Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis

BACKGROUND: The plant cuticle is an extracellular lipophilic biopolymer covering leaf and fruit surfaces. Its main function is the protection of land-living plants from uncontrolled water loss. In the past, the permeability of the cuticle to water and to non-ionic lipophilic molecules (pesticides, herbicides and other xenobiotics) was studied intensively, whereas cuticular penetration of polar ionic compounds was rarely investigated. RECENT PROGRESS: Recent work measuring cuticular penetration of inorganic and organic ions is presented; the effects of molecular size of ions, temperature, wax extraction, humidity and plasticizers strongly support the conclusion that ions penetrate cuticles via water-filled pores. The cuticle covering stomata and trichomes forms the preferential site of ion penetration. This indicates that cuticles possess a pronounced lateral heterogeneity: the largest fraction of the cuticle surface is covered by the lipophilic domains of cutin and wax, but to a certain extent polar domains are also present in the cuticle, which form preferential sites of penetration for polar compounds. THE FUTURE: The chemical nature of these polar domains awaits detailed characterization, which will be of major importance in agriculture and green biotechnology, since polar paths of diffusion represent the most important transport routes for foliar-applied nutrients. Furthermore, many compounds acting as inducers of gene expression in transgenic plants are ionic and need to penetrate the cuticle via polar paths in order to be active.     

The Influences of Epicuticular Wax Disruption and Cutinase Resistance on Penetration of Tomatoes by Colletotrichum gloeosporioides

Conidia of Colletotrichum gloeosporioides germinated on green and ripe tomato fruit with intact epicuticular wax, and formed penetration pegs below melanized appressoria. If the delicate layer of epicuticular wax was distupted by abrasion or removed by solvents before inoculation, apparent increased diffusion of fruit substances into the inoculum stimulated fungal growth, hyphal anastomosis and the production of penetration pegs from hyaline appressoria. This was followed by cutlcle erosion centred on the penetration pegs in green fruit allowing sec-ondary growth of infection hyphae. Due to the development of cutinase resistance when the cuticle became yellow at ripening, no cuticle erosion occurred at penetrations on ripe fruit Since cuticle erosion followed penetration of the cutinase-susceptible cuticle and since penetration peg formation was not hindered by the cutinase cuticle, the process of primary penetration is regarded as mechanical.     

Magnaporthe grisea cutinase2 mediates appressorium differentiation and host penetration and is required for full virulence

The rice blast fungus Magnaporthe grisea infects its host by forming a specialized infection structure, the appressorium, on the plant leaf. The enormous turgor pressure generated within the appressorium drives the emerging penetration peg forcefully through the plant cuticle. Hitherto, the involvement of cutinase(s) in this process has remained unproven. We identified a specific M. grisea cutinase, CUT2, whose expression is dramatically upregulated during appressorium maturation and penetration. The cut2 mutant has reduced extracellular cutin-degrading and Ser esterase activity, when grown on cutin as the sole carbon source, compared with the wild-type strain. The cut2 mutant strain is severely less pathogenic than the wild type or complemented cut2/CUT2 strain on rice (Oryza sativa) and barley (Hordeum vulgare). It displays reduced conidiation and anomalous germling morphology, forming multiple elongated germ tubes and aberrant appressoria on inductive surfaces. We show that Cut2 mediates the formation of the penetration peg but does not play a role in spore or appressorium adhesion, or in appressorial turgor generation. Morphological and pathogenicity defects in the cut2 mutant are fully restored with exogenous application of synthetic cutin monomers, cAMP, 3-isobutyl-1-methylxanthine, and diacylglycerol (DAG). We propose that Cut2 is an upstream activator of cAMP/protein kinase A and DAG/protein kinase C signaling pathways that direct appressorium formation and infectious growth in M. grisea. Cut2 is therefore required for surface sensing leading to correct germling differentiation, penetration, and full virulence in this model fungus.     

