Comprehensive identification, characterization and expression analysis of genes underpinning heat acclimatization in Triticum durum and Aegilops tauschii.

Wheat (Triticum aestivum L.) is an important cereal crop cultivated and consumed worldwide. Global warming-induced escalation of temperature during the seedling and grain-filling phase adversely affects productivity. To survive under elevated temperatures, most crop plants develop natural mechanisms at molecular level by activating heat shock proteins. However, other heat stress-related proteins like heat acclimatization (HA) proteins are documented in hexaploid wheat but have not been explored in detail in its diploid and tetraploid progenitors, which might help to overcome elevated temperature regimes for short periods. Our study aims to explore the potential HA genes in progenitors Triticum durum and Aegilops tauschii that perform well at higher temperatures. Seven genes were identified and phylogenetically classified into three families: K homology (KH), Chloroplast protein-enhancing stress tolerance (CEST), and heat-stress-associated 32 kDa (HSA32). Protein-protein interaction network revealed partner proteins that aid mRNA translation, protein refolding, and reactive species detoxification. Syntenic analysis displayed highly conserved relationships. RT-qPCR-based expression profiling revealed HA genes to exhibit diverse and dynamic patterns under high-temperature regimes, suggesting their critical role in providing tolerance to heat stress. The present study furnishes genetic landscape of HA genes that might help in developing climate-resilient wheat with higher acclimatization potential.

The F-box protein encoding genes of the leaf-rust fungi Puccinia triticina: genome-wide identification, characterization and expression dynamics during pathogenesis.

The F-box proteins in fungi perform diverse functions including regulation of cell cycle, circadian clock, development, signal transduction and nutrient sensing. Genome-wide analysis revealed 10 F-box genes in Puccinia triticina, the causal organism for the leaf rust disease in wheat and were characterized using in silico approaches for revealing phylogenetic relationships, gene structures, gene ontology, protein properties, sequence analysis and gene expression studies. Domain analysis predicted functional domains like WD40 and LRR at C-terminus along with the obvious presence of F-box motif in N-terminus. MSA showed amino acid replacements, which might be due to nucleotide substitution during replication. Phylogenetic analysis revealed the F-box proteins with similar domains to be clustered together while some sequences were spread out in different clades, which might be due to functional diversity. The clustering of Puccinia triticina GG705409 with Triticum aestivum TaAFB4/TaAFB5 in a single clade suggested the possibilities of horizontal gene transfer during the coevolution of P. triticina and wheat. Gene ontological annotation categorized them into three classes and were functionally involved in protein degradation through the protein ubiquitination pathway. Protein-protein interaction network revealed F-box proteins to interact with other components of the SCF complex involved in protein ubiquitination. Relative expression analysis of five F-box genes in a time course experiment denoted their involvement in leaf rust susceptible wheat plants. This study provides information on structure elucidation of F-box proteins of a basidiomycetes plant pathogenic fungi and their role during pathogenesis.

An accretive detection method for in silico identification and validation of circular RNAs in wheat (Triticum aestivum L.) using RT-qPCR.

BACKGROUND: Circular RNAs (circRNAs) are novel class of non-coding RNAs, which are involved in various functions at the transcriptional and post-transcriptional level in response to a fungal pathogen (Puccinia triticina), including microRNA (miRNA) sponge, RNA binding proteins sponge, regulation of parental gene and biomarkers. Detailed analysis of wheat circRNAs is essential to accelerate the regulated expression of fungal miRNAs. Therefore, we suggest a protocol to aid circRNA identification through RNA-Seq data using various algorithms based on perl script followed by validation through divergent primer designing, standard PCR, and RT-qPCR assays. METHODS AND RESULT: The divergent primer has been widely used to detect, validate, and quantify back-spliced junction (BSJ) of circRNAs. The procedure covers index file formation, circRNA identification and BSJ detections. However, the laboratory validation of circRNA includes wheat genomic DNA isolation, RNA isolation and its cDNA conversion upto validation. In this study, we identified 28 circRNAs from RNA-Seq of S0 and R0, wherein six circRNAs are commonly present and 75% of the identified circRNAs were belongs to inter-genic, 14% were exonic and intronic category were 11%. Divergent primer designing method successfully validated the two circRNAs via RT-qPCR assay, where circRNA_2 showed less relative expression pattern than circRNA_1 in contrast with housekeeping genes. CONCLUSION: Thus, our results of identified and validated circRNAs showed that, this protocol is quite helpful, relatively easy, reliable, and accurate for large datasets as other algorithms need various dependencies and have complex scripts with high chances of error occurrence. Additionally, analysis time will vary depending on the expertise level and the number of RNA-Seq data. This proposed protocol can also be used for a wide range of monocotyledons belonging to the Poaceae plant family.

