Amolkumar U. Solanke

Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes

Cotton is an important source of natural fibre used in the textile industry and the productivity of the crop is adversely affected by drought stress. High throughput transcriptomic analyses were used to identify genes involved in fibre development. However, not much information is available on cotton genome response in developing fibres under drought stress. In the present study a genome wide transcriptome analysis was carried out to identify differentially expressed genes at various stages of fibre growth under drought stress. Our study identified a number of genes differentially expressed during fibre elongation as compared to other stages. High level up-regulation of genes encoding for enzymes involved in pectin modification and cytoskeleton proteins was observed at fibre initiation stage. While a large number of genes encoding transcription factors (AP2-EREBP, WRKY, NAC and C2H2), osmoprotectants, ion transporters and heat shock proteins and pathways involved in hormone (ABA, ethylene and JA) biosynthesis and signal transduction were up-regulated and genes involved in phenylpropanoid and flavonoid biosynthesis, pentose and glucuronate interconversions and starch and sucrose metabolism pathways were down-regulated during fibre elongation. This study showed that drought has relatively less impact on fibre initiation but has profound effect on fibre elongation by down-regulating important genes involved in cell wall loosening and expansion process. The comprehensive transcriptome analysis under drought stress has provided valuable information on differentially expressed genes and pathways during fibre development that will be useful in developing drought tolerant cotton cultivars without compromising fibre quality.

Plant RNA Interference Pathways: Diversity in Function, Similarity in Action

Small non-coding RNA-mediated gene-silencing pathways, collectively called RNA interference (RNAi), are involved in regulation of endogenous gene expression and plant defence. It is manifested through two broad classes of small non-coding regulatory RNAs, small interfering RNA (siRNA) and microRNA (miRNA). siRNAs, generated from cleavage of long hairpin RNA by RNase III-class endonuclease, Dicer-like, mediate transcriptional or post-transcriptional gene silencing. At transcriptional level, 24-nucleotide (nt)-long-siRNAs guide an effector complex for DNA methylation, which leads to heterochromatinisation of target loci and consequently transcriptional silencing. At post-transcriptional level, a different size class of 21-nt-long siRNAs guides a silencing complex, called RNA-induced silencing complex, for cleavage of target mRNA. cis-acting siRNAs are involved in plant defence against viruses and transposons, and trans-acting siRNAs regulate endogenous genes involved in plant growth. miRNAs are generated from processing of imperfect stem-loop RNA precursors by Dicer-like. They regulate plant growth and adaptive stress responses by either degradation or translational repression of target mRNAs.

Draft Genome of the Wheat Rust Pathogen (Puccinia triticina) Unravels Genome-Wide Structural Variations during Evolution

Abstract Leaf rust is one of the most important diseases of wheat and is caused by Puccinia triticina, a highly variable rust pathogen prevalent worldwide. Decoding the genome of this pathogen will help in unraveling the molecular basis of its evolution and in the identification of genes responsible for its various biological functions. We generated high quality draft genome sequences (approximately 100- 106 Mb) of two races of P. triticina; the variable and virulent Race77 and the old, avirulent Race106. The genomes of races 77 and 106 had 33X and 27X coverage, respectively. We predicted 27678 and 26384 genes, with average lengths of 1,129 and 1,086 bases in races 77 and 106, respectively and found that the genomes consisted of 37.49% and 39.99% repetitive sequences. Genome wide comparative analysis revealed that Race77 differs substantially from Race106 with regard to segmental duplication (SD), repeat element, and SNP/InDel characteristics. Comparative analyses showed that Race 77 is a recent, highly variable and adapted Race compared with Race106. Further sequence analyses of 13 additional pathotypes of Race77 clearly differentiated the recent, active and virulent, from the older pathotypes. Average densities of 2.4 SNPs and 0.32 InDels per kb were obtained for all P. triticina pathotypes. Secretome analysis demonstrated that Race77 has more virulence factors than Race 106, which may be responsible for the greater degree of adaptation of this pathogen. We also found that genes under greater selection pressure were conserved in the genomes of both races, and may affect functions crucial for the higher levels of virulence factors in Race77. This study provides insights into the genome structure, genome organization, molecular basis of variation, and pathogenicity of P. triticina. The genome sequence data generated in this study have been submitted to public domain databases and will be an important resource for comparative genomics studies of the more than 4000 existing Puccinia species.

