Genome-wide identification and characterization of NBLRR genes in finger millet (Eleusine coracana L.) and their expression in response to Magnaporthe grisea infection.

BACKGROUND: The nucleotide binding site leucine rich repeat (NBLRR) genes significantly regulate defences against phytopathogens in plants. The genome-wide identification and analysis of NBLRR genes have been performed in several species. However, the detailed evolution, structure, expression of NBLRRs and functional response to Magnaporthe grisea are unknown in finger millet (Eleusine coracana (L.) Gaertn.). RESULTS: The genome-wide scanning of the finger millet genome resulted in 116 NBLRR (EcNBLRRs1-116) encompassing 64 CC-NB-LRR, 47 NB-LRR and 5 CCR-NB-LRR types. The evolutionary studies among the NBLRRs of five Gramineae species, viz., purple false brome (Brachypodium distachyon (L.) P.Beauv.), finger millet (E. coracana), rice (Oryza sativa L.), sorghum (Sorghum bicolor L. (Moench)) and foxtail millet (Setaria italica (L.) P.Beauv.) showed the evolution of NBLRRs in the ancestral lineage of the target species and subsequent divergence through gene-loss events. The purifying selection (Ka/Ks < 1) shaped the expansions of NBLRRs paralogs in finger millet and orthologs among the target Gramineae species. The promoter sequence analysis showed various stress- and phytohormone-responsive cis-acting elements besides growth and development, indicating their potential role in disease defence and regulatory mechanisms. The expression analysis of 22 EcNBLRRs in the genotypes showing contrasting responses to Magnaporthe grisea infection revealed four and five EcNBLRRs in early and late infection stages, respectively. The six of these nine candidate EcNBLRRs proteins, viz., EcNBLRR21, EcNBLRR26, EcNBLRR30, EcNBLRR45, EcNBLRR55 and EcNBLRR76 showed CC, NB and LRR domains, whereas the EcNBLRR23, EcNBLRR32 and EcNBLRR83 showed NB and LRR somains. CONCLUSION: The identification and expression analysis of EcNBLRRs showed the role of EcNBLRR genes in assigning blast resistance in finger millet. These results pave the foundation for in-depth and targeted functional analysis of EcNBLRRs through genome editing and transgenic approaches.

Exploring the diversity of virulence genes in the Magnaporthe population infecting millets and rice in India.

Blast pathogen, Magnaporthe spp., that infects ancient millet crops such pearl millet, finger millet, foxtail millet, barnyard millet, and rice was isolated from different locations of blast hotspots in India using single spore isolation technique and 136 pure isolates were established. Numerous growth characteristics were captured via morphogenesis analysis. Among the 10 investigated virulent genes, we could amplify MPS1 (TTK Protein Kinase) and Mlc (Myosin Regulatory Light Chain edc4) in majority of tested isolates, regardless of the crop and region where they were collected, indicating that these may be crucial for their virulence. Additionally, among the four avirulence (Avr) genes studied, Avr-Pizt had the highest frequency of occurrence, followed by Avr-Pia. It is noteworthy to mention that Avr-Pik was present in the least number of isolates (9) and was completely absent from the blast isolates from finger millet, foxtail millet, and barnyard millet. A comparison at the molecular level between virulent and avirulent isolates indicated observably large variation both across (44%) and within (56%) them. The 136 Magnaporthe spp isolates were divided into four groups using molecular markers. Regardless of their geographic distribution, host plants, or tissues affected, the data indicate that the prevalence of numerous pathotypes and virulence factors at the field level, which may lead to a high degree of pathogenic variation. This research could be used for the strategic deployment of resistant genes to develop blast disease-resistant cultivars in rice, pearl millet, finger millet, foxtail millet, and barnyard millet.

Composition and Codon Usage Pattern Results in Divergence of the Zinc Binuclear Cluster (Zn(II)2Cys6) Sequences among Ascomycetes Plant Pathogenic Fungi.

Zinc binuclear cluster proteins (ZBC; Zn(II)2Cys6) are unique to the fungi kingdom and associated with a series of functions, viz., the utilization of macromolecules, stress tolerance, and most importantly, host-pathogen interactions by imparting virulence to the pathogen. Codon usage bias (CUB) is the phenomenon of using synonymous codons in a non-uniform fashion during the translation event, which has arisen because of interactions among evolutionary forces. The Zn(II)2Cys6 coding sequences from nine Ascomycetes plant pathogenic species and model system yeast were analysed for compositional and codon usage bias patterns. The clustering analysis diverged the Ascomycetes fungi into two clusters. The nucleotide compositional and relative synonymous codon usage (RSCU) analysis indicated GC biasness toward Ascomycetes fungi compared with the model system S. cerevisiae, which tends to be AT-rich. Further, plant pathogenic Ascomycetes fungi belonging to cluster-2 showed a higher number of GC-rich high-frequency codons than cluster-1 and was exclusively AT-rich in S. cerevisiae. The current investigation also showed the mutual effect of the two evolutionary forces, viz. natural selection and compositional constraints, on the CUB of Zn(II)2Cys6 genes. The perseverance of GC-rich codons of Zn(II)2Cys6 in Ascomycetes could facilitate the invasion process. The findings of the current investigation show the role of CUB and nucleotide composition in the evolutionary divergence of Ascomycetes plant pathogens and paves the way to target specific codons and sequences to modulate host-pathogen interactions through genome editing and functional genomics tools.

Lipopolysaccharide-induced priming enhances NO-mediated activation of defense responses in pearl millet challenged with Sclerospora graminicola.

