Host Plant Cells Research Articles

Immunocytochemical localization of fusarium toxins in infected wheat spikes by Fusarium culmorum

Two antisera raised against deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-ADON) were used to investigate the subcellular localization of the fusarium toxins, DON, 3-ADON and 15-ADON, in Fusarium culmorum infected wheat spikes and kernels by means of the immunogold labelling technique. The hyphae of the pathogen produced the toxins when they grew on the surface of the lemma and the ovary as early as 36 h after inoculation, after this incubation time the toxins were also found in the parenchyma cells. When the hyphae grew in the host tissues, more toxins were detected in the host cells, especially in the cells in close contact with the hyphae. Toxin accumulation showed a very close relationship with pathogenic changes in host cells, symptom appearance and colonization of host tissues by hyphae, suggesting that the toxins might play an important role in the disease development. The toxin labelling patterns in hyphal cells and in the infected host tissues for DON antiserum were very similar to that for 3-ADON antiserum, but the labelling density for DON was obviously higher than that for 3-ADON. In hyphal cells, the toxins were localized in the cytoplasm, mitochondria, vacuoles and the cell wall. The young hyphal cells usually showed more gold labelling in the mitochondria and the dense cytoplasm. In the host plant cells, the toxins were detected in the cytoplasm, chloroplasts, plasmalemma, cell wall and vacuoles, and sometimes gold particles were found associated with endoplasmic reticulum and ribosomes in the host cytoplasm. Four–6 days after inoculation of spikelets, hyphae reached the rachis and spread inside and outside of vascular bundles of the rachis. The toxins were present in the xylem vessels, phloem sieve tubes, paratracheal parenchyma cells and the parenchyma cells outside the vascular bundles. By 10 days after inoculation no hyphae were found in the rachis, glume, lemma or young kernels which were located three spikelets above the inoculated spikelets. However, immunolabelling of these tissues showed that toxins were present, especially in xylem vessels and phloem sieve tubes. Ten days after inoculation toxins were also detected in phloem sieve tubes of the rachis three spikelets below the inoculated spikelets. These results suggest that the toxins can be translocated upwards through the xylem vessels and phloem sieve tubes, and downwards through the phloem sieve tubes. In the infected wheat kernels, the toxins were shown not only in the hyphae, but also in the pericarp tissues, pigment strand, aleurone cells and starchy endosperm in different concentrations.

Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora.

Erwinia amylovora is a Gram-negative pathogenic bacterium that infects pear and apple trees as well as other plants from the Rosaceae family. E. amylovora pathogenicity is dependent on a functional Hrp type III secretion system. Harpin, a protein playing a major role in virulence, has been shown to be exported in vitro via the type III secretion apparatus. The data presented here focus on harpin detection in planta after infection of apple seedlings with the wild-type strain CFPB 1430. Using a specific harpin antiserum, harpin was not detected inside the host plant cells, but was found associated with the bacteria and secreted. The extracellular localization of harpin is in agreement with the physiological effects induced by purified harpin when applied as an exogenous elicitor. Harpin was not found associated with the host plant cell wall, a result that weakens its postulated role in cell wall loosening. A differential labelling was observed at the bacterial level: for some bacteria, harpin was exclusively cytoplasmic, whereas in others, it appeared as small patches over the bacterial outer membrane or associated with extracellular linear structures. All the bacteria present within the same area were similarly labelled, suggesting co-ordination in the secretion process. All observations suggest that harpin is synthesized in the bacterial cytoplasm and that secretion occurs from this cytoplasmic pool upon sensing of a plant or bacterial signal.

Histological And Ultrastructural Changes In Leaves Of Resistant And Susceptible Chickpea Cultivars To Ascochyta Rabiei (Pass.) Labr

The Ascochyta blight caused by Ascochyta rabiei ( Pass.) Labr. is a serious foliar fungal disease of chickpea (Cicer arientinum L.). The histo- and cytopathological examination of hostpathogen interactions was conducted in resistant ( ELC-195) and susceptible (Canitez-87) cultivars of chickpea at 3, 5, 7 days after inoculation with A. rabiei. Several histological studies have been made with A. Rabiei. No ultrastructural studies comparing the resistant and susceptible reactions of chickpea to A. rabiei have been reported.The hypersensitive response (HR) is a very common reaction in resistant chickpea plants challenged with A. Rabiei. The FfR has also been associated with defence reactions which is phytoalexin synthesis. Phytoalexins are compounds, synthesised in host plant cells, after infection, which have antimicrobial properties. Fligh concentrations are often found in nearby necrotic cells which prevents further growth of the A. Rabiei in resistance chickpea.A. rabiei produce typical appressorium formation for cuticle penetration but stomata penetration have been observed in some micrographs (Fig.l).

