Toshikazu Tani

The role of avenalumin in the resistance of oat to crown rust, Puccinia coronata f. sp. avenae

The relationship between the degree of expression of resistance and the production of avenalumins was investigated in the interactions of 2 physiological races of Puccinia coronata avenae and 21 oat Pc lines each carrying a different single major gene for resistance. Avenalumins accumulated in all incompatible interactions. More rapid and abundant accumulation of avenalumins was found in the more incompatible interactions where fungal growth was more rapidly restricted. Large accumulation of avenalumins coincided with the time of detection of retardation of hyphal growth within leaf tissues. Very little accumulation of avenalumins was found in any compatible interaction. Investigations with the incompatible Shokan 1-race 226 combination showed that the amount of accumulated avenalumins was linearly correlated with infection rate through stomata, and that no avenalumins were detected in areas adjacent to or apart from the infected area. It was thus considered that avenalumins accumulate locally in infected stomatal sites. In highly resistant interactions, the estimated concentration of avenalumins in an infected stomatal site greatly exceeded the inhibitory level for growth of germ tubes from uredospores. Avenalumins could also diffuse into intercellular spaces of infected leaves. It is postulated that the production of avenalumins is regulated as a result of resistance gene expression and is effective against the development of crown rust fungus in oat leaves.

The production of phytoalexins by oat in response to crown rust, Puccinia coronata f. sp. avenae

The antifungal compounds accumulated only in the incompatible host-parasite combinations; the time of rapid accumulation coincided with the time of the retardation of the growth of infection hyphae. Three major compounds were isolated. They are highly hydrophilic nitrogen-containing phenolics. Their antifungal activities against Puccinia coronata avenae and P. graminis tritici were demonstrated. They were regarded as phytoalexins and given the trivial names avenalumins I, II, and III. Avenalumins are the first nitrogen-containing phytoalexins found in plants and they are the first chemically-identified ones found in cereal plants in response to rust fungi.

Differentiation of Rhizoctonia AG-D isolates from turfgrass into subgroups I and II based on rDNA and RAPD analyses

Binucleate Rhizoctonia anastomosis group (AG) D is the cause of rhizoctonia-patch and elephant-footprint diseases of zoysiagrass, and winter-patch disease of bentgrass. Rhizoctonia AG-D is also known as the causal pathogen of other diseases such as sharp-eye-spot of cereals, foot-rot of cereals and winter-stem-rot of mat rush. Isolates of AG-D have been divided into the two subgroups AG-D (I) and AG-D (II), based on the results of cultural characteristics and pathogenicity tests. Isolates obtained from zoysiagrass exhibiting symptoms of rhizoctonia-patch disease, from bentgrass with winter-patch disease, from wheat with foot-rot disease, and from mat rush with winter-stem-rot disease were reported to belong to subgroup AG-D (I). On the other hand, isolates obtained from zoysiagrass with elephant-footprint disease were assigned to subgroup AG-D (II). To confirm the existence of these two subgroups in AG-D, the genetic structure of AG-D isolates from turfgrass and other crops was compared. RFLP analysis of the ITS region from rDNA after digestion with the restriction enzymes EcoRI, HaeIII, HhaI, HinfI, and MboI separated AG-D isolates into two groups corresponding to AG-D (I) and AG-D (II). Furthermore, other AGs except AG-Q (AGs-A, Ba, Bb, C, E, F, G, I, K, L, O, P, and R. solani AG1-IC) did not have the same patterns that were seen for the two AG-D subgroups. AG-Q isolates from bentgrass showed the same patterns as AG-D (I). The results of the RAPD analysis also revealed the existence of two groups that corresponded to AG-D (I) and AG-D (II). These analyses revealed that Rhizoctonia AG-D isolates from turfgrass could be divided into two subgroups consistent with those based on cultural characteristics and pathogenicity. In addition, isolates of foot-rot disease of wheat and isolates of winter-stem-rot disease of mat rush whose cultural characteristics were the same as those of AG-D (I) also showed similar RFLP and RAPD patterns to those of AG-D (I) isolates from turfgrass.

