Members of the tomato LeEIL (EIN3-like) gene family are functionally redundant and regulate ethylene responses throughout plant development.

The plant hormone ethylene regulates many aspects of growth, development and responses to the environment. The Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) protein is a nuclear-localized component of the ethylene signal-transduction pathway with DNA-binding activity. Loss-of-function mutations in this protein result in ethylene insensitivity in Arabidopsis. To gain a better understanding of the ethylene signal-transduction pathway in tomato, we have identified three homologs of the Arabidopsis EIN3 gene (LeEILs). Each of these genes complemented the ein3-1 mutation in transgenic Arabidopsis, indicating that all are involved in ethylene signal transduction. Transgenic tomato plants with reduced expression of a single LeEIL gene did not exhibit significant changes in ethylene response; reduced expression of multiple tomato LeEIL genes was necessary to reduce ethylene sensitivity significantly. Reduced LeEIL expression affected all ethylene responses examined, including leaf epinasty, flower abscission, flower senescence and fruit ripening. Our results indicate that the LeEILs are functionally redundant and positive regulators of multiple ethylene responses throughout plant development.

Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria.

The hypersensitive response (HR) involves rapid death of cells at the site of pathogen infection and is thought to limit pathogen growth through the plant. Ethylene regulates senescence and developmental programmed cell death, but its role in hypersensitive cell death is less clear. Expression of two ethylene receptor genes, NR and LeETR4, is induced in tomato (Lycopersicon esculentum cv. Mill) leaves during an HR to Xanthomonas campestris pv. vesicatoria, with the greatest increase observed in LeETR4. LeETR4 antisense plants previously were shown to exhibit increased sensitivity to ethylene. These plants also exhibit greatly reduced induction of LeETR4 expression during infection and an accelerated HR at inoculum concentrations ranging from 10(5) to 10(7) CFU/ml. Increases in ethylene synthesis and pathogenesis-related gene expression are greater and more rapid in infected LeETR4 antisense plants, indicating an enhanced defense response. Populations of avirulent X. campestris pv. vesicatoria decrease more quickly and to a lower level in the transgenic plants, indicating a greater resistance to this pathogen. Because the ethylene action inhibitor 1-methylcyclopropene alleviates the enhanced HR phenotype in LeETR4 antisense plants, these changes in pathogen response are a result of increased ethylene sensitivity.

Ethylene-dependent salicylic acid regulates an expanded cell death response to a plant pathogen.

The molecular events associated with susceptible plant responses to disease-causing organisms are not well understood. We have previously shown that ethylene-insensitive tomato plants infected with Xanthomonas campestris pv. vesicatoria have greatly reduced disease symptoms relative to wild-type cultivars. Here we show that salicylic acid (SA) is also an important component of the susceptible disease response. SA accumulates in infected wild-type tissues and is correlated with necrosis but does not accumulate in ethylene-insensitive plants. Exogenous feeding of SA to ethylene-deficient plants restores necrosis, indicating that reduced disease symptoms are associated with failure to accumulate SA. These results indicate a mechanism for co-ordination of phytohormone signals that together constitute a susceptible response to pathogens.

The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family.

The plant hormone ethylene is involved in many developmental processes, including fruit ripening, abscission, senescence, and leaf epinasty. Tomato contains a family of ethylene receptors, designated LeETR1, LeETR2, NR, LeETR4, and LeETR5, with homology to the Arabidopsis ETR1 ethylene receptor. Transgenic plants with reduced LeETR4 gene expression display multiple symptoms of extreme ethylene sensitivity, including severe epinasty, enhanced flower senescence, and accelerated fruit ripening. Therefore, LeETR4 is a negative regulator of ethylene responses. Reduced expression of this single gene affects multiple developmental processes in tomato, whereas in Arabidopsis multiple ethylene receptors must be inactivated to increase ethylene response. Transgenic lines with reduced NR mRNA levels exhibit normal ethylene sensitivity but elevated levels of LeETR4 mRNA, indicating a functional compensation of LeETR4 for reduced NR expression. Overexpression of NR in lines with lowered LeETR4 gene expression eliminates the ethylene-sensitive phenotype, indicating that despite marked differences in structure these ethylene receptors are functionally redundant.

