Comparison of microRNA populations in SACMV infected

tolerant and susceptible cassava landraces

9th 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) * 1 000 000

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)* 1 000 000

Fold Change•Log2(Normalised SACMV/Normalised Mock)

Statistical significance

•Students t-test

12dpi 32dpi 67dpi

T200 susceptible

TME3 Tolerant

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

families

mir166

mir156

mir319

mir408

mir157

mir858

mir168

mir160

mir169

mir170

mir397

mir530

mir164

mir390

mir2111

mir477

mir827

mir384

mir4995

mir6300

0

5

10

15

20

25

30

35

40

The number of individual miRNA members belonging to each miRNA family in TME3

miRNA families

Num

ber o

f ind

ivid

ual m

iRN

As b

elon

gig

to e

ach

fam

ily

Results TME3 – Tolerant

12dpi• All miRNA families had log2 fold

changes between 2 and -2

32dpi• 11 miRNA families (27%) had log2

fold changes greater than 2 and less than -2

• 8 upregulation• 3 downregulation

TME3 – Tolerant

Floral organ identify; 50%

Adaptive responses to stress;

25%

Auxin signalling; 12%

Leaf de-velopment;

12%

Flowering time; 12%

Role of conserved plant miRNAs

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

TME3 – Tolerant

mir166

mir396

mir167

mir319

mir159

mir398

mir535

mir858

mir160

mir170

mir6445

mir390

mir164

mir2950

mir530

mir827

mir1511

mir394

mir482

mir5139

mir6300

0

5

10

15

20

25

30

35

The number of individual miRNA members belonging to each miRNA family identified in T200

miRNA Famlies

The

num

ber o

f ind

ivid

ula

miR

NA

mem

bers

bel

ongi

ng to

eac

h fa

mily

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 log2 fold

changes greater than 2 (upregulation)

Adaptive responses to stress; 