Occurrence of African cassava mosaic virus (ACMV) and East African cassava mosaic virus – Uganda (EACMV-UG) in Jatropha curcas

Poster presentation from IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery Arraial dAjuda, Bahia, Brazil. 26 June - 2 July 2011(VLID)90654

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

<p>Abstract</p> <p>Background</p> <p>Geminiviruses infect a wide range of plant species including <it>Jatropha </it>and cassava both belonging to family <it>Euphorbiaceae</it>. Cassava is traditionally an important food crop in Sub - Saharan countries, while <it>Jatropha </it>is considered as valuable biofuel plant with great perspectives in the future.</p> <p>Results</p> <p>A total of 127 <it>Jatropha </it>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. <it>Jatropha </it>samples from 4 different districts in Kenya and Ethiopia (analyzed by ELISA) were negative for all three RNA viruses tested: <it>Cassava brown streak virus </it>(CBSV), <it>Cassava common mosaic virus</it>, <it>Cucumber mosaic virus</it>, 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 <it>African cassava mosaic virus </it>(ACMV) and <it>East African cassava mosaic virus </it>- <it>Uganda</it>. 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 <it>Jatrophia</it>; b) primers amplifying shorter sequences represent a reliable diagnostic tool. This is the first report of the two Begomoviruses in <it>J. curcas</it>. 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 <it>Jatropha</it>.</p> <p>Conclusion</p> <p>Cassava geminiviruses occurring in <it>Jatropha </it>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.</p

Investigation of genetic variation in Jatropha curcas by Ecotilling and ISSR

The ability of species to adapt to different environments resides in their genetic diversity. This diversity, most commonly manifested as Single Nucleotide Polymorphisms (SNPs), can provide clues to the adaptive processes and population histories that have played a role in the species ’ evolution. A number of different techniques for identifying SNPs have been developed, all having their limitations. Reverse genetics approaches rely on the detection of sequence alterations in target genes to identify allelic variations in natural or mutant populations. Ecotilling, a variant of TILLING (Targeting Induced Local Lesions IN Genomes) technique, allows high-throughput analyses of natural genetic diversity in plants [1], particularly in species with limited genetic diversity. Jatropha curcas L. is a perennial, monoecious shrub of the Euphorbiaceae family, native to America but distributed widely in the tropical and subtropical areas [2]. Wild or semi-cultivated types of J. curcas can grow well under unfavourable climatic and soil conditions [3]. J. curcas has attracted a great deal of attention worldwide, regarding its potential as a new energy plant. The seeds of J. curcas contain 30-45 % oil [4] with a high percentage of monounsaturated oleic and polyunsaturated linoleic acid [5]. For genomic analyses, J. curcas is an interesting model species, since it has a relatively small genome (2C DNA content of 0.850 ± 0.006 pg or C DNA content of 0.416 × 109 bp) [6]. However, to achieve specific breeding goals in Jatropha for wider ecological adaptation, disease resistance and novel seed quality, the use of germplasm from different group and regions is necessary. Understandin

Determinants of dietary diversity among women of reproductive age in two different agro-ecological zones of Rongai Sub-County, Nakuru, Kenya

This study demonstrated that the diets of women were of poor quality despite the differences in agro-ecological zones and availability of food from the farm. Education level influenced women’s dietary diversity positively in low agricultural potential areas while household gender, education level, age and family size were important determinants in high potential areas. Nutrition interventions focusing on improving dietary quality of women should therefore pay special attention to developing region-specific interventions instead of generalizing interventions

Biotechnologische Ans\ue4tze zur z\ufcchterischen und phytosanit\ue4ren Verbesserung von Euphorbiaceen

