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Comparison of microRNA populations in SACMV infected tolerant and susceptible cassava landraces

Comparison of microRNA populations in SACMV infected tolerant and susceptible cassava landraces . 9 th Regional Plant Biotechnology Forum . RNA Silencing . Plants live in fluctuant and unpredictable environments Exposed to a large amount of potential stressors

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Comparison of microRNA populations in SACMV infected tolerant and susceptible cassava landraces

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  1. Comparison of microRNA populations in SACMV infected tolerant and susceptible cassava landraces 9th Regional Plant Biotechnology Forum

  2. RNA Silencing Plants live in fluctuant and unpredictable environments Exposed to a large amount of potential stressors Physiological flexibility is a crucial attribute Regulation of gene expression is a key element in remaining adaptive to variable stresses Such regulation can impact all transcriptional levels To facilitate genome integrity – Plants employ RNA silencing Evolutionary conserved gene regulation mechanism Mediated by sRNAs 19-30nt in length

  3. RNA Silencing RNA Silencing TGS PTGS Plants use RNA silencing for three purposes: Creating and maintaining heterochromatin of repetitive DNA and transposons Regulating development, stress response and other endogenous regulatory functions Defending against pathogenic infections Affects gene expression by degradation of mRNAor repression of RNA translation Initiates and maintains the heterochromatic state of certain DNA regions NAT-siRNA ta-siRNA miRNA

  4. MicroRNAs • Second most abundant plant sRNAs • Short non-coding RNAs 18-24nt in length • Originate from ssRNAs transcribed from MIR loci distinct from protein coding regions • MiRNAs play critical roles in diverse aspects of plant development, nutrient acquisition and use, and adaption to biotic and abiotic stresses • Perform their functions by binding to target mRNA and causing cleavage or translational repression

  5. MicroRNAs and Plant Virus infection • Role of miRNAs in antiviral defense remains elusive • Viral infections have been shown to trigger changes in miRNA transcriptomes of several plant species: • Bazzini et al., 2007 • Infection of tobacco plants with TMV, Tomato MV, Tobacco etch virus, PVY and PVX changed the abundance of 10 conserved miRNAs • Tagamiet al., 2007 • TMV-infected Arabidopsis: levels of certain miRNAs increased significantly and 4 novel miRNAs identified • Amin et al., 2011 • Ten developmental miRNA studied in N. benthamiana inoculated with begomoviruses including ACMV • miR156, 160 decreased while miR159, 164, 165, 166, 167, 168 and 169 increased • Singh et al., 2012 • Grapevine infected with Grapevine vein-clearing virus triggered changes to the miRNA profile. miR169 and 398 were downregulated, whereas miR168 and miR3623 were upregulated • Exact contribution to defense mechanisms still remains unknown

  6. Cassava • Cassava (Manihot esculenta Crantz) is a staple food for approximately 700 million people living in developing countries • Grown for its starchy tuberous roots • In the developing world, cassava is amongst the top four most important crops • Also used as a raw material in paper, textile and adhesive production, animal feed, and biofuel production

  7. SACMV • Many pathogens and pests reduce cassava yields, especially in Africa • Cassava mosaic disease (CMD) is the most economically important disease • The casual agents are whitefly-transmitted geminiviruses belonging to the family Geminiviridae, genus Begomovirus • Members of the family Geminiviridae are circular bipartite single-stranded DNA (ssDNA) viruses that infect a wide range of plant species and are responsible for economically devastating diseases • SACMV is a member the Geminiviridae family

  8. Aims • Identify conserved and novel miRNAs in cassava using deep sequencing (NGS) data 2. Determine changes in expression levels in susceptible (T200) and tolerant (TME3) landraces infected with SACMV

  9. Methods 12dpi 32dpi 67dpi T200 susceptible TME3 Tolerant

  10. Results – Conserved miRNAs • TME3 – Tolerant: • Identified 290 individual conserved miRNAs belonging to 40 miRNA families

  11. Results TME3 – Tolerant • 12dpi • All miRNA families had log2 fold changes between 2 and -2

  12. TME3 – Tolerant • 32dpi • 11 miRNA families (27%) had log2 fold changes greater than 2 and less than -2 • 8 upregulation • 3 downregulation

  13. TME3 – Tolerant • 67dpi • 3 miRNA families (7%) had log2 fold change less than -2 (down regulation) • 2 (66%) are known to be involved in Adaptive responses to stress

  14. T200 – susceptible: • Identified 317 individual conserved miRNAs belonging to 42 miRNA families • Two miRNA families present in T200 that were not observed in TME3 • miR1507 and miR482

  15. T200 – Susceptible • 12dpi • 4 (10%) miRNAs had log2 fold changes greater than 2 (upregulation)

  16. T200 – Susceptible • 32dpi • 23 miRNA families (55%) had log2 fold changes greater than 2 and less than -2 • 18 upregulated • 5 downregulated

  17. T200 – Susceptible • Regulation of miRNAs and siRNAs • miR162 – DCL1 • miR168 – AGO1 • miR403 – AGO2 • miR395 - targets R gene transcripts • miR398 - Expression was the most significantly altered (up at 12 and down at 32 dpi) • In previous study in Grapevine infected with Grapevine Vein-clearing virus: • miR168 ; miR169 ; miR398

  18. T200 – Susceptible • 67dpi • 10 miRNA families (23%) had log2fold changes greater than 2 and less than -2 • 6 upregulated • 4 downregulated

  19. Results – Novel miRNAs • 46 novel miRNA Families were identified and named mes-1 to mes-46 • More landrace-specific miRNA families were observed in the novel miRNAs compared to the conserved miRNAs (only 2)

  20. TME3 - Tolerant • 33 miRNA members belonging to 32 miRNA families • 21 landrace specific • 12dpi • 7 (21%) of the miRNAs had log2 fold changes greater than 2 and less than -2 • 2 upregulated • 5 downregulated • 32dpi • 10 (30%) of the miRNAs had log2fold changes greater than 2 and less than -2 • 8 upregulated • 2 downregulated • 67dpi • 6 (18%) of the miRNAs had log2 fold changes greater than 2 and less than -2 • 3 upregulated • 3 downregulated

  21. T200 – susceptible • 27 miRNA members belonging to 26 miRNA families • 15 landrace specific • 12dpi • 5 (18%) of the miRNAs had log2 fold changes greater than 2 and less than -2 • 4 upregulated • 1 downregulated • 32dpi • 3 (11%) of the miRNAs had log2 fold changes greater than 2 • 67dpi • 2 (7%) of the miRNAs had log2 fold changes greater than 2 and less than -2 • 1 upregulated • 1 downregulated

  22. Conclusion • In TME3 40 and T200 42 conserved miRNA families were identified • In T200, compared to TME3, the changes in expression levels were more drastic • TME3 range of expression log2 fold change values: 3.9 to -4.6 • T200 range of expression log2 fold change values: 61.5 to -273.1 (15X and 59X) • Expression of miR398 was the most significantly altered (up at 12 (40)and down at 32 (273) dpi) • 46 Novel miRNA Cassava families were identified in this study • Conserved and Novel landrace specific miRNAs were identified

  23. Future Work • All results need to be confirmed by Real-time PCR and 5’ RACE-PCR • This discovery and characterisation of pathogen-regulated miRNAs may help to elucidate the molecular mechanisms of cassava disease resistace and defense response

  24. Acknowledgements • NRF • Casquip Starch Manufacturing Co. • Cassava Biotech lab members • My supervisor Prof Rey for her guidance and support

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