Invaginated Sites Research Articles

Direct interaction between glyoxysomes and lipid bodies in cotyledons of the Arabidopsis thaliana ped1 mutant.

During germination and subsequent growth of fatty seeds, higher plants obtain energy from the glyconeogenic pathway in which fatty acids are converted to succinate in glyoxysomes, which contain enzymes for fatty acid beta-oxidation and the glyoxylate cycle. The Arabidopsis thaliana ped1 gene encodes a 3-ketoacyl-CoA thiolase (EC 2.3.1.16) involved in fatty acid beta-oxidation. The ped1 mutant shows normal germination and seedling growth under white light. However, etiolated cotyledons of the ped1 mutant grow poorly in the dark and have small cotyledons. To elucidate the mechanisms of lipid degradation during germination in the ped1 mutant, we examined the morphology of the ped1 mutant. The glyoxysomes in etiolated cotyledons of the ped1 mutant appeared abnormal, having tubular structures that contained many vesicles. Electron microscopic analysis revealed that the tubular structures in glyoxysomes are derived from invagination of the glyoxysomal membrane. By immunoelectron microscopic analysis, acyl-CoA synthetase (EC 6.2.1.3), which was located on the membrane of glyoxysomes in wild-type plants, was located on the membranes of the tubular structures in the glyoxysomes in the ped1 mutant. These invagination sites were always in contact with lipid bodies. The tubular structure had many vesicles containing substances with the same electron density as those in the lipid bodies. From these results, we propose a model in which there is a direct mechanism of transporting lipids from the lipid bodies to glyoxysomes during fatty acid beta-oxidation.

Heterogeneous distribution of AMPA glutamate receptor subunits at the photoreceptor synapses of rodent retina

In the retina the segregation of different aspects of visual information starts at the first synapse in signal transfer from the photoreceptors to the second-order neurons, via the neurotransmitter glutamate. We examined the distribution of the four AMPA glutamate receptor subunits GluR1-GluR4 at the photoreceptor synapses in mouse and rat retinae by light and immunoelectron microscopy and serial section reconstructions. On the dendrites of OFF-cone bipolar cells, which make flat, noninvaginating contacts postsynaptic at cone synaptic terminals, the subunits GluR1 and GluR2 were predominantly found. Horizontal cell processes postsynaptic at both rod and cone synaptic terminals preferentially expressed the subunits GluR2, GluR2/3 and GluR4. An intriguing finding was the presence of GluR2/3 and GluR4 subunits on dendrites of putative rod bipolar cells, which are thought to signal through the sign-inverting metabotropic glutamate receptor 6, mGluR6. Furthermore, at the rod terminals, horizontal cell processes and rod bipolar cell dendrites showed labelling for the AMPA receptor subunits at the ribbon synaptic site or perisynaptically at their site of invagination into the rod terminal. The wide distribution of AMPA receptor subunits at the photoreceptor synapses suggests that AMPA receptors play an important role in visual signal transfer from the photoreceptors to their postsynaptic partners.

Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding.

Many intracellular compartments of eukaryotic cells do not adopt a spherical shape, which would be expected in the absence of mechanisms organizing their structure. However, little is known about the principles determining the shape of organelles. We have observed very defined structural changes of vacuoles, the lysosome equivalents of yeast. The vacuolar membrane can form a large tubular invagination from which vesicles bud off into the lumen of the organelle. Formation of the tube is regulated via the Apg/Aut pathway. Its lumen is continuous with the cytosol, making this inverse budding reaction equivalent to microautophagocytosis. The tube is highly dynamic, often branched, and defined by a sharp kink of the vacuolar membrane at the site of invagination. The tube is formed by vacuoles in an autonomous fashion. It persists after vacuole isolation and, therefore, is independent of surrounding cytoskeleton. There is a striking lateral heterogeneity along the tube, with a high density of transmembrane particles at the base and a smooth zone devoid of transmembrane particles at the tip where budding occurs. We postulate a lateral sorting mechanism along the tube that mediates a depletion of large transmembrane proteins at the tip and results in the inverse budding of lipid-rich vesicles into the lumen of the organelle.

Photoreceptor fine structure in Oreochromis niloticus L. (Cichlidae; Teleostei) in light- and dark-adaptation.

