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Association of bacteria with fungi is a major area of research in infection biology, however, very few strains of bacteria have been reported that can invade and reside within fungal hyphae. Here, we report the characterization of an endofungal bacterium Serratia marcescens D1 from Mucor irregularis SS7 hyphae. Upon re-inoculation, colonization of the endobacterium S. marcescens D1 in the hyphae of Mucor irregularis SS7 was demonstrated using stereo microscopy. However, S. marcescens D1 failed to invade into the hyphae of the tested Ascomycetes (except Fusarium oxysporum) and Basidiomycetes. Remarkably, Serratia marcescens D1 could invade and spread over the culture of F. oxysporum that resulted in mycelial death. Prodigiosin, the red pigment produced by the Serratia marcescens D1, helps the bacterium to invade fungal hyphae as revealed by the increasing permeability in fungal cell membrane. On the other hand, genes encoding the type VI secretion system (T6SS) assembly protein TssJ and an outer membrane associated murein lipoprotein also showed significant up-regulation during the interaction process, suggesting the involvement of T6SS in the invasion process.

Alternaria blight is a devastating disease that causes significant crop losses in oilseed Brassicas every year. Adoption of conventional breeding to generate disease-resistant varieties has so far been unsuccessful due to the lack of suitable resistant source germplasms of cultivated Brassica spp. A thorough understanding of the molecular basis of resistance, as well as the identification of defense-related genes involved in resistance responses in closely related wild germplasms, would substantially aid in disease management. In the current study, a comparative transcriptome profiling was performed using Illumina based RNA-seq to detect differentially expressed genes (DEGs) specifically modulated in response to Alternaria brassicicola infection in resistant Sinapis alba , a close relative of Brassicas, and the highly susceptible Brassica rapa . The analysis revealed that, at 48 hpi (hours post inoculation), 3396 genes were upregulated and 23239 were downregulated, whereas at 72 hpi, 4023 genes were upregulated and 21116 were downregulated. Furthermore, a large number of defense response genes were detected to be specifically regulated as a result of Alternaria infection. The transcriptome data was validated using qPCR-based expression profiling for selected defense-related DEGs, that revealed significantly higher fold change in gene expression in S. alba when compared to B. rapa . Expression of most of the selected genes was elevated across all the time points under study with significantly higher expression towards the later time point of 72 hpi in the resistant germplasm. S. alba activates a stronger defense response reaction against the disease by deploying an array of genes and transcription factors involved in a wide range of biological processes such as pathogen recognition, signal transduction, cell wall modification, antioxidation, transcription regulation, etc. Overall, the study provides new insights on resistance of S. alba against A. brassicicola , which will aid in devising strategies for breeding resistant varieties of oilseed Brassica.

The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be -616.989 and -16.9749 kJ mol-1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2b produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme's regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

The 1‐deoxy‐d‐xylulose‐5‐phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH‐dependent reductase, plays a pivotal role in the methylerythritol 4‐phosphate pathway (MEP), in the conversion of 1‐deoxy‐d‐xylulose‐5‐phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full lengthdxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized throughin silico and tissue‐specific expression studies to explain its structure–function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three‐domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate‐binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue‐specific differential expression analysis using semi‐quantitative RT‐PCR and qRT‐PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future. We sequenced the complete coding sequence of citronelladxr gene. Analysis of CwDXR revealed the plastidial nature of the enzyme. Comparative modelling of CwDXR was performed. Molecular dynamics simulation and docking explained the mode of cofactor recognition. Tissue‐specific expression of CwDXR was performed using RT‐PCR and qRT‐PCR.

RNA-seq analysis of B . megaterium exposed to pH 7.0 and pH 4.5 showed differential expression of 207 genes related to several processes. Among the 207 genes, 11 genes displayed increased transcription exclusively in pH 4.5. Exposure to pH 4.5 induced the expression of genes related to maintenance of cell integrity, pH homeostasis, alternative energy generation and modification of metabolic processes. Metabolic processes like pentose phosphate pathway, fatty acid biosynthesis, cysteine and methionine metabolism and synthesis of arginine and proline were remodeled during acid stress. Genes associated with oxidative stress and osmotic stress were up-regulated at pH 4.5 indicating a link between acid stress and other stresses. Acid stress also induced expression of genes that encoded general stress-responsive proteins as well as several hypothetical proteins. Our study indicates that a network of genes aid B . megaterium G18 to adapt and survive in acid stress condition.

Background In rice, drought stress at reproductive stage drastically reduces yield, which in turn hampers farmer’s efforts towards crop production. The majority of the rice varieties have resistance genes against several abiotic and biotic stresses. Therefore, the traditional landraces were studied to identify QTLs/candidate genes associated with drought tolerance. Methods and results A high-density SNP-based genetic map was constructed using a Genotyping-by-sequencing (GBS) approach. The recombinant inbred lines (RILs) derived from crossing ‘Banglami × Ranjit’ were used for QTL analysis. A total map length of 1306.424 cM was constructed, which had an average inter-marker distance of 0.281 cM. The phenotypic evaluation of F 6 and F 7 RILs were performed under drought stress and control conditions. A total of 42 QTLs were identified under drought stress and control conditions for yield component traits explaining 1.95–13.36% of the total phenotypic variance (PVE). Among these, 19 QTLs were identified under drought stress conditions, whereas 23 QTLs were located under control conditions. A total of 4 QTLs explained a PVE ≥ 10% which are considered as the major QTLs. Moreover, bioinformatics analysis revealed the presence of 6 candidate genes, which showed differential expression under drought and control conditions. Conclusion These QTLs/genes may be deployed for marker-assisted pyramiding to improve drought tolerance in the existing rice varieties.

