Renesh Bedre

Genome-Wide Transcriptome Analysis of Cotton (Gossypium hirsutum L.) Identifies Candidate Gene Signatures in Response to Aflatoxin Producing Fungus Aspergillus flavus.

Aflatoxins are toxic and potent carcinogenic metabolites produced from the fungi Aspergillus flavus and A. parasiticus. Aflatoxins can contaminate cottonseed under conducive preharvest and postharvest conditions. United States federal regulations restrict the use of aflatoxin contaminated cottonseed at >20 ppb for animal feed. Several strategies have been proposed for controlling aflatoxin contamination, and much success has been achieved by the application of an atoxigenic strain of A. flavus in cotton, peanut and maize fields. Development of cultivars resistant to aflatoxin through overexpression of resistance associated genes and/or knocking down aflatoxin biosynthesis of A. flavus will be an effective strategy for controlling aflatoxin contamination in cotton. In this study, genome-wide transcriptome profiling was performed to identify differentially expressed genes in response to infection with both toxigenic and atoxigenic strains of A. flavus on cotton (Gossypium hirsutum L.) pericarp and seed. The genes involved in antifungal response, oxidative burst, transcription factors, defense signaling pathways and stress response were highly differentially expressed in pericarp and seed tissues in response to A. flavus infection. The cell-wall modifying genes and genes involved in the production of antimicrobial substances were more active in pericarp as compared to seed. The genes involved in auxin and cytokinin signaling were also induced. Most of the genes involved in defense response in cotton were highly induced in pericarp than in seed. The global gene expression analysis in response to fungal invasion in cotton will serve as a source for identifying biomarkers for breeding, potential candidate genes for transgenic manipulation, and will help in understanding complex plant-fungal interaction for future downstream research.

Identification of cold-responsive genes in energycane for their use in genetic diversity analysis and future functional marker development.

Breeding for cold tolerance in sugarcane will allow its cultivation as a dedicated biomass crop in cold environments. Development of functional markers to facilitate marker-assisted breeding requires identification of cold stress tolerance genes. Using suppression subtractive hybridization, 465 cold-responsive genes were isolated from the cold-tolerant energycane Ho02-144. Predicted gene interactions network indicated several associated pathways that may coordinately regulate cold tolerance responses in energycane. Expression analysis of a select set of genes, representing signaling and transcription factors, genes involved in polyamine and antioxidant biosynthesis, protein degradation and in the repair of damaged proteins in the cytosol, showed their time-dependent regulation under cold-stress. Comparative expression profiles of these genes between Ho02-144 and a cold-sensitive clone (L79-1002) showed that almost all genes were induced immediately upon imposition of cold stress and maintained their expression in Ho02-144 whereas they were either downregulated or their upregulation was very low in L79-1002. Simple sequence repeat markers derived from 260 cold-responsive genes showed allelic diversity among the cold-sensitive commercial hybrids that were distinct from the Saccharum spontaneum clones. Future efforts will target sequence polymorphism information of these genes in our ongoing QTL and association mapping studies to identify functional markers associated with cold tolerance in sugar/energycane.

Salt Adaptation Mechanisms of Halophytes: Improvement of Salt Tolerance in Crop Plants

Soil salinity is one of the most serious environmental factors that affect crop productivity worldwide. Inevitable global climate change leading to rise in sea water level would exacerbate degradation of irrigation systems and contamination of ground water resources, which render conventional agricultural practices impossible due to the sensitivity of most crops to salinity. Breeding for development of salt-tolerant crop plants has been a major challenge due to the complexity and multigenic control of salt tolerance traits. Halophytes are capable of surviving and thriving under salt at concentrations as high as 5 g/L, by maintaining negative water potential. Physiological and molecular studies have suggested that halophytes, unlike glycophytes, have evolved mechanisms, such as ion homeostasis through ion extrusion and compartmentalization, osmotic adjustments, and antioxidant production for adaptation to salinity. Employment of integrated approaches involving different omics tools would amplify our understanding of the biology of stress response networks in the halophytes. Translation of the knowledge and resources generated from halophyte relatives of crop plants through functional genomics will lead to the development of new breeds of crops that are suitable for saline agriculture.

Sequencing and expression analysis of salt-responsive miRNAs and target genes in the halophyte smooth cordgrass (Spartina alternifolia Loisel).

MicroRNAs have been shown to be involved in regulating plant’s response to environmental stresses, including salinity. There is no report yet on the miRNA-mediated posttranscriptional regulation of salt stress response of a grass halophyte by miRNAs. Here we report on the deep-sequencing followed by expression validation through (s)qRT-PCR of a selected set of salt-responsive miRNAs and their targets of the salt marsh monocot halophyte smooth cordgrass (Spartina alterniflora Loisel). Expression kinetics study of 12 miRNAs showed differential up/down-regulation in leaf and root tissues under salinity. Induction of expression of six putative novel microRNAs with high read counts in the sequence library suggested that the halophyte grass may possess different/novel gene posttranscriptional regulation of its salinity adaptation. Similarly, expression analysis of target genes of four selected miRNAs showed temporal and spatial variation in the up/down-regulation of their transcript accumulation under salt stress. The expression levels of miRNAs and their respective targets were coherent, non-coherent, or semi-coherent type. Understanding the gene regulation mechanism(s) at the miRNA level will broaden our fundamental understanding of the biology of the salt stress tolerance of the halophyte and provide novel positive regulators of salt stress tolerance for downstream research.

