Conversion of superior bread wheat genotype HD3209 carrying Lr19/Sr25 into CMS line for development of rust-resistant wheat hybrids.

Hybrid development is one of the most promising strategies for boosting crop yields. Parental lines used to create hybrids must have good per se performance and disease resistance for developing superior hybrids. Indian wheat line HD3209 was developed by introducing the rust resistance genes Lr19/Sr25 into the background of popular wheat variety HD2932. The wheat line HD3209 carrying Lr19/Sr25 has been successfully and rapidly converted to the CMS line A-HD3209, with 96.01% background genome recovery, based on selection for agro-morphological traits, rust resistance, pollen sterility, and foreground and background analyses utilizing SSR markers. The converted CMS line A-HD3209 was completely sterile and nearly identical to the recurrent parent HD3209. Based on high per se performance and rust resistance, the study concludes that the derived CMS line A-HD3209 is promising and can be employed successfully in hybrid development.

Transcriptome analysis of Bipolaris sorokiniana - Hordeum vulgare provides insights into mechanisms of host-pathogen interaction.

Spot blotch disease incited by Bipolaris sorokiniana severely affects the cultivation of barley. The resistance to B. sorokiniana is quantitative in nature and its interaction with the host is highly complex which necessitates in-depth molecular analysis. Thus, the study aimed to conduct the transcriptome analysis to decipher the mechanisms and pathways involved in interactions between barley and B. sorokiniana in both the resistant (EC0328964) and susceptible (EC0578292) genotypes using the RNA Seq approach. In the resistant genotype, 6,283 genes of Hordeum vulgare were differentially expressed out of which 5,567 genes were upregulated and 716 genes were downregulated. 1,158 genes of Hordeum vulgare were differentially expressed in the susceptible genotype, out of which 654 genes were upregulated and 504 genes were downregulated. Several defense-related genes like resistant gene analogs (RGAs), disease resistance protein RPM1, pathogenesis-related protein PRB1-2-like, pathogenesis-related protein 1, thaumatin-like protein PWIR2 and defensin Tm-AMP-D1.2 were highly expressed exclusively in resistant genotype only. The pathways involved in the metabolism and biosynthesis of secondary metabolites were the most prominently represented pathways in both the resistant and susceptible genotypes. However, pathways involved in MAPK signaling, plant-pathogen interaction, and plant hormone signal transduction were highly enriched in resistant genotype. Further, a higher number of pathogenicity genes of B. sorokiniana was found in response to the susceptible genotype. The pathways encoding for metabolism, biosynthesis of secondary metabolites, ABC transporters, and ubiquitin-mediated proteolysis were highly expressed in susceptible genotype in response to the pathogen. 14 and 11 genes of B. sorokiniana were identified as candidate effectors from susceptible and resistant host backgrounds, respectively. This investigation will offer valuable insights in unraveling the complex mechanisms involved in barley- B. sorokiniana interaction.

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.

Curcumin-capped gold nanorods as optical sensing platform for sequence specific detection of DNA based on their self-assembly.

We are reporting curcumin-induced gold nanorods as an optical sensing platform for the detection of sequence-specific DNA target through their self-assembly. The combined effect of eco-friendly reducing agent (i.e., curcumin) and silver nitrate in a basic medium (i.e., pH 10) has been attributed for the formation of small gold nanorods (AuNRs) having approximate length and diameter i.e., 19.7 ± 0.8 nm and 6.0 ± 0.5 nm, respectively, and lower longitudinal surface plasmon resonance (SPR) enable to detect and analyse different biomarkers. Further, for evaluating cellular uptake of as-synthesized AuNRs, the cytotoxicity study has been carried out by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on A549 cells and HEPG2 cell lines, respectively, and shown approximately similar cytotoxicity. Interestingly, as-synthesized optically and electronically active AuNRs based nanobiosensing platform enable to detect sequence-specific DNA targets with low level of detection limit i.e., LOD 8.6 ± 0.15 pM for complimentary target (CT) DNA with higher sensitivity and better selectivity. Finally, this study is suggesting a simplistic bio-mediated approach of tuning the shape and size of AuNRs for sensitive, selective and reliable nanobiosensing platform for sequence-specific DNA detection related to cancer cells.

Unravelling structural, functional, evolutionary and genetic basis of SWEET transporters regulating abiotic stress tolerance in maize.

