Carol J. Bayles

Inhibition by 2-deoxy-D-glucose of callose formation, papilla deposition, and resistance to powdery mildew in an ml-o barley mutant☆

Abstract Resistance to powdery mildew in the ml - o mutant barley line, Riso 5678/3 ∗ Carlsberg II, is characterized by a high frequency of early formed, callose-containing papillae. Treatment of ml - o resistant barley coleoptiles with 10 −5 m 2-deoxy-D-glucose, an inhibitor of callose formation in plants, resulted in a decrease in papilla frequency from 92-41% and an increase in penetration efficiency from 15–78%. Time-course analysis showed that papilla formation was also delayed by 2-deoxy-D-glucose treatment and that sites with late-forming papillae were penetrated by the fungus whereas sites with early-forming papillae were not. These results support previous evidence that early papilla formation is the mechanism of ml - o resistance. Use of the callose-specific dye, sirofluor, showed that nearly all papillae in both the controls and the 2-deoxy-D-glucose-treated cells contained callose, suggesting that callose formation may greatly facilitate papilla deposition in this ml - o barley mutant. Papilla frequency also decreased in the susceptible barley isoline, and an increase in penetration efficiency occurred at 4 × 10 −5 m 2-deoxy-D-glucose. We infer that the mutation at the ml - o locus enhanced a constitutive, but originally ineffective, resistance mechanism, namely papilla formation.

Evidence that molecular components of papillae may be involved in ml-o resistance to barley powdery mildew☆

Abstract We demonstrate that papillae in a resistant barley isoline, Riso 5678 (R), contain a light-absorbing component that is not characteristic of papillae in the corresponding susceptible isoline. In resistant coleoptiles incubated on a standard Ca(NO 3 ) 2 solution, penetration attempts by the powdery mildew fungus were typically unsuccessful and papillae contained the light-absorbing component, whereas in the susceptible coleoptiles the reverse occurred. Chlortetracycline strongly inhibited both the resistance to penetration and the incorporation of the light-absorbing component into papillae in the resistant isoline but had little or no effect in the susceptible isoline. When data from all treatments and genotypes were combined, the penetration efficiency was 5% where papillae contained the light-absorbing component, 99% where they did not, and 47% where the amount of light absorbance was intermediate. Autofluorescence, UV absorbance and lacmoid staining all suggested that the light-absorbing component is rich in phenylpropanoids. Acid fuchsin revealed a basic staining material in papillae in resistant but not in susceptible coleoptiles, and in resistant coleoptiles the basic staining material was specifically associated with penetration failures. Thus, the light-absorbing component and basic staining material in papillae may be molecular components of the ml-o resistance mechanism.

Apparent calcium mediation of resistance of an ml-o barley mutant to powdery mildew

Abstract Resistance to Erysiphe graminis f. sp. hordei conditioned by the ml - o gene in barley was inhibited with treatments that were expected to lower the concentration of cytoplasmic, ionized calcium in the host cells. When partially dissected coleoptiles were incubated on 10 m m Ca(NO 3 ) 2 or CaCl 2 , resistance was very high, whereas lowering the exogenous calcium concentration to c . 0.25 m m decreased resistance by 50%. Adding indoleacetic acid to the low levels of calcium enhanced this effect, and resistance was almost totally inhibited. Treatments with the calcium ion chelators, chlortetracycline and anhydrochlortetracycline, also inhibited resistance, whereas treatment with the nonchelating analogue, isochlortetracycline, did not. This inhibition could be overcome by adding calcium to the tetracycline solution. These results lead us to hypothesize that calcium is required for activation of the resistance mechanism and that the ml - o mutation affects calcium regulation in the cell, resulting in an elevated cytosolic calcium ion level in the resistant isoline.

A papilla-regulating extract that induces resistance to barley powdery mildew

Abstract A partially purified aqueous extract from healthy barley leaves, composed apparently of a mixture of peptides and oligosaccharides, was found to regulate papilla formation in inoculated barley coleoptiles. This papilla-regulating extract, the first of its kind to be reported, induced the plant to form oversize papillae in response to attempted fungal penetration, and made susceptible barley resistant to powdery mildew disease. It also induced the plant to deposit more autofluorescent compounds, presumably phenylpropanoid derivatives, in the papillae and host cell walls. Functionally similar extracts were obtained from several plants, including cauliflower, cucumber and onion. All of the extracts induced the formation of oversize papillae in response to fungal attack in wheat coleoptiles as well as in barley. These substances are apparently constitutive in many kinds of plants, and they may play a key role in the regulation of widespread, non-specific defence reactions such as papilla formation and phenylpropanoid deposition.

International Test Results for Objective Lens Quality, Resolution, Spectral Accuracy and Spectral Separation for Confocal Laser Scanning Microscopes

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

Measurement of nuclear DNA contents of Mexican isolates of Phytophthora infestans

The nuclear DNA contents of 145 isolates of Phytophthora infestans from the central highlands of Mexico (the centre of origin of this oomycete), northwestern Mexico, and northeastern Mexico were measured by quantifying the fluorescence from nuclei stained with 4,6-diamidino-2-phenyl-indole (DAPI). Isolates were collected over several different years. Isolates that had been repeatedly subcultured tended to have higher DNA contents than had isolates from storage. Therefore, the DNA contents were measured on isolates soon after isolation from the field or soon after retrieval from storage (under oil at 18 °C or cryogenically). In all three locations, P. infestans isolates mainly contained low quantities of DNA (consistent with presumed diploidy). However, a few isolates with larger quantities of DNA were also detected — primarily in populations from northern Mexico. There appeared to be no strong association between level of DNA content and mating type or year of collection.