Jesse Machuka

Interspecific RNA Interference of SHOOT MERISTEMLESS-Like Disrupts Cuscuta pentagona Plant Parasitism

The authors demonstrate that parasite gene-specific silencing signals originating from a transgenic host are transferred into the invading parasite, leading to reduced parasite yield, stature, and infectivity. This article also refreshes the debate on the origin of haustoria as the authors use morphological and molecular evidence to show that haustoria have both stem and root characteristics. Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA–mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

Long-distance transport of mRNA via parenchyma cells and phloem across the host-parasite junction in Cuscuta.

It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes a continuous vascular system through which water and nutrients are drawn. Along with solutes, viruses and proteins, mRNA transcripts are transported from the host to the parasite. The path of the transcripts and their stability in the parasite have yet to be revealed. To discover the route of mRNA transportation, the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to locally amplify host transcript within parasitic tissue. The stability of host mRNA molecules was also checked by monitoring specific transcripts along the growing dodder thread. Four mRNAs, alpha and beta subunits of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells and to the phloem. LePFP transcripts were found in the growing dodder stem up to 30 cm from the tomato-dodder connection. These results suggest that mRNA molecules are transferred from host to parasite via symplastic connections between parenchyma cells, move towards the phloem, and are stable for a long distance in the parasite. This may allow developmental coordination between the parasite and its host.

Striga parasitizes transgenic hairy roots of zea Mays and provides a tool for studying plant-plant interactions

BackgroundStriga species are noxious root hemi-parasitic weeds that debilitate cereal production in sub-Saharan Africa (SSA). Control options for Striga are limited and developing Striga resistant crop germplasm is regarded as the best and most sustainable control measure. Efforts to improve germplasm for Striga resistance by a non-Genetic Modification (GM) approach, for example by exploiting natural resistance, or by a GM approach are constrained by limited information on the biological processes underpinning host-parasite associations. Additionaly, a GM approach is stymied by lack of availability of candidate resistance genes for introduction into hosts and robust transformation methods to validate gene functions. Indeed, a majority of Striga hosts, the world’s most cultivated cereals, are recalcitrant to genetic transformation. In maize, the existing protocols for transformation and regeneration are tedious, lengthy, and highly genotype-specific with low efficiency of transformation.ResultsWe used Agrobacterium rhizogenes strain K599 carrying a reporter gene construct, Green Fluorescent Protein (GFP), to generate transgenic composite maize plants that were challenged with the parasitic plant Striga hermonthica. Eighty five percent of maize plants produced transgenic hairy roots expressing GFP. Consistent with most hairy roots produced in other species, transformed maize roots exhibited a hairy root phenotype, the hallmark of A. rhizogenes mediated transformation. Transgenic hairy roots resulting from A. rhizogenes transformation were readily infected by S. hermonthica. There were no significant differences in the number and size of S. hermonthica individuals recovered from either transgenic or wild type roots.ConclusionsThis rapid, high throughput, transformation technique will advance our understanding of gene function in parasitic plant-host interactions.

Molecular improvement of tropical maize for drought stress tolerance in sub-Saharan Africa

