A quantitative histological and ultrastructural analysis of interactions between the flax rust and near-isogenic host lines varying in their degree of incompatibility

1983 ◽  
Vol 61 (7) ◽  
pp. 1831-1850 ◽  
Author(s):  
Michael D. Coffey ◽  
Frances H. E. Allen
Keyword(s):  
Phosphotungstic Acid ◽  
Rust Fungus ◽  
Periodic Acid ◽  
Fungal Growth ◽  
Host Cells ◽  
Similar Proportion ◽  
Thin Sections ◽  
Melampsora Lini ◽  
Uninfected Host ◽  
Electron Lucent

Histological differences were evident in the leaves of eight near-isogenic lines of flax infected with the rust fungus Melampsora lini. Compared with the compatible L9 and M1 interactions, fungal growth was progressively more restricted in the moderately incompatible K and M4, incompatible M and P, and highly incompatible L and M3 interactions. This restriction took place in advance of appreciable necrosis of host cells in K, M4, M, and P. At 72 h the proportion of haustoria-containing cells which were necrotic was only 10–15% in K and M4. In M at 72 h necrosis was 80% or more at infection sites with small colonies but was negligible at sites with large colonies. In P, by contrast, a similar proportion of necrosis, 40% at 72 h, was present at all infection sites. However, in L and M3 host necrosis was much more rapid and the fungus was restricted to a few host cells. An early ultrastructural event was the appearance of extensive fibrillar deposits in the initially electron-lucent extrahaustorial matrices of both the incompatible M and P and moderately incompatible M4 and K interactions. A positive reaction with silver proteinate indicated that these matrical deposits contained carbohydrate, possibly a mucopolysaccharide or glycoprotein, but they were not extracted by either cellulase, pectinase, or chitinase. The extrahaustorial membrane, surrounding the haustoria in the compatible L9 and M1 interactions and the moderately incompatible K interaction was not stained by the periodic acid – phosphotungstic acid – chromic acid (PACP) procedure believed specific for the plasmalemma. In incompatible reactions clusters of electron-dense particles sometimes replaced starch in plastids of infected host cells. This event usually coincided with the appearance of extensive matrical deposits around fungal haustoria. At the same time particles were also found in plastids in uninfected host cells immediately adjacent to infection sites, particularly in the M and P interactions. These particles were extracted from thin sections by using pullulanase followed by α-amylase, indicating that they consisted of highly branched amylopectin.

Flax rust resistance involving the K gene: an ultrastructural survey

1976 ◽  
Vol 54 (13) ◽  
pp. 1443-1457 ◽  
Author(s):  
Michael D. Coffey
Keyword(s):  
Major Gene ◽  
Rust Fungus ◽  
Progressive Increase ◽  
Host Cells ◽  
Electron Microscopic ◽  
Susceptible Host ◽  
Melampsora Lini ◽  
Major Gene Resistance ◽  
Complete Layer ◽  
Extrahaustorial Matrix

An electron microscopic study of major gene resistance involving the flax K gene and the rust fungus Melampsora lini revealed several interesting ultrastructural features. Up to 9 days after infection, most haustoria-containing resistant host cells appeared viable. During this period there was a progressive increase in a fibrillar deposit in the extrahaustorial matrix which was not detected in the susceptible host. This material was in direct continuity with the distal wall of the haustorial neck. It frequently constituted an apparently complete layer or 'encapsulation' around the haustorium and was present in 75% of the extrahaustorial matrices as early as 4 days after infection. Initially, callose-like encasements formed around only a small percentage of haustoria in the resistant host; however, by day 9, about 20% of the haustoria were encased. No encasements were found around susceptible haustoria at this stage. Hypersensitive host cell collapse occurred in a small percentage of resistant infections.

ELECTRON MICROSCOPY OF UREDOSPORES OF MELAMPSORA LINI AND OF RUST-INFECTED FLAX

1967 ◽  
Vol 45 (9) ◽  
pp. 1575-1582 ◽  
Author(s):  
M. S. Manocha ◽  
Michael Shaw
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Linum Usitatissimum ◽  
Room Temperature ◽  
Rust Fungus ◽  
Thin Sections ◽  
Infection Structures ◽  
Linum Usitatissimum L ◽  
Melampsora Lini ◽  
Germ Tubes