Cutinase in Cryphonectria parasitica, the chestnut blight fungus: suppression of cutinase gene expression in isogenic hypovirulent strains containing double-stranded RNAs.

Plant-pathogenic fungi produce cutinase, an enzyme required to degrade plant cuticles and facilitate penetration into the host. The absence of cutinase or a decrease in its production has been associated with a decrease in pathogenicity of the fungus. A set of isogenic strains of Cryphonectria parasitica, the chestnut blight fungus, was tested for the presence and amounts of cutinase activity. The virulent strain of C. parasitica produced and secreted significantly higher amounts of cutinase than the hypovirulent strains. Use of both nucleic acid and polyclonal antibody probes for cutinase from Fusarium solani f. sp. pisi showed that cutinase in C. parasitica is 25 kDa in size and is coded by a 1.1-kb mRNA. Both mRNA and protein were inducible by cutin hydrolysate, while hypovirulence agents suppressed the level of mRNA and the enzyme. Since all the strains had the cutinase gene, the suppression of expression was due to the hypovirulence agents. The data presented are the first report indicating that hypovirulence agents in C. parasitica regulate a gene associated with pathogenicity in other plant-pathogenic fungi.     

SELECTIVITY MECHANISM FOR THE DIFFERENTIAL DESTRUCTION OF PLANT TISSUES BY METHYL DECANOATE EMULSION

Methyl decanoate emulsified with Tween 20 and sprayed on chrysanthemum plants kills the immature tissues. The mechanism of selectivity was found to be a property of the cuticle which acts as a penetration barrier to the emulsion. Disruption of cuticles before emulsion application resulted in death of underlying cells due to membrane destruction, as shown by electron microscopy. Small quantities of methyl decanoate, however, do penetrate the unaltered cuticle, resulting in moderate, reversible alterations to the ultrastructure of the cell. These alterations appear as aggregates of vesicles in the cytoplasm and larger aggregates of vesicles in the vacuole. No destruction of organelle membranes occurred, and visual symptoms were not seen. The marginal cuticular penetration was verified by isolated cuticle tests.     

Cutinase gene disruption in Fusarium solani f sp pisi decreases its virulence on pea.

Fusarium solani f sp pisi (Nectria haematococca) isolate 77-2-3 with one cutinase gene produced 10 to 20% of the cutinase produced by isolate T-8 that has multiple cutinase genes, whereas cutinase gene-disrupted mutant 77-102 of isolate 77-2-3 did not produce cutinase. On the surface of pea stem segments, lesion formation was most frequent and most severe with T-8, less frequent and less severe with 77-2-3, and much less frequent and much milder with the gene-disrupted mutant. Microscopic examination of the lesions caused by the mutant strongly suggest that it penetrated the host mostly via the stomata. In seedling assays, 77-2-3 caused severe lesions on every seedling and stunted growth, whereas the mutant showed very mild lesions on one-third of the seedlings with no stunting. Thus, cutinase gene disruption resulted in a significant decrease in the pathogenicity of F. s. pisi on pea.     

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.     

Appressorium-localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host-specific,toxin-producing fungus Alternaria alternata Japanese pear pathotype

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant–pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.     

Proof for the Production of Cutinase by Fusarium solani f. pisi during Penetration into Its Host, Pisum sativum

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this ferritin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.     

Cutinase is not required for fungal pathogenicity on pea.

Cutinase, a fungal extracellular esterase, has been proposed to be crucial in the early events of plant infection by many pathogenic fungi. To test the long-standing hypothesis that cutinase of Nectria haematococca (Fusarium solani f sp pisi) is essential to pathogenicity, we constructed cutinase-deficient mutants by transformation-mediated gene disruption of the single cutinase gene of a highly virulent N. haematococca strain. Four independent mutants were obtained lacking a functional cutinase gene, as confirmed by gel blot analyses and enzyme assays. Bioassays of the cutinase-deficient strains showed no difference in pathogenicity and virulence on pea compared to the wild type and a control transformant. We conclude that the cutinase of N. haematococca is not essential for the infection of pea.     

Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassiana as revealed by scanning electron microscopy

Conidia from highly pathogenic mutants of Beauveria bassiana germinate quickly (within 18 hr) on the surface of corn earworm larvae (Heliothis zea) and immediately begin penetration of the cuticle. Enzymes produced by the penetrating hyphae degrade the cuticle since holes are formed at the point of entry. Clustering of conidia around nodules of larvae is often seen, but penetration is not restricted to such areas. Direct evidence is presented to show that conidia can also germinate inside of spiracle openings and could invade larvae by this route. Once inside the hemocoel, the fungus multiplies extensively; however, larval death occurs with only minimal breakdown of internal tissues. During mummification, outgrowths of fungal hyphae occur first and most extensively in the intersegmental regions of larvae. Conidia from mutants exhibiting low levels of pathogenicity are either significantly delayed or do not germinate on larval surfaces. When germination does occur, hyphae from such mutants do not penetrate immediately; instead, extensive surface hyphal growth, with or without eventual penetration of the integument, is evident.     

Cuticular defects lead to full immunity to a major plant pathogen

In addition to its role as a barrier, the cuticle is also a source of signals perceived by invading fungi. Cuticular breakdown products have been shown previously to be potent inducers of cutinase or developmental processes in fungal pathogens. Here the question was addressed as to whether plants themselves can perceive modifications of the cuticle. This was studied using Arabidopsis thaliana plants with altered cuticular structure. The expression of a cell wall-targeted fungal cutinase in A. thaliana was found to provide total immunity to Botrytis cinerea. The response observed in such cutinase-expressing plants is independent of signal transduction pathways involving salicylic acid, ethylene or jasmonic acid. It is accompanied by the release of a fungitoxic activity and increased expression of members of the lipid transfer protein, peroxidase and protein inhibitor gene families that provide resistance when overexpressed in wild-type plants. The same experiments were made in the bodyguard (bdg) mutant of A. thaliana. This mutant exhibits cuticular defects and remained free of symptoms after inoculation with B. cinerea. The expression of resistance was accompanied by the release of a fungitoxic activity and increased expression of the same genes as observed in cutinase-expressing plants. Structural defects of the cuticle can thus be converted into an effective multi-factorial defence, and reveal a hitherto hidden aspect of the innate immune response of plants.     

Differences in penetration routes and establishment rates of four entomopathogenic nematode species into four white grub species

We compared the penetration of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), S. glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and H. bacteriophora (GPS11 strain) into third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis. When larvae were exposed to nematodes for 6-72 h larval mortality and nematode establishment rate and occasionally speed of kill often showed the same pattern within nematode-white grub combinations. But no two nematodes or white grub species had the same pattern for these observations for all white grub or nematode species, respectively. Mortality, establishment, and speed of kill followed a similar pattern for H. zealandica, S. glaseri, and S. scarabaei, but there was no clear relationship for H. bacteriophora. Significant nematode establishment was only observed after at least 48 h exposure in most nematode-white grub combinations. Faster establishment was observed only for H. zealandica in A. orientalis and R. majalis (after 24 h) and for S. scarabaei in P. japonica and R. majalis (after 12 h). Nematode establishment after 72 h in the different scarab species was generally low for S. glaseri (<1.5%) and H. bacteriophora (<3%), higher for H. zealandica (2-5%), and the highest for S. scarabaei (1-14%). However, in another experiment establishment was generally higher after 96h exposure. Nematode penetration sites were determined by comparing nematode establishment in larvae with mouth, anus, mouth+anus, or none sealed with glue. The trends for each nematode species were very similar in the different white grub species. H. zealandica and H. bacteriophora showed excellent cuticular penetration ability but may also penetrate through mouth and/or anus. S. glaseri also penetrated through the cuticle but lower establishment in larvae with mouth or mouth+anus sealed suggested that the mouth is an important penetration site. S. scarabaei showed a preference for the mouth as a penetration site, but it showed some cuticular penetration ability and may also use the anus as a penetration site. The methodology used cannot exclude that cuticular penetration also included penetration through the spiracles. To fully understand the effect of nematode and white grub species on nematode virulence, future studies will have to compare host immune response to the penetrating IJs and the role of the symbiotic bacteria in these interactions.     