Genetic Mapping of Quantitative Trait Loci for Grain Yield under Drought in Rice under Controlled Greenhouse Conditions

Drought stress is a constant threat to rice production worldwide. Most modern rice cultivars are sensitive to drought, and the effect is severe at the reproductive stage. Conventional breeding for drought resistant (DR) rice varieties is slow and limited due to the quantitative nature of the DR traits. Identification of genes (QTLs)/markers associated with DR traits is a prerequisite for marker-assisted breeding. Grain yield is the most important trait and to this end drought yield QTLs have been identified under field conditions. The present study reports identification of drought yield QTLs under controlled conditions without confounding effects of other factors prevalent under natural conditions. A linkage map covering 1,781.5 cM with an average resolution of 9.76 cM was constructed using an F2 population from a cross between two Japonica cultivars, Cocodrie (drought sensitive) and Vandana (drought tolerant) with 213 markers distributed over 12 rice chromosomes. A subset of 59 markers (22 genic SSRs and 37 SNPs) derived from the transcriptome of the parents were also placed in the map. Single marker analysis using 187 F2 : 3 progeny identified 6 markers distributed on chromosomes 1, 5, and 8 to be associated with grain yield under drought (GYD). Composite interval mapping identified six genomic regions/quantitative trait loci (QTL) on chromosome 1, 5, 8, and 9 to be associated with GYD. QTLs located on chromosome 1 (qGYD1.2, qGYD1.3), chromosome 5 (qGYD5.1) and chromosome 8 (qGYD8.1) were contributed by Vandana alleles, whereas the QTLs, qGYD1.1 and qQYD9.1 were contributed by Cocodrie alelles. The additive positive phenotypic variance explained by the QTLs ranged from 30.0 to 34.0%. Candidate genes annotation within QTLs suggested the role of transcription factors and genes involved in osmotic potential regulation through catalytic/metabolic pathways in drought tolerance mechanism contributing to yield.

An actin-depolymerizing factor from the halophyte smooth cordgrass, Spartina alterniflora (SaADF2), is superior to its rice homolog (OsADF2) in conferring drought and salt tolerance when constitutively overexpressed in rice

Summary Actin‐depolymerizing factors (ADFs) maintain the cellular actin network dynamics by regulating severing and disassembly of actin filaments in response to environmental cues. An ADF isolated from a monocot halophyte, Spartina alterniflora (SaADF2), imparted significantly higher level of drought and salinity tolerance when expressed in rice than its rice homologue OsADF2. SaADF2 differs from OsADF2 by a few amino acid residues, including a substitution in the regulatory phosphorylation site serine‐6, which accounted for its weak interaction with OsCDPK6 (calcium‐dependent protein kinase), thus resulting in an increased efficacy of SaADF2 and enhanced cellular actin dynamics. SaADF2 overexpression preserved the actin filament organization better in rice protoplasts under desiccation stress. The predicted tertiary structure of SaADF2 showed a longer F‐loop than OsADF2 that could have contributed to higher actin‐binding affinity and rapid F‐actin depolymerization in vitro by SaADF2. Rice transgenics constitutively overexpressing SaADF2 (SaADF2‐OE) showed better growth, relative water content, and photosynthetic and agronomic yield under drought conditions than wild‐type (WT) and OsADF2 overexpressers (OsADF2‐OE). SaADF2‐OE preserved intact grana structure after prolonged drought stress, whereas WT and OsADF2‐OE presented highly damaged and disorganized grana stacking. The possible role of ADF2 in transactivation was hypothesized from the comparative transcriptome analyses, which showed significant differential expression of stress‐related genes including interacting partners of ADF2 in overexpressers. Identification of a complex, differential interactome decorating or regulating stress‐modulated cytoskeleton driven by ADF isoforms will lead us to key pathways that could be potential target for genome engineering to improve abiotic stress tolerance in agricultural crops.

Identification of candidate resistance genes of cotton against Aspergillus flavus infection using a comparative transcriptomics approach

A comparative transcriptome analysis was performed using the genes significantly differentially expressed in cotton, corn and peanut in response to aflatoxin producing fungus Aspergillus flavus with an objective of identifying candidate resistance genes in cotton. Two-way analyses identified 732 unique genes to be differentially regulated by the fungus with only 26 genes common across all three crops that were considered candidate A. flavus resistance genes with an assumption that these genes have specific roles in conferring the resistance trait. Genes of membrane cellular component involved in DNA binding with involvement in defense responses were highly represented among the differentially expressed unique genes. Most (six) of these genes coded for 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily proteins. Genes encoding helix loop helix protein, alcohol dehydrogenase and UDP glycosylation transferase which were upregulated in response to both atoxigenic and toxigenic strains of A. flavus, could be potential resistance candidate genes for downstream functional manipulation to confer resistance.