Thin sections of germinated and ungerminated uredospores of the flax rust fungus (Melampsora lini (Pers.) Lev.) and rust-infected cotyledons of flax (Linum usitatissimum L., variety Bison) were examined in a Philips 100B electron microscope. The line structure of the mature uredospores, the formation of germ tubes including the development of cross walls, and of the intercellular mycelium, haustoria, sporogenous hyphae, and developing uredospores are all briefly described. Germinated and ungerminated uredospores were fixed in KMnO4 followed by OsO4 at room temperature. Infected tissue was fixed in KMnO4 at room temperature or in glutaraldehyde at 4 °C followed by OsO4 at 4 °C. Nucleoli were not observed in the nuclei of mature uredospores or germ tubes but were present in the intercellular mycelium, sporogenous hyphae, and developing uredospores and were particularly prominent in the two haustorial nuclei. The results are discussed and attention is drawn to the need for further work on the nuclei and nucleoli in germinating uredospores under conditions conducive to the development of infection structures.

Ultrastructure of the Parotid Gland of the Nine-Banded Armadillo

1977 ◽  
Vol 35 ◽  
pp. 640-641
Author(s):  
J. R. Ruby
Keyword(s):  
Endoplasmic Reticulum ◽  
Parotid Gland ◽  
Periodic Acid ◽  
Acinar Cells ◽  
Duct System ◽  
Parotid Glands ◽  
Dasypus Novemcinctus ◽  
Anterior Portion ◽  
Periodic Acid Schiff ◽  
Thin Sections

Parotid glands were obtained from five adult (four male and one female) armadillos (Dasypus novemcinctus) which were perfusion-fixed. The glands were located in a position similar to that of most mammals. They extended interiorly to the anterior portion of the submandibular gland.In the light microscope, it was noted that the acini were relatively small and stained strongly positive with the periodic acid-Schiff (PAS) and alcian blue techniques, confirming the earlier results of Shackleford (1). Based on these qualities and other structural criteria, these cells have been classified as seromucous (2). The duct system was well developed. There were numerous intercalated ducts and intralobular striated ducts. The striated duct cells contained large amounts of PAS-positive substance.Thin sections revealed that the acinar cells were pyramidal in shape and contained a basally placed, slightly flattened nucleus (Fig. 1). The rough endoplasmic reticulum was also at the base of the cell.

Cytochemical Localization of Polysaccharides in Diplodia Maydis With Thiosemicarbazide : Evaluation of Three Fixatives For Pa:Tsc:Os Reaction

1976 ◽  
Vol 34 ◽  
pp. 48-49 ◽  
Author(s):  
Judith A. Murphy ◽  
Mary R. Thompson ◽  
A.J. Pappelis
Keyword(s):  
Reaction Center ◽  
Osmium Tetroxide ◽  
Periodic Acid ◽  
Cytochemical Localization ◽  
Thin Sections ◽  
Selective Reaction ◽  
Amorphous Character

In an attempt to identify polysaccharide components in thin sections of D. maydis, procedures were employed such that a PAS localization could be carried out. Three different fixatives were evaluated ie. glutaraldehyde, formaldehyde and paraformaldehyde. These were used in conjunction with periodic acid (PA), thiosemicarbazide(TSC), and osmium tetroxide(Os) to localize polysaccharides in V. maydis using a pre-embedded reaction procedure. Polysaccharide localization is based on the oxidation of vic-glycol groups by PA, and the binding of TSC as a selective reaction center for the formation of osmium black. The reaction product is sufficiently electron opaque, insoluble in lipids, not altered when tissue is embedded, and has a fine amorphous character.

scholarly journals New methods for confocal imaging of infection threads in crop and model legumes

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Angus E. Rae ◽  
Vivien Rolland ◽  
Rosemary G. White ◽  
Ulrike Mathesius
Keyword(s):  
Cell Wall ◽  
Root Hair ◽  
Periodic Acid ◽  
Confocal Imaging ◽  
Wall Material ◽  
Future Research ◽  
Thin Sections ◽  
New Methods ◽  
Infection Threads ◽  
Imaging Of Infection

Abstract Background The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. Results We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. Conclusions Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

Histological studies on host-cell necrosis conditioned by the Sr6 gene for resistance in wheat to stem rust

