Bacterial lipopolysaccharides as inducers of disease resistance in tobacco.

The cell wall component of Pseudomonas solanacearum that induces disease resistance in tobacco was highly heat stable at neutral or alkaline pH but highly labile at acid pH. Activity was unaffected by nucleases and proteases but destroyed by a mixture of beta-glycosidases. Washing of bacterial cell walls released a lipopolysaccharide (LPS) fraction with high inducer activity. Purified LPS, extracted by a variety of procedures from whole cells, isolated cell walls, and culture filtrates of both smooth and rough forms of P. solanacearum, induced disease resistance in tobacco at concentrations as low as 50 microgram/ml. The LPS from the non-plant pathogens Escherichia coli B, E. coli K, and Serratia marcescens was also active. Cell wall protein, free phospholipid, and nucleic acids were not necessary for activity. Moreover, since LPS from rough forms was active, the O-specific polysaccharide of the LPS was not required for activity. Hydrolysis of the remaining core-lipid A linkage or deacylation of lipid A destroyed inducer activity. When injected into tobacco leaves, purified LPS attached to tobacco mesophyll cell walls and induced ultrastructural changes in the host cell similar to those induced by attachment of whole heat-killed bacteria.     

Breeding tobacco for disease resistance

Resistance toward eight fungal, four virus, two nematode and one parasitic seed-plant disease have almost exclusively occupied the attention of tobacco breeders, and improvement in quality has so far received only negligible consideration.     

Bacterial lipopolysaccharides activate immune suppressor cells in newborn mice

Neonatal injection of C57B1/6 mice with bacterial LPS results in an impairment of the ability of splenic lymphocytes to respond to erythrocyte antigens in vitro 4 weeks later. This impairment is due either to a de novo activation of suppressor cells or to an enhancement of the longevity of “naturally occurring” suppressor cells found in the newborn spleen since cells from LPS-injected mice also inhibited normal control responses. The suppressor cells from LPS-injected mice are not macrophages and, by conventional criteria, appear to be T lymphocytes. Results of this study raise questions concerning the effects of suppressor cells on LPS-potentiated antibody formation and the multiplicity of pathways for activation of antibody-forming precursor cells.     

Bacterial amphiphiles as amyloid inducers: Effect of Rhamnolipid and Lipopolysaccharide on FapC fibrillation

Pseudomonas species export the amyloid-forming protein FapC to strengthen bacterial biofilm. P. species also produce the biosurfactant rhamnolipid (Rhl) and its outer membrane contains lipopolysaccharide (LPS). Given the possible contacts between FapC, Rhl and LPS, we here investigate how Rhl and LPS affect FapC fibrillation compared with SDS, known to promote fibrillation of proteins at sub-micellar concentrations. Micelles of all three surfactants help FapC bypass the nucleation lag phase, leading to rapid fibrillation, which persists even at high concentrations of micelles and incorporates almost all available FapC monomers. Fibrils formed at high micellar concentrations of Rhl and SDS seed fibrillation at low surfactant concentrations while retaining the original fibril structure. FapC interacts strongly with SDS to form a dense network of narrow fibrils. Small angle X-ray scattering (SAXS) analyses reveal that surfactants reduce the population of intermediates in the fibrillation process and detect a fast aggregation step over the first 2–4 h which precedes the main fibrillation monitored by Thioflavin T. An additional SAXS-detected rearrangement of early aggregates occurs after 4–10 h. At high Rhl concentrations, the micelles are decorated with protein fibrils. SDS induces FapC fibrillation so efficiently that epigallocatechin-3-gallate (EGCG) is unable to inhibit this process. However, EGCG stimulates FapC oligomer formation and inhibits fibrillation both on its own and in the presence of Rhl and LPS. This oligomer could be modelled as a compact core with a flexible shell. This suggests that EGCG can override the natural amyloid-stimulatory properties of these biosurfactants and thus target biofilm.     

Fungal nucleic acids as interferon inducers.

Nucleic acids isolated from the fungi Aspergillus niger x11, Piptoporus betulinus and Ganoderma applanatum reduced the number of vaccinia virus plaques in chick embryo fibroblast (CEF) tissue culture and when administered intravenously to white mice protected them against lethal infection with tick borne encephalitis virus strain K5 (TBE). In CEF tissue culture the nucleic acids of the studied fungi were found to induce small but detectable amounts of a substance with the character of interferon. In vivo only ribonucleic acid from G. applanatum induced a substance showing interferon properties in the spleen of mice.     

Isolation and selection of plant growth-promoting rhizobacteria as inducers of systemic resistance in melon

Backgroud and aims

Powdery mildew elicited by Podosphaera fusca is an important threat to cucurbits. In order to find alternatives to the current use of chemicals, we examined the potential use of plant growth-promoting rhizobacteria (PGPR) for controlling the disease by induction of systemic resistance in the host plant.

Methods

A collection of Bacillus and Pseudomonas strains from different origins was studied, including strains isolated from roots of disease-free melon plants obtained from a greenhouse plagued by powdery mildew. The selection of best candidates was based on the evaluation of different traits commonly associated with PGPR, such as antifungal and siderophore production, swimming and swarming motilities, biofilm formation, auxin production and promotion of root development.