The structure and arrangement of both light- and dark-adapted retinal photoreceptors of Oreochromis niloticus L. were studied. Eyes of four light-adapted and four dark-adapted O. niloticus were fixed routinely for light and transmission electron microscopy. Rods, single cones, and double (twin) cones were present in a ratio of 30:1:2, respectively. Light-adapted rods were tall, extending into the retinal epithelial layer. Rod inner segments showed a distal ellipsoid of mitochondria that narrowed dramatically in the myoid region. Dark-adapted rod inner segments were much shorter with a thicker myoid region, indicating photomechanical movement. Rod synaptic spherules were small, with both superficial synapses and invaginated sites. Single cones were similar to individual members of a double cone. Cone outer segments consisted of uniform discs with a single incisure. All cones displayed a short, tapering outer segment, a large ellipsoid of mitochondria, and a myoid region rich in organelles. Both members of double cones had extensive subsurface cisternae along their contiguous surfaces. Cone inner segments changed little throughout the circadian cycle, suggesting an absence of significant retinomotor movements. Large, vesicular cone nuclei were located adjacent to or through the external limiting membrane. The cones' synaptic pedicles had larger synapses than rod spherules, with more of both invaginated (ribbon) and conventional (superficial) synaptic sites. Cone photoreceptors were arranged in a repeating square mosaic pattern with a single cone surrounded by four double (twin) cones. The photoreceptors of the Nile tilapia presented basic piscine characteristics, and also some more species-specific features.

THREE CASES OF INTUSSUSCEPTION OF THE SMALLINTESTINE CAUSED BY AN ILEUS TUBE

Adult cases of intussusception often have organic diseases including tumors and inflammation of the intestine. We rarely see adult intussusception probably due to an ileus tube in the literature. This paper describes our experience with three cases of such rare intussusception. In one case intussusception occurred during indwelling of an ileus tube for aspiration therapy of intestinal obstruction. In the remaining two cases, intussusception developed after drawing out an ileus tube which was inserted preoperatively as splinting tube to prevent postoperative intestinal obstruction and was left untouched. All invaginated sites were found out in the jejunum. One case demanded excision of the intestine, but the remaining two cases were successfully reduced by Hutchinson's procedure. No organic diseases were noted in the lead of the intussusception, and hence it was thought that the indwelt ileus tube might cause intussusception in these cases. One of these cases was treated 3.5 months after the onset of the deseases. The ileus tube should be used cautiously by observing symptomatic changes even after removal of the tube in terms of possible association of intussusception.

Cytochemical localization of adenylate cyclase activity in the symbiotic protocorms of Spiranthes sinensis

A cytochemical method has been used to investigate the localization of adenylate cyclase [EC 4.6.1.1] activity in the protocorms of Spiranthes sinensis developing with one of its endomycorrhizal fungi, Ceratobasidium cornigerum and in seeds cultured without fungi. In both symbiotic protocorms and asymbiotic seeds, the reaction products precipitated on the exterior side of the host plasma membrane in contact with the host cell wall. The invagination sites of the plasma membrane and vesicles near the membrane showed a strong reaction. Non-electron-dense deposits were found on the host membrane enveloping the hyphae and on the fungal plasma membrane. The host membrane enveloping the hyphae stained with the two selective stains for plasma membrane of plant cells, phosphotungstic acid-chromic acid and alkaline bismuth. The detection of the adenylate cyclase activity on the host plasma membrane in contact with host cell wall suggested the presence of cAMP in orchid tissues, and the absence of activity on the host plasma membrane enveloping the hyphae suggested that cAMP played no role in the interface between host and fungus.

Sine oculis is a homeobox gene required for Drosophila visual system development.

The somda (sine oculis-medusa) mutant is the result of a P element insertion at position 43C on the second chromosome. somda causes aberrant development of the larval photoreceptor (Bolwig's) organ and the optic lobe primordium in the embryo. Later in development, adult photoreceptors fail to project axons into the optic ganglion. Consequently optic lobe development is aborted and photoreceptor cells show age-dependent retinal degeneration. The so gene was isolated and characterized. The gene encodes a homeodomain protein expressed in the optic lobe primordium and Bolwig's organ of embryos, in the developing adult visual system of larvae, and in photoreceptor cells and optic lobes of adults. In addition, the SO product is found at invagination sites during embryonic development: at the stomadeal invagination, the cephalic furrow, and at segmental boundaries. The mutant somda allele causes severe reduction of SO embryonic expression but maintains adult visual system expression. Ubiquitous expression of the SO gene product in 4-8-hr embryos rescues all somda mutant abnormalities, including the adult phenotypes. Thus, all deficits in adult visual system development and function results from failure to properly express the so gene during embryonic development. This analysis shows that the homeodomain containing SO gene product is involved in the specification of the larval and adult visual system development during embryogenesis.