Insights of auxin signaling F-box genes in wheat (Triticum aestivum L.) and their dynamic expression during the leaf rust infection.

The TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) protein serves as auxin receptor and links with Aux/IAA repressor protein leading to its degradation via SKP-Cullin-F box (SCFTIR1/AFB) complex in the auxin signaling pathway. Present study revealed 11 TIR1/AFB genes in wheat by genome-wide search using AFB HMM profile. Phylogenetic analysis clustered these genes in two classes. Several phytohormone, abiotic, and biotic stress responsive cis-elements were detected in promoter regions of TIR1/AFB genes. These genes were localized on homoeologous chromosome groups 2, 3, and 5 showing orthologous relation with other monocot plants. Most genes were interrupted by introns and the gene products were localized in cytoplasm, nucleus, and cell organelles. TaAFB3, TaAFB5, and TaAFB8 had nuclear localization signals. The evolutionary constraint suggested paralogous sister pairs and orthologous genes went through strong purifying selection process and are slowly evolving at protein level. Functional annotation revealed all TaAFB genes participated in auxin activated signaling pathway and SCF-mediated ubiquitination process. Furthermore, in silico expression study revealed their diverse expression profiles during various developmental stages in different tissues and organs as well as during biotic and abiotic stress. QRT-PCR based studies suggested distinct expression pattern of TIR1-1, TIR1-3, TaAFB1, TaAFB2, TaAFB3, TaAFB4, TaAFB5, TaAFB7, and TaAFB8 displaying maximum expression at 24 and 48 h post inoculation in both susceptible and resistant near isogenic wheat lines infected with leaf rust pathogen. Importantly, this also reflects coordinated responses in expression patterns of wheat TIR1/AFB genes during progression stages of leaf rust infection.

Enhanced vanillin production from eugenol by Bacillus cereus NCIM-5727.

Biovanillin production by a wild strain of Bacillus cereus NCIM-5727 is studied using eugenol as the precursor aiming to achieve maximum vanillin productivity. Based on shake flask optimization, molar yield and global volumetric productivity of vanillin reached up to 71.2% (6.6 gL-1) and 0.18 g(Lh)-1, respectively, at 36 h by resting cells of B. cereus NCIM-5727 at the optimum cell concentration of 3 gL-1 using eugenol concentration of 10 gL-1 at 37 ºC, buffer pH 7.0, buffer volume 10%, and shaking speed 180 rpm. Furthermore, small-scale optimization in a bioreactor at the controlled aeration rate of 0.5 Lmin-1, agitation rate of 210 rpm, and pH 7.0 enhanced the global volumetric productivity of vanillin up to 0.28 g(Lh)-1 at 25 h of bioconversion. The highest vanillin molar yield (75.2%) is reported using resting cells of B. cereus NCIM-5727 upon eugenol biotransformation and found stable for 10 h.

Comprehensive molecular dissection of TIFY Transcription factors reveal their dynamic responses to biotic and abiotic stress in wheat (Triticum aestivum L.).

