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 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

RNA Silencing TGS PTGS Initiates and maintains the heterochromatic state of certain DNA regions Affects gene expression by degradation of mRNA or repression of RNA translation miRNA ta-siRNA NAT- siRNA Plants use RNA silencing for three purposes: 1.Creating and maintaining heterochromatin of repetitive DNA and transposons 2.Regulating development, stress response and other endogenous regulatory functions 3.Defending against pathogenic infections RNA Silencing

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 MicroRNAs

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 –Tagami et 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 MicroRNAs and Plant Virus infection

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 Cassava

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 SACMV

1.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 Aims

Methods TME3 and T200 plantlets mock inoculated or infected with SACMV at 4- 6 leaf stage Apical developing leaves collected at 12, 32 and 67dpi Total RNA Extraction (HMWPEG Protocol) Filtered for sRNAs using Ambion MirVana Kit Illumina HiSeq2000 (LGC Genomics) miRProf Conserved miRNAs Determines normalised expression levels of sRNAs matching known miRNAs in miRBase Weighted Count Raw count/Genome matches Normalised Count (Weighted count/Total number of reads in this sample) * Fold Change Log2(Normalised SACMV/Normalised Mock) Statistical significance Students t-test miRCat Novel miRNAs and Conserved miRNAs Predicts miRNAs from high-throughput sRNA sequencing data without requiring a putative precursor Input genome and miRNA identification criteria – miRNA* Weighted Count Raw count/Genome matches Normalised Count (Weighted count/Total number of reads in this sample)* Fold Change Log2(Normalised SACMV/Normalised Mock) Statistical significance Students t-test 12dpi32dpi67dpi T200 susceptible TME3 Tolerant

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

Results TME3 – Tolerant 12dpi All miRNA families had log 2 fold changes between 2 and -2

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

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

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

T200 – Susceptible 12dpi 4 (10%) miRNAs had log 2 fold changes greater than 2 (upregulation)

32dpi 23 miRNA families (55%) had log 2 fold changes greater than 2 and less than upregulated 5 downregulated 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 T200 – Susceptible

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

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) Results – Novel miRNAs

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

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

In TME3 40 and T conserved miRNA families were identified In T200, compared to TME3, the changes in expression levels were more drastic –TME3 range of expression log 2 fold change values: 3.9 to -4.6 –T200 range of expression log 2 fold change values: 61.5 to (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 Conclusion

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

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