Mesendoderm cell and archenteron formation in isolated blastomeres from the shrimp Sicyonia ingentis

The fate map of 2- and 4-cell-stage Sicyonia ingentis embryos was determined by microinjection of lysyl-rhodamine-dextran into single blastomeres. Microinjected embryos were cultured to the limb bud stage, when the body plan of the nauplius larva was evident. The animal blastomere, AB, gave rise to anterior ectoderm, while the vegetal blastomere, CD, gave rise to posterior structures, including the invagination site during gastrulation. The A blastomere gave rise to mirror-image patterns of dorsal-lateral ectoderm, while the B blastomere gave rise to anterior, ventral ectoderm. The C blastomere gave rise to posterior, dorsal-lateral ectoderm, complementary to the A pattern, as well as some naupliar mesoderm. The D blastomere gave rise to mesendoderm, naupliar mesoderm, and some posterior ectoderm. To study the specification of the early blastomeres, they were microsurgically separated and cultured in isolation. Two mesendoderm cells formed in 1/2, 1/4, 1/8, and 1/16 blastomeres in embryos dissociated at the 2-, 4-, 8-, and 16-cell stages, respectively. CD and D blastomeres could be distinguished by their larger size and gave rise to the mesendoderm cells. Archenteron formation and elongation of the embryo occurred in CD but not in AB isolates. Isolated blastomeres were recombined in various ways to determine whether their state of commitment could be altered in different cellular environments. Duplicated mesendoderm cells and archenterons formed in CD + CD recombinations, while AB + AB recombinations formed blastulae but did not produce mesendoderm cells and did not invaginate. The normal number of mesendoderm cells and a single archenteron formed in D + AB recombinations, while C + AB recombinations remained as blastulae and did not form mesendoderm cells. The results suggest that the mesendoderm cells are autonomously specified, possibly by cytoplasmic localization at the vegetal pole. The mesendoderm may also function as a signaling region to organize other developmental events.

The endoscopic localization of endometrial implants in the ovarian chocolate cyst

To describe the characteristics of the endometrial cyst and to locate the implants for selective biopsy. Prospective study. Fifty-one women with one or two ovarian chocolate cysts of 3 cm or more were investigated. Laparoscopy and random biopsy versus a new technique of ovarioscopy and selective biopsy. Visual characteristics and histopathology of endometrial cysts. The clinical suspicion of an endometrioma was confirmed in a series of 59 hemorrhagic cysts by histopathology in 89% and 42%, respectively, of typical and atypical cases and in 27% of recurrent chocolate cysts in the presence of postoperative adhesions. The atraumatic technique of ovarioscopy allowed description of the typical characteristics of the inner wall of the endometrioma and location of the active implants for biopsy. Endometrial tissue was obtained by small ovarioscopy-guided biopsies in 82% of the cases versus 42% in large random biopsies. Red lesions were highly significant for a mucosa-type implant and were predominantly located at the site of invagination stigma and adhesions with the pelvic wall. Endoscopy of ovarian chocolate cysts allows observation of typical features of the wall that differentiates it from other benign cysts of the ovary. Microbiopsies obtained under endo-ovarian endoscopy provided significantly more active, endometrial tissue than random biopsies. The data confirm that in most cases the endometrioma is formed by invagination of the cortex and that active implants are located at the site of invagination. Ovarioscopy is therefore proposed as a useful tool to differentiate in doubtful cases between a hemorrhagic functional and an endometriotic cyst and to select the sites for biopsies.