The plant specific TIFY (previously known as ZIM) transcription factor (TF) family plays crucial roles in cross talk between Jasmonic Acid and other phytohormones like gibberellins, salicylic acid, abscisic acid, auxin, and ethylene signaling pathways. Wheat yield is severely affected by rust diseases and many abiotic stresses, where different phytohormone signaling pathways are involved. TIFYs have been studied in many plants yet reports describing their molecular structure and function in wheat are lacking. In the present study, we have identified 23 novel TIFY genes in wheat genome using in silico approaches. The identified proteins were characterized based on their conserved domains and phylogenetically classified into nine subfamilies. Chromosomal localization of the identified TIFY genes showed arbitrary distribution. Forty cis-acting elements including phytohormone, stress and light receptive elements were detected in the upstream regions of TIFY genes. Seventeen wheat microRNAs targeted the identified wheat TIFY genes. Gene ontological studies revealed their major contribution in defense response and phytohormone signaling. Secondary structure of TIFY proteins displayed the characteristic alpha-alpha-beta fold. Synteny analyses indicated all wheat TIFY genes had orthologous sequences in sorghum, rice, maize, barley and Brachypodium indicating presence of similar TIFY domains in monocot plants. Six TIFY genes had been cloned from wheat genomic and cDNA. Sequence characterization revealed similar characteristics as the in silico identified novel TIFY genes. Tertiary structures predicted the active sites in these proteins to play critical roles in DNA binding. Expression profiling of TIFY genes showed their contribution during incompatible and compatible leaf rust infestation. TIFY genes were also highly expressed during the initial hours of phytohormone induced stress. This study furnishes fundamental information on characterization and putative functions of TIFY genes in wheat.

Saccharomyces boulardii ameliorates gut dysbiosis associated cognitive decline.

Saccharomyces boulardii, a probiotic yeast is well prescribed for various gastrointestinal disorders accompanied by gut dysbiosis such as inflammatory bowel disease, bacterial diarrhea and antibiotic associated diarrhea. Gut dysbiosis has been associated with central nervous system via gut brain axis primarily implied in the modulation of psychiatric conditions. In the current study we use Saccharomyces boulardii as a therapeutic agent against gut dysbiosis associated cognitive decline. In mice, gut dysbiosis was induced by oral Ampicillin Na (250 mg/kg twice-daily) for 14 days. While in the treatment group S. boulardii (90 mg/kg once a day) was administered orally for 21 days along with 14 days of antibiotic treatment. Gene expression studies revealed antibiotic mediated decrease in the Lactobacillus, Bifidobacterium, Firmicutes and Clostridium which were restored by S. boulardii treatment. Cognitive behavioral studies showed a parallel reduction in fear conditioning, spatial as well as recognition memory which were reversed upon S. boulardii treatment in these animals. S. boulardii treatment reduced myeloperoxidase enzyme, an inflammatory marker, in colon as well as brain which was increased after antibiotic administration. Similarly, S. boulardii reduced the brain acetylcholine esterase, oxidative stress and inflammatory cytokines and chemokines which were altered due to antibiotic treatment. S. boulardii treatment also protected hippocampal neuronal damage and restored villus length and crypt depth thus normalizing gut permeability in antibiotic treated animals. Hence, we conclude that S. boulardii prevented antibiotic associated gut dysbiosis leading to reduced intestinal and brain inflammation and oxidative stress thus preventing hippocampal neuronal damage and eventually reversing gut dysbiosis associate cognitive decline in mice.

Flavonoid-rich wheatgrass (Triticum aestivum L.) diet attenuates diabetes by modulating antioxidant genes in streptozotocin-induced diabetic rats.

Wheatgrass, young germinated shoots of Triticum aestivum L., is proclaimed as antidiabetic nutraceutical by traditional medicines across the world. In this study, the effects of the wheatgrass diet in ameliorating oxidative stress (OS) induced during diabetes were investigated. Total phenolic and flavonoid contents (TPC and TFC) and in vitro antioxidant activity of wheatgrass extract were estimated at different days (5, 7, 9, 11, 13, and 15) after germination. Correlating the TPC and TFC with in vitro antioxidant activity, 9th DAG wheatgrass was found to possess maximum antioxidant potential. UHPLC-MS/MS analysis also revealed the presence of nine flavonoids. For in vivo studies, diabetes was induced by streptozotocin in Wistar rats fed with a high-fat diet. Concomitant administration of 9th-day wheatgrass diet (200 and 400 mg/kg) for 60 days exhibited significant improvements in hyperglycemia, body weight, lipid profile, biochemical indices (AST, ALT, GSH, GPx), and restoration of tissue architectures equivalent to normal rats. Further, qRT-PCR-based expression profiling revealed a significant modulation of major antioxidant marker genes and insulin gene which substantiated that the wheatgrass diet is effective in reducing OS during diabetes. Therefore, flavonoid-rich 9th-day wheatgrass could be used as a functional food to control diabetes. PRACTICAL APPLICATIONS: The present research supported that wheatgrass protects against oxidative stress and therefore could be utilized to ameliorate diabetes. The findings may contribute to the development and formulation of wheatgrass-based functional food or dietary supplement for diabetes by nutraceutical industries.