Understanding Host-Pathogen Interactions with Expression Profiling of NILs Carrying Rice-Blast Resistance Pi9 Gene

Magnaporthe oryzae infection causes rice blast, a destructive disease that is responsible for considerable decrease in rice yield. Development of resistant varieties via introgressing resistance genes with marker-assisted breeding can eliminate pesticide use and minimize crop losses. Here, resistant near-isogenic line (NIL) of Pusa Basmati-1(PB1) carrying broad spectrum rice blast resistance gene Pi9 was used to investigate Pi9-mediated resistance response. Infected and uninfected resistant NIL and susceptible control line were subjected to RNA-Seq. With the exception of one gene (Pi9), transcriptional signatures between the two lines were alike, reflecting basal similarities in their profiles. Resistant and susceptible lines possessed 1043 (727 up-regulated and 316 down-regulated) and 568 (341 up-regulated and 227 down-regulated) unique and significant differentially expressed loci (SDEL), respectively. Pathway analysis revealed higher transcriptional activation of kinases, WRKY, MYB, and ERF transcription factors, JA-ET hormones, chitinases, glycosyl hydrolases, lipid biosynthesis, pathogenesis and secondary metabolism related genes in resistant NIL than susceptible line. Singular enrichment analysis demonstrated that blast resistant NIL is significantly enriched with genes for primary and secondary metabolism, response to biotic stimulus and transcriptional regulation. The co-expression network showed proteins of genes in response to biotic stimulus interacted in a manner unique to resistant NIL upon M. oryzae infection. These data suggest that Pi9 modulates genome-wide transcriptional regulation in resistant NIL but not in susceptible PB1. We successfully used transcriptome profiling to understand the molecular basis of Pi9-mediated resistance mechanisms, identified potential candidate genes involved in early pathogen response and revealed the sophisticated transcriptional reprogramming during rice-M. oryzae interactions.

Expression of Finger Millet EcDehydrin7 in Transgenic Tobacco Confers Tolerance to Drought Stress

One of the critical alarming constraints for agriculture is water scarcity. In the current scenario, global warming due to climate change and unpredictable rainfall, drought is going to be a master player and possess a big threat to stagnating gene pool of staple food crops. So it is necessary to understand the mechanisms that enable the plants to cope with drought stress. In this study, effort was made to prospect the role of EcDehydrin7 protein from normalized cDNA library of drought tolerance finger millet in transgenic tobacco. Biochemical and molecular analyses of T0 transgenic plants were done for stress tolerance. Leaf disc assay, seed germination test, dehydration assay, and chlorophyll estimation showed EcDehydrin7 protein directly link to drought tolerance. Northern and qRT PCR analyses shows relatively high expression of EcDehydrin7 protein compare to wild type. T0 transgenic lines EcDehydrin7(11) and EcDehydrin7(15) shows superior expression among all lines under study. In summary, all results suggest that EcDehydrin7 protein has a remarkable role in drought tolerance and may be used for sustainable crop breeding program in other food crops.

Chloroplast Genome Sequence of Clusterbean (Cyamopsis tetragonoloba L.): Genome Structure and Comparative Analysis

Clusterbean (Cyamopsis tetragonoloba L.), also known as guar, belongs to the family Leguminosae, and is an annual herbaceous legume. Guar is the main source of galactomannan for gas mining industries. In the present study, the draft chloroplast genome of clusterbean was generated and compared to some of the previously reported legume chloroplast genomes. The chloroplast genome of clusterbean is 152,530 bp in length, with a quadripartite structure consisting of large single copy (LSC) and small single copy (SSC) of 83,025 bp and 17,879 bp in size, respectively, and a pair of inverted repeats (IRs) of 25,790 bp in size. The chloroplast genome contains 114 unique genes, which includes 78 protein coding genes, 30 tRNAs, 4 rRNAs genes, and 2 pseudogenes. It also harbors a 50 kb inversion, typical of the Leguminosae family. The IR region of the clusterbean chloroplast genome has undergone an expansion, and hence, the whole rps19 gene is included in the IR, as compared to other legume plastid genomes. A total of 220 simple sequence repeats (SSRs) were detected in the clusterbean plastid genome. The analysis of the clusterbean plastid genome will provide useful insights for evolutionary, molecular and genetic engineering studies.

Isolation and characterization of drought responsive EcDehydrin7 gene from finger millet (Eleusine coracana (L.) Gaertn.)

