Epidermal Cell Junctions Research Articles

Targeted delivery of oxytetracycline to the epidermal cell junction and stomata for crop protection

A micronutrient-based nanosystem was utilized to target the delivery of oxytetracycline to the stomata and epidermal cell junctions. The findings have potential to significantly improve crop protection against surface-restricted pathogens.

A fungal Bipolaris bicolor strain as a potential bioherbicide for goosegrass (Eleusine indica) control.

Tea, one of the most important commercial crops on earth, is strongly affected by weeds on productivity and quality. Bioherbicides are shedding new light on weed control in tea gardens in an economical and safe manner. A pathogenic strain SYNJC-2-2 was isolated from diseased leaves of a noxious weed, goosegrass (Eleusine indica), from a tea garden in Zhejiang Province, China. It was identified as the fungal species Bipolaris bicolor based on the morphological characteristics and phylogenetic analysis. The potential of the B. bicolor strain SYNJC-2-2 as a bioherbicide was assessed by determining its efficacy to control weeds and selectivity to crops, its infection process and the influence of environmental conditions on conidial production and germination. The ED90 (effective dose of conidia resulting in 90 disease index) of SYNJC-2-2 on goosegrass was 2 × 104 conidia mL-1 . Additionally, three Poaceae weeds, Setaria viridis, Microstegium vimineum and Pennisetum alopecuroides, were also extremely susceptible to SYNJC-2-2. SYNJC-2-2 was safe to 14 out of 17 crop species in nine families, especially tea plants. Conidial germination, hyphal growth and appressorial formation occurred within 3 to 6h on goosegrass leaves. Hyphae invaded leaf tissues mainly through epidermal cell junctions and cracks, causing cell death and necrotic lesions within 2 days on inoculated leaves and killing goosegrass plants within 7 days. Furthermore, SYNJC-2-2 has a strong adaptability to environmental variables and high conidial production capacity on goosegrass juice agar media. Bipolaris bicolor strain SYNJC-2-2 has the potential to be developed as a bioherbicide for controlling goosegrass, especially in tea gardens.

Emergence of self-organizational patterning at the mesoscopic scale.

In this issue of Developmental Cell, Chuyen etal. (2021) suggest that the Scf/Kit pathway controls mutual repulsion of multiciliated cells and their affinity for epidermal cell junctions through soluble and membrane-associated Scf ligands, respectively. Effective self-organizational patterning emerges at the mesoscopic scale as a small set of effective behaviors.

A high-content imaging approach to profile C. elegans embryonic development.

The Caenorhabditis elegans embryo is an important model for analyzing mechanisms of cell fate specification and tissue morphogenesis. Sophisticated lineage-tracing approaches for analyzing embryogenesis have been developed but are labor intensive and do not naturally integrate morphogenetic readouts. To enable the rapid classification of developmental phenotypes, we developed a high-content method that employs two custom strains: a Germ Layer strain that expresses nuclear markers in the ectoderm, mesoderm and endoderm/pharynx; and a Morphogenesis strain that expresses markers labeling epidermal cell junctions and the neuronal cell surface. We describe a procedure that allows simultaneous live imaging of development in 80-100 embryos and provide a custom program that generates cropped, oriented image stacks of individual embryos to facilitate analysis. We demonstrate the utility of our method by perturbing 40 previously characterized developmental genes in variants of the two strains containing RNAi-sensitizing mutations. The resulting datasets yielded distinct, reproducible signature phenotypes for a broad spectrum of genes that are involved in cell fate specification and morphogenesis. In addition, our analysis provides new in vivo evidence for MBK-2 function in mesoderm fate specification and LET-381 function in elongation.