Movement of surface markers along the pinocytotic pseudopodia ofAmoeba proteus

The movement of latex beads over pinocytotic pseudopodia produced byAmoeba proteus was recorded in the presence of 117.65 mM EGTA as an inducer of pinocytosis. The results show that all particles flow in the direction of pseudopodial growth, with a slightly higher velocity than the advancing frontal edge. This means that markers are removed from the base of a pinocytotic pseudopodium and gradually approach the pseudopodium tip. Two particles on the surface of the same pseudopodium can move at the same rate or differ slightly in the velocity of their forward flow. A bead can move even if another blocks the channel orifice. Retrograde particle movement has never been observed. Whether all latex spheres bound to pinocytotic pseudopodia flow with the laterally mobile plasma membrane fraction, which slides over submembranous contractile layer, or whether the whole cortical complex, the actin network and the plasma membrane, move together towards the invagination site is discussed.

The six fold symmetry of the B880 light-harvesting complex and the structure of the photosynthetic membranes of Rhodopseudomonas marina.

The reaction center free light-harvesting core complex of Rp. marina was purified by DEAE 52 ion exchange chromatography in the presence of the detergent OG. The protein complex was crystallised by microdialysis yielding two-dimensional crystals with a diameter of up to 10 microns. The crystals were negatively stained with uranyl acetate or prepared in vitrified ice and electron micrographs were taken. They exhibited a hexagonal lattice with a lattice constant of 102 +/- 3 A. The optical diffraction pattern of the best ordered areas of electron micrographs showed spots up to a resolution of 29 A. Image processing revealed a six fold symmetry of the ring like B880-complex. The protein ring is hexagonal with one subunit in each corner of the hexagon and two subunits forming the connection site to the neighbouring B880-complex in the crystal. In freeze fracture preparations of whole cells the intra-cytoplasmic photosynthetic membranes are seen to be organised into large stacks that affect the organisation of the photosynthetic complexes. Most notably, the stacked membrane regions exhibit hexagonally packed photosynthetic complexes with a repeat of approximately 100 A, which is very similar to the lattice of the artificial B880-complex crystals. The same quasi-crystalline structure appeared in the cytoplasmic membrane of the contact sites with the intra-cytoplasmic membrane stack, but was absent from the end membrane of the stack. Thus, membrane stacking appears to induce the formation of the crystalline arrays, presumably through interactions between the cytoplasmic surface domains of the photosynthetic complexes. Tight packing of the photosynthetic particles is not sufficient to induce the crystalline order. The intra cytoplasmic membranes form a continuum with the cytoplasmic membrane via their origins at the round invagination sites.

Retinal photoreceptor fine structure in the velvet cichlid (Astronotus ocellatus).

The structure and arrangement of the retinal photoreceptors of the velvet cichlid fish (Astronotus ocellatus) have been studied by light and electron microscopy. Rods, single cones and double (twin) cones are present. In the light-adapted state, rods are very tall cells that reach deep into the retinal epithelial (RPE) layer. The long outer segment is composed of discs of uniform diameter displaying one or two incisures. The rod inner segment shows a distal ellipsoid of mitochondria, and then narrows dramatically in the myoid region. Rod nuclei are electron dense and located deep in the outer nuclear layer. Rod synaptic spherules are small and show two to three invaginated synaptic sites as well as superficial synapses. Single cones are similar to the individual members of a double cone and all display a short tapering outer segment, a large ellipsoid of mitochondria and a myoid rich in rough endoplasmic reticulum, polysomes, Golgi zones and autophagic vacuoles. Double cones have extensive subsurface cisternae along their entire contiguous surfaces. Cone nuclei are large and vesicular and located close to or through the external limiting membrane. The synaptic pedicles of cones are larger, more electron lucent, and display more invaginated (ribbon) synapses as well as conventional (superficial) synaptic sites than do the rod spherules. Rod photoreceptors certainly undergo retinomotor movements and it is probable that cones do as well. In the light-adapted state the cone photoreceptors are arranged in a repeating square mosaic pattern with one single cone surrounded by four double (twin) cones.

Components of phagocytosis of non-edible materials induced in amoeba proteus

Amoebae sensitized by etnanol ingest non-edible participate materials, which produce chemotactic and mechanical stimuli. At the particle capture stage the initial pseudopodium is guided by positive chemotactic signals and later retracted under the negative (contracting) influence of mechanical contact. The internalization stage is controlled by interference of both stimuli: the mechanically induced retraction provokes invagination beneath the particle, while the simultaneous positive chemotactic response of the cell surface around the invagination site produces secondary pseudopodia which encircle the particle and form the phagosome. All components of this mechanism are also known in locomotion, motor response to stimuli and pinocytosis.

