Application of Integrated Computational Approaches in Prediction of Plant Virus Encoded miRNAs and Their Targeted Plant Genes.

This chapter presents a comprehensive approach to predict novel miRNAs encoded by plant viruses and identify their target plant genes, through integration of various ab initio computational approaches. The predictive process begins with the analysis of plant viral sequences using the VMir Analyzer software. VMir Viewer software is then used to extract primary hairpins from these sequences. To distinguish real miRNA precursors from pseudo miRNA precursors, MiPred web-based software is employed. Verified real pre-miRNA sequences with a minimum free energy of < -20 Kcal/mol, are further analyzed using the RNAshapes software. Validation of predictions involves comparing them with available Expressed Sequence Tags (ESTs) from the relevant plant using BlastN. Short sequences with lengths ranging from 19 to 25 nucleotides and exhibiting <5 mismatches are prioritized for miRNA prediction. The precise locations of these short sequences within pre-miRNA structures generated using RNAshapes are meticulously identified, with a focus on those situated on the 5' and 3' arms of the structures, indicating potential miRNAs. Sequences within the arms of pre-miRNA structures are used to predict target sites within the ESTs of the specific plant, facilitated by psRNA Target software, revealing genes with potential regulatory roles in the plant. To confirm the outcome of target prediction, results are individually submitted to the RNAhybrid web-based software. For practical demonstration, this approach is applied to analyze African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda (EACMV-UG) viruses, as well as the ESTs of Jatropha and cassava.

Phenotypic and Genotypic Methods for the Identification and Quantification of Cyanobacteria in Lake Water.

Early monitoring of Microcystis, a cyanobacterium that produces microcystin, is paramount in order to confirm the presence of Microcystis spp. Both phenotypic and genotypic methods have been used. The phenotypic methods provide the presence of the microcystis but do not confirm its species type and toxin produced. Additionally, phenotypic methods cannot differentiate toxigenic from non-toxigenic Microcystis. Therefore, the current protocol also describes genetic methods based on PCR to detect toxigenic Microcystis spp. based on microcystin synthetase E (mcy E) gene and 16-23S RNA genes for species-specific identification, which can effectively comprehend distinct lineages and discrimination of potential complexity of microcystin populations. The presence of these microcystin toxins in blood, in most cases, indicates contamination of drinking water by cyanobacteria. The methods presented herein are used to identify microcystin toxins in drinking water and blood.

Medicinal Plants Used in Traditional Management of Cancer in Uganda: A Review of Ethnobotanical Surveys, Phytochemistry, and Anticancer Studies.

The burden of neoplastic diseases is a significant global health challenge accounting for thousands of deaths. In Uganda, about 32,617 cancer cases were reported in 2018, accompanied by 21,829 deaths. In a view to identify some potential anticancer plant candidates for possible drug development, the current study was designed to compile the inventory of plants with reported anticancer activity used in rural Uganda and the evidences supporting their use in cancer therapy. An electronic survey in multidisciplinary databases revealed that 29 plant species belonging to 28 genera distributed among 24 families have been reported to be used in the management of cancer in Uganda. Anticancer plants were majorly from the families Bignoniaceae (7%), Caricaceae (7%), Fabaceae (7%), Moraceae (7%), and Rutaceae (7%). Most species occur in the wild (52%), though some are cultivated (48%). The growth habit of the plants is as trees (55%) or herbs (45%). Anticancer extracts are usually prepared from leaves (29%), bark (24%), roots (21%), and fruits (13%) through decoctions (53%), as food spices (23%) or pounded to produce ointments that are applied topically (10%). Prunus africana (Hook.f.) Kalkman, Opuntia species, Albizia coriaria (Welw. ex Oliver), Daucus carota L., Cyperus alatus (Nees) F. Muell., Markhamia lutea (Benth.) K. Schum., and Oxalis corniculata L. were the most frequently encountered species. As per global reports, Allium sativum L., Annona muricata L., Carica papaya L., Moringa oleifera Lam., Opuntia species, Prunus africana (Hook.f.) Kalkman, and Catharanthus roseus (L.) G. Don. are the most studied species, with the latter having vincristine and vinblastine anticancer drugs developed from it. Prostate, cervical, breast, and skin cancers are the top traditionally treated malignancies. There is a need to isolate and evaluate the anticancer potential of the bioactive compounds in the unstudied claimed plants, such as Cyperus alatus (Nees) F. Muell., Ficus dawei Hutch., Ficus natalensis Hochst., and Lovoa trichilioides Harms, and elucidate their mechanism of anticancer activity.