Herpobasidium filicinum (Eocronartiaceae, Platygloeales) occurs on Dennstaedtia wilfordii (Dennstaedtiaceae) in Japan

A fungus parasitic on a fern, Dennstaedtia wilfordii (Dennstaedtiaceae), was found at the foothill of Mt. Fuji, Yamanashi Prefecture, Japan. Its hyphae spread within host mesophyll cells and through intercellular spaces, forming coiled haustoria in the epidermal and mesophyll cells. The hyphae emerged either through stomata or by disrupting epidermal cell junctions. The hyphae spreading over the abaxial leaf surface generated one-septate, thin-walled basidia. All the morphological features observed were characteristic of the genus Herpobasidium. The species identification of the fungus as H. filicinum by morphology was supported by molecular phylogenetic analyses of the D1/D2 region of the large subunit rRNA gene.

Epidermal cell junctions and their regulation by p63 in health and disease.

As the outermost tissue of the body, the epidermis is the first physical barrier for any pressure, stress or trauma. Several specialized cell-matrix and cell-cell adhesion structures, together with an intracellular network of dedicated intermediate filaments, are required to confer critical resilience to mechanical stress. The transcription factor p63 is a master regulator of gene expression in the epidermis and in other stratified epithelia. It has been extensively demonstrated that p63 positively controls a large number of tissue-specific genes, including those encoding a large fraction of tissue-restricted cell adhesion molecules. Consistent with p63 functions in cell adhesion and in epidermal differentiation, heterozygous mutations clustered mainly in the p63 C-terminus are causative of AEC syndrome, an autosomal dominant disorder characterized by cleft palate, ankyloblepharon and ectodermal dysplasia associated with severe skin erosions, bleeding and infections. The molecular basis of skin erosions in AEC patients is not fully understood, although defects in desmosomes and in other cell junctions are likely to be involved. Here, we provide an extensive review of the different epidermal cell junctions that cooperate to withstand mechanical stress and on the mechanisms by which p63 regulates gene expression of their components in healthy skin and in AEC syndrome. Collectively, advancement in understanding the molecular mechanisms by which epidermal cell junctions precisely exert their functions and how p63 orchestrates their coordinated expression, will ultimately lead to insight into developing future strategies for the treatment of AEC syndrome and more in generally for diseases that share an overlapping phenotype.

Confocal microscopy study to understand Clonostachys rosea and Botrytis cinerea interactions in tomato plants

Clonostachys rosea, a biological control agent for plant diseases, is found in a variety of habitats and colonises and survives in different tissues. This antagonist is effective at controlling grey mould, which is caused by Botrytis cinerea, in different plant species. Despite the existing knowledge regarding the efficiency of C. rosea at biologically controlling grey mould, there are few studies concerning this interaction at the histological level. Therefore, we studied the antagonist–pathogen interactions using confocal microscopy. C. rosea survived in tomato tissues for at least 30 days between 18–30ºC. The antagonist colonised the wounded tomato stems faster and more efficiently than the pathogen. The colonisation of the leaf tissues by C. rosea was slow, and the spore concentration was poor in this experiment. Combined with the pathogen’s direct penetration into the leaves, this slow colonisation could cause the biological control to fail. C. rosea also preyed parasitically upon the pathogen’s hyphae, penetrated the tomato’s leaf tissue through the stomata and colonised the stem’s intercellular spaces. Root colonisation was abundant, with a dense hyphae network forming between epidermal cell junctions. This observation provided evidence that the fungus can penetrate via the roots. This paper will help to better define an application strategy for C. rosea in tomato propagation, with the goal of biological control or growth promotion, because to understand how the antagonist survives and interacts in its habitat will define how and when to apply it.

Genetic transformation, infection process and qPCR quantification of Verticillium dahliae on smoke-tree Cotinus coggygria