Photoreceptor fine structure in the archerfish (Toxotes jaculatrix)

The fine structure and arrangement of the photoreceptor cells of the archerfish (Toxotes jaculatrix) have been studied by electron microscopy.Rods, twin cones, and single cones are present. In the light-adapted state, the rods are very tall cells reaching almost to the base of the retinal epithelial cells. The outer segment is composed of membranous discs of uniform diameter displaying a single incisure. The rod inner segment displays a distal small ellipsoid and an extremely thin myoid region. The nuclei of rods are electrondense,and the synaptic spherule displays two or three invaginated sites. The single cone is similar to the individual members of a twin cone and displays a tapering outer segment and accompanying accessory outer segment. The wider cone inner segment contains a large, centrally located ellipsoid and a peripheral region rich in endoplasmic reticulum, polysomes, and microfilaments.Twin cones display subsurface cisternae along their entire contiguous surfaces.The cone nuclei are large and vesicular and located vitread to the external limiting membrane. The synaptic pedicle of cones is larger and more electronlucent and contains more invaginated "ribbon" synaptic sites (ten to 12) than do rods. In addition, small "coated" invaginations and larger synaptic vesiclefilled processes are also seen within cone pedicles. In the light-adapted state the cone photoreceptors are arranged in a repeating square mosaic pattern with one single cone surrounded by four twin cones.

Intracellular localization of photosynthetic membrane growth initiation sites in Rhodopseudomonas sphaeroides

Putative membrane invagination sites at which intracytoplasmic photosynthetic membrane growth is initiated in Rhodopseudomonas sphaeroides can be isolated in an upper pigmented fraction by rate-zone sedimentation. The intracellular localization of membranes present in the isolated fraction was investigated with the impermeant surface-labeling reagent pyridoxal 5'-phosphate, which has been shown to diffuse into the periplasmic space and to label proteins of both the peripheral cytoplasmic membrane and the mature intracytoplasmic membrane. A comparison of the extent of labeling at 25 and 0 degrees C was consistent with the possibility that membranes present in the upper pigmented fraction arise from sites near the cell periphery. Pronase digestion of the surface-labeled membranes suggested further that the purified upper fraction consisted largely of open membrane fragments and that the majority of the intracytoplasmic membrane is labeled by this procedure. The pigmented membrane growth initiation sites were separated partially from undifferentiated respiratory cytoplasmic membrane also present in the upper fraction.

Differential protein insertion into developing photosynthetic membrane Regions of Rhodopseudomonas sphaeroides

Previous studies have suggested that much of the B800-850 light-harvesting bacteriochlorophyll a-protein complex is inserted directly into the intracytoplasmic photosynthetic membrane of Rhodopseudomonas sphaeroides. In contrast, the B875 light-harvesting and reaction center complexes are assembled preferentially at peripheral sites of photosynthetic membrane growth initiation. The basis for this apparent site-specific polypeptide insertion was examined during the inhibition of RNA and protein syntheses. The pulse labeling of polypeptides at the membrane growth initiation sites was significantly less sensitive to inhibition by rifampicin, chloramphenicol, or kasugamycin than in the intracytoplasmic or outer membranes. This suggests increased stability for the translation machinery at these membrane invagination sites. Similar differential effects in polypeptide insertion were observed during inhibition of bacteriochlorophyll synthesis through deprival of delta-aminolevulinate to R sphaeroides mutant H-5, which requires this porphyrin precursor. The pulse-labeling patterns observed during the inhibition of both RNA and pigment syntheses were consistent with the uncoupling of polypeptide insertion into the membrane invagination sites from their growth and maturation into intracytoplasmic membranes.

Photoreceptor fine structure in the goldeye (Hiodon alosoides) (teleost).