Die Familie Euphorbiaceae ist von gro fer Bedeutung, da sie wirtschaftlich wertvolle Pflanzenarten wie Maniok, der vor allem als Grundnahrungsmittel zur Ern\ue4hrungssicherheit von Millionen in Asien und Afrika verwendet wird, und Jatropha enth\ue4lt. Jatropha wird haupts\ue4chlich als Bio-Treibstoff und Arzneimittel verwendet, und bietet die M\uf6glichkeit zur R\ufcckgewinnung von Ruderalb\uf6den, zur Reduzierung der Risiken von Erosion und W\ufcstenbildung. Jatropha und Maniok stehen vor Herausforderungen durch biotische und abiotische Stressfaktoren. In dieser Arbeit wurden verschiedene Strategien eingesetzt, die zur Verbesserung der Euphorbiaceae beitragen k\uf6nnen: 1) Die Untersuchung der genetischen Variation in Jatropha durch ISSR und EcoTILLING zeigte Nukleotid-Polymorphismen auf und lieferten Hinweise auf adaptive Prozesse und die Populationsgeschichte. 2) Die Verwendung von molekularen Nachweismethoden f\ufchrte zur Identifikation von Viren in Jatropha und Maniok. Verbesserte diagnostische Ans\ue4tze erlaubten die Sequenzierung des gesamten DNA-Molek\ufcle A von 40 kenianischen Isolate von African cassava mosaic virus (ACMV) und East African cassava mosaic virus Uganda (EACMV-UG). 3) Der in silico Ansatz wurde angewendet, um miRNA die von ACMV und EACMV-UG kodiert werden, zu identifizieren. Au ferdem wurden miRNAs aus Pflanzen vorhergesagt, die die F\ue4higkeit besitzen, an DNA- A Genome von ACMV und EACMV UG zu binden. Dies f\ufchrte zur Identifizierung von miRNA in viralen ORFs, einschlie flich der AC2 und AC4, die als Unterdr\ufccker von RNA-Silencing und \u201ePathogenesis-related Proteins\u201c bekannt sind. Mapping ihrer Zielsequenzen auf ESTs aus Jatropha und Maniok zeigten, dass einige dieser Gene an Stoffwechselwegen zur Pathogenabwehr beteiligt sind. Dar\ufcber hinaus wurden Pflanzen miRNAs, die ORFs sowohl ACMV und EACMV-UG-Genom adressieren, identifiziert.The family Euphorbiaceae is of significant importance as it comprises economically valuable crop species such as cassava, which is mainly used as a staple food and serves for food security of millions in Asia and Africa, and Jatropha, which serves mainly as a bio-fuel and pharmaceutical crop, with possibility of reclaiming marginal soils, reducing risks of erosion and desertification. Jatropha and cassava face challenges of both biotic and abiotic stresses. In this work, different strategies which may contribute towards the improvement of Euphorbiaceae were employed: 1) The investigation of the genetic variation in Jatropha through ISSR and Ecotilling shed light on the nucleotide polymorphisms and provided clues to adaptive processes and populations history. 2) The use of different molecular detection techniques led to the identification of viruses infecting Jatropha and cassava. Improved diagnostic approaches allowed the detection of pathogens in Jatropha 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 (EACMV-UG). 3) The in silico approach was applied to identify miRNA encoded by ACMV and EACMV-UG. Furthermore, plant miRNA with ability to bind ACMV and EACMV-UG DNA-A genomes were predicted. This led to identification of virus miRNA encoded in ORFs including the AC2 and AC4 which are suppressors of RNA silencing and pathogenesis related proteins. Mapping of their targets on the Jatropha and cassava ESTs showed that some of these targets are pathways involved in plant defense. In addition, plant miRNAs that could target the ORFs of both ACMV and EACMV-UG were identified.submitted by Rose Chepchirchir RamkatAbweichender Titel laut cbersetzung der Verfasserin/des VerfassersWien, Univ. f\ufcr Bodenkultur, Diss., 201

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

<div><p>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 <i>African cassava mosaic virus</i> (ACMV) and <i>East African cassava mosaic virus</i>-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 <i>Jatropha</i> 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 <i>Euphorbiaceous</i> crop plants.</p></div

The novel virus miR/miR* sequences predicted from ACMV and EACMV-UG real pre-miRNA hairpins.

<p>The miR/miRs* were predicted using <i>Jatropha</i> and cassava sequence hits from BlastN. The length, location on 5′ or 3′ arms of hairpins and sequence of the nucleotides 2–8 at the 5′ end (highlighted in bold) using RNAShape are shown.</p

Outline of the strategy to identify plant miRNAs from miRBase that potentially target regions of DNA-A in ACMV and EACMV-UG.

<p>Computational approaches were identified plant miRs/miRs* with potential to target viral genomic regions.</p

Nucleotide content of 7 real ACMV and 2 real EACMV-UG miRNA hairpins.

<p>Nucleotide content of 7 real ACMV and 2 real EACMV-UG miRNA hairpins.</p

End point PCR amplification of plant miRNAs on cassava and <i>Jatropha</i>.

<p>PCR products of 60-infected cassava plant samples, respectively: S4C4, S2C6, S4C6, B2C15, –RT control. Lanes 6–7 are one infected and one non-infected <i>Jatropha</i> plant samples, respectively: K5J5, S4J12. <i>Actin</i> (76 bp) was used as internal control.</p