In Silico Identification of Cassava Genome-Encoded MicroRNAs with Predicted Potential for Targeting the ICMV-Kerala Begomoviral Pathogen of Cassava.

Cassava mosaic disease (CMD) is caused by several divergent species belonging to the genus Begomovirus (Geminiviridae) transmitted by the whitefly Bemisia tabaci cryptic species group. In India and other parts of Asia, the Indian cassava mosaic virus-Kerala (ICMV-Ker) is an emergent begomovirus of cassava causing damage that results in reduced yield loss and tuber quality. Double-stranded RNA-mediated interference (RNAi) is an evolutionary conserved mechanism in eukaryotes and highly effective, innate defense system to inhibit plant viral replication and/or translation. The objective of this study was to identify and characterize cassava genome-encoded microRNAs (mes-miRNA) that are predicted to target ICMV-Ker ssDNA-encoded mRNAs, based on four in silico algorithms: miRanda, RNA22, Tapirhybrid, and psRNA. The goal is to deploy the predicted miRNAs to trigger RNAi and develop cassava plants with resistance to ICMV-Ker. Experimentally validated mature cassava miRNA sequences (n = 175) were downloaded from the miRBase biological database and aligned with the ICMV-Ker genome. The miRNAs were evaluated for base-pairing with the cassava miRNA seed regions and to complementary binding sites within target viral mRNAs. Among the 175 locus-derived mes-miRNAs evaluated, one cassava miRNA homolog, mes-miR1446a, was identified to have a predicted miRNA target binding site, at position 2053 of the ICMV-Ker genome. To predict whether the cassava miRNA might bind predicted ICMV-Ker mRNA target(s) that could disrupt viral infection of cassava plants, a cassava locus-derived miRNA-mRNA regulatory network was constructed using Circos software. The in silico-predicted cassava locus-derived mes-miRNA-mRNA network corroborated interactions between cassava mature miRNAs and the ICMV-Ker genome that warrant in vivo analysis, which could lead to the development of ICMV-Ker resistant cassava plants.

In silico identification of sugarcane (Saccharum officinarum L.) genome encoded microRNAs targeting sugarcane bacilliform virus.

Sugarcane bacilliform virus (SCBV) is considered one of the most economically damaging pathogens for sugarcane production worldwide. Three open reading frames (ORFs) are characterized in the circular, ds-DNA genome of the SCBV; these encode for a hypothetical protein (ORF1), a DNA binding protein (ORF2), and a polyprotein (ORF3). A comprehensive evaluation of sugarcane (Saccharum officinarum L.) miRNAs for the silencing of the SCBV genome using in silico algorithms were carried out in the present study using mature sugarcane miRNAs. miRNAs of sugarcane are retrieved from the miRBase database and assessed in terms of hybridization with the SCBV genome. A total of 14 potential candidate miRNAs from sugarcane were screened out by all used algorithms used for the silencing of SCBV. The consensus of three algorithms predicted the hybridization site of sof-miR159e at common locus 5534. miRNA-mRNA interactions were estimated by computing the free-energy of the miRNA-mRNA duplex using the RNAcofold algorithm. A regulatory network of predicted candidate miRNAs of sugarcane with SCBV-ORFs, generated using Circos-is used to identify novel targets. The predicted data provide useful information for the development of SCBV-resistant sugarcane plants.

A novel computational approach to the silencing of Sugarcane Bacilliform Guadeloupe A Virus determines potential host-derived MicroRNAs in sugarcane (Saccharum officinarum L.).

Sugarcane Bacilliform Guadeloupe A Virus (SCBGAV, genus Badnavirus, family Caulimoviridae) is an emerging, deleterious pathogen of sugarcane which presents a substantial barrier to producing high sugarcane earnings. Sugarcane bacilliform viruses (SCBVs) are one of the main species that infect sugarcane. During the last 30 years, significant genetic changes in SCBV strains have been observed with a high risk of disease incidence associated with crop damage. SCBV infection may lead to significant losses in biomass production in susceptible sugarcane cultivars. The circular, double-stranded (ds) DNA genome of SCBGAV (7.4 Kb) is composed of three open reading frames (ORFs) on the positive strand that replicate by a reverse transcriptase. SCBGAV can infect sugarcane in a semipersistent manner via the insect vectors sugarcane mealybug species. In the current study, we used miRNA target prediction algorithms to identify and comprehensively analyze the genome-wide sugarcane (Saccharum officinarum L.)-encoded microRNA (miRNA) targets against the SCBGAV. Mature miRNA target sequences were retrieved from the miRBase (miRNA database) and were further analyzed for hybridization to the SCBGAV genome. Multiple computational approaches-including miRNA-target seed pairing, multiple target positions, minimum free energy, target site accessibility, maximum complementarity, pattern recognition and minimum folding energy for attachments-were considered by all algorithms. Among them, sof-miR396 was identified as the top effective candidate, capable of targeting the vital ORF3 of the SCBGAV genome. miRanda, RNA22 and RNAhybrid algorithms predicted hybridization of sof-miR396 at common locus position 3394. The predicted sugarcane miRNAs against viral mRNA targets possess antiviral activities, leading to translational inhibition by mRNA cleavage. Interaction network of sugarcane-encoded miRNAs with SCBGAV genes, created using Circos, allow analyze new targets. The finding of the present study acts as a first step towards the creation of SCBGAV-resistant sugarcane through the expression of the identified miRNAs.