Background Drought stress is a major constraint for rice production worldwide. Reproductive stage drought stress (RSDS) leads to heavy yield losses in rice. The prospecting of new donor cultivars for identification and introgression of QTLs of major effect (Quantitative trait locus) for drought tolerance is crucial for the development of drought-resilient rice varieties. Methods and results Our study aimed to map QTLs associated with yield and its related traits under RSDS conditions. A saturated linkage map was constructed using 3417 GBS (Genotyping by sequencing) derived SNP (Single nucleotide polymorphism) markers spanning 1924.136 cM map length with an average marker density of 0.56 cM, in the F 3 mapping population raised via cross made between the traditional ahu rice cultivar, Koniahu (drought tolerant) and a high-yielding variety, Disang (drought susceptible). Using the Inclusive composite interval mapping approach, 35 genomic regions governing yield and related traits were identified in pooled data from 198 F 3 and F 4 segregating lines evaluated for two consecutive seasons under both RSDS and irrigated control conditions. Of the 35 QTLs, 23 QTLs were identified under RSDS with LOD (Logarithm of odds) values ranging between 2.50 and 7.83 and PVE (phenotypic variance explained) values of 2.95–12.42%. Two major QTLs were found to be linked to plant height (qPH1.29) and number of filled grains per panicle (qNOG5.12) under RSDS. Five putative QTLs for grain yield namely, qGY2.00, qGY5.05, qGY6.16, qGY9.19, and qGY10.20 were identified within drought conditions. Fourteen QTL regions having ≤ 10 Mb QTL interval size were further analysed for candidate gene identification and a total of 4146 genes were detected out of these 2263 (54.63%) genes were annotated to at least one gene ontology (GO) term. Conclusion Several QTLs associated with grain yield and yield components and putative candidate genes were identified. The putative QTLs and candidate genes identified could be employed to augment drought resilience in rice after further validation through MAS strategies.

We employed an Illumina-based high-throughput metagenomics sequencing approach to unveil the rhizosphere and root endosphere microbial community associated with an organically grown Camellia population located at the Experimental Garden for Plantation Crops, Assam (India). The de novo assembled tea root endosphere metagenome contained 24,231 contigs (total 7,771,089 base pairs with an average length of 321 bps), while tea rhizosphere soil metagenome contained 261,965 sequences (total 230,537,174 base pairs, average length 846). The most prominent rhizobacteria belonged to the genera, viz ., Bacillus (10.35%), Candidatus Solibacter (6.36%), Burkholderia (5.19%), Pseudomonas (3.9%), Streptomyces (3.52%), and Bradyrhizobium (2.77%), while the root endosphere was dominated by bacterial genera, viz ., Serratia (46.64%), Methylobacterium (8.02%), Yersinia (5.97%), Burkholderia (2.05%), etc. The presence of few agronomically important bacterial genera, Bradyrhizobium , Rhizobium (each 0.93%), Sinorhizobium (0.34%), Azorhizobium, and Flavobacterium (0.17% each), was also detected in the root endosphere. KEGG pathway mapping indicated the presence of microbial metabolic pathway genes related to tyrosine metabolism, tryptophan metabolism, glyoxylate, and dicarboxylate metabolism which play important roles in endosphere activities, including survival, growth promotion, and host adaptation. The root endosphere microbiome also contained few important plant growth promoting traits related to phytohormone production, abiotic stress alleviation, mineral solubilization, and plant disease suppression.

Forest ecosystems of Northeast India are rich in the diversity of wild mushrooms. Few reports have demonstrated the diversity of wild mushrooms from this region on the basis of their morphology. This study reports the characterization of wild mushrooms collected from different locations of Northeast India based on their molecular information of the internal transcribed spacer (ITS) region. The identified samples belonged to the genera Fomitopsis, Ganoderma, Hexagonia, Lentinus, Lenzites, Leucoagaricus, Leucocoprinus, Lycoperdon, Microporus, Panus, Physisporinus, Pleurotus, Polyporus and Trametes. Phylogenetic analysis using Maximum Likelihood method revealed close evolutionary relationships among samples of the same species collected from different locations. There was a positive correlation between phylogenetic distances and heterogeneity in the ITS sequences. High nucleotide variations were observed in the ITS1 and ITS2 segments of ITS region, while the 5.8S ribosomal RNA segment was found as relatively constant throughout different species. The intra-specific distances were recorded from 0 (Lentinus squarrosulus between AAU1 and DH1; Lycoperdon scabrum: between RB6 and KM7) to 0.106 (Lentinus squarrosulus: between AAU4 and KB2), while inter-specific distances were recorded from 0.054 (between Ganoderma tropicum RB1 and G. lucidum RB2) to 0.725 (between Pleurotus giganteus KM1 and Lycoperdon scabrum DIM8). It was concluded that the ITS region can be used as a good genomic marker for DNA barcoding of mushrooms. The barcodes for the samples used in this study are available at BOLD (Barcode of Life Data systems; www.boldsystems.org).