1977 ◽  
Vol 55 (11) ◽  
pp. 1445-1452 ◽  
Author(s):  
D. J. Samborski ◽  
W. K. Kim ◽  
R. Rohringer ◽  
N. K. Howes ◽  
R. J. Baker
Keyword(s):  
Triticum Aestivum ◽  
Stem Rust ◽  
Fungal Growth ◽  
Triticum Aestivum L ◽  
Host Cells ◽  
Puccinia Graminis ◽  
Near Isogenic Lines ◽  
Cell Necrosis ◽  
Calcofluor White ◽  
Histological Studies

Seedlings of resistant (Sr6) and susceptible (sr6) near-isogenic lines of wheat (Triticum aestivum L.) were inoculated with a race of stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn.) that was avirulent on the line with Sr6 and they were kept at 19, 25, 26, and 27 °C. Fluorescence microscopy was used to detect autofluorescing necrotic host cells and rust colonies after these were stained with a fiuorochrome (Calcofluor White M2R New).In leaves containing the Sr6 gene, a smaller percentage of colonies grown at 25 °C had necrotic cells associated with them than those that were grown at 19 °C. The incidence of colony-associated necrosis in these leaves could be further reduced by increasing the temperature to 26 °C and 27 °C. Similarly, the number of necrotic host cells per colony decreased with an increase in temperature. Colonies in genotypically resistant leaves were usually smaller than those in genotypically susceptible leaves, but the differences in colony sizes between these two lines decreased at the higher temperatures.When infected plants containing the Sr6 gene were kept for varying times at 25 °C and then were transferred to 19 °C, there was significantly less fungal growth and more necrosis than in plants kept continuously at 25 °C. This necrosis occurred largely in those cells that were invaded after the transfer to 19 °C, when the Sr6 gene was activated.

Membranes in lupin root nodules. II. Preparation and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules

1978 ◽  
Vol 30 (1) ◽  
pp. 151-174
Author(s):  
J.G. Robertson ◽  
M.P. Warburton ◽  
P. Lyttleton ◽  
A.M. Fordyce ◽  
S. Bullivant
Keyword(s):  
Sucrose Gradient ◽  
Phosphotungstic Acid ◽  
Nadh Oxidase ◽  
Osmotic Shock ◽  
Root Nodules ◽  
Freeze Fracture ◽  
Electrophoretic Analysis ◽  
Thin Sections ◽  
Peribacteroid Space ◽  
Gradient Fractionation

Peribacteroid membranes and bacteroid envelope inner membranes have been isolated from developing lupin nodules. Isolation of the peribacteroid membranes was achieved by first preparing membrane-enclosed bacteroids free from other plant organelles or membranes. The peribacteroid membranes were then released by osmotic shock and purified by centrifugation to equilibrium on sucrose gradients. The bacteroids were broken in a pressure cell and the bacteroid envelope inner membranes were isolated using sucrose gradient fractionation of the bacteroid total envelope preparation. The density of the peribacteroid membranes decreased during the period of development of N2-fixation in lupin nodules from 1.148 g/ml for nodules from 12-day plants to 1.137 g/ml for nodules from 18-day plants. The density of the bacteroid envelope inner membranes from nodules from 18-day plants was 1–153 g/ml. The identity and homogeneity of the isolated membranes was established, by comparison with membranes in intact nodules, using phosphotungstic acid and silver staining of thin sections and particle densitites on faces of freeze-fracture replicas of the membranes. Analyses for NADH oxidase and succinate dehydrogenase, spectral analyses and gel-electrophoretic analysis of proteins were also used to characterize the membrane and soluble protein fractions from the nodules. The ratio of lipid to protein was 6.1 for the peribacteroid membranes and 2.5 for the bacteroid envelope inner membranes. Leghaemoglobin was localized in the plant cytoplasm in lupin nodules and not in the peribacteroid space.

scholarly journals New Isolates of Pandoraviruses: Contribution to the Study of Replication Cycle Steps

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Ana Cláudia dos Santos Pereira Andrade ◽  
Paulo Victor de Miranda Boratto ◽  
Rodrigo Araújo Lima Rodrigues ◽  
Talita Machado Bastos ◽  
Bruna Luiza Azevedo ◽  
...  
Keyword(s):  
Time Course ◽  
Replication Cycle ◽  
Host Cells ◽  
Comprehensive Description ◽  
Genomic Features ◽  
New Information ◽  
Viral Particles ◽  
Infected Cells ◽  
Giant Viruses ◽  
Electron Lucent