The smoke-tree vascular wilt fungus, Verticillium dahliae, was successfully transformed via PEG-mediated protoplasts using cassettes for eGFP and hygromycin B resistance (hph) gene expression. The transformants were stable after alternative sub-culturing on selective and non-selective medium for 6 months. The transformed isolates did not differ markedly from wild-type strains in terms of growth, morphological characteristics, asexual conidiation and pathogenicity. Fluorescence observations clearly indicated strong expression of the green fluorescent protein in fungal structures. The colonization and penetration of smoke-tree roots by eGFP-tagged V. dahliae was monitored by fluorescence, and the early infection processes were clearly visualized. At 24-hour-post-inoculation (hpi), hyphae were found to be predominantly colonizing the fibers rather than the taproots, and subsequently, hyphae started to grow parallel to the longitudinal axis of the root along the junctions of epidermal cells at 36 hpi. At 48 hpi, the elongated hyphae were interspersed among the root hairs and the first observation of central cylinder colonization occurred at 5 dpi. Elevated amounts of fungal DNA were detectable in stems 6 dpi, in branches 12 dpi, and in leaves 14 dpi. The data indicated that once V. dahliae enters smoke-tree, the pathogen rapidly spreads and increases in planta, and GFP can be used as a vital and stable marker for studying the interaction between V. dahliae and woody plants. Furthermore, the establishment of a PEG-mediated transformation system will contribute to functional genomics studies of smoke-tree Verticillium wilt fungus.

δ-Aminolevulinate synthase is required for apical transcellular barrier formation in the skin of the Drosophila larva

Animals construct a layered skin to prevent dehydration and pathogen entrance. The barrier function of the skin relies on the extensive cross-linking of specialised components. In insects, for instance, epidermal cells produce an apical extracellular cuticle that consists of a network of proteins, chitin and lipids. We have identified mutations in the Drosophila gene coding for the δ-aminolevulinate synthase (Alas) that cause massive water loss. The cuticle of alas mutant larvae detaches from the epidermis and its basal region is frayed suggesting that an Alas dependent pathway is needed to organise the contact between the cuticle and the epidermis and anchor the cuticle to the apical surface of epidermal cells. Concomitantly, reduction of Alas function results in weakening of the extracellular dityrosines network in the cuticle, whereas glutamyl–lysine isopeptide bonds are not affected. The lateral septate junctions of epidermal cells that serve as a paracellular plug are intact, as well. Taken together, we hypothesise that Alas activity, which initiates heme biosynthesis in the mitochondrion, is needed for the formation of a dityrosine-based barrier that confers resistance to the internal hydrostatic pressure protecting both the cuticle from transcellular infiltration of body fluid and the animal from dehydration. We conclude that at least two modules – an apical protein–chitin lattice and the lateral septate junctions, act in parallel to ensure Drosophila skin impermeability.

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata ( Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum ( Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10 5 CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance ( hph) and β-glucuronidase ( uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.

Symptomatic Evidence for Differential Root Invasion by Fusarium Crown and Root Rot Pathogens Between Common Tomato Lycopersicon esculentum and Its Varieties

AbstractThe pathogenic isolates (Kin2001a, Kin2001b and Kin2003) of Fusarium oxysporum f. sp. radicis‐lycopersici were obtained from hydroponically cultured seedlings of pear tomato (Lycopersicon esculentum var. pyriforme) infected at different times and their pathogenicity examined in an in vitro assay system on cotyledonal seedlings of pear tomato, cherry tomato (L. esculentum var. cerasiforme) and common tomato (L. esculentum). With the in vitro assay, infection and subsequent disease progress could be microscopically observed. Pear and cherry tomatoes suppressed invasion by all isolates at the junctions of epidermal cells along the root, comparable with the resistant cultivars of common tomato. The pathogen entered pear and cherry tomatoes at the tips of lateral roots and tap roots, in contrast to infection of susceptible cultivars of common tomato. In Kin2003‐inoculated roots, the top of the lateral rootlets first became discoloured, followed by the cortical parenchyma, central xylem vessel and finally the crown. This dark‐brown discolouration expanded rapidly and severe rot developed in the discoloured regions. In contrast, the dark‐brown discolouration in Kin2001b‐infected roots expanded into the cortical parenchyma cells abutting the originally infected lateral rootlets and at a much slower rate. Kin2001a was in a new group that entered via the cortical cleavage formed by the emergence of lateral rootlets, in addition to the tips of taproots and lateral roots. In this in vitro assay system, the Japanese pathogenic isolates collected from different districts of Japan were characterized and classified by the mode of host invasion. Of 13 isolates, four were placed with Kin2003, six with Kin2001a and three with Kin2001b.