Virus Adaptation to Plants.

Bromoviruses are a group of spherical plant RNA viruses. Their genome consists of approximately 8, 000 nucleotides in total and contains only four genes: 1a and 2a genes necessary for viral RNA replication, 3a gene required for viral cell-to-cell movement and the coat protein gene. Bromoviruses produce a large number of progeny virus particles in host plants, but never induce death. Therefore, we suppose that these viruses are adapted to host plant cells in various ways. Here, I describe molecular mechanisms of bromovirus adaptation to host plant cells and non-host plants.

Ferrous iron is transported across the peribacteroid membrane of soybean nodules

Symbiosomes and bacteroids isolated from soybean (Glycine max Merr.) nodules are able to take up ferrous iron. This uptake activity was completely abolished in the presence of ferrous-iron chelators. The kinetics of uptake were characterized by initially high rates of iron internalization, but no saturation was observed with increasing iron concentration. This process does not appear to involve the ferric reductase of the peribacteroid membrane. The transport of ferrous iron was inhibited by other transition metals, particularly copper. Ferrous iron was taken up by symbiosomes more efficiently than the ferric form. This indicates that the iron transport from the plant host cell to the microsymbiont in vivo may occur mainly as the ferrous form.

Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid.

The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchöe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.

Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses.

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1-1Delta mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1-1Delta mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1-1Delta mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

Biological characterization of citrus tristeza virus isolates by in vitro tissue cultures

Stem segments from Mexican lime, sweet orange, grapefruit, Citrus excelsa and alemow, infected with five citrus tristeza virus (CTV) isolates, were cultured in vitro. Regeneration of roots and shoots were modified as a result of infection. The effect of CTV on the morphogenesis of stem segments cultured in vitro depended on the CTV isolate and the plant host, and showed a correlation with the in vivo effects observed in biological indexing. Evaluation of the morphogenic response of stem segments of Mexican lime and grapefruit can be used as an additional tool for the biological characterization of CTV isolates. The symptoms on sweet orange plants obtained from regenerated shoots indicated that CTV is unevenly distributed in the host plant cells and that the regeneration process may be utilized as a tool to separate strains from complex field isolates.

Induction of Crown Gall on Carrot Slices

Laboratory experiments on the transfer of a plasmid to a bacterium, whether by conjugation, transformation or transduction, are available for use in the teaching laboratory. However, the transfer of plasmid from a bacterium (prokaryote) to a plant cell (eukaryote) has received little attention. The following is an experiment for studying this latter type of genetic transformation. The Gram negative soil bacterium, Agrobacterium tumefaciens L., is the causative agent of crown gall disease, a malignant tumor, occurring on the stems and leaves of infected dicotyledonous plants. Induction of this tumor is mediated by the presence in the bacterium of the large (200 kbp in length) tumor-inducing (Ti) plasmid. Upon bacterial infection of a wounded plant, phenolic defense chemicals released from the plant activate virulence genes on the Ti plasmid, leading to the processing and transferring of a small (20-23 kbp) segment of the Ti plasmid into the host plant cell. The bacterium remains outside the plant cell, living in the intercellular spaces of the plant. By transference through a conjugation tube initiated by the bacterium, this small segment of the Ti plasmid, designated transferred DNA or T-DNA, penetrates the host plant cell and subsequently incorporates into the plant's genome. T-DNA contains genes for the syntheses of opines, chemicals used as sources of carbon and nitrogen by the infecting bacterium, and phytohormones. Overproduction of these phytohormones (i.e. auxin and cytokinin) results in uncontrolled plant cell growth and hence the development of a tumor (Gelvin & Karcher 1996; Russell 1996). In this exercise, we induce crown