The C4 grass Zea mays (maize or corn) is the third most important food crop globally after wheat and rice in terms of production and the second most widespread genetically modified (GM) crop, after soybean. Its demand is predicted to increase by 45% by the year 2020. In sub-Saharan Africa, tropical maize has traditionally been the main staple of the diet, 95% of the maize grown is consumed directly as human food and as an important source of income for the resource—poor rural population. However, its growth, development and production are greatly affected by environmental stresses such as drought and salinization. In this respect, food security in tropical sub-Saharan Africa is increasingly dependent on continuous improvement of tropical maize through conventional breeding involving improved germplasm, greater input of fertilizers, irrigation, and production of two or more crops per year on the same piece of land. Integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding practices opens tremendous avenues for genetic modifications and fundamental research in tropical maize. The ability to transfer genes into this agronomically important crop might enable improvement of the species with respect to enhanced characteristics, such as enriched nutritional quality, high yield, resistance to herbicides, diseases, viruses, and insects, and tolerance to drought, salt, and flooding. These improvements in tropical maize will ultimately enhance global food production and human health. Molecular approaches to modulate drought stress tolerance are discussed for sub-Saharan Africa, but widely applicable to other tropical genotypes in Central and Latin America. This review highlights abiotic constraints that affect growth, development and production of tropical maize and subsequently focuses on the mechanisms that regulate drought stress tolerance in maize. Biotechnological approaches to manage abiotic stress tolerance in maize will be discussed. The current status of tropical maize transformation using Agrobacterium as a vehicle for DNA transfer is emphasized. This review also addresses the present status of genetically modified organisms (GMOs) regulation in sub-Saharan Africa.

Agricultural genomics and sustainable development: perspectives and prospects for Africa

The genomes of various organisms have now been fully sequenced, including human and representative microbial, insect, animal and plant genomes. The research challenge in the post-genome era is to establish how genes and proteins function to bring about changes in phenotype. Some of these phenotypes, and products obtainable through modern biotechnology, are of crucial importance within the context of sustainable development of African economies. The greatest ultimate impact will be in agricultural genomics, especially for marker assisted selection and breeding programs in crop and animal agriculture, development of animal disease diagnostics and vaccines, crop genetic engineering to overcome abiotic and biotic stresses and for improvement of the nutritional quality of major food staples. It is imperative that African countries become key players in the “gene revolution” since the cost of leaving them behind may be higher than the cost of empowering them to become players in mastering and benefiting from biotechnology. This paper highlights the potential impact of the latest advances in modern biotechnology, including genomics and bioinformatics, on sustainable development, in line with the goals of the New Partnership for Africas Development (NEPAD). These include acceleration of economic growth, eradication of widespread and severe poverty and efforts to halt the marginalization of Africa in the globalization process.

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity.

Regeneration of Sudanese maize inbred lines and open pollinated varieties

Eight maize inbred lines and three open pollinated varieties from Sudan were evaluated for their response to tissue culture. Immature embryos obtained 16 days after pollination were used as explants for callus induction. Calli were induced on LS medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid. Callus induction capacity was highest in inbred lines IL3, IL15 and IL1. The Varieties Hudiba-2 and Hudiba-1 were not statistically different (p >0.05) in callus induction. The capacity for embryogenic callus formation was highest in inbred line IL3 followed by IL1 and IL38 and in varieties Hudiba-2 and Hudiba-1. Inbred lines IL16, IL42, IL43 and IL28 had the lowest embryogenic callus formation capacity. Plant regenerating genotypes were IL3, IL38, IL15, IL1, Hudiba-2 and Mojtamaa-45. Inbred line IL3 was the most regenerable genotype with a shoot formation frequency of 76% averaging 6 shoots per callus. The highest regenerating variety was Mojtamaa-45, which averaged 5 shoots per callus.Characterization of genetic diversity among cultivated cowpea [Vigna unguiculata (L.) Walp.] varieties is important to optimize the use of available genetic resources by farmers, local communities, researchers and breeders. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity in 70 cowpea accessions collected throughout Benin. Nine random primers were screened on 24 accessions to assess their ability to reveal polymorphisms in cowpea and four of them were selected for use in characterizing the total sample. A total of 32 amplified bands were generated by the four primers. The number of loci detected varied from 5 to 11. RAPD profiles were analysed and amplified polymorphic DNA fragments were used to construct a dendrogram, clustering the accessions into nine groups at a similarity index of 71% based on the Unweighted Pair-Group Method using Arithmetic Averages. The genetic diversity among the cowpea cultivars investigated was large and the RAPD proved to be a useful technique to characterise it. Based on the molecular variance, the fixation index suggests a large differentiation of cowpea cultivars in Benin.