Genome-Scale Identification, in Silico Characterization and Interaction Study Between Wheat SNARE and NPSN Gene Families Involved in Vesicular Transport.

Wheat is an important cereal crop grown worldwide but it's yield is severely affected by various biotic and abiotic stresses. SNAREs are key regulators of vesicle trafficking and are present in abundance in higher plant species suggesting their prominence in growth and development. Novel Plant SNAREs (NPSN) are found exclusively in plants. Hence, a comprehensive analysis of these two gene families in wheat genome was accomplished in this study. We report here 27 SNAREs and eight NPSN genes. These genes and their respective proteins were investigated for gene structure, physiochemical properties, domain and motif architecture, phylogeny, chromosomal localization and possible interactions. Phylogenetic and motif analysis confirmed SNARE domain in all the proteins. Functional annotation revealed participation in biological process like vesicle fusion, exocytosis, protein targeting to vacuole and SNAP receptor activity. At subcellular level, SNAREs were localized in multiple organelles whereas NPSN proteins were localized in cytoplasm where they regulate vesicle fusion. The 3-D structures built with Modeller proved the presence of SNARE motifs in the identified proteins. Possible protein-protein interactions between SNARE and NPSN proteins were determined and docking was performed. The results augmented our understanding about molecular function, evolutionary relation, location inside the cell and their interactions.

Characterization of the leaf rust responsive ARF genes in wheat (Triticum aestivum L.).

KEY MESSAGE: Genome-wide identification, classification, functional characterization and expression analysis of Auxin Responsive Factor (ARF) gene family in wheat reveal their attributes and role during leaf rust infection. Auxins are important plant growth regulators that also impact plant-pathogen interaction. Auxin responsive factors (ARF) are plant specific transcription factors that control responses to auxins. Whole genome investigation of ARF gene family is limited in allohexaploid wheat (Triticum aestivum L.). Comprehensive study of this gene family was carried out by employing the currently available reference genome sequence of wheat. In total, 27 ARF genes were identified and located on the wheat genome as well as were positioned on wheat chromosome arms. Additionally, examination of the predicted genes unveiled a decent degree of relatedness within and among the phylogenetic clades. Leaf rust, caused by the obligate biotrophic fungal pathogen Puccinia triticina, is responsible for drastic loss of wheat crop worldwide reducing grain yield by 10-90%. Expression profiling of ARF genes in retort to leaf rust infection indicated their differential regulation during this plant-pathogen interaction. Highest expression of ARF genes were observed at 12 hpi that was maintained up to 72 hpi during incompatible interaction, whereas the high expression levels receded at 48 hpi during compatible interactions. Few of the identified ARF genes were likely to be post-transcriptionally regulated by microRNAs. Many light and stress responsive elements were detected in the promoter regions of ARF genes. Microsynteny analysis showed the conservation of ARF genes within the members of the Poaceae family. This study provides fundamental details for understanding the different types of ARF genes in wheat and there putative roles during leaf rust-wheat interaction.

A rapid and simple ultra high performance liquid chromatography method for the simultaneous determination of methoxyphenol derivatives involved in the eugenol catabolic pathway.