Sugars Will Eventually be Exported Transporters (SWEETs) are the novel sugar transporters widely distributed among living systems. SWEETs play a crucial role in various bio-physiological processes, viz., plant developmental, nectar secretion, pollen development, and regulation of biotic and abiotic stresses, in addition to their prime sugar-transporting activity. Thus, in-depth structural, evolutionary, and functional characterization of maize SWEET transporters was performed for their utility in maize improvement. The mining of SWEET genes in the latest maize genome release (v.5) showed an uneven distribution of 20 ZmSWEETs. The comprehensive structural analyses and docking of ZmSWEETs with four sugars, viz., fructose, galactose, glucose, and sucrose, revealed frequent amino acid residues forming hydrogen (asparagine, valine, serine) and hydrophobic (tryptophan, glycine, and phenylalanine) interactions. Evolutionary analyses of SWEETs showed a mixed lineage with 50-100 % commonality of ortho-groups and -sequences evolved under strong purifying selection (Ka/Ks < 0.5). The duplication analysis showed non-functionalization (ZmSWEET18 in B73) and neo- and sub-functionalization (ZmSWEET3, ZmSWEET6, ZmSWEET9, ZmSWEET19, and ZmSWEET20) events in maize. Functional analyses of ZmSWEET genes through co-expression, in silico expression and qRT-PCR assays showed the relevance of ZmSWEETs expression in regulating drought, heat, and waterlogging stress tolerances in maize. The first ever ZmSWEET-regulatory network revealed 286 direct (ZmSWEET-TF: 140 ZmSWEET-miRNA: 146) and 1226 indirect (TF-TF: 597; TF-miRNA: 629) edges. The present investigation has given new insights into the complex transcriptional and post-transcriptional regulation and the regulatory and functional relevance of ZmSWEETs in assigning stress tolerance in maize.

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.

Leaf rolling in bread wheat (Triticum aestivum L.) is controlled by the upregulation of a pair of closely linked/duplicate zinc finger homeodomain class transcription factors during moisture stress conditions.

Zinc finger-homeodomain (ZF-HDs) class IV transcriptional factors (TFs) is a plant-specific transcription factor and play a key role in stress responses, plant growth, development, and hormonal signaling. In this study, two new leaf rolling TFs genes, namely TaZHD1 and TaZHD10, were identified in wheat using comparative genomic analysis of the target region that carried a major QTL for leaf rolling identified through multi-environment phenotyping and high throughput genotyping of a RIL population. Structural and functional annotation of the candidate ZHD genes with its closest rice orthologs reflects the species-specific evolution and, undoubtedly, validates the notions of remote-distance homology concept. Meanwhile, the morphological analysis resulted in contrasting difference for leaf rolling in extreme RILs between parental lines HD2012 and NI5439 at booting and heading stages. Transcriptome-wide expression profiling revealed that TaZHD10 transcripts showed significantly higher expression levels than TaZHD1 in all leaf tissues upon drought stress. The relative expression of these genes was further validated by qRT-PCR analysis, which also showed consistent results across the studied genotypes at the booting and anthesis stage. The contrasting modulation of these genes under drought conditions and the available evidenced for its epigenetic behavior that might involve the regulation of metabolic and gene regulatory networks. Prediction of miRNAs resulted in five Tae-miRs that could be associated with RNAi mediated control of TaZHD1 and TaZHD10 putatively involved in the metabolic pathway controlling rolled leaf phenotype. Gene interaction network analysis indicated that TaZHD1 and TaZHD10 showed pleiotropic effects and might also involve other functions in wheat in addition to leaf rolling. Overall, the results increase our understanding of TaZHD genes and provide valuable information as robust candidate genes for future functional genomics research aiming for the breeding of wheat varieties tolerant to leaf rolling.

Genome-wide characterization and identification of cyclophilin genes associated with leaf rust resistance in bread wheat (Triticum aestivum L.).

Cyclophilins (CYPs) are a group of highly conserved proteins involved in host-pathogen interactions in diverse plant species. However, the role of CYPs during disease resistance in wheat remains largely elusive. In the present study, the systematic genome-wide survey revealed a set of 81 TaCYP genes from three subfamilies (GI, GII, and GIII) distributed on all 21 wheat chromosomes. The gene structures of TaCYP members were found to be highly variable, with 1-14 exons/introns and 15 conserved motifs. A network of miRNA targets with TaCYPs demonstrated that TaCYPs were targeted by multiple miRNAs and vice versa. Expression profiling was done in leaf rust susceptible Chinese spring (CS) and the CS-Ae. Umbellulata derived resistant IL "Transfer (TR). Three homoeologous TaCYP genes (TaCYP24, TaCYP31, and TaCYP36) showed high expression and three homoeologous TaCYP genes (TaCYP44, TaCYP49, and TaCYP54) showed low expression in TR relative to Chinese Spring. Most of the other TaCYPs showed comparable expression changes (down- or upregulation) in both contrasting TR and CS. Expression of 16 TaCYPs showed significant association (p < 0.05) with superoxide radical and hydrogen peroxide abundance, suggesting the role of TaCYPs in downstream signaling processes during wheat-leaf rust interaction. The differentially expressing TaCYPs may be potential targets for future validation using transgenic (overexpression, RNAi or CRISPR-CAS) approaches and for the development of leaf rust-resistant wheat genotypes.

G-DIRT: a web server for identification and removal of duplicate germplasms based on identity-by-state analysis using single nucleotide polymorphism genotyping data.