Nuclear Import and Export of Viruses and Virus Genomes

Many viruses replicate in the nucleus of their animal and plant host cells. Nuclear import, export, and nucleo-cytoplasmic shuttling play a central role in their replication cycle. Although the trafficking of individual virus proteins into and out of the nucleus has been well studied for some virus systems, the nuclear transport of larger entities such as viral genomes and capsids has only recently become a subject of molecular analysis. In this review, the general concepts emerging are discussed and a survey is provided of current information on both plant and animal viruses. Summarizing the main findings in this emerging field, it is evident that most viruses that enter or exit the nucleus take advantage of the cell's nuclear import and export machinery. With a few exceptions, viruses seem to cross the nuclear envelope through the nuclear pore complexes, making use of cellular nuclear import and export signals, receptors, and transport factors. In many cases, they capitalize on subtle control systems such as phosphorylation that regulate traffic of cellular components into and out of the nucleus. The large size of viral capsids and their composition (they contain large RNA and DNA molecules for which there are few precedents in normal nuclear transport) make the processes unique and complicated. Prior capsid disassembly (or deformation) is required before entry of viral genomes and accessory proteins can occur through nuclear pores. Capsids of different virus families display diverse uncoating programs which culminate in genome transfer through the nuclear pores.

Attachment of Agrobacterium tumefaciens to carrot cells and Arabidopsis wound sites is correlated with the presence of a cell-associated, acidic polysaccharide.

An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells. A. tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells. The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria. Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide. Anion-exchange chromatography followed by 1H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar. Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide. Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant. This indicates that the acidic polysaccharide may play a role in the attachment of A. tumefaciens to host soma plant cells.

Transient energy coupling between rhizobia and legume cells mediated by the peribacteroid membrane ATPase proton pump.

We present a model for the metabolic coupling between rhizobia and plant cell in the nitrogen-fixing legume root nodules. The symbiosome, an organelle-like structure formed by the modified rhizobia (the bacteroids) enclosed by a plant cell derived peribacteroid membrane, is an unique structure in which two energized membranes are closely packed: the inner bacteroid membrane and the peribacteroid membrane that possesses an ATPase proton pump. The model is based on the following points: (i) The permeability for hydrogen ions of the outer membrane of the rhizobia. (ii) The reversibility of the ATPase proton pump of the peribacteroid membrane [Szafran, M.M. and Haaker, H. (1995) Plant Physiol. 108, 1227-1232]. (iii) The relative affinites for oxygen of the bacteroid and plant mitochondria terminal oxidases, and the prevailing oxygen concentration inside the nodule, which results in aerobic metabolism for the bacteroid, but in quite fermentative catabolism for the host plant cell. We propose that the bacteroid can transiently supply free energy to the plant cell in the form of protonmotive force by the movement of hydrogen ions from the bacteroid periplasmic space to the plant cytoplasm through the peribacteroid membrane ATPase. The proposed hydrogen ion flux could be dependent on the phosphorylation potential in both the plant cell cytoplasm and the bacteroid, and the simultaneous ion movements to avoid the development of opposite delta psi. It could be important in situations of transient ATP depletion inside plant cell, which involves the block of ammonia assimilation and, subsequently, the inhibition of bacteroid nitrogenase.

Scanning electron microscopic observation of resting spore clusters of Polymyxa spp.

Resting spore clusters ofPolymyxa graminis andP. betae, fungal vectors of virus diseases, were observed using the scanning electron microscope. The spore clusters in host plant cells were uncovered using the styrene resin cracking method. Resting spores ofP. graminis were found to be spherical, whileP. betae spores were more irregular in shape.

Inheritance of host plant effect on latent period of wheat leaf rust in single-seed descent F8 lines

Latent period (LP) and infection type (IT) were determined in single-seed descent F8 wheat lines from the crosses Westphal 12A (PR = partially resistant) × BH1146 (PR), Akabozu (PR) × BH1146 (PR), and BH1146 (PR) × Little Club (susceptible), inoculated with the Flamingo isolate of the wheat leaf rust pathogen. Each parental line showed a significantly longer LP than the highly susceptible standard Morocco indicating that each parent carried at least one LP-prolonging gene. Significant transgression was found for shorter LP in each cross, indicating that BH1146 differed from Westphal 12A, Akabozu, and Little Club at least partly in genes affecting LP. Significant transgression for longer LP was observed only in the cross Akabozu × BH1146 in one F8 line. In each cross, significantly more lines were found that showed a shorter LP than the most susceptible parent, than lines that showed a longer LP than the most resistant parent, indicating non-additive gene effects on LP. BH1146 was estimated to carry at least five genes which are involved in prolonging the LP. LP and IT were negatively correlated. Correlation coefficients were low and lines were present showing similar ITs but different LPs which suggested that collapse of host plant cells could not be the only reason for growth retardation of colonies of the wheat leaf rust pathogen.