Isolation and structure elucidation of genuine oat phytoalexin, avenalumin I

Abstract The major phytoalexin from oat leaves has been identified as 2-[2-(4-hydroxyphenyDethenyl] -6-hydroxy-4H-3,1-benzoxazin-4-one ( A ).

Initiation of resistance and host cell collapse in the hypersensitive reaction of oat leaves against Puccinia coronata avenae

Abstract The relationship between expression of resistance and host cell collapse was studied for the hypersensitive resistance of oat cultivar Shokan 1 infected by incompatible race 226 of Puccinia coronata avenae. Microscopic examination of primary leaves of seedlings treated by several methods involving heat treatment, stem detachment and double inoculation indicated that initial events determining the host resistance occurred between 8 and 12 h after inoculation, when the fungus produced substomatal vesicles. The initial events were followed by the reduction of growth of infection hyphae to half that in susceptible leaves. During the period of prehaustorial growth of the fungus, neither host cell collapse at infected sites nor permeability increase of leaf tissues was observed. The cessation of hyphal growth and haustorial development appeared to be normally concomitant with host cell collapse at 35 h after inoculation. However, study of leaves treated with blasticidin S or ethidium bromide or heat revealed that the limitation of fungal development in resistant reactions was not correlated with the degree of host cell collapse. The development of a normally compatible race was restricted without any collapse of mesophyll cells when the leaves were previously inoculated with the incompatible race and then heated. We propose that in the Shokan 1-race 226 host-parasite system, the initial determinative events for resistance are induced independently from the process of host cell collapse. Host cell collapse, although a normal consequence of the resistant reaction, therefore has no determinative role in the expression of resistance of Shokan 1 leaves to race 226.

Microspectrophotometric analysis of the location of ] avenalumin accumulation in oat leaves in response to fungal infection

Abstract Cellular localization of avenalumins accumulated in oat leaves infected by either crown rust through stomata or rice blast fungus through the cuticular layer was demonstrated by microspectrophotometry using the fluorescence emission and u.v. absorption spectra characteristic of avenalumins. Thin sections of incompatible rust-infected leaves revealed collapsed mesophyll cells which emitted autofluorescence and absorbed u.v. light with spectral characteristics close to those of the avenalumins. Autofluorescent and u.v.-absorbing cells were detected only in incompatible rust race-oat combinations. Epidermal cells accumulated quite high levels of avenalumins in response to cuticular infection by the facultative pathogen. Spectrophotometry of the freshly stripped epidermis showed the presence of autofluorescent materials, with spectra characteristic of avenalumins, in infected and the directly adjacent cells. U.v.-absorption spectra close to those of avenalumins were also detected in fluorescent cells. Extraction of epidermal strips with methanol reduced fluorescence emission and u.v.-absorption in fluorescent cells by c . 40% of the original, thus suggesting that avenalumins could be partially eluted out of the fluorescent cells. Epidermal cells which were injected with aqueous solution of avenalumin I became fluorescent. Results suggest that avenalumins accumulate in the fluorescent mesophyll and epidermal cells formed in oat leaves at infection sites of fungal pathogens.

Nucleic acid and protein synthesis in association with the resistance of oat leaves to crown rust

Abstract Causal association of ribonucleic acid (RNA) and protein synthesis with expression of resistance in the oat crown rust disease was studied using primary leaves of cv. Shokan 1 and incompatible race 226 and compatible race 203 of Puccinia coronata avenae. Fungal development of the incompatible race was stimulated by the treatment of leaves with either the RNA synthesis inhibitor, cordycepin, or the protein synthesis inhibitor, puromycin. The effect of cordycepin was fully expressed when it was supplied within 10 h after inoculation, but gradually declined as the time of supply was delayed. The effect of puromycin was fully expressed even when supplied to leaves at 12 h after inoculation, but declined when supplied later than 14 h after inoculation. Double-labelling with [3H]uridine and [14C]leucine showed that the synthesis of total RNA and protein increased between 12 and 16 h and 14 and 20 h, respectively, after inoculation of leaves with the incompatible race. Incorporation of [3H]uridine into messenger RNA (mRNA) also increased between 12 and 16 h after inoculation with the incompatible race. Such increases were not observed in leaves inoculated with the compatible race. It was postulated that synthesis of RNA, including mRNA, is initiated at 12 h after inoculation and continues for several hours, leading to increased synthesis of proteins involved in expression of resistance.