The retinal photoreceptors of the goldeye (Hiodon alosoides) are arranged in large bundles of 40-50 cells optically isolated from other bundles by the retinal epithelial cells. Within each bundle are found both rods and cones in roughly equal numbers. Rod photoreceptors show marked retinomotor responses to project beyond the photoreceptor bundle in light-adaptation and to lie entirely within the bundle in dark-adaptation. In all stages of the light cycle cone outer segments remain at the apex of the photoreceptor bundle. In light-adaptation, rod inner segments display an apical ellipsoid separated from a basal ellipsoid by the greatly elongated myoid. In dark-adaptation the rod inner segment is much the same diameter throughout its length. In both rods and cones, profiles of rough and smooth endoplasmic reticulum and Golgi zones are present in a supranuclear location. The nuclei of rods display little heterochromatin and are located vitreal to the external limiting membrane in light-adaptation, whereas in dark-adaptation more heterochromatin is noted and the nuclei lie scleral to the external limiting membrane. Cone nuclei display the same changes in chromatin pattern as rods but they show changes in nuclear location opposite that of the rods. Throughout its length, the rod photoreceptor cytoplasm is more electron dense than that of the cone. The synaptic spherule of rods displays 2-3 invaginated synaptic sites while the cone pedicle is larger and presents 8-10 invaginated synaptic sites. Both rods and cones also appear to have superficial synaptic sites. Membrane specializations are found along the length of the inner segments where rods and cones are contiguous. These may act as sites of intercellular communication and the whole photoreceptor bundle may therefore be considered as a macroreceptor.

Freeze fracture studies of reaction centers from Rhodospirillum rubrum in chromatophores and liposomes

In freeze-fractures of chromatophores of Rhodospirillum rubrum the reaction centers are seen as hexagonal arranged particles of 13 nm diameter with a density of around 5,500 particles per μm2. Similar regions on the cytoplasmic membrane suggest that these parts are the prospective invagination sites.

Circus movements in dissociated cells in normal and hybrid frog embryos.

Circus movements, involving circumferential rotation of a hyaline cytoplasmic blister and endoplasmic flow, occur in EDTA-dissociated gastrula stage Rana pipiens embryos. Such cell movements occur in very few cells taken from pre-gastrula stage embryos. During gastrulation, there is a progressive increase in the proportion of a population of cells that is engaged in circus movements. Circus movements do not occur in dividing cells. Individual cells in culture, as well as small clusters of cells in vitro, are jostled about in an apparently aimless fashion over short distances by circus movements, although the translocation of masses of cells over long distances is substantially greater than the translocation of isolated cells. In an early gastrula stage normal embryo, cells from around the site of blastopore invagination are most active in circus movements. Cells taken from different stages of arrested hybrid embryos show variable depression in the formation of rotating hyaline blebs. Aggregates of cells from arrested hybrid embryos are also relatively immobile in culture. The morphogenetic significance of circus movements in normal embryos and gastrula-arrest hybrid embryos is discussed.

Two action sites of AM-toxin I produced by apple pathotype of Alternaria alternata in host cells: an ultrastructural study

The localization of primary action sites of AM-toxin I in host cells was examined by ultrastructural investigation and electron microscopic autoradiography. In susceptible apple leaves, the first effect of the toxin appeared 1 h after treatment in the plasma membranes and chloroplasts of mesophyll and vascular bundle sheath cells and in the plasma membranes of phloem and epidermal cells. Membranes and vesicles which were stained positively with a specific staining solution for grana lamellae were found in the matrix of the chloroplasts, showing that the membranous materials were derived from the disrupted grana. Cell wall lesions were formed around plasmodesmata where plasma membranes were invaginated. The invaginated sites were filled with amorphous materials from degraded cell walls, including membranes derived from plasma membranes and the desmotubules extending from plasmodesmata. The modified chloroplasts and plasma membranes were observed more often as the time after the toxin treatment was prolonged. Modified plastids were not found in the leaf cells. The other cellular membranes appeared normal even 10 h after the treatment. Resistant leaf cells were rarely affected by the toxin. Not all tissues from susceptible apples were sensitive as the toxin caused no necrosis or ultrastructural changes in petal cells. Resistant petal cells were also insensitive to the toxin, but the toxin causes necrosis and ultrastructural changes in moderately resistant petal cells in which the primary effect of the toxin appeared as plasma membrane modifications. Plastids were not affected by the toxin. These results indicate that the action sites of the toxin may be located on the plasma membrane – cell wall association in susceptible leaf cells and in moderately resistant petal cells and also on the chloroplasts of susceptible cells. The results of electron microscopic autoradiography also provided evidence that the action sites of the toxin were present on chloroplasts and the plasma membrane –cell wall association of susceptible leaf cells.