Salicylic acid seed priming instigates defense mechanism by inducing PR-Proteins in Solanum melongena L. upon infection with Verticillium dahliae Kleb.

Salicylic acid (SA) is a hormone connected with various cellular functions including the fight against invading pathogens. Priming of seeds pre-sowing is a very simple method to the farmers' to produce better growth, yield and manage the pathogens. The present study was aimed to determine the growth and disease resistance ability in brinjal seeds primed with different concentrations (0.25, 0.5, 0.75 and 1.0 mM) of SA under greenhouse conditions. Priming of seeds with SA significantly increased seed germination and seedling vigor with a maximum of 84% and 859.18, respectively at 0.5 mM concentration. Seed priming with SA also reduced Verticillium wilt incidence to 39.25% (at 0.5 mM) under greenhouse conditions and also enhanced the vegetative growth parameters of the plant compared to control. The induced resistance obtained with SA was in line with higher expression of PR-protein (ß-1,3-glucanase and chitinase) related defense enzymes. Further, an increase of 1.7, 2.9, 2.1, 2.5 and 2-fold increase in gene expression of IAA27, MPK1, GPX, chitinase and ß-1,3-glucanase, respectively were observed in SA primed challenge inoculated seedlings than non-primed susceptible inoculated controls. The higher expression of IAA27, MPK1, GPX, chitinase and ß-1,3-glucanase correlates with the plant growth promoting and disease protection studies as these genes are vital for increasing plant growth and inducing resistance during host-pathogen interaction. Enhanced activation of defense-related activities in plants upon priming with SA suggests that it alters plant physiology which in turn is useful for production and protection of brinjal.

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants.

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor ß-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction.

Proteomic analysis of elicitation of downy mildew disease resistance in pearl millet by seed priming with ß-aminobutyric acid and Pseudomonas fluorescens.

Downy mildew is one of the severe diseases of pearl millet, globally affecting its commercial production. Priming of seeds of a susceptible cultivar of pearl millet with ß-aminobutyric acid (BABA) and Pseudomonas fluorescens has reduced the downy mildew disease incidence level under field studies. In the current study, proteomic approach was used to elucidate the poorly studied resistance mechanism in these elicitor primed pearl millet seeds in response to Sclerospora graminicola infection. 2DE-MS/MS based proteomic approach revealed that majority of the 63 differentially accumulated (p≤0.05) proteins associated with energy and metabolism followed by stress and defense category. Multivariate statistics disclosed that infection caused by the pathogen rather than elicitor treatment had a major influence on the dynamics of protein abundance. Mechanism of priming mediated by BABA and P. fluorescens were different from each other as evident by the protein abundance profile of hierarchical clustering analysis. Over-representation of proteins pertaining to glucose metabolism suggests that seed priming ensures plant protection against disease without compromising its normal growth and development. In addition the study forms a basis for future investigation by functional analysis of these differentially accumulated proteins to further unravel the resistance mechanism of elicitor primed plant against the S. graminicola. BIOLOGICAL SIGNIFICANCE: The study is based on the comparative proteomic analysis between BABA and P. fluorescens mediated resistance in pearl millet, in response to downy mildew causing biotroph - S. graminicola. To our knowledge, this article is the first to report on seedling proteome of pearl millet whose genome is not yet sequenced. In addition, the study also provides clue for the plausible antagonistic cross-talk that might exist between jasmonic acid signaling and salicylic acid signaling in SAR and ISR mediated resistance by BABA and P. fluorescens against the downy mildew pathogen. Furthermore, pearl millet seedling proteome being perturbed by pathogen inoculation was more apparent than that caused by elicitor treatment, as revealed by multivariate statistics like PCA. Analysis by gene enrichment tools further revealed that the glucose metabolism pathway was majorly being affected in our study. This could be attributed to the essential balance that is being maintained in energy diversion towards stress and normal physiological process due to the priming effect of the elicitors against biotic stress.

Immuno-affinity purification of PglPGIP1, a polygalacturonase-inhibitor protein from pearl millet: studies on its inhibition of fungal polygalacturonases and role in resistance against the downy mildew pathogen.

Polygalacturonase-inhibitor proteins (PGIPs) are important plant defense proteins which modulate the activity of microbial polygalacturonases (PGs) leading to elicitor accumulation. Very few studies have been carried out towards understanding the role of PGIPs in monocot host defense. Hence, present study was taken up to characterize a native PGIP from pearl millet and understand its role in resistance against downy mildew. A native glycosylated PGIP (PglPGIP1) of ~43 kDa and pI 5.9 was immunopurified from pearl millet. Comparative inhibition studies involving PglPGIP1 and its non-glycosylated form (rPglPGIP1; recombinant pearl millet PGIP produced in Escherichia coli) against two PGs, PG-II isoform from Aspergillus niger (AnPGII) and PG-III isoform from Fusarium moniliforme, showed both PGIPs to inhibit only AnPGII. The protein glycosylation was found to impact only the pH and temperature stability of PGIP, with the native form showing relatively higher stability to pH and temperature changes. Temporal accumulation of both PglPGIP1 protein (western blot and ELISA) and transcripts (real time PCR) in resistant and susceptible pearl millet cultivars showed significant Sclerospora graminicola-induced accumulation only in the incompatible interaction. Further, confocal PGIP immunolocalization results showed a very intense immuno-decoration with highest fluorescent intensities observed at the outer epidermal layer and vascular bundles in resistant cultivar only. This is the first native PGIP isolated from millets and the results indicate a role for PglPGIP1 in host defense. This could further be exploited in devising pearl millet cultivars with better pathogen resistance.

The pearl millet mitogen-activated protein kinase PgMPK4 is involved in responses to downy mildew infection and in jasmonic- and salicylic acid-mediated defense.

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor ß-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

Involvement of mitogen-activated protein kinase signalling in pearl millet-downy mildew interaction.

Mitogen-activated protein kinase (MAPK) cascade-mediated signalling is essential in the establishment of resistance towards pathogens. The present study compared MAPK activities in a compatible and incompatible interaction between pearl millet [Pennisetum glaucum (L.) R. Br.] and downy mildew pathogen Sclerospora graminicola. Differential expression was observed with rapid and increased activation of MAPKs, PgMPK1 (48kDa) and PgMPK2 (44kDa), in the incompatible interaction; with a weak activity of only PgMPK1 in the compatible interaction. Immunoblot analysis showed PgMPK1 and PgMPK2 to be orthologs of salicylic acid-induced protein kinase and wound-induced protein kinase, respectively. Immunocytochemical analysis revealed pathogen-induced accumulation and nuclear localisation of PgMPKs only in the incompatible interaction with highest signals in the vascular tissues. Maximum PgMPKs activation correlated with the activation of several defence-related enzymes. In addition, inhibition of MAPK-activation by kinase cascade inhibitors correlated with the suppression of defence-related enzyme activities and pathogen-induced H2O2 accumulation. Treatment of pearl millet seedlings with abiotic and biotic elicitors led to a strong early induction of only PgMPK1. ß-Amino butyric acid and H2O2 were found to be best activators of PgMPK1. These results suggest that in pearl millet MAPK signalling is involved in mediating several defence mechanisms in response to pathogen infection.