An epidermis/papilla-specific oxalate oxidase-like protein in the defence response of barley attacked by the powdery mildew fungus.

A cDNA clone of a defence response transcript was isolated from a library prepared from barley leaves expressing papilla resistance towards the powdery mildew fungus, Blumeria (syn. Erysiphe) graminis f.sp. hordei (Bgh). The 904 bp sequence encodes a 229 amino acid polypeptide with a putative signal peptide of 23 amino acids. After cleavage, the protein has a mass of 22.3 kDa and exhibits up to 60% amino acid identity to certain dicot proteins, and 46% amino acid identity to barley oxalate oxidase; therefore we designated it HvOxOLP (for Hordeum vulgare oxalate oxidase-like protein). Single-base substitutions among several cDNA and RACE clones demonstrate a gene of many copies. Both the transcript and protein accumulate from 3 h after inoculation with Bgh. The transcript level peaks at 18-24 h and subsequently decreases, whereas the protein level is stable from 24 h after inoculation. The accumulation patterns are independent of the outcome of the barley/powdery mildew interaction, unlike that of PR proteins, for example. The transcript accumulates specifically in the inoculated epidermal tissue. This temporal and spatial expression pattern suggests a very close relationship to papilla formation. Immunoblot analyses have facilitated a demonstration that HvOxOLP, like oxalate oxidase, is a water-soluble 100 kDa oligomeric protein. The oligomer is heat-stable and SDS-tolerant, and it can be denatured into a 25 kDa monomer. Attempts to demonstrate oxalate oxidase activity for this protein have failed. However, the relationships to oxalate oxidase suggests that HvOxOLP may be involved in H2O2 generation necessary for, for example, cross-linking of cell wall components during formation of papillae.

Seedborne Infection of Rice by Pyricularia oryzae and Its Transmission to Seedlings.

Seedborne infection of rice by Pyricularia oryzae and its transmission to seedlings were studied quantitatively with naturally infected seeds of three rice cultivars collected from three locations in Nepal. A linear relationship on a logistic scale was found between panicle symptoms and seed infection, i.e., the more symptoms the higher seed infection. However, healthy-looking panicles and branches of panicles could also yield infected seeds. Postharvest measures such as winnowing and sun-drying significantly reduced seed infection by P. oryzae and filled grains had a lower degree of infection than unfilled grains. Sporulation of P. oryzae was most often confined to the embryonal end of germinating seeds. In contrast, most of the nongerminating seeds had sporulation all over the seed surface. Transmission of P. oryzae from seeds to seedlings, studied under various seeding conditions, showed that the transmission rate was always low. Thus, a seed sample with 21% seed infection resulted in less than 4% seedlings with blast lesions. Seed transmission was found for light covering of the seeds with soil or for moist seeding without covering. Transmission was rarely found when seeds were completely covered, and never in seedlings raised under water seeding conditions. Lower infection frequency was observed in seedlings raised in unsterilized soil than in seedlings raised in sterilized soil. Also, percent recovery of P. oryzae from infected seeds was higher in sterilized soil than in unsterilized soil and declined with time. Seedlings grown under low temperature (15 to 20°C) conditions did not develop blast lesions but when the same plants were transferred to high temperature (25 to 30°C) conditions, blast lesions were detected. This confirmed the latent infection in seedlings by P. oryzae grown under low temperature conditions.

Mechanisms of Induced Resistance in Barley Against Drechslera teres.

ABSTRACT Quantitative and qualitative histopathological methods and molecular analyses were used to study the mechanisms by which preinoculation with either of the nonbarley pathogens, Bipolaris maydis and Septoria nodorum, inhibited growth of Drechslera teres. Collectively, our data suggest that induced resistance is the principal mechanism responsible for impeding the pathogen. The enhancement of resistance in the host was primarily manifested during penetration by D. teres, and after penetration, where growth of D. teres ceased soon after development of infection vesicles. Thus, 24 h after pretreatment with B. maydis or S. nodorum, the penetration frequency from D. teres appressoria was reduced from 42.7% in the controls to 9.5 and 14.8%, respectively. The reductions were associated with increased formation of fluorescent papillae in induced cells (early defense reaction). The postpenetrational inhibition of D. teres completely stopped fungal growth and was apparently linked to an enhancement of multicellular hypersensitive responses in inducer-treated leaves (late defense reaction). Papillae formation and multicellular hypersensitive reactions were also observed in fully susceptible, noninduced control leaves, but they were inadequate to stop fungal progress. Northern blots from leaves treated with either inducer alone support the conclusion that induced resistance is involved in suppression of D. teres by increased formation of papillae and hypersensitive reactions. Thus, the blots showed strong expression of several defense response genes that are involved in these reactions in barley attacked by Erysiphe graminis f. sp. hordei.

Suppression of Rice Blast by Preinoculation with Avirulent Pyricularia oryzae and the Nonrice Pathogen Bipolaris sorokiniana.

ABSTRACT Avirulent isolates of Pyricularia oryzae and isolates of Bipolaris sorokiniana, a nonrice pathogen, were used to suppress rice blast caused by P. oryzae. In greenhouse experiments, both fungi substantially reduced leaf blast when applied 24 h or more before the pathogen. B. sorokiniana, but not avirulent isolates of P. oryzae, systemically reduced disease in leaf 5 when applied to whole plants at the four-leaf stage. In field experiments, both fungi were able to reduce neck blast significantly. No increase in grain yield was obtained by using avirulent isolates of P. oryzae, whereas five sprays with B. sorokiniana from seedling to heading stages increased the grain yield in two of three experiments conducted at two locations in Nepal. The significant increase in yield was observed under high inoculum pressure of P. oryzae. Induced resistance is suggested to be involved in the suppression of disease.

Characterization of the transcript of a new class of retroposon-type repetitive element cloned from the powdery mildew fungus, Erysiphe graminis.

The putative master transcript of a novel class of repetitive element has been cloned from the fungus erysiphe graminis f.sp. hordei. Sequence analysis of the cDNA revealed that the element, designated Eg-R1, is a member of the retroposon superfamily with properties in common with SINEs and LINEs (short or long interspersed elements). SINE-like properties include the transcript size (approximately 700 bp), and the lack of major open reading frames. In contrast, the fact that the transcript is polyadenylated and is most probably transcribed by RNA polymerase II, suggests a functional relationship to LINEs. Except for a short, but striking, sequence identity to a published SINE from the same fungus, no similar sequence was found in database searches. A constitutively high transcript level is found throughout the asexual life cycle of the fungus. Small differences in band patterns of Southern blots were observed between two isolates of E. graminis f.sp. hordei, while the band patterns in an isolate of the very close relative E. graminis f.sp. tritici in general appear dissimilar. This may imply that the element is currently active. Recent dispersal is confirmed by the observation that an approximately 550 bp internal hinfI fragment is conserved in the majority of the copies in all three isolates. Approximately 50 copies are present in E. graminis f.sp. hordei.