Characterization of aqueous pores in plant cuticles and permeation of ionic solutes

Plant cuticles are lipid membranes with separate diffusion paths for lipophilic non-electrolytes and hydrated ionic compounds. Ions are lipid insoluble and require an aqueous pathway across cuticles. Based on experimental data, the aqueous pathway in cuticles has been characterized. Aqueous pores arise by hydration of permanent dipoles and ionic functional groups. They can be localized using ionic fluorescent dyes, silver nitrate, and mercuric chloride. Aqueous pores preferentially occur in cuticular ledges, at the base of trichomes, and in cuticles over anticlinal walls. Average pore radii ranged from 0.45 to 1.18 nm. Penetration of ions was a first order process as the fraction of the salt remaining on the cuticle surface decreased exponentially with time. Permeability of cuticles to ions depended on humidity and was highest at 100% humidity. Wetting agents increased rate constants by factors of up to 12, which indicates that the pore openings are surrounded by waxes. The pores in cuticular ledges of Helxine soleirolii allowed passage of berberine sulphate, which has a molecular weight of 769 g mol(-1). Increasing the molecular weight of solutes from 100 to 500 g mol(-1) decreased the rate constants of penetration by factors of 7 (Vicia faba) and 13 (Populus canescens), respectively. Half-times of penetration of inorganic salts and organic ions across Populus cuticles and Vicia leaf surfaces varied between 1 and 12 h. This shows that penetration of ionic compounds can be fairly rapid, and ions with molecular weights of up to 800 g mol(-1) can penetrate cuticles that possess aqueous pores.     

Periclinal penetration of potassium permanganate into mature cuticular membranes ofAgave andClivia leaves: new implications for plant cuticle development

Staining cuticular membranes ofAgave americana andClivia miniata en bloc with potassium permanganate results in a strong contrast in the interior cuticular layer while the exterior part remains unstained. This is not caused by a selective chemical reaction with the interior part but by the unidirectional penetration of the reagent from the interior side, the outside being protected by the cuticle proper. In transverse cryosections of the cuticular membrane, permanganate penetrates nearly as easily into the exterior cuticular layer as into the interior one giving the same contrast. However, compared with the periclinal penetration into the cuticle proper this penetration is accelerated five-to tenfold by the polysaccharide network within the cuticular layer which serves as a distribution-channel system. Periclinal penetration into the cuticle proper occurs independently in each cutin penetration unit included between two obvious lucent lamellae and further divided into subunits.     

The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis

The outermost epidermal cell wall is specialized to withstand pathogens and natural stresses, and lipid-based cuticular polymers are the major barrier against incursions. The Arabidopsis thaliana mutant bodyguard (bdg), which exhibits defects characteristic of the loss of cuticle structure not attributable to a lack of typical cutin monomers, unexpectedly accumulates significantly more cell wall-bound lipids and epicuticular waxes than wild-type plants. Pleiotropic effects of the bdg mutation on growth, viability, and cell differentiation are also observed. BDG encodes a member of the alpha/beta-hydrolase fold protein superfamily and is expressed exclusively in epidermal cells. Using Strep-tag epitope-tagged BDG for mutant complementation and immunolocalization, we show that BDG is a polarly localized protein that accumulates in the outermost cell wall in the epidermis. With regard to the appearance and structure of the cuticle, the phenotype conferred by bdg is reminiscent of that of transgenic Arabidopsis plants that express an extracellular fungal cutinase, suggesting that bdg may be incapable of completing the polymerization of carboxylic esters in the cuticular layer of the cell wall or the cuticle proper. We propose that BDG codes for an extracellular synthase responsible for the formation of cuticle. The alternative hypothesis proposes that BDG controls the proliferation/differentiation status of the epidermis via an unknown mechanism.     