Conidial morphology, virulence, molecular characterization, and host–parasite interactions of selected Alternaria panax isolates on American ginseng

The fungus Alternaria panax Whetzel causes leaf and stem blight of American ginseng ( Panax quinquefolius L.). Six isolates of A. panax, collected from widely separated ginseng-growing areas in North America, were characterized on the basis of their colony and conidial morphology, molecular characteristics, and virulence. Colony morphology on standard media and conidial morphology based on light microscopy showed some variation with geographic locality: Wisconsin isolates were slightly different from Ontario and British Columbia isolates. Isolates from each locality were equally virulent on American ginseng. Ginseng phytotoxin was present in the conidial germination fluid of all isolates. Sequences of the β-tubulin gene from all six isolates were identical as were the sequences of the internal transcribed spacer (ITS) region of the rDNA gene. The ITS1 and ITS2 gene sequences of the six isolates were also identical to those of American and Korean isolates of A. panax deposited in GenBank. Cellulase and pectinase were detected by agar biodiffusion assays of a representative isolate. Germination of conidia of this isolate on ginseng leaflets began within 2 h of application to leaf surfaces, and over 80% of conidia germinated within 12 h of application. Conidia produced one to several germ tubes that grew in all directions over the leaflet surface. The germ tubes formed terminal appressoria over stomatal openings, epidermal cells, and epidermal cell junctions. The frequency of direct penetration into ginseng leaflets was greater than penetration through stomata. Both retraction of plasma membrane and disruption of chloroplast membranes were observed 12 h after leaflet inoculation. At 24 h after inoculation, hyphae were present in both intercellular spaces and inside mesophyll cells. Severe disruption of plasma membrane, chloroplasts, and mitochondria was observed between 24 and 48 h after inoculation.

Stable phylloplane colonization by entomopathogenic bacterium Pseudomonas fluorescens KPM-018P and biological control of Phytophagous ladybird beetles Epilachna vigintioctopunctata (Coleoptera: Coccinellidae)

An entomopathogenic bacterium was isolated from tomato leaves and used as a microbial agent to control larvae of phytophagous ladybird beetles Epilachna vigintioctopunctata. The isolate was identified as Pseudomonas fluorescens KPM-018P on the basis of its bacteriological characteristics. KPM-018P produced extracellular chitinase to form a transparent zone around their colonies by hydrolyzing chitin in a minimal medium. Pale-yellow colonies turned red after a change of incubation temperature. These characteristics were availed as markers for tracking KPM-018P. The bacteria produced biosurfactants that enabled the bacteria to stably colonize the hydrophobic leaf surface; they were recovered without any considerable decrease even after a suspension of KPM-018P was sprayed onto leaves. KPM-018P, transformed with the gfp gene and observed with fluorescence microscopy, stably dwelled in the junctions of epidermal cells of bacteria-sprayed leaves. Ingestion of KPM-018P-sprayed leaves by the larvae caused prompt death of these insects to eventually suppress their pupation. This method is thus effective for decreasing the population of larvae and adult insect pests in the subsequent generation. The study provides an experimental basis for the biocontrol of herbivorous insect pests using a leaf-inhabiting, entomopathogenic strain of P. fluorescens.

Leaf wax layer may prevent appressorium differentiation but does not influence orientation of the leaf rust fungus Puccinia hordei on Hordeum chilense leaves