Identification and characterization of miRNA169 family members in banana (Musa acuminata L.) that respond to fusarium oxysporum f. sp. cubense infection in banana cultivars.

MicroRNAs (miRNAs) play an important role in plant resistance to pathogen infections. However, little is known about the role of miRNAs in banana Fusarium wilt, which is the most economically devastating disease in banana production. In the present study, we identified and characterized a total of 18 miR169 family members in banana (Musa acuminata L.) based on small RNA sequencing. The banana miR169 family clustered into two groups based on miRNA evolutionary analysis. Multiple sequence alignment indicated a high degree of sequence conservation in miRNA169 family members across 28 plant species. Computational target prediction algorithms were used to identify 25 targets of miR169 family members in banana. These targets were enriched in various metabolic pathways that include the following molecules: glycine, serine, threonine, pentose, glycerolipids, nucleotide sugars, starch, and sucrose. Through miRNA transcriptomic analysis, we found that ma-miR169a and ma-miR169b displayed high expression levels, whereas the other 16 ma-miR169 members exhibited low expression in the HG and Baxi banana cultivars. Further experiments indicate that there were negative relationships between ma-miR169a, ma-miR169b and their targets basing on their expression levels to Foc4 (Fusarium oxysporum f. sp. cubense tropical race 4) infection in resistant cultivars. But they were low expressed in susceptive cultivars. These results suggested that the expression levels of ma-miR169a and ma-miR169b were consistent with the resistance degree of the banana cultivars to Foc4. The analysis presented here constitutes a starting point to understand ma-miR169-mediated Fusarium wilt resistance at the transcriptional level in banana and predicts possible candidate targets for the genetic improvement of banana resistance to Foc4.

Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs.

Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly 'gut' genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or 'gut' of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit α, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalase1, and Trehalose transporter1. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit α and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.

Herbicide-resistant cotton (Gossypium hirsutum) plants: an alternative way of manual weed removal.

BACKGROUND: Cotton yield has been badly affected by different insects and weed competition. In Past Application of multiple chemicals is required to manage insects and weed control was achieved by different conventional means, such as hand weeding, crop rotation and polyculture, because no synthetic chemicals were available. The control methods shifted towards high input and target-oriented methods after the discovery of synthetic herbicide in the 1930s. To utilise the transgenic approach, cotton plants expressing the codon-optimised CEMB GTGene were produced in the present study. RESULTS: Local cotton variety CEMB-02 containing Cry1Ac and Cry2A in single cassette was transformed by synthetic codon-optimised 5-enolpyruvylshikimate-3-phosphate synthase gene cloned into pCAMBIA 1301 vector under 35S promoter with Agrobacterium tumifaciens. Putative transgenic plants were screened in MS medium containing 120 µmol/L glyphosate. Integration and expression of the gene were evaluated by PCR from genomic DNA and ELISA from protein. A 1.4-kb PCR product for Glyphosate and 167-bp product for Cry2A were obtained by amplification through gene specific primers. Expression level of Glyphosate and Bt proteins in two transgenic lines were recorded to be 0.362, 0.325 µg/g leaf and 0.390, 0.300 µg/g leaf respectively. FISH analysis of transgenic lines demonstrates the presence of one and two copy no. of Cp4 EPSPS transgene respectively. Efficacy of the transgene Cp4 EPSPS was further evaluated by Glyphosate spray (41 %) assay at 1900 ml/acre and insect bioassay which shows 100 %mortality of insect feeding on transgenic lines as compared to control. CONCLUSION: The present study shows that the transgenic lines produced in this study were resistant not only to insects but also equally good against 1900 ml/acre field spray concentration of glyphosate.

Functional characterization of a bidirectional plant promoter from cotton leaf curl Burewala virus using an Agrobacterium-mediated transient assay.

The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional gene promoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant.

Cloning and chloroplast-targeted expression studies of insect-resistant gene with ricin fusion-gene under chloroplast transit peptide in cotton

Background: Transgenic plants inhabiting single Bt gene are prone to develop insect resistance and this resistance has been reported in case of some important yield-devastating insect larvae of commercial crops, such as cotton and rice. Therefore, it has become essential to adapt new strategies to overcome the problem of insect resistance and these new strategies should be sophisticated enough to target such resistant larvae in broad spectrum. Among these, plants may be transformed with Bt gene tagged with some fusion-protein gene that possesses lectin-binding capability to boost the binding sites for crystal protein gene within insect mid-gut in order to overcome any chances of insect tolerance against Bt toxin. Enhanced chloroplast-targeted Bt gene expression can also help in the reduction of insect resistance. Results: In the present investigation, a combined effect of both these strategies was successfully used in cotton (G. hirsutum). For this purpose, plant expression vector pKian-1 was created, after a series of cloning steps, carrying Cry1Ac gene ligated with chloroplast transit peptide towards N-terminal and Ricin B-Chain towards C-terminal, generating TP-Cry1Ac-RB construct. Conclusions: Efficacy of pKian-1 plasmid vector was confirmed by in-planta Agrobacterium-mediated leaf GUS assay in tobacco. Cotton (G. hirsutum) local variety MNH-786 was transformed with pKian-1 and the stable integration of TP-Cry1Ac-RB construct in putative transgenic plants was confirmed by PCR; while fusion-protein expression in cytosol as well as chloroplast was substantiated by Western blot analysis. Whereas, confocal microscopy of leaf-sections of transgenic plants exposed that hybrid-Bt protein was expressing inside chloroplasts.