Correction: Virus versus Host Plant MicroRNAs: Who Determines the Outcome of the Interaction?

[This corrects the article DOI: 10.1371/journal.pone.0098263.].

Virus versus host plant microRNAs: who determines the outcome of the interaction?

Considering the importance of microRNAs (miRNAs) in the regulation of essential processes in plant pathogen interactions, it is not surprising that, while plant miRNA sequences counteract viral attack via antiviral RNA silencing, viruses in turn have developed antihost defense mechanisms blocking these RNA silencing pathways and establish a counter-defense. In the current study, computational and stem-loop Reverse Transcription - Polymerase Chain Reaction (RT-PCR) approaches were employed to a) predict and validate virus encoded mature miRNAs (miRs) in 39 DNA-A sequences of the bipartite genomes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus-Uganda (EACMV-UG) isolates, b) determine whether virus encoded miRs/miRs* generated from the 5'/3' harpin arms have the capacity to bind to genomic sequences of the host plants Jatropha or cassava and c) investigate whether plant encoded miR/miR* sequences have the potential to bind to the viral genomes. Different viral pre-miRNA hairpin sequences and viral miR/miR* length variants occurring as isomiRs were predicted in both viruses. These miRNAs were located in three Open Reading Frames (ORFs) and in the Intergenic Region (IR). Moreover, various target genes for miRNAs from both viruses were predicted and annotated in the host plant genomes indicating that they are involved in biotic response, metabolic pathways and transcription factors. Plant miRs/miRs* from conserved and highly expressed families were identified, which were shown to have potential targets in the genome of both begomoviruses, representing potential plant miRNAs mediating antiviral defense. This is the first assessment of predicted viral miRs/miRs* of ACMV and EACMV-UG and host plant miRNAs, providing a reference point for miRNA identification in pathogens and their hosts. These findings will improve the understanding of host- pathogen interaction pathways and the function of viral miRNAs in Euphorbiaceous crop plants.

Biotechnological approaches to determine the impact of viruses in the energy crop plant Jatropha curcas.

BACKGROUND: Geminiviruses infect a wide range of plant species including Jatropha and cassava both belonging to family Euphorbiaceae. Cassava is traditionally an important food crop in Sub - Saharan countries, while Jatropha is considered as valuable biofuel plant with great perspectives in the future. RESULTS: A total of 127 Jatropha samples from Ethiopia and Kenya and 124 cassava samples from Kenya were tested by Enzyme-Linked Immunosorbent Assay (ELISA) for RNA viruses and polymerase chain reaction for geminiviruses. Jatropha samples from 4 different districts in Kenya and Ethiopia (analyzed by ELISA) were negative for all three RNA viruses tested: Cassava brown streak virus (CBSV), Cassava common mosaic virus, Cucumber mosaic virus, Three cassava samples from Busia district (Kenya) contained CBSV. Efforts to develop diagnostic approaches allowing reliable pathogen detection in Jatropha, involved the amplification and sequencing of the entire DNA A molecules of 40 Kenyan isolates belonging to African cassava mosaic virus (ACMV) and East African cassava mosaic virus - Uganda. This information enabled the design of novel primers to address different questions: a) primers amplifying longer sequences led to a phylogenetic tree of isolates, allowing some predictions on the evolutionary aspects of Begomoviruses in Jatrophia; b) primers amplifying shorter sequences represent a reliable diagnostic tool. This is the first report of the two Begomoviruses in J. curcas. Two cassava samples were co - infected with cassava mosaic geminivirus and CBSV. A Defective DNA A of ACMV was found for the first time in Jatropha. CONCLUSION: Cassava geminiviruses occurring in Jatropha might be spread wider than anticipated. If not taken care of, this virus infection might negatively impact large scale plantations for biofuel production. Being hosts for similar pathogens, the planting vicinity of the two crop plants needs to be handled carefully.