Finger millet is known for its drought tolerance ability.The present investigation was carried out to explore drought responsive genes from finger millet. To generate more genetic and genomic information, a highly normalized cDNA library was prepared from 12 days old stress imposed seedlings and utilized it for identification of drought responsive genes using cDNA macroarray. Differentially expressing six genes (Dehydrin7, Hypothetical protein1, Chloroplast envelope membrane protein, Hypothetical protein2, S-adenosyl methionine decarboxylase2 and unknown gene) were selected for qRT-PCR analysis under different stress conditions. Gene EcDehydrin7 (Accession No. KM096446) showed high expression under drought and heat stress. Functional validation of this gene can provide more information about drought tolerance mechanism in crop plants.

Genetic Engineering for Tolerance to Climate Change-Related Traits

Climate change is expected to introduce new challenges for sustainable crop production worldwide. High temperature, less water availability, and emergence of new pests and pathogens calls for changing strategies and using biotechnological interventions to meet these challenges to sustaining food supply. Engineering biotic and abiotic stress tolerance will require concerted and combined efforts by plant breeders and biotechnologists alike. Several genes have been identified to have potential in mitigating climate change effects. These can be broadly classified as single-action genes and multiple action genes. Single action genes include osmoprotectants, detoxifying, LEA, HSP, ANPs, and ion transporters which have incremental roles in providing abiotic stress tolerance. Multiaction regulatory genes provide an attractive strategy to improve crop plants as these genes activate a cascade of genes which act together to enhance stress tolerance. CBF/DREB, SNAC, MYB, HSF, and AREB are some candidate genes of this category. Signal transduction genes such as osmosensors, AHK1, SNF1-related kinases are potential candidate genes for engineering stress tolerance in the near future. For insect resistance cry genes will remain the ideal choice however, engineering biotic resistance will involve new technologies such as RNAi and micro RNAs for combating insects and pests. Regulatory genes and genes involved in signal transduction will assume great importance in developing cultivars adapted to the threats of climate change. Here we review the target traits and potential genes for engineering stress tolerance in crop plants to meet climate change challenges for food production.

High Quality Unigenes and Microsatellite Markers from Tissue Specific Transcriptome and Development of a Database in Clusterbean (Cyamopsis tetragonoloba, L. Taub)

Clusterbean (Cyamopsis tetragonoloba L. Taub), is an important industrial, vegetable and forage crop. This crop owes its commercial importance to the presence of guar gum (galactomannans) in its endosperm which is used as a lubricant in a range of industries. Despite its relevance to agriculture and industry, genomic resources available in this crop are limited. Therefore, the present study was undertaken to generate RNA-Seq based transcriptome from leaf, shoot, and flower tissues. A total of 145 million high quality Illumina reads were assembled using Trinity into 127,706 transcripts and 48,007 non-redundant high quality (HQ) unigenes. We annotated 79% unigenes against Plant Genes from the National Center for Biotechnology Information (NCBI), Swiss-Prot, Pfam, gene ontology (GO) and KEGG databases. Among the annotated unigenes, 30,020 were assigned with 116,964 GO terms, 9984 with EC and 6111 with 137 KEGG pathways. At different fragments per kilobase of transcript per millions fragments sequenced (FPKM) levels, genes were found expressed higher in flower tissue followed by shoot and leaf. Additionally, we identified 8687 potential simple sequence repeats (SSRs) with an average frequency of one SSR per 8.75 kb. A total of 28 amplified SSRs in 21 clusterbean genotypes resulted in polymorphism in 13 markers with average polymorphic information content (PIC) of 0.21. We also constructed a database named ‘ClustergeneDB’ for easy retrieval of unigenes and the microsatellite markers. The tissue specific genes identified and the molecular marker resources developed in this study is expected to aid in genetic improvement of clusterbean for its end use.

Identification of long non-coding RNA in rice lines resistant to Rice blast pathogen Maganaporthe oryzae

Rice blast disease caused by a fungus Magnaporthae oryzae is one of the most important biotic factors that severely damage the rice crop. Several molecular approaches are now being applied to tackle this issue in rice. It is of interest to study long non-coding RNA (lncRNA) in rice to control the disease. lncRNA, a non-coding transcript that does not encode protein, is known to play an important role in gene regulation of various biological processes. Here we describe a computational pipeline to identify lncRNA from a resistant rice line. The number of lncRNA found in resistant line was 1429, 1927 and 1981 in mock and M. oryzae (ZB13 and Zhong) inoculated samples, respectively. Functional classification of these lncRNA reveals a higher number of long intergenic non-coding RNA compared to antisense lncRNA in both mock and M. oryzae inoculated resistant rice lines. Many intergenic lncRNA candidates were identified from resistant rice line and their role to regulate the resistance mechanism in rice during M. oryzae invasion is implied.