Response to Xanthomonas campestris pv. vesicatoria in tomato involves regulation of ethylene receptor gene expression.

Although ethylene regulates a wide range of defense-related genes, its role in plant defense varies greatly among different plant-microbe interactions. We compared ethylene's role in plant response to virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria in tomato (Lycopersicon esculentum Mill.). The ethylene-insensitive Never ripe (Nr) mutant displays increased tolerance to the virulent strain, while maintaining resistance to the avirulent strain. Expression of the ethylene receptor genes NR and LeETR4 was induced by infection with both virulent and avirulent strains; however, the induction of LeETR4 expression by the avirulent strain was blocked in the Nr mutant. To determine whether ethylene receptor levels affect symptom development, transgenic plants overexpressing a wild-type NR cDNA were infected with virulent X. campestris pv. vesicatoria. Like the Nr mutant, the NR overexpressors displayed greatly reduced necrosis in response to this pathogen. NR overexpression also reduced ethylene sensitivity in seedlings and mature plants, indicating that, like LeETR4, this receptor is a negative regulator of ethylene response. Therefore, pathogen-induced increases in ethylene receptors may limit the spread of necrosis by reducing ethylene sensitivity.

Separation of cis elements responsive to ethylene, fruit development, and ripening in the 5'-flanking region of the ripening-related E8 gene.

The E8 gene is expressed at a high level during fruit ripening, and is transcriptionally activated by ethylene. We have identified a 428 bp fragment of the E8 5'-flanking region, from -1528 to -1100, that makes a minimal 35S promoter responsive to ethylene. This fragment confers ethylene-responsiveness only in the 5'-to-3' orientation; in the reverse orientation it results in increased expression in unripe fruit. Interestingly, this ethylene-responsive construct does not have high levels of expression during fruit ripening, indicating that sequences required for high level expression during fruit ripening are separate from sequences required for ethylene response. The ethylene-responsive sequences of the E8 5'-flanking region interact with the same DNA-binding protein that interacts with sequences required for ethylene responsiveness of the coordinately regulated E4 gene. We also conducted experiments to test the function of a second DNA-binding protein that interacts with both E4 and E8 5'-flanking sequences, the E4/E8-binding protein (E4/E8BP). We examined the effect of an internal deletion from -1088 to -863, which includes the binding site for E4/E8BP, on gene expression. This deletion did not affect expression in ripening fruit, and did not impair ethylene responsiveness. The deletion had a negative effect on expression in unripe fruit, but resulted in increased expression in leaves. These results suggest that the E4/E8BP is not critical for high levels of expression during fruit ripening or for ethylene response, but may play a role in organ-specific gene transcription.

The influence of saliva on infection of the human mouth by mutans streptococci.

The relationship between oral implantation of Streptococcus mutans IB1600 (serotype c) and Streptococcus sobrinus OMZ65 (serotype g), the aggregating activity of saliva, and its influence on the adherence of these bacterial strains in vitro was examined in seven human subjects. All the saliva samples aggregated strain IB1600 but not strain OMZ65 cells. Whole saliva from subjects with low levels of infection by Strep. mutans aggregated strain IB1600 to a greater degree than did whole saliva from those who were readily infected. Whole saliva from subjects most resistant to infection supported the adsorption of the highest number of either strain IB1600 or OMZ65 to hydroxyapatite surfaces. Thus the ability of whole saliva to aggregate Strep. mutans may influence the ability of these microorganisms to infect the mouth.

Glucan-binding factor in saliva.