ABSTRACT Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are some of the most intriguing members, exhibiting giant particles and genomes presenting at up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopic methods, the replication cycle steps of three new pandoravirus isolates from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Various patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by the opposite side. On the basis of the counting of viral particles during the infection time course, we observed that pandoravirus particles could undergo exocytosis after their morphogenesis in a process that involved intense recruitment of membranes that wrapped the just-formed particles. The treatment of infected cells with brefeldin affected particle exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributed to viral release. This work reinforces knowledge of and reveals important steps in the replication cycle of pandoraviruses. IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the biology of the virus. For this report, we describe new pandoraviruses and used different methods to better characterize the steps of the replication cycle of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.

scholarly journals Effect of activity on performance and morphology in ischaemic rat slow muscles

1990 ◽  
Vol 152 (1) ◽  
pp. 265-279
Author(s):  
A. Corsi ◽  
A. L. Granata ◽  
O. Hudlicka
Keyword(s):  
Blood Supply ◽  
Periodic Acid ◽  
Capillary Density ◽  
Muscle Performance ◽  
Similar Proportion ◽  
Periodic Acid Schiff ◽  
Rat Soleus Muscle ◽  
Improved Performance ◽  
Isometric Twitch ◽  
Schiff Reaction

Muscle performance and structure was studied in rat soleus muscle with limited blood supply in combination with chronic muscle stimulation. Blood supply to the lower leg was restricted by ligation of the common iliac artery, electrodes were implanted in the vicinity of the sciatic nerve and ankle flexors were denervated. Three days later, soleus and gastrocnemius muscles were stimulated at 4 Hz four times a day for a period of 20 min with 2 h intervals between stimulations; this procedure was continued for 4 days. Muscle performance, histochemistry and ultrastructure were studied on the eighth day after operation in these muscles and in ischaemic unstimulated muscles with denervated ankle flexors. Both were compared with control animals. Muscles with limited blood supply developed less isometric twitch tension than control muscles (peak twitch tension in ischaemic muscle was 60.3 +/− 4.8 g g-1 muscle, mean +/− S.E.M., compared to 79.7 +/− 6.9 g g-1 in control muscle; tensions after 5 min contraction were 54.5 +/− 5.5 g g-1 in ischaemic muscle compared to 70.6 +/− 6 g g-1 in controls). Stimulated muscles with limited blood supply had higher peak (85 +/− 16.6 g g-1) and final (87 +/− 12 g g-1) tensions, and also fatigued less than muscles with limited blood supply but no stimulation. Histochemical estimation of capillary density (by staining for alkaline phosphatase) and slow (SO) and fast (FOG) fibres (by myosin ATPase staining) revealed similar capillary to fibre ratios (2.5) and a similar proportion of FOG fibres (around 18%) in all muscles. The proportion of glycogen-depleted fibres (estimated from the periodic acid Schiff reaction, PAS) in muscles removed from animals 10 min after a 5 min period of isometric twitches was significantly lower in ischaemic muscles (45.1 +/− 1.9%) than in control (80.5 +/− 1.5%) or chronically stimulated ischaemic muscles (67.3 +/− 4.0%). Electron microscopy showed disorganised myofibrils with Z-line streaming in 7.48 +/− 3.04% of fibres in muscles with limited blood supply. Swollen and degenerated mitochondria, dilated sarcoplasmic reticulum and areas of disrupted sarcolemma were also observed. Stimulated ligated muscles showed a significantly lower proportion of fibres with disorganised filaments (0.65 +/− 0.32%) and other signs of damage were much less frequent. The reduced damage and improved performance of chronically stimulated slow muscle may be the result of improved microcirculation, preventing accumulation of lactate.

scholarly journals Structural genomic applied on the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine-knot and nuclear transport factor 2-like protein folds

2019 ◽  
Author(s):  
Karine de Guillen ◽  
Cécile Lorrain ◽  
Pascale Tsan ◽  
Philippe Barthe ◽  
Benjamin Petre ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Nuclear Transport ◽  
Sequence Similarity ◽  
Rust Fungus ◽  
Parasitic Infection ◽  
Effector Proteins ◽  
Host Cells ◽  
Dimensional Structure ◽  
Structural Genomic ◽  
Transport Factor

ABSTRACTRust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins or domains. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we used a strategy based on recombinant protein production in Escherichia coli to study eleven candidate effectors of the leaf rust fungus Melampsora larici-populina. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both proteins show no sequence similarity with known proteins, they exhibit structural similarities to knottin and nuclear transport factor 2-like proteins, respectively. Altogether, our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize rust effector candidates.

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