from those described in other fungi.

scholarly journals Isolation and Characterization of Mini-Tn5Km2 Insertion Mutants of Brucella abortus Deficient in Internalization and Intracellular Growth in HeLa Cells

2003 ◽  
Vol 71 (6) ◽  
pp. 3020-3027 ◽  
Author(s):  
Suk Kim ◽  
Masahisa Watarai ◽  
Yuki Kondo ◽  
Janchivdorj Erdenebaatar ◽  
Sou-ichi Makino ◽  
...  
Keyword(s):  
Hela Cells ◽  
Brucella Abortus ◽  
Kanamycin Resistance ◽  
Host Cells ◽  
Class Iii ◽  
Wild Type ◽  
Intracellular Growth ◽  
Isolation And Characterization ◽  
Bacterial Genes

ABSTRACT Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.

scholarly journals Minimising severity of dengue serotype 1 infection by transmissible interfering particles

2020 ◽  
Author(s):  
A. Shausan ◽  
J. Aaskov ◽  
C. Drovandi ◽  
K. Mengersen
Keyword(s):  
Therapeutic Agent ◽  
Antiviral Agent ◽  
Host Cells ◽  
Wild Type Virus ◽  
Parameter Estimates ◽  
Type Virus ◽  
Wild Type ◽  
Significant Difference ◽  
Serotype 1

AbstractTransmissible interfering dengue particles (DENV–TIPs) are engineered dengue virus mutants which are defective and can replicate only with the help of dengue wild–type virus (DENV). In vitro studies have found that when DENV–TIPs and DENV coinfect a cell, they compete for viral genomes and cell proteins for replication and packaging, and DENV–TIPs outperform DENV in this process. Thus, it is hypothesised that DENV–TIPs may be used as a novel therapeutic agent. However, the effectiveness of DENV–TIPs as an antiviral agent is yet to be explored at an epidemiological scale. We present a mathematical model for the replication of DENV and DENV–TIPs as they interact with human host cells, accounting for the effectiveness of DENV–TIPs in blocking DENV from coinfected cells. We fit the model to sequentially measured plasma viral titre data from primary and secondary dengue serotype 1 infected patients in Vietnam. We show that variation in initial DENV load is sufficient to recreate the observed variation between patients. Parameter estimates, differing in primary and secondary infections, do not confirm a significant difference between these two types of infection. We use our model to investigate the potential impact of DENV–TIPs as an antiviral agent. We conclude that, when the effectiveness of DENV–TIPs in inhibiting DENV from coinfected cells is at least 80%, a dose as high as 1012 copies per millilitre of blood is required to reduce duration of infection and peak DENV serotype 1 infection level at any time point of infection. This work provides a quantitative understanding of the relationship between DENV–TIPs levels and their efficiency in clearing dengue viral infection. It will guide future development of mechanistic models of how DENV–TIPs might contribute as an antiviral agent in limiting natural dengue infection.Author summaryInhibition of dengue wild–type virus (DENV) by transmissible interfering dengue particles (DENV–TIPs) is seen in some in vitro studies, and it is hypothesised that DENV–TIPs may be used as a therapeutic agent. However, the efficiency of DENV–TIPs in limiting DENV infection in patients is yet to be explored at an epidemiological scale. Using data collected from dengue serotype 1 infected patients, we model how DENV replicates in an infected patient and how effective DENV–TIPs are in controlling that replication. Our results are of use in the evaluation of DENV–TIPs as a potential antiviral agent.

scholarly journals Inactivation of Lgt Allows Systematic Characterization of Lipoproteins from Listeria monocytogenes

2006 ◽  
Vol 189 (2) ◽  
pp. 313-324 ◽  
Author(s):  
Maja Baumgärtner ◽  
Uwe Kärst ◽  
Birgit Gerstel ◽  
Martin Loessner ◽  
Jürgen Wehland ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Culture Supernatant ◽  
Eukaryotic Cell ◽  
Signal Peptidase ◽  
Wild Type ◽  
Intracellular Growth ◽  
Extracellular Proteome ◽  
Insight Into

ABSTRACT Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Δlgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Δlgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Δlgt ΔprfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.

Control of cellular differentiation in maize leaves

1995 ◽  
Vol 350 (1331) ◽  
pp. 53-57 ◽  
Keyword(s):  
Cellular Differentiation ◽  
Functional Characterization ◽  
Cell Types ◽  
Bundle Sheath ◽  
Gene Products ◽  
Mesophyll Cells ◽  
Bundle Sheath Cells ◽  
Maize Leaves ◽  
Single Cell Type

Mature maize leaves exhibit a series of parallel veins that are surrounded by concentric rings of bundle sheath and mesophyll cells. To identify genes that control cellular differentiation patterns in the leaf, we have isolated a group of mutations that specifically disrupt the differentiation of a single cell-type. In bundle sheath defective ( bsd ) mutant plants, bundle sheath cells fail to differentiate yet mesophyll and all other leaf cell-types develop normally. Morphological and functional characterization of specific bsd mutants ( bsd1, bsd2, bsd3, pg14 and g2 ) reveals that they differ in the degree to which bundle sheath cell differentiation is perturbed. Mutant analysis predicts roles for BSD gene products in normal development.

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