Maintaining duplicate germplasms in genebanks hampers effective conservation and utilization of genebank resources. The redundant germplasm adds to the cost of germplasm conservation by requiring a large proportion of the genebank financial resources towards conservation rather than enriching the diversity. Besides, genome-wide-association analysis using an association panel with over-represented germplasms can be biased resulting in spurious marker-trait associations. The conventional methods of germplasm duplicate removal using passport information suffer from incomplete or missing passport information and data handling errors at various stages of germplasm enrichment. This limitation is less likely in the case of genotypic data. Therefore, we developed a web-based tool, Germplasm Duplicate Identification and Removal Tool (G-DIRT), which allows germplasm duplicate identification based on identity-by-state analysis using single-nucleotide polymorphism genotyping information along with pre-processing of genotypic data. A homozygous genotypic difference threshold of 0.1% for germplasm duplicates has been determined using tetraploid wheat genotypic data with 94.97% of accuracy. Based on the genotypic difference, the tool also builds a dendrogram that can visually depict the relationship between genotypes. To overcome the constraint of high-dimensional genotypic data, an offline version of G-DIRT in the interface of R has also been developed. The G-DIRT is expected to help genebank curators, breeders and other researchers across the world in identifying germplasm duplicates from the global genebank collections by only using the easily sharable genotypic data instead of physically exchanging the seeds or propagating materials. The web server will complement the existing methods of germplasm duplicate identification based on passport or phenotypic information being freely accessible at http://webtools.nbpgr.ernet.in/gdirt/.

Identification of Novel Broad-Spectrum Leaf Rust Resistance Sources from Khapli Wheat Landraces.

Wheat leaf rust caused by Puccinia triticina Eriks is an important disease that causes yield losses of up to 40% in susceptible varieties. Tetraploid emmer wheat (T. turgidum ssp. Dicoccum), commonly called Khapli wheat in India, is known to have evolved from wild emmer (Triticum turgidum var. dicoccoides), and harbors a good number of leaf rust resistance genes. In the present study, we are reporting on the screening of one hundred and twenty-three dicoccum wheat germplasm accessions against the leaf rust pathotype 77-5. Among these, an average of 45.50% of the germplasms were resistant, 46.74% were susceptible, and 8.53% had mesothetic reactions. Further, selected germplasm lines with accession numbers IC138898, IC47022, IC535116, IC535133, IC535139, IC551396, and IC534144 showed high level of resistance against the eighteen prevalent pathotypes. The infection type varied from ";", ";N", ";N1" to ";NC". PCR-based analysis of the resistant dicoccum lines with SSR marker gwm508 linked to the Lr53 gene, a leaf rust resistance gene effective against all the prevalent pathotypes of leaf rust in India and identified from a T. turgidum var. dicoccoides germplasm, indicated that Lr53 is not present in the selected accessions. Moreover, we have also generated 35K SNP genotyping data of seven lines and the susceptible control, Mandsaur Local, to study their relationships. The GDIRT tool based on homozygous genotypic differences revealed that the seven genotypes are unique to each other and may carry different resistance genes for leaf rust.

Genetic Dissection of Seedling Root System Architectural Traits in a Diverse Panel of Hexaploid Wheat through Multi-Locus Genome-Wide Association Mapping for Improving Drought Tolerance.

Cultivars with efficient root systems play a major role in enhancing resource use efficiency, particularly water absorption, and thus in drought tolerance. In this study, a diverse wheat association panel of 136 wheat accessions including mini core subset was genotyped using Axiom 35k Breeders' Array to identify genomic regions associated with seedling stage root architecture and shoot traits using multi-locus genome-wide association studies (ML-GWAS). The association panel revealed a wide variation of 1.5- to 50-fold and were grouped into six clusters based on 15 traits. Six different ML-GWAS models revealed 456 significant quantitative trait nucleotides (QTNs) for various traits with phenotypic variance in the range of 0.12-38.60%. Of these, 87 QTNs were repeatedly detected by two or more models and were considered reliable genomic regions for the respective traits. Among these QTNs, eleven were associated with average diameter and nine each for second order lateral root number (SOLRN), root volume (RV) and root length density (RLD). A total of eleven genomic regions were pleiotropic and each controlled two or three traits. Some important candidate genes such as Formin homology 1, Ubiquitin-like domain superfamily and ATP-dependent 6-phosphofructokinase were identified from the associated genomic regions. The genomic regions/genes identified in this study could potentially be targeted for improving root traits and drought tolerance in wheat.

Spectroscopic and molecular docking studies for the binding and interaction aspects of curcumin-cysteine conjugate and rosmarinic acid with human telomeric G-quadruplex DNA.

The binding and interaction aspects of potential anticancer ligands like: curcumin-cysteine (CC) and rosmarinic acid (RA) with human telomeric G-quadruplex DNA, a novel anticancer target, have been probed by spectroscopic and molecular docking approach. The circular dichroism study unravels the conformational switching from mixed hybrid to parallel structure for the short sequence of human telomeric G-quadruplex structure in the presence of both the ligands. Further a good correlation for binding affinity has been established from the emission and absorption binding spectrum analysis. Further our spectroscopic and molecular docking studies have suggested that the CC having better binding capability than RA to human telomeric G-quadruplex. The presence of L-cysteine moiety in CC ligand is responsible factor for its binding via both minor as well as major groove of human telomeric G-quadruplex DNA where-as RA binds only via minor groove of telomeric G-DNA.

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.

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.