Effects of elevated temperature and α-aminooxyacetate on the accumulation of avenalumins in oat leaves infected with Puccinia coronata f. sp. avenae☆

Abstract The rapid accumulation of avenalumins in incompatible crown rust-infected Shokan 1 oat leaves which occurred at 20 °C was greatly inhibited when infected plants were grown at 25 to 35 °C. At 30 °C, avenalumin accumulation was almost totally inhibited and much greater hyphal growth occurred than at 20 °C. When infected plants were grown at 20 °C for 36 h and then transferred to 30 °C, most of the previously-accumulated avenalumins disappeared rapidly within 48 h and hyphal growth continued. However, no subsequent hyphal growth resulted when the plants were transferred to 30 °C at 72 h after inoculation. Treatment of plants with α-aminooxyacetate, a potent competitive inhibitor of phenylalanine ammonia-lyase, greatly inhibited the rapid accumulation of avenalumins in normally incompatible interactions. This was correlated with an increase in susceptibility of the host plants. Significant inhibition of avenalumin accumulation was observed 48 h after inoculation when inoculation was followed by application of the inhibitor at 10 μ m , but much less inhibition of hypersensitive host cell collapse occurred in the treated leaves. These facts suggest that rapid and large accumulation of the avenalumins is crucial for resistance expression in oat leaves against an incompatible crown rust race.

Two-dimensional analysis of enhanced synthesis of proteins in oat leaves responding to the crown rust infection

Abstract Changes in protein composition of primary leaves of oat (Avena sativa L.) cultivar Shokan 1 were studied in relation to resistance expression at the early stage of infection (16 to 20 h after inoculation) with incompatible race 226 of Puccinia coronata avenae. The incorporation ratio of [14C]-leucine into the acid-insoluble fraction was increased by inoculation with race 226 in both the leaf homogenate and the cytoplasmic fraction, while in the microsomal fraction it was unchanged. The pre-treatment of inoculated leaves with blasticidin S, a protein synthesis inhibitor, largely inhibited this incorporation into the cytoplasmic fraction, but only slightly into the microsomal fraction. Two-dimensional polyacrylamide gels stained with Coomassie blue revealed no changes in protein composition in leaves inoculated with either race 226 or race 203. However, the tracer experiments made with [14C]-leucine revealed that the number of protein spots detectable by fluorography were 87, 77 and 67 for cytoplasmic fractions from leaves inoculated with races 226 and 203 and uninoculated leaves respectively. Eleven spots were found to be specific for the incompatible interaction but were not detected in leaves treated with blasticidin S. Sixteen spots labeled with [14C]-leucine were detected in microsomal fractions from leaves inoculated with race 226 and from the uninoculated control but no difference was observed between them. It is suggested that enhanced synthesis of cytoplasmic proteins is involved in the resistance expression of oat leaves against the incompatible race of the crown rust fungus.

Selective enhancement of ribosomal RNA synthesis of crown rust-infected oat leaves by stem excision

Abstract In leaves of Avena sativa infected by a compatible race of Puccinia coronata avenae , a rapid increase of ribosomal ribonucleic acid (rRNA) was found when the stem was excised 4 days after inoculation, at which time the fungus was in the beginning of differentiation toward uredosorus formation. Concentrations increased up to 1·5-fold within 4 h after excision, while in the attached control the rRNA concentration increased gradually and reached the same level 16 h later. Stem excision did not affect the concentration of soluble RNA (sRNA) and deoxyribonucleic acid (DNA). This selective increase of rRNA by stem excision was observed neither in the infected leaves 3 and 5 days after inoculation, nor in the noninoculated control leaves. Experiments using [ 32 P]orthophosphate indicated that the increase of rRNA was due to enhanced rRNA synthesis. No appreciable changes were shown for the synthesis of sRNA, tenaciously bound RNA and DNA. Polyacrylamide gel electrophoretic separation of rRNA species revealed that the increase of rRNA was due to cytoplasmic rRNA and not due to chloroplast rRNA.