An efficient ultra high performance liquid chromatography method of separation was developed for the analysis of six important methoxyphenol derivatives involved in the eugenol catabolic pathway. In the present study, an Acquity UPLC BEH C18 column was used for the chromatographic separation of the industrially important phenolic compounds such as vanillin, vanillic acid, ferulic acid, coniferyl alcohol, and coniferyl aldehyde obtained during microbial transformation of eugenol. Eluted components were identified using the dual wavelength (254 and 310 nm) UV detector. A gradient method of elution using mobile phase of aqueous 1 mM trifluoroacetic acid (Solvent A) and methanol (Solvent B) at a flow rate of 0.3 mL/min separated all the five intermediate methoxyphenol derivatives along with their precursor eugenol within 15 min with stable baseline resolution. Method validation was performed for the accurate quantification of vanillin, coniferyl aldehyde, and eugenol using the parameters of linearity, specificity, precision, limit of detection, limit of quantification, and robustness. The developed method would be helpful for clear separation and identification of the five most important intermediate metabolites of the eugenol catabolism pathway.

Development of a rapid RNA extraction procedure from urediniospores of the leaf rust fungus, Puccinia triticina.

Obtaining high quality RNA in good quantities is often a requirement for plant-pathogen interaction studies, so it becomes very essential that a highly efficient method should be deployed to isolate RNA from minute quantities of fungal spores. The methods available to date, either require a high quantity of spores or the use of expensive chemicals. The protocol discussed here for RNA isolation from Puccinia triticina pathotype 77-5 urediniospores utilizes TRI Reagent as extraction buffer that is widely used for RNA isolation from plant tissues. Urediniospores have a tough cell wall as compared to other plant cells. Therefore, the protocol was optimized keeping the primary focus on quickly disrupting cell walls. Two different methods, one using a combination of liquid nitrogen and ultrasonic water-bath and the other method using micro-homogenizer were utilized for crushing the spores in the present study. The developed methods do not utilize mortar and pestle, instead they promote direct crushing of urediniospores in tubes; thereby minimizing sample loss and enhancing quality.

Leaf rust (Puccinia triticina) mediated RNAi in wheat (Triticum aestivum L.) prompting host susceptibility.

Significance of microRNAs in regulating gene expression in higher eukaryotes as well as in pathogens like fungi to suppress host defense is a well-established phenomenon. The present study focuses on leaf rust fungi Puccinia triticina (Pathotype 77-5) mediated RNAi to make wheat (Triticum aestivum L.) more susceptible. To reach such conclusions, we first confirmed the presence of argonaute (AGO) and dicer-like protein (DCL) family sequences in Puccinia. Bioinformatic tools were applied to retrieve the sequences from Puccinia genome followed by cloning and sequencing from P. triticina pathotype 77-5 cDNA to obtain the specific sequences. Their homologs were searched in other 14 Puccinia races to relate them with pathogenesis. Further, precursor sequences for three miRNA-like RNA molecules (milRs) were cloned from P. triticina cDNA. Their target genes like MAP kinase were successfully predicted and validated through degradome mapping and qRT-PCR. Gradual increase in milR2 (milR and milR*) expression over progressive time point of infection and positive expression for all the milRs within 77-5 urediniospores confirmed a complete host- independent RNAi activity by P. triticina.

Functional and DNA-protein binding studies of WRKY transcription factors and their expression analysis in response to biotic and abiotic stress in wheat (Triticum aestivum L.).

WRKY, a plant-specific transcription factor family, plays vital roles in pathogen defense, abiotic stress, and phytohormone signalling. Little is known about the roles and function of WRKY transcription factors in response to rust diseases in wheat. In the present study, three TaWRKY genes encoding complete protein sequences were cloned. They belonged to class II and III WRKY based on the number of WRKY domains and the pattern of zinc finger structures. Twenty-two DNA-protein binding docking complexes predicted stable interactions of WRKY domain with W-box. Quantitative real-time-PCR using wheat near-isogenic lines with or without Lr28 gene revealed differential up- or down-regulation in response to biotic and abiotic stress treatments which could be responsible for their functional divergence in wheat. TaWRKY62 was found to be induced upon treatment with JA, MJ, and SA and reduced after ABA treatments. Maximum induction of six out of seven genes occurred at 48 h post inoculation due to pathogen inoculation. Hence, TaWRKY (49, 50, 52, 55, 57, and 62) can be considered as potential candidate genes for further functional validation as well as for crop improvement programs for stress resistance. The results of the present study will enhance knowledge towards understanding the molecular basis of mode of action of WRKY transcription factor genes in wheat and their role during leaf rust pathogenesis in particular.