Results

Three Bacillus strains, B. subtilis UMAF6614 and UMAF6639 and B. cereus UMAF8564, and two Pseudomonas fluorescens strains, UMAF6031 and UMAF6033, were selected after ranking the strains using a nonparametric statistics test. Applied to melon seedlings, the selected strains were able to promote plant growth, increasing fresh weight up to 30%. Furthermore, these strains provided protection against powdery mildew and also against angular leaf spot caused by Pseudomonas syringae pv. lachrymans, with disease reductions of up to 60%.

Conclusions

These results suggest that the use of ISR-promoting PGPR could be a promising strategy for the integrated control of cucurbit powdery mildew and other cucurbit diseases.     

Elicitor-mediated oligomerization of the tobacco N disease resistance protein

Plant nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins are similar to the nucleotide binding oligomerization domain (NOD) protein family in their domain structure. It has been suggested that most NOD proteins rely on ligand-mediated oligomerization for function, and we have tested this possibility with the N protein of tobacco (Nicotiana tabacum). The N gene for resistance to Tobacco mosaic virus (TMV) is a member of the Toll-interleukin receptor (TIR)-NBS-LRR class of plant disease resistance (R) genes that recognizes the helicase domain from the TMV replicase. Using transient expression followed by immunoprecipitation, we show that the N protein oligomerizes in the presence of the elicitor. The oligomerization was not affected by silencing Nicotiana benthamiana ENHANCED DISEASE SUSCEPTIBILITY1 and N REQUIREMENT GENE1 cofactors of N-mediated resistance, but it was abolished by a mutation in the P-loop motif. However, loss-of-function mutations in the RNBS-A motif and in the TIR domain retain the ability to oligomerize. From these results, we conclude that oligomerization is an early event in the N-mediated resistance to TMV.     

Modification of disease resistance of tobacco callus tissues by cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a “hypersensitive” reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.     

Polyacrylic acid-induced resistance to tobacco mosaic virus in tobacco cv. Xanthi

Polyacrylic acid (PA) of molecular weight 1700A, 1700B and 3500 caused resistance to infection with tobacco mosaic virus in tobacco cv. Xanthi-nc, when sprayed on the leaves or watered into the soil. The numbers of lesions produced in the treated plants were between 27 and 97% fewer than in the untreated plants depending on the concentration of PA, its molecular weight and the method of application. Some resistance was caused against potato virus X and potato virus Y but only at concentrations that were harmful to the plants. It appears that PA activates a mechanism responsible for localizing viruses in hypersensitive plants.     

Multiplication of tobacco mosaic virus in tobacco callus tissues and in vitro selection for viral disease resistance

Tobacco mosaic virus-resistant tobacco was selected in vitro using callus tissues induced from axillary buds of systemically infected tobacco plants. Callus lines in which the virus was continuously multiplying were first isolated and redifferentiated into shoots. By the procedure, non-diseased, healthy shoots were successfully isolated from diseased shoots, which showed typical mosaic symptoms of the virus, and regenerated into intact plants.These regenerated plants showed resistance to virus inoculation, and selfed progeny of virus-resistant regenerants segregated the resistance and susceptibility according to the Mendelian system.     

Control of Pratylenchus brachyurus in soybean with Trichoderma spp. and resistance inducers

Trichoderma spp. is a fungus with nematode control potential; however, its potential to control the root lesion nematode Pratylenchus brachyurus remains poorly studied. Thus, the aim of this study was to select Trichoderma spp. isolates and assess their ability to control P. brachyurus in soybean crops. Different experiments were conducted aiming at selecting isolates, assessing whether they were able to reduce nematode penetration in plants or cause mortality in vitro, and whether they were able to induce resistance in soybean, as well as at studying the possibility of using the selected isolates associated with resistance inducers (acibenzolar‐S‐methyl, Ecolife^? and AgroMos^?). The selection experiment found three isolates showing satisfactory results, namely GF422, GF425 and GF427; the GF362 isolate was assessed in the subsequent experiments. These four isolates reduced P. brachyurus penetration in soybean roots and promoted nematode mortality in vitro. Increased total protein and catalase activity were recorded, mainly in the 72‐hr assessments. Overall, the protein production was different between isolates. The best results were found in the combination between the GF362 isolate and the three resistance inducers, between GF427 and Ecolife^?, between GF427 and AgroMos^? and between GF422 and Ecolife^?.     

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.     

Polycations as prostaglandin sythesis inducers. II. structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (>80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. Full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Polycations as prostaglandin synthesis inducers. II. Structure-activity relationships

Moderately high molecular weight polycations stimulate arachidonic acid release with concomitant synthesis and release of prostaglandins in cultured 3T3 mouse fibroblasts. We have examined a series of synthetic polycations for prostaglandin synthesis-inducing activity as an approach to defining the structural features required for activity. Extensive (greater than 80%) acetylation of poly(vinylamine) was tolerated without loss of activity, indicating that a uniform high density of charges is not required. However, complete acetylation of poly(vinylamine) abolished activity, indicating that some positive charges are required for activity. full activity was observed for charge densities in the range of one per two to one per six atoms of polymer backbone. Branched and linear polycations activated equally well. Location of the charge with respect to the polymer backbone did not affect activity in polymers bearing charges located up to seven atoms away from the backbone. Polycations lacking primary or secondary amino groups exhibited full activity, indicating that Schiff base formation is not required for activity. These results are consistent with a model in which activation involves electrostatic interactions with discrete anionic sites on the target cell.     

Brassinosteroid functions in a broad range of disease resistance in tobacco and rice

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.