Jonathan P. Ride

A rapid method for the chemical estimation of filamentous fungi in plant tissue

Abstract A sensitive colour reaction is described for estimating fungi which contain chitin. The method is based on the alkaline deacetylation of chitin to chitosan, the glucosamine residues of which are susceptible to deamination with nitrous acid. This yields an aldehyde which is determined colorimetrically with 3-methyl-2-benzothiazolone hydrazone. The observed level of aldehyde, expressed as glucosamine, was related to fungal dry weight by experiments using different fungi grown in vitro. The method, requiring 5 h to complete, has been tested on five phytopathogenic fungi and three hosts, and should be applicable to a wide range of host-pathogen systems.

Lignification in wounded wheat leaves in response to fungi and its possible rôle in resistance

Abstract Lignin was synthesized rapidly around wounds in wheat leaves following inoculation with the non-pathogenic fungi Botrytis cinerea and Mycosphaerella pinodes and subsequent fungal growth was limited to the wounds. Wounding alone did not stimulate lignification. Spectroscopy, nitrobenzene oxidation and staining methods indicated that newly synthesized lignin had a different structure from the polymer in healthy leaves. The rate of lignification was slower in response to the pathogenic Septoria nodorum and Septoria tritici and these fungi were not restricted to the wounds. Preinoculation with a non-pathogen delayed attack by either pathogen. The possible role of lignin in controlling the spread of fungi from wounds is discussed.

Isolation and characterization of two phytoalexins from rice as momilactones A and B

Abstract Two phytoalexins were isolated as chromatographically homogeneous amorphous solids from UV-irradiated, dark-grown rice coleoptiles. From their mass and 1 H NMR spectra, the compounds were characterized as the known diterpenes, momilactones A and B. The same compounds were also produced in blast-infected, WL 28325-treated rice leaves. They appear to be the first clearly identified cereal phytoalexins.

Chitin and related compounds as elicitors of the lignification response in wounded wheat leaves

Abstract Chitin, an important component of most fungal cell walls, elicited lignification in wheat leaves when applied as a suspension to wounds. The soluble derivatives ethylene glycol, chitin and chitosan were also active, but glucosamine, N -acetylglucosamine and small chitin oligomers did not elicit the response. The fungal glucan pullulan lacked activity, but laminarin, an algal glucan, was a weak elicitor. The response could also be elicited by fungal cell wall preparations. The possible role of chitin and related wall polymers in inducing host-defence mechanisms in wheat is discussed.

The effect of induced lignification on the resistance of wheat cell walls to fungal degradation

Abstract Lignified papillae and haloes from Botrytis cinerea inoculated wheat leaves were highly resistant to in vitro degradation by fourteen fungal species. Wheat pathogens were generally no more capable of degrading the structures than non-pathogens. Papillae and haloes from leaves taken as early as 12 h after inoculation showed substantial resistance indicating the possible importance of relatively small amounts of lignin. Quantitative measurements of the release of carbohydrate from lignified and non-lignified walls by fungal culture filtrates supported these visual observations. The activities of laccase and p -coumaryl esterase in culture filtrates did not correlate with pathogenicity. The significance of the results is discussed in relation to the proposed role of lignification in the resistance of wheat to fungi.

Lignification and papilla formation at sites of attempted penetration of wheat leaves by non-pathogenic fungi

Abstract Failure of Botrytis cinerea to penetrate the intact surface of primary wheat leaves was associated closely with the production of papillae and alterations in the upper epidermal walls (“haloes”) and adjacent lateral walls. Autoradiography, spectroscopy, fluorescence microscopy and histochemical tests indicated that lignification occurred at these sites. Papillae also contained a substantial amount of polysaccharide, callose and cellulose being indicated by histochemical tests. The apparent time of initiation of lignification was dependent on the detection method employed. Autoradiography of the surface of extracted epidermal strips taken from leaves fed with 3H-phenylalanine or 14C-cinnamic acid indicated that lignification of the upper epidermal wall occurred at an early stage, being detectable as soon as papillae were visible. Papillae, haloes and altered lateral walls were resistant to enzymes which degrade normal epidermal walls. This resistance, which was detectable as soon as papillae were formed, could be destroyed by chemical delignification but not by removal of wall-esterified phenolic acids. Papillae, haloes and lignification were also associated with attempted penetrations by Alternaria tenuis, Septoria apiicola, Botrytis allii and B. fabae. Drop-diffusate tests for phytoalexins using B. cinerea were negative. The possible ways in which lignification, papillae and haloes may be involved in resistance are discussed.

Cellular lignification as a factor in the hypersensitive resistance of wheat to stem rust