The infection process of most rust fungi start with spore germination, directional growth of the germ tube towards a stoma, differentiation of an appressorium over the stoma, and penetration into the substomatal cavity. In the South American wild barley Hordeum chilense Roem. & Schult., wide variation occurs in the degree to which several rust fungal species are able to form appressoria over the stomata. Apparently, features of the plant may hamper early stages of the infection process. Such an early defence is called avoidance. In order to find out how germ tube growth is directed towards stomata, and whether the cuticular wax layer plays a role in this orientated growth and in appressorium differentiation, several orientation and differentiation parameters of Puccinia hordei germ tubes were measured on H. chilense leaves with and without the wax layer. Orientated growth of the germ tubes started upon contact with the epidermal cell junctions. The growth of lateral branches of the germ tube over the first epidermal cell junction that it meets, may help the germ tube to grow along the transverse axis of the leaf. No evidence was found of attraction of the germ tube to stomata. Removal of the cuticular wax layer did not result in loss of germ tube orientation. This suggests that the leaf wax layer has no role in the guidance of germ tubes. On high avoidance accessions, removal of the wax layer allowed appressoria to develop over stomata that would otherwise be overgrown. No effect of the cell widths in stomatal complexes was found on the chance that stomata were overgrown. This suggests that the overgrowth of stomata on H. chilense leaves by P. hordei germ tubes is mainly caused by the wax covering of the stomatal apparatus.

Whats new in genodermatoses?

Recent genetic analysis of the genodermatoses, in particular the palmoplantar keratodermas, has identified the important role of proteins involved in the regulation and formation of epidermal cell junctions. There are four major types of junction, of which three have been demonstrated to be important in skin, and in which component proteins such as desmoplakin and connexins are mutated in epidermal disease. These are the gap junctions, desmosomes and adherens junctions. These junctions are responsible for cell-cell adhesion and communication, key properties to maintain the normal cellular phenotype and tissue architecture.

Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris.

Pemphigus foliaceus and pemphigus vulgaris are skin diseases in which antibodies against the cell surface of keratinocytes destroy the adhesion between epidermal cells, producing blisters. Patients with pemphigus foliaceus have antibodies to a complex of three polypeptides of 260, 160, and 85 kd (the foliaceus complex), whereas patients with pemphigus vulgaris have antibodies to a complex of 210-kd, 130-kd, and 85-kd polypeptides (the vulgaris complex). The 160-kd polypeptide of the foliaceus complex has been identified as desmoglein, a desmosomal glycoprotein. We suspected that the 85-kd component in both these antigenic complexes might be plakoglobin, another molecule in the adhering junctions of cells. To characterize these antigenic complexes, we used the serum of five patients with pemphigus foliaceus, that of four patients with pemphigus vulgaris, and monoclonal antiplakoglobin antibodies. We found that monoclonal antibodies to plakoglobin immunoprecipitated the 85-kd polypeptide from the dissociated foliaceus and vulgaris complexes and precipitated both complexes from epidermal extracts. Serum from patients with pemphigus foliaceus or pemphigus vulgaris (but not from four normal controls) bound desmoglein and the 130-kd polypeptide, respectively, showing that these peptides (and not plakoglobin) are the antigenic binding sites in these disorders. We conclude that plakoglobin, a protein of the adhering junctions of epidermal cells, is the 85-kd molecule in the antigenic complexes found in both pemphigus foliaceus and pemphigus vulgaris, although it is not the binding site in either disorder.

Penetration of Zea mays by Helminthosporium carbonum

Light microscopy showed that on corn leaves, 81–93% of appressoria of Helminthosporium carbonum races I and II are formed over junctions of epidermal cells. During the early stages of penetration of corn plants resistant and susceptible to race I, 63–83% of appressoria have an epidermal cell nucleus within 10 μm. Transmission electron microscopy of race II on inbred W187R showed that appressoria are attached to the cuticle by fibrillar material. Vesicles are present in the appressorium at the site of cuticle penetration and initial cuticle penetration appears to be enzymic; subsequent rupture may be mechanical as the penetration peg widens. A septum forms between the appressorium and the subcuticular hyphae. Epidermal cell cytoplasm is thicker beneath penetration sites than elsewhere under the epidermal wall. Changes in epidermal cytoplasm were observed 8 h after inoculation; by 18 h epidermal cells beneath subcuticular hyphae have electron-opaque contents. Hyphae are mainly subcuticular up to 48 h after inoculation, and underlying epidermal and mesophyll cells are frequently collapsed. Results indicate that H. carbonum races I and II have similar initial reactions on susceptible and resistant genotypes and that penetration occurs by degradation of the cuticle and host cell walls.