Insights of Lr28 mediated wheat leaf rust resistance: Transcriptomic approach.

Leaf rust is a fungal disease that causes severe yield losses in wheat. Resistant varieties with major and quantitative resistance genes are the most effective method to control the disease. However, the main problem is inadequate information for understanding resistance mechanism and its usefulness. This paper presents Lr28 mediated genome-wide response of known and unknown genes during wheat-Puccinia triticina interaction. In this study, we prepared Serial Analysis of Gene Expression (SAGE) libraries using seedling wheat mRNA for infected and mock conditions. The libraries were sequenced on Sequencing by Oligonucleotide Ligation and Detection (SOLiD) system generating 37-48 million reads. After mapping and gene expression analysis of ~6-12 million trimmed reads/library, we revealed five major categories comprised of Lr28 controlled transcripts in resistant (+Lr28) isoline (39), transcripts specific to susceptible (-Lr28) isoline (785), transcripts specific to hypersensitive-response (HR) (375), transcripts specific for basal-defense (153) and transcripts for establishment of pathogen (1616). We estimated the impact of specific genes and pathways through mapping on plant resistant gene database (PRGdb), reactive oxygen species (ROS) and phytohormone database. Functional annotation results revealed, receptor binding, homeostatic processes and cytoskeletal components as the major discriminating factors between susceptibility and resistance. We validated 28 key genes using qRT-PCR and found positive results. These findings were projected on hypothetical interaction model to demonstrate interaction mechanism. The study might have significant impact on future rust-resistance breeding through knowledge based smart genetic selection of quantitative resistance genes besides major effect R-gene.

Identification and molecular characterization of a trans-acting small interfering RNA producing locus regulating leaf rust responsive gene expression in wheat (Triticum aestivum L.).

MAIN CONCLUSION: A novel leaf rust responsive ta-siRNA-producing locus was identified in wheat showing similarity to 28S rRNA and generated four differentially expressing ta-siRNAs by phasing which targeted stress responsive genes. Trans-acting-small interfering RNAs (Ta-siRNAs) are plant specific molecules generally involved in development and are also stress responsive. Ta-siRNAs identified in wheat till date are all responsive to abiotic stress only. Wheat cultivation is severely affected by rusts and leaf rust particularly affects grain filling. This study reports a novel ta-siRNA producing locus (TAS) in wheat which is a segment of 28S ribosomal RNA but shows differential expression during leaf rust infestation. Four small RNA libraries prepared from wheat Near Isogenic Lines were treated with leaf rust pathogen and compared with untreated controls. A TAS with the ability to generate four ta-siRNAs by phasing events was identified along with the microRNA TamiR16 as the phase initiator. The targets of the ta-siRNAs included α-gliadin, leucine rich repeat, trans-membrane proteins, glutathione-S-transferase, and fatty acid desaturase among others, which are either stress responsive genes or are essential for normal growth and development of plants. Expression of the TAS, its generated ta-siRNAs, and their target genes were profiled at five different time points after pathogen inoculation of susceptible and resistant wheat isolines and compared with mock-inoculated controls. Comparative analysis of expression unveiled differential and reciprocal relationship as well as discrete patterns between susceptible and resistant isolines. The expression profiles of the target genes of the identified ta-siRNAs advocate more towards effector triggered susceptibility favouring pathogenesis. The study helps in discerning the functions of wheat genes regulated by ta-siRNAs in response to leaf rust.

Genome-wide identification and characterization of NB-ARC resistant genes in wheat (Triticum aestivum L.) and their expression during leaf rust infection.