Fluorescence microscopy, histochemistry and autoradiography all indicated the cellular accumulation of phenolic compounds during hypersensitive necrosis induced by Puccinia graminis f. sp. tritici in resistant wheat cultivars. Hypersensitivity and phenolic accumulation were observed in near-isogenic lines carrying the Sr 5 or Sr 6 (at 19°C) alleles for resistance, but not in the susceptible reactions of the parent cultivar Marquis ( sr 5, sr 6), or the Sr 6 line when tested at 26°C. Toluidine blue staining and fluorescence microscopy indicated the accumulation of a phenolic material in hypersensitively necrotic cells. Removal of wall-bound phenolic acids with hot alkali caused little loss in fluorescent intensity, but subsequent delignification with chlorine dioxide reduced fluorescence to negligible levels. Autoradiography, using the lignin precursors [ 3 H]-phenylalanine and [ 14 C]-cinnamic acid, showed incorporation of label into cytoplasmic regions of necrotic and degenerating host cells, corresponding to the sites of autofluorescence and histochemical staining. [ 3 H]-Phenylalanine incorporated into healthy cells and fungal structures was lost from both resistant and susceptible tissue on alkali treatment, but some label was retained in necrotic cells. Necrotic regions from infected resistant cultivars were resistant to cell wall degrading enzymes, but extraction of lignin allowed subsequent tissue maceration. The accumulation of a phenolic material which is alkali-insoluble, enzyme resistant and chlorine dioxide extractable indicates that lignification occurs in necrotic cells of the hypersensitive response. A role for cellular lignification as an induced barrier is postulated and its direct or indirect effect on fungal growth in resistant tissue is discussed.

A chemical method for estimating Fusarium oxysporum f. lycopersici in infected tomato plants

Abstract The amount of Fusarium oxysporum f. lycopersici in stems of infected tomato was estimated by measuring N -acetylglucosamine derived from fungal mycelium by enzymic hydrolysis. Hydrolysis, with an enzyme system induced from a soil bacterium, was more convenient and subject to fewer interferences than acid hydrolysis. The variation in the glucosamine/dry weight of F. oxysporum f. lycopersici with morphology, age and cultural conditions was determined on fungus grown in vitro ; 80 μg glucosamine/mg dry weight of fungus was then adopted as a standard for in vivo determinations. The method compares favourably with plating techniques, and is capable of estimating, under optimum conditions, 5 μg fungus/g fresh weight tomato.

The aldo-keto reductase AKR1C3 contributes to 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol mediated oxidative DNA damage in myeloid cells: Implications for leukemogenesis

The aldo-keto reductase AKR1C3, has been shown to regulate myelopoiesis via its ability to metabolise prostaglandin D2 (PGD2). Other studies have demonstrated the oxidative activation of polycyclic aromatic hydrocarbon (PAH) procarcinogens by AKR1C3 in cell-free systems. This is the first study that addresses whether AKR1C3 mediates carcinogen activation within intact living cells following manipulation of AKR1C3 by molecular intervention. Quantitative RT-PCR identified AKR1C3 as the predominant AKR1C isoform expressed in acute myeloid leukemia (AML). Exposure of K562 and KG1a myeloid cell lines to the known AKR1C3 substrate 7,12-dimethylbenz(a)anthracene-3,4-dihydrodiol (7,12-DMBA-3,4-diol) resulted in both single strand DNA breaks and oxidative DNA damage as measured using conventional and FPG-modified comet assays respectively. PGD2-keto reductase activity was shown to be correlated with relative AKR1C3 expression and together with quantitative real time PCR was used to validate the RNAi-knockdown of AKR1C3 in K562 cells. Knockdown of AKR1C3 did not alter single strand DNA breaks following 7,12-DMBA-3,4-diol exposure but significantly decreased oxidative DNA damage. A similar interrelationship between AKR1C3 activity and 7,12-DMBA-3,4-diol mediated oxidative DNA damage but not single strand breaks was observed in KG1a cells. Finally, AKR1C3 knockdown also resulted in spontaneous erythroid differentiation of K562 cells. Since K562 cells are a model of AML blast crisis of chronic myeloid leukemia (CML) the data presented here identify AKR1C3 as a novel mediator of carcinogen-induced initiation of leukemia, as a novel regulator of erythroid differentiation and paradoxically as a potential new target in the treatment of CML.

11β-Hydroxysteroid Dehydrogenase Type 1 Regulation by Intracellular Glucose 6-Phosphate Provides Evidence for a Novel Link between Glucose Metabolism and Hypothalamo-Pituitary-Adrenal Axis Function

Microsomal glucose-6-phosphatase-α (G6Pase-α) and glucose 6-phosphate transporter (G6PT) work together to increase blood glucose concentrations by performing the terminal step in both glycogenolysis and gluconeogenesis. Deficiency of the G6PT in liver gives rise to glycogen storage disease type 1b (GSD1b), whereas deficiency of G6Pase-α leads to GSD1a. G6Pase-α shares its substrate (glucose 6-phosphate; G6P) with hexose-6-phosphate-dehydrogenase (H6PDH), a microsomal enzyme that regenerates NADPH within the endoplasmic reticulum lumen, thereby conferring reductase activity upon 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). 11β-HSD1 interconverts hormonally active C11β-hydroxy steroids (cortisol in humans and corticosterone in rodents) to inactive C11-oxo steroids (cortisone and 11-dehydrocorticosterone, respectively). In vivo reductase activity predominates, generating active glucocorticoid. We hypothesized that substrate (G6P) availability to H6PDH in patients with GSD1b and GSD1a will decrease or increase 11β-HSD1 reductase activity, respectively. We investigated 11β-HSD1 activity in GSD1b and GSD1a mice and in two patients with GSD1b and five patients diagnosed with GSD1a. We confirmed our hypothesis by assessing 11β-HSD1 in vivo and in vitro, revealing a significant decrease in reductase activity in GSD1b animals and patients, whereas GSD1a patients showed a marked increase in activity. The cellular trafficking of G6P therefore directly regulates 11β-HSD1 reductase activity and provides a novel link between glucose metabolism and function of the hypothalamo-pituitary-adrenal axis.