Steven Runo

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.

Praziquantel sensitivity of Kenyan Schistosoma mansoni isolates and the generation of a laboratory strain with reduced susceptibility to the drug

Schistosomiasis is a neglected tropical disease caused by blood-dwelling flukes of the genus Schistosoma. While the disease may affect as many as 249 million people, treatment largely relies on a single drug, praziquantel. The near exclusive use of this drug for such a prevalent disease has led to concerns regarding the potential for drug resistance to arise and the effect this would have on affected populations. In this study, we use an in vitro assay of drug sensitivity to test the effect of praziquantel on miracidia hatched from eggs obtained from fecal samples of Kenyan adult car washers and sand harvesters as well as school children. Whereas in a previous study we found the car washers and sand harvesters to harbor Schistosoma mansoni with reduced praziquantel sensitivity, we found no evidence for the presence of such strains in any of the groups tested here. Using miracidia derived from seven car washers to infect snails, we used the shed cercariae to establish a strain of S. mansoni with significantly reduced praziquantel sensitivity in mice. This was achieved within 5 generations by administering increasing doses of praziquantel to the infected mice until the parasites could withstand a normally lethal dose. This result indicates that while the threat of praziquantel resistance may have diminished in the Kenyan populations tested here, there is a strong likelihood it could return if sufficient praziquantel pressure is applied.

Engineering host-derived resistance against plant parasites through RNA interference: Challenges and opportunities

RNA interference (RNAi) has rapidly advanced to become a powerful genetic tool and holds promise to revolutionizing agriculture by providing a strategy for controlling a wide array of crop pests. Numerous studies document RNAi efficacy in achieving silencing in viruses, insects, nematodes and weeds parasitizing crops. In general, host derived pest resistance through RNAi is achieved by genetically transforming host plants with double stranded RNA constructs targeted at essential parasite genes leading to generation of small interfering RNAs (siRNAs). Small interfering RNAs formed in the host are then delivered to the parasite and transported to target cells. Delivery can be oral – worms and insects, viral infections, viruses – or through a vascular connections – parasitic plants, while delivery to target cells is by cell to cell systemic movement of the silencing signal. Despite the overall optimism in generating pest resistant crops through RNAi-mediated silencing, some hurdles have recently begun to emerge. Presently, the main challenge is delivery of sufficient siRNAs, in the right cells, and at the right time to mount; a strong, durable, and broad-spectrum posttranscriptional gene silencing (PTGS) signal. This review highlights the novel strategies available for improving host derived RNAi resistance in downstream applied agriculture.

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.

Novel Sources of Witchweed (Striga) Resistance from Wild Sorghum Accessions

Sorghum is a major food staple in sub-Saharan Africa (SSA), but its production is constrained by the parasitic plant Striga that attaches to the roots of many cereals crops and causes severe stunting and loss of yield. Away from cultivated farmland, wild sorghum accessions grow as weedy plants and have shown remarkable immunity to Striga. We sought to determine the extent of the resistance to Striga in wild sorghum plants. Our screening strategy involved controlled laboratory assays of rhizotrons, where we artificially infected sorghum with Striga, as well as field experiments at three sites, where we grew sorghum with a natural Striga infestation. We tested the resistance response of seven accessions of wild sorghum of the aethiopicum, drummondii, and arundinaceum races against N13, which is a cultivated Striga resistant landrace. The susceptible control was farmer-preferred variety, Ochuti. From the laboratory experiments, we found three wild sorghum accessions (WSA-1, WSE-1, and WSA-2) that had significantly higher resistance than N13. These accessions had the lowest Striga biomass and the fewest and smallest Striga attached to them. Further microscopic and histological analysis of attached Striga haustorium showed that wild sorghum accessions hindered the ingression of Striga haustorium into the host endodermis. In one of the resistant accessions (WSE-1), host and parasite interaction led to the accumulation of large amounts of secondary metabolites that formed a dark coloration at the interphase. Field experiments confirmed the laboratory screening experiments in that these same accessions were found to have resistance against Striga. In the field, wild sorghum had low Area under the Striga Number Progressive curve (AUSNPC), which measures emergence of Striga from a host over time. We concluded that wild sorghum accessions are an important reservoir for Striga resistance that could be used to expand the genetic basis of cultivated sorghum for resistance to the parasite.

Regenerability of elite tropical maize ( Zea mays L.) inbred lines using immature zygotic embryo explants

Five elite tropical maize inbred lines; CML395, CML443, CML442, MAS [MSR/312]-117-2-2-1-B-5-B) and CML216 as a control, were evaluated for their regenerability making use of calli derived from immature zygotic embryos. Murashige and Skoog basal salts supplemented with 1.0, 1.5, 2.0 and 2.5 mg/L 2,4dichlorophenoxyacetic acid were used to induce callus. Callus induction frequency and formation of embryogenic callus varied significantly (p<0.01) depending on genotype and level of 2,4dichlorophenoxyacetic acid. Shoot regeneration efficiency also differed significantly (p<0.01) depending on genotype. Significantly (p<0.05) higher callus induction and frequency of embryogenic callus were obtained at 1 mg/L 2,4-dichlorophenoxyacetic acid, indicating this as the optimal level for regenerating these inbred lines. CML395 and CML442 revealed significantly (p<0.05) higher callus induction and embryogenic callus frequency compared to CML443 and MAS [MSR/312]-117-2-2-1-B-5B), while they were at par with the control inbred line CML216. Plants were regenerated from all the inbred lines except CML443 and were successfully acclimatized and grown to maturity. CML395 was the best regenerable line with significantly (p<0.05) higher regeneration efficiency of 109.3%. It was concluded that CML395, CML216 and CML442 can be used in in vitro genetic transformation.

Drought tolerant tropical maize ( Zea mays L.) developed through genetic transformation with isopentenyltransferase gene

Maize is a staple food crop for millions of Africans. Despite this fact, African farmers have been harvesting average grain yield of not more than 2 t/ha while there is a potential of producing more than 10 t/ha. Drought is one of the major abiotic constraints contributing to this low productivity. Drought diminishes crop productivity mainly by causing premature leaf senescence. The ipt gene codes for isopentenyltransferase (IPT) enzyme which catalyzes the rate limiting step in the biosynthesis of cytokinin and has been shown to enhance tolerance to drought in transgenic crops by delaying drought-induced leaf senescence. This created interest to investigate if ipt gene can be useful in enhancing drought tolerance in locally adapted African tropical maize genotypes. The tropical maize inbred line CML216 was transformed with ipt gene using Agrobacterium-mediated transformation method. Five transgenic lines which were proved to be stably transformed through Southern blot analysis with copy number of 2 to 4 per event were developed. In drought assay carried out in the glass house, transgenic lines expressing the ipt gene showed tolerance to drought as revealed by delayed leaf senescence compared to the wild type plants. Transgenic plants maintained higher relative water content and total chlorophyll during the drought period and produced significantly higher mean grain yield of 44.3 g/plant while the wild type plants produced mean grain yield of 1.43 g/plant. It is proposed that the transgenic lines developed in this study can be further tested for tolerance to drought under contained field trials. Furthermore, transgenic lines developed can be used in breeding programs to improve drought tolerance in other commercial tropical maize genotypes through conventional breeding. Keywords: Cytokinin, delayed leaf senescence, drought inducible, CML216, ipt gene

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.