KEY MESSAGE: NB-ARC domain-containing resistance genes from the wheat genome were identified, characterized and localized on chromosome arms that displayed differential yet positive response during incompatible and compatible leaf rust interactions. Wheat (Triticum aestivum L.) is an important cereal crop; however, its production is affected severely by numerous diseases including rusts. An efficient, cost-effective and ecologically viable approach to control pathogens is through host resistance. In wheat, high numbers of resistance loci are present but only few have been identified and cloned. A comprehensive analysis of the NB-ARC-containing genes in complete wheat genome was accomplished in this study. Complete NB-ARC encoding genes were mined from the Ensembl Plants database to predict 604 NB-ARC containing sequences using the HMM approach. Genome-wide analysis of orthologous clusters in the NB-ARC-containing sequences of wheat and other members of the Poaceae family revealed maximum homology with Oryza sativa indica and Brachypodium distachyon. The identification of overlap between orthologous clusters enabled the elucidation of the function and evolution of resistance proteins. The distributions of the NB-ARC domain-containing sequences were found to be balanced among the three wheat sub-genomes. Wheat chromosome arms 4AL and 7BL had the most NB-ARC domain-containing contigs. The spatio-temporal expression profiling studies exemplified the positive role of these genes in resistant and susceptible wheat plants during incompatible and compatible interaction in response to the leaf rust pathogen Puccinia triticina. Two NB-ARC domain-containing sequences were modelled in silico, cloned and sequenced to analyze their fine structures. The data obtained in this study will augment isolation, characterization and application NB-ARC resistance genes in marker-assisted selection based breeding programs for improving rust resistance in wheat.

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

MAIN CONCLUSION: Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30 % of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust resistance breeding programs.

De Novo Assembled Wheat Transcriptomes Delineate Differentially Expressed Host Genes in Response to Leaf Rust Infection.

Pathogens like Puccinia triticina, the causal organism for leaf rust, extensively damages wheat production. The interaction at molecular level between wheat and the pathogen is complex and less explored. The pathogen induced response was characterized using mock- or pathogen inoculated near-isogenic wheat lines (with or without seedling leaf rust resistance gene Lr28). Four Serial Analysis of Gene Expression libraries were prepared from mock- and pathogen inoculated plants and were subjected to Sequencing by Oligonucleotide Ligation and Detection, which generated a total of 165,767,777 reads, each 35 bases long. The reads were processed and multiple k-mers were attempted for de novo transcript assembly; 22 k-mers showed the best results. Altogether 21,345 contigs were generated and functionally characterized by gene ontology annotation, mining for transcription factors and resistance genes. Expression analysis among the four libraries showed extensive alterations in the transcriptome in response to pathogen infection, reflecting reorganizations in major biological processes and metabolic pathways. Role of auxin in determining pathogenesis in susceptible and resistant lines were imperative. The qPCR expression study of four LRR-RLK (Leucine-rich repeat receptor-like protein kinases) genes showed higher expression at 24 hrs after inoculation with pathogen. In summary, the conceptual model of induced resistance in wheat contributes insights on defense responses and imparts knowledge of Puccinia triticina-induced defense transcripts in wheat plants.

Deltamethrin-induced oxidative stress and mitochondrial caspase-dependent signaling pathways in murine splenocytes.

Deltamethrin (DLM) is a well-known pyrethroid insecticide used extensively in pest control. Exposure to DLM has been demonstrated to cause apoptosis in various cells. However, the immunotoxic effects of DLM on mammalian system and its mechanism is still an open question to be explored. To explore these effects, this study has been designed to first observe the interactions of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has strong binding affinity toward the CD45 and CD28 receptors. In vitro study revealed that DLM induces apoptosis in murine splenocytes in a concentration-dependent manner. The earliest markers of apoptosis such as enhanced reactive oxygen species and caspase 3 activation are evident as early as 1 h by 25 and 50 µM DLM. Western blot analysis demonstrated that p38 MAP kinase and Bax expression is increased in a concentration-dependent manner, whereas Bcl 2 expression is significantly reduced after 3 h of DLM treatment. Glutathione depletion has been also observed at 3 and 6 h by 25 and 50 µM concentration of DLM. Flow cytometry results imply that the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. N-acetyl cysteine effectively reduces the percentage of apoptotic cells, which is increased by DLM. In contrast, buthionine sulfoxamine causes an elevation in the percentage of apoptotic cells. Phenotyping data imply the effect of DLM toxicity in murine splenocytes. In brief, the study demonstrates that DLM causes apoptosis through its interaction with CD45 and CD28 receptors, leading to oxidative stress and activation of the mitochondrial caspase-dependent pathways which ultimately affects the immune functions. This study provides mechanistic information by which DLM causes toxicity in murine splenocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 808-819, 2016.