A new double right border binary vector for producing marker-free transgenic plants

BackgroundOnce a transgenic plant is developed, the selectable marker gene (SMG) becomes unnecessary in the plant. In fact, the continued presence of the SMG in the transgenic plant may cause unexpected pleiotropic effects as well as environmental or biosafety issues. Several methods for removal of SMGs that have been reported remain inaccessible due to protection by patents, while development of new ones is expensive and cost prohibitive. Here, we describe the development of a new vector for producing marker-free plants by simply adapting an ordinary binary vector to the double right border (DRB) vector design using conventional cloning procedures.FindingsWe developed the DRB vector pMarkfree5.0 by placing the bar gene (representing genes of interest) between two copies of T-DNA right border sequences. The β-glucuronidase (gus) and nptII genes (representing the selectable marker gene) were cloned next followed by one copy of the left border sequence. When tested in a model species (tobacco), this vector system enabled the generation of 55.6% kanamycin-resistant plants by Agrobacterium-mediated transformation. The frequency of cotransformation of the nptII and bar transgenes using the vector was 66.7%. Using the leaf bleach and Basta assays, we confirmed that the nptII and bar transgenes were coexpressed and segregated independently in the transgenic plants. This enable separation of the transgenes in plants cotransformed using pMarkfree5.0.ConclusionsThe results suggest that the DRB system developed here is a practical and effective approach for separation of gene(s) of interest from a SMG and production of SMG-free plants. Therefore this system could be instrumental in production of “clean” plants containing genes of agronomic importance.

Enhanced Utilization of Biotechnology Research and Development Innovations in Eastern and Central Africa for Agro-ecological Intensification

The Association for Strengthening Agricultural Research in Eastern and Central Africa (ASARECA) through its Agrobiodiversity and Biotechnology Programme is enhancing the utilization of biotechnology research and development innovations in Eastern and Central Africa (ECA). We present successes in the application of biotechnology to enhance the productivity of cassava, sweet potato, banana, maize and sorghum in ECA. These products—drought tolerant maize, sorghum resistant to striga, as well as the technology for producing and distributing disease free planting materials of cassava, sweet potato and banana to farmers—are central for the agro-ecological intensification of farming systems in the central African highlands.

Glossina Proteolytic Lectin Does Not Require a Carbohydrate Moiety for Enzymatic or Trypanosome-Transforming Activities

Abstract The developmental cycle of the cyclically transmitted African trypanosome involves an obligatory passage through the tsetse fly, Glossina spp. This intricate relationship requires the presence of molecules within the insect vector, including a midgut lectin, that interact with the trypanosome. Recently, a gene encoding for a proteolytic lectin, with trypanosome-transforming activity, was isolated from a midgut cDNA library of Glossina fuscipes fuscipes Austen in our laboratory. Using the same approach, we have identified a similar gene from a midgut cDNA library of Glossina austeni (Newstead). The protein encoded by this gene was expressed in bacteria and a baculovirus-based expression system. The baculovirus-expressed lectin was found in the medium of baculovirus-infected Sf-21 cell cultures, indicating that the tsetse fly-derived signal peptide was recognized and cleaved by the Sf-21 cells. The baculovirus-expressed protein also was glycosylated despite the absence of classical O-linked and N-linked sugar attachment motifs. Both the baculovirus- and bacterium-expressed lectin proteins were shown to agglutinate trypanosomes and rabbit red blood cells in vitro. This agglutination was strongly inhibited by d-glucosamine. d-Glucosamine also inhibited the action of the authentic and recombinant lectins upon the chromogenic substrate Chromozym TRY. Interestingly, both baculovirus- and bacterium-expressed lectins showed no significant differences in terms of these activities, indicating that a sugar moiety is not essential for biological activity. Our results provide an important molecular tool for further characterization of Glossina proteolytic lectin.