High-molecular-weight polymers of alpha-1,6-linked D-glucans are insoluble in alcohol solutions. Whole, but not parotid, saliva prevented the precipitation of D-glucans by 80% (vol/vol) ethanol, showing that the whole saliva contained a factor which complexed with the glucan to render it alcohol soluble. The glucan-binding factor was retained on a column of Sephacryl S-200 which had been preequilibrated with 80% ethanol. The factor was then eluted with water. Passive hemagglutination assays revealed that the glucan-binding factor could sensitize erythrocytes to agglutination with anti-poly(glycerolphosphate), suggesting that the active glucan-binding component with lipoteichoic acid. The glucan-solubilizing factor was resistant to heat (100 degrees C), proteases, sialidase, lysozyme, lactoperoxidase, trichloroacetic acid, and Triton X-100. When sucrose was added to saliva, a suspension of Streptococcus cricetus AHT, or a suspension of Streptococcus sanguis 10556, relatively large amounts of glucan-binding factor were released in a soluble form. In addition, penicillin G caused the release of the glucan-solubilizing component from a suspension of S. cricetus AHT. It is suggested that whole saliva contains a component, tentatively identified as lipoteichoic acid, which can complex with glucans in a relatively hydrophobic solvent. This type of complex formation may be important in the adhesion of oral streptococci to saliva-coated surfaces.

Altered bacterial aggregation and adherence associated with changes in rat parotid-gland salivary proteins induced in vivo by beta-adrenergic stimulation.

Reduced adherence and aggregation were associated with protein alterations in parotid saliva after chronic treatment with the beta-adrenergic agonist isoproterenol. In contrast, saliva from animals treated with the beta-antagonist, propranolol, did not cause such changes; the protein composition of this saliva was similar to that of controls. SDS-polyacrylamide gel electrophoresis of protein in saliva samples before and after they were mixed with 10 mg of spheroidal hydroxyapatite beads (HA), as well as protein adsorbed and recovered from the HA, showed that an acidic, proline-rich protein with a molecular weight of approx. 40,000 was the predominant protein adsorbed. This protein was significantly diminished in saliva from isoproterenol-treated rats. Proteins with molecular weights between 44,000 and 48,000 and unique to the saliva from isoproterenol-treated animals were also adsorbed to HA. Thus alterations in proline-rich proteins of parotid saliva may influence the adherence and aggregation of oral bacteria, two processes considered important for in-vivo colonization of oral surfaces.

Cell-to-cell interaction of Streptococcus sanguis and Propionibacterium acnes on saliva-coated hydroxyapatite.

Cell-to-cell interaction (coaggregation) between Propionibacterium acnes PK93 and Streptococcus sanguis DL1 was measured on saliva-coated hydroxyapatite beads (SHA) at bacterial concentrations between 1.3 X 10(6) and 6.7 X 10(8) cells per ml. Four hundredfold more DL1 than PK93 cells adhered to the saliva-coated beads, and the adherence of S. sanguis was proportional to cell input. SHA precoated with 3 X 10(8) DL1 cells bound 75 to 80% of available PK93 cells at all input amounts tested, up to an input of 8 X 10(7) cells. Adherence of PK93 to DL1-coated SHA approached saturation at an input of approximately 10(9) PK93 cells, when 1.5 X 10(8) bound. The coaggregation on SHA occurred either in buffer or saliva and was inhibited by N-acetylgalactosamine and by lactose; the attachment of DL1 to SHA was not inhibited by these sugars. S. sanguis 34 and heat-treated DL1 cells, neither of which form coaggregates with PK93, attached to SHA, but such cells did not bind PK93 cells. The findings of this study indicate that bacteria unable to attach to saliva-coated hydroxyapatite can indeed adhere to such a surface by strong lectin-mediated cell-to-cell interactions with bacteria already attached to the surface.

Role of sucrose in plaque formation.