In vitro Techniques and Plant Morphogenesis: Fundamental Aspects and Practical Applications.

Plant cell and tissue culture together with the recent advances in recombinant DNA technology are getting great attention because these are the sources of innovation for the major agricultural industries and practice. However, despite the many successful applications of plant tissue culture in agriculture and horticulture, differentiation and especially plant regeneration, remain a major problem with a number of crop species. Relatively few crop species are capable of predictable regeneration. Even in those instances where regeneration has been reported, e. g. Nicotiana, Petunia and Arabidopsis, through empirical selection of explants and media, problems are often encountered with particular cultivars within a species. Hence, these problems constitute a serious block to crop improvement. Moreover, a detailed characterization of the competent host plant cell will be required if we are to further exploit the Agrobacterium system for transformation of recalcitrant plant species. In this review article we describe some possible opportunities which may lead to rational plant improvement.

Molecular Basis of Gene-for-Gene Specificity in Bacterial Speck Disease of Tomato

Transient expression of the Pseudomonas syringae avirulence gene avrPto in plant cells resulted in a Pto-dependent necrosis. The AvrPto avirulence protein was observed to interact directly with the Pto resistance protein in the yeast two-hybrid system. Mutations in the Pto and avrPto genes which reduce in vivo activity had parallel effects on association in the two-hybrid assay. These data suggest that during infection the pathogen delivers AvrPto into the plant host cell and that resistance is specified by direct interaction of Pto with AvrPto.

Recognition of the Bacterial Avirulence Protein AvrBs3 Occurs inside the Host Plant Cell

The molecular mechanism by which bacterial avirulence genes mediate recognition by resistant host plants has been enigmatic for more than a decade. In this paper we provide evidence that the Xanthomonas campestris pv. vesicatoria avirulence protein AvrBs3 is recognized inside the plant cell. Transient expression of avrBs3 in pepper leaves, using Agrobacterium tumefaciens for gene delivery, results in hypersensitive cell death, specifically on plants carrying the resistance gene Bs3. In addition, for its intracellular recognition, AvrBs3 requires nuclear localization signals that are present in the C-terminal region of the protein. We propose that AvrBs3 is translocated into plant cells via the Xanthomonas Hrp type III secretion system and that nuclear factors are involved in AvrBs3 perception.

Kinetics, energetics and specificity of a general amino acid transporter from the ectomycorrhizal fungus Paxillus involutus

The kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves. The apparent K m derived from the Eadie-Hofstee plots ranged from 7 μM for alanine to 27 μM for glutamate. Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide, vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus. Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast. Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive. The physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed.

Stringently and developmentally regulated levels of a cytoplasmic double-stranded RNA and its high-efficiency transmission via egg and pollen in rice.

A very restricted amount of high-molecular-weight double-stranded RNA (dsRNA) has been found in healthy japonica rice plants. We discriminated dsRNA-carrying rice plants from noncarriers. The endogenous dsRNA was localized in the cytoplasm (about 100 copies per cell) and was transmissible to progeny plants by mating. In crosses between carriers and noncarriers, the RNA was transmitted efficiently to F1 plants via both egg and pollen. The rice dsRNA was maintained at an almost constant level by host plant cells from generation to generation. The high-efficiency transmission of the endogenous dsRNA to progeny plants appears to depend on the autonomously controlled replication of the dsRNA localized in cytoplasmic vesicles. However, an increase in copy number (about 10-fold) of the dsRNA was observed during the suspension culture of host cells. The number of copies of dsRNA returned to the original low value in regenerated plants, suggesting that the copy number is stringently and developmentally regulated in rice cells.

Tissue and intra‐cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.