Isolation and characterisation of a protein elicitor from Sclerospora graminicola and elicitor-mediated induction of defence responses in cultured cells of Pennisetum glaucum.

Sclerospora graminicola (Sacc.) Schroet., an oomycete pathogen of Pennisetum glaucum (L.) R.Br. infects the meristematic tissues of young seedlings. The motile zoospores from the sporangia encyst, germinate and penetrate the plant tissue. Resistance to the invading pathogen is governed by the specific recognition of conserved pathogen-associated proteins or elicitors. In the present study, a zoospore protein was isolated and purified to homogeneity by a combination of size exclusion and high-performance liquid chromatography (HPLC). The crude fractionated protein was able to elicit an array of defence responses in resistant and susceptible cells of pearl millet. Treatment of cultured cells of pearl millet with partially purified elicitor protein resulted in a rapid loss of cell viability in the resistant cells and the percentage of cell death was higher in the resistant than in the susceptible cells. Cultures of resistant cells showed a sharp increase in the extra cellular pH compared with susceptible cells when treated with the crude elicitor. Increased oxidative burst was also recorded in the cells treated with the crude elicitor. The purified elicitor showed unique properties. The purified protein was acidic with a pI of 5.6 as revealed by isoelectric focusing (IEF) and matrix-assisted laser desorption ionisation (MALDI) analysis showed that the elicitor had a molecular mass of 7040 daltons. The primary structure determined by N-terminal Edman degradation and searches with BLAST did not reveal similarities to any known plant pathogenic or oomycete elicitor. Higher activities of the important defence-related enzymes phenylalanine ammonia lyase (PAL) and peroxidase in the resistant cell cultures than in the susceptible cell cultures treated with the purified elicitor were clearly evident. Studies of gene expression by northern blotting with heterologus peroxidase, PAL and oxalate oxidase probes showed that the mRNA transcripts were strongly up-regulated in resistant cell cultures within 30 min of elicitor treatment. The purified elicitor also demonstrated a very strong concentration-dependent sterol binding. The purified elicitor protein belongs to a class of low molecular weight oomycete elicitors with sterol carrier properties. The identified low molecular weight protein elicitor displays unique properties that can be exploited for synthesis of novel molecules for eco-friendly crop protection.

Induction and accumulation of polyphenol oxidase activities as implicated in development of resistance against pearl millet downy mildew disease.

Polyphenol oxidase (PPO) activity was analysed in seedlings of resistant and susceptible pearl millet [Pennisetum glaucum (L.) R.Br] cultivars with or without inoculation of the downy mildew pathogen Sclerospora graminicola (Sacc.) Schroet. Seedlings of resistant varieties had greater PPO activity than susceptible seedlings, and inoculated seedlings had significantly higher PPO levels than uninoculated seedlings. Temporal accumulation of PPO showed a maximum activity at 24 h post-inoculation in resistant seedlings, whereas in susceptible seedlings it peaked at 48 h. PPO activity was positively correlated with levels of downy mildew resistance in different pearl millet cultivars under field conditions. Native PAGE staining showed four isoforms of PPO, which were differentially induced in relation to the time of appearance and intensities in the uninoculated seedlings, whereas a fifth PPO isoform appeared after inoculation with S. graminicola. PPO activity was significantly higher in the shoot and leaves of pearl millet than in the root. Tissue printing analysis of the enzyme expression showed that the enzyme is predominantly expressed after pathogen inoculation and is localised in the epidermal and vascular regions. Temporal analysis of transcript accumulation showed that in resistant seedlings PPO mRNAs was expressed earlier and more abundantly than in susceptible seedlings. Our studies demonstrate, for the first time, that PPO is actively involved in plant defence and can be used as a marker of resistance to downy mildew infection in pearl millet.

Seed treatment with beta-aminobutyric acid protects Pennisetum glaucum systemically from Sclerospora graminicola.

beta-Aminobutyric acid (BABA) treatment of pearl millet [Pennisetum glaucum (L) R Br] seeds influenced seedling vigour and protected the seedlings from downy mildew disease caused by the oomycetous biotropic fungus Sclerospora graminicola (Sacc) Schroet. Of the different concentrations of BABA tested, viz 25, 50, 75 and 100 mM, seeds treated with 50 mM for 6 h resulted in the maximum of 1428 seedling vigour and showed 23% disease incidence in comparison with the control which recorded a seedling vigour of 1260 and 98% disease incidence i.e. 75% protection from disease. Seeds treated with BABA when challenged for downy mildew disease using zoospores of S graminicola required 48 h after inducer treatment to develop maximum resistance. Durability of induced resistance was also tested in plants raised from seeds treated with the inducer and identified as resistant, by second challenge inoculation with the downy mildew pathogen at tillers and inflorescence axes. Reduced disease incidence of only 10 and 12% in these plants, compared with 71 and 76% disease in control plants inoculated at the tillers and inflorescence axes, respectively, suggested that resistance induced in seeds with BABA remained operative through vegetative and reproductive growth of pearl millet plants. Induction of resistance by seed treatment with BABA enhanced vegetative growth, viz height, fresh weight, leaf area and tillering, and reproductive growth, viz early flowering, number of productive ear heads and 1000 seed weight. Studies on induction of resistance in different cultivars of pearl millet with varying resistance reaction to downy mildew indicated that the protection offered by BABA is independent of the nature of cultivars used and not dependent on their constitutive resistance.