Results are presented which support the concept that the bacterial enzyme glucosyltransferase (GTF) plays a crucial role in sucrose induced plaque formation. GTF was shown to adhere strongly to anionic, hydrophobic and polysaccharide solid materials, and to be able to produce glucans in the adsorbed state. It appears conceivable that GTF adsorb to teeth and produce glucans. Glucan chains on the surface of the bacteria and glucans on the tooth surfaces interact (pack) and form a strong binding mechanism. The rigid alpha 1,3 linked glucans produced by Streptococcus mutans are particularly suited for interaction of this kind. This mechanism could account for sucrose-induced binding of bacteria to enamel, pellicle covered enamel and preformed plaque. S. mutans would adhere particularly strongly to tooth surfaces in the presence of sucrose, according to this model.

Effect of antibody preparations on glucose uptake by a cariogenic Streptococcus.

The effect of antisera to whole cells or cell wall components on glucose uptake by S. mutans 6715 was examined. Early stationary phase 6715 cells were treated with test serum and incubated at 37 degrees C in the presence of 14C-glucose. Samples were removed at timed intervals and measured in a liquid scintillation counter for radioactive uptake. Antisera to both whole cells and components known to be present on the surface of the cells reduced glucose uptake relative to normal serum. It is suggested that inhibition of glucose uptake may be one mechanism by which a caries vaccine may operate.

The effect of xylitol on plaque metabolism.

Evidence is presented which shows that Strep. mitior and Strep. mutans (which are unable to metabolize xylitol) take yp xylitol and transfer it to xylitol phosphate. Dental plaque also takes up xylitol. In this case a xylitol/protein complex is formed in addition to xylitol phosphate and also some labelled components yet to be identified. It is suggested that accumulation of xylitol phosphate inside the cells may "poison" the bacteria and possibly explain the caries therapeutic effect of xylitol observed in some laboratories.

Effect of topical application of stannous fluoride, stannous chloride and stannous tartrate on rat caries.

Topical application of 10 mM aqueous solutions of stannous fluoride inhibited caries in rats to a higher degree than 20 mM sodium fluoride, although the difference was not statistically significant. Furthermore, stannous fluoride reduced the number of Strep. mutans in plaque significantly; stannous ions have an antibacterial effect. Stannous chloride and stannous tartrate did not reduce caries in the rats, probably because of the low concentrations of available stannous ions in these solutions at low pH. The high concentration of stannous ions in solutions of stannous fluoride is probably partly due to the reduced hydroxide formation resulting from the buffering effect of HF formed at low pH in this solution.

Identification of IgA, IgG, lysozyme, albumin, alpha-amylase and glucosyltransferase in the protein layer adsorbed to hydroxyapatite from whole saliva.

IgA, IgG, albumin, lysozyme, alpha-amylase and glucosyltransferase were identified in the saliva coat which forms on hydroxyapatite exposed to whole saliva. It is suggested that these proteins may be involved in binding microorganisms to saliva coated hydroxyapatite in model studies.

Adsorption of glucosyltransferase to saliva coated hydroxyapatite. Possible mechanism for sucrose dependent bacterial colonization of teeth.

Glucosyltransferase (GTF) adsorbed to hydroxyapatite and to saliva coated hydroxyapatite in vitro. Several proteins which are known to be present in the "pellicle" which forms on hydroxyapatite when this mineral is exposed to whole saliva were shown to stimulate or inhibit GTF. It is suggested that these proteins may interact with GTF and cause binding of the enzyme to saliva coated hydroxyapatite. A model is suggested where GTF adsorbed to tooth surfaces may induce binding of microorganisms to tooth surfaces.

Rapid quantitative determination of the effect of antiplaque agents and antisera on the growth, acid production, and adherence of Streptococcus mutans.

A rapid quantitative in vitro assay measured the effects of antiplaque agents and antiserum on growth and sucrose-mediated adherence of radio-labeled S. mutans 6715. Acid production was measured by change in pH. In this assay the primary effect of chlorhexidine and fluoride was bacteriostasis and inhibition of acid production, respectively; the primary effect of dextran, dextranase, and specific antiserum was inhibition of sucrose-mediated adherence.