Lipopolysaccharide (LPS) elicitors isolated from Pseudomonas fluorescens UOM SAR 14 effectively induced systemic and durable resistance against pearl millet downy mildew disease caused by the oomycete Sclerospora graminicola. Rapid and increased callose deposition and H2O2 accumulation were evidenced in downy mildew susceptible seeds pre-treated with LPS (SLPS) in comparison with the control seedlings, which also correlated with expression of various other defense responses. Biochemical analysis of enzymes and quantitative real-time polymerase chain reaction data suggested that LPS protects pearl millet against downy mildew through the activation of plant defense mechanisms such as generation of nitric oxide (NO), increased expression, and activities of defense enzymes and proteins. Elevation of NO concentrations was shown to be essential for LPS-mediated defense manifestation in pearl millet and had an impact on the other downstream defense responses like enhanced activation of enzymes and pathogen-related (PR) proteins. Temporal expression analysis of defense enzymes and PR-proteins in SLPS seedlings challenged with the downy mildew pathogen revealed that the activity and expression of peroxidase, phenylalanine ammonia lyase, and the PR-proteins (PR-1 and PR-5) were significantly enhanced compared to untreated control. Higher gene expression and protein activities of hydroxyproline-rich glycoproteins (HRGPs) were observed in SLPS seedlings which were similar to that of the resistant check. Collectively, our results suggest that, in pearl millet-downy mildew interaction, LPS pre-treatment affects defense signaling through the central regulator NO which triggers the activities of PAL, POX, PR-1, PR-5, and HRGPs.

Draft genome sequence of Sclerospora graminicola, the pearl millet downy mildew pathogen.

Sclerospora graminicola pathogen is the most important biotic production constraints of pearl millet in India, Africa and other parts of the world. We report a de novo whole genome assembly and analysis of pathotype 1, one of the most virulent pathotypes of S. graminicola from India. The draft genome assembly contained 299,901,251 bp with 65,404 genes. This study may help understand the evolutionary pattern of pathogen and aid elucidation of effector evolution for devising effective durable resistance breeding strategies in pearl millet.

Analysis of genetic and aflatoxin diversity among Aspergillus flavus isolates collected from sorghum seeds.

Thirty-four Aspergillus flavus isolates were recovered from sorghum seeds sampled across five states in India. Our study included (1) species confirmation through PCR assay, (2) quantification of total aflatoxin concentrations by the indirect competitive-ELISA (ic-ELISA) method, and (3) analysis of molecular diversity among the A. flavus isolates using ß-tubulin, ITS, and ISSR markers. Among the isolates studied, 28 were found to be positive for the production of aflatoxins. ITS and ß-tubulin phylogenetic analysis segregated the A. flavus sample population into two major groups or clades with little to no subdivision based on geography. In contrast, ISSR analysis also separated the A. flavus isolates into two main clusters, showing a distance of 0.0-0.5, with one cluster exhibiting a high level of diversity though no geographic or chemotype subdivision could be observed. The majority of sampled A. flavus isolates were highly toxigenic, and also highly diversified in terms of toxin-producing potential in-vitro. Genetic diversity among the sorghum isolates of A. flavus further warrants the development of appropriate farming management practices as well as improved aflatoxin detection measures in India.

Hepatoprotective action of Orthosiphon diffusus (Benth.) methanol active fraction through antioxidant mechanisms: an in vivo and in vitro evaluation.

ETHNOPHARMACOLOGICAL RELEVANCE: Preparations of Orthosiphon diffusus (Benth.) have been used by folk medicinal practitioners in the Western Ghats of India for treating inflammation, hepatitis and jaundice for many years and their effectiveness is widely acclaimed among the tribal communities. AIM OF THE STUDY: To evaluate the mechanisms behind the antioxidant and hepatoprotective potential of Orthosiphon diffusus methanol active fraction (MAF) using in vivo (rat) and in vitro (cell culture) models. MATERIALS AND METHODS: Neutralization of CCl4-induced hepatotoxicity by MAF was evaluated in rats. Towards this, serum levels of hepatic injury markers (lactate dehydrogenase and alkaline phosphatase), antioxidant enzymes in the liver homogenates, and histological examination were performed. In in vitro studies, mechanisms of neutralization of H2O2-induced toxicity by MAF using MTT, Comet assay and up-regulation of antioxidant enzymes at genetic level (RT-PCR) was performed in HepG2 cells. RESULTS: Rats pre-treated with Orthosiphon diffusus MAF demonstrated significantly reduced levels of serum LDH (1.3-fold, p<0.05) and ALP (1.6-fold, p<0.05). Similarly, multiple dose MAF administration demonstrated significantly enhanced levels (p<0.05) of antioxidant enzymes in the liver homogenates. Histological analysis revealed complete neutralization of CCl4-induced liver injury by the extract. The in vitro studies demonstrated that, pre-treatment of MAF effectively prevented H2O2-induced oxidative stress, genotoxicity and significantly enhanced (~6-fold, p<0.01) expression of genes for antioxidant enzymes. CONCLUSIONS: Orthosiphon diffusus MAF demonstrated significant hepatoprotection against CCl4-induced hepatotoxicity by antioxidant mechanisms comparable to silymarin. H2O2-induced oxidative stress was completely neutralized by MAF through enhanced expression of genes for antioxidant enzymes. Therefore, this study validates the use of Orthosiphon diffusus by folk medicinal practitioners in India. Further, MAF of Orthosiphon diffusus can serve as a strong candidate for the development of herbal hepatoprotective agents.