A gene involved in modifying transfer RNA is required for fungal pathogenicity and stress tolerance of Colletotrichum lagenarium

7-Methylguanosine (m7G) modification of tRNA occurs widely in prokaryotes and eukaryotes, although information about its biological roles is limited. Here, we report that a gene involved in m7G modification of tRNA is required for infection by the phytopathogenic fungus Colletotrichum lagenarium. Analysis of the infection-deficient mutant of C. lagenarium, produced by plasmid insertional mutagenesis, identified a tagged gene that is designated APH1. The aph1 mutants, generated by targeted gene disruption, exhibit significant reduction in pathogenicity on the host plants. We conclude that APH1 is required for fungal infection in C. lagenarium. Aph1 showed a strong similarity to Saccharomyces cerevisiae Trm8 involved in m7G modification of tRNA. The m7G content of tRNA from the aph1 deletion mutant was severely reduced compared with that from the wild type, indicating that APH1 is required for m7G methyltransferase activity. Appressoria formed by the aph1 mutants developed penetration hyphae into cellophane, suggesting that appressoria of the mutants retain basic function for penetration. However, the aph1 mutants failed to develop intracellular penetration hyphae into epidermis of the host plants, suggesting a specific requirement of APH1 for appressorium-mediated host invasion. The mutants also had increased sensitivity to salinity and H2O2 stresses. Interestingly, a heat shock treatment on the host plants enabled the aph1 mutant to penetrate them. These data suggest that the APH1 is required for the plant invasion, probably to overcome environmental stresses derived from basal preinvasion (penetration) defence of the host plants.     

Ultrastructure of the epidermis in the ice worm, Mesenchytraeus solifugus

The ice worm is adapted for life at O°C. A survey of the ultrastructure of the cuticle, epidermal epithelium and basement membrane does not reveal any features which self-evidently correlate with such metabolic specialization; instead, these tissues are much like those of the earthworm and some freshwater oligochaetes. The cuticular fibers are unstriated. Epithelial cells aresuggested as the source of cuticular material. Epithelial microvilli penetrate the cuticle. There is an array of membrane bound bodies on the cuticle surface. The basement membrane fibers are transversely striated and are oriented in crossed lamellae. The junctional complex is represented by azonula adhaerens and septate desmosome.     

Sensitivity of an insect mechanoreceptor during moulting

ABSTRACT. The fine structure and the behavioural threshold for vibration sensitivity of the eight thoracic filiform hairs of Barathra brassicae caterpillars were investigated through an intermoult/moult cycle. Associated with each filiform hair is one bipolar sensory cell and three enveloping cells. The outer dendritic segment terminates in an ecdysial canal in the hair base and a tubular body lies at its distal end. Shortly before apolysis the dendrite elongates. By this means the connection between the sensory cell and the old cuticular apparatus is maintained while the epithelium and the old thoracic cuticle are separating. The new cuticular apparatus of the filiform hair is formed in the second half of the larval stage by the three enveloping cells. A second tubular body in the elongated outer dendritic segment is formed at the base of the replacement hair 10 h before next ecdysis, so that the new hair functions as soon as ecdysis is completed, the old cuticular apparatus with the old tubular bodies being torn away with the exuvia during ecdysis. Sensitivity to a 300 Hz tone was tested in the standing wave of a Kundt's tube. Throughout most of the larval instar the threshold was 2.0 ± 0.3 μm particle displacement amplitude until 1–2h before ecdysis when it rose to 6.8 ± 1.3 μm and at 10–30 min before the beginning of ecdysis no reaction to sound could be detected. Once the old cuticle was shed maximum sensitivity returned as soon as the replacement hairs were erect. The sensilla are therefore physiologically functional at all developmental stages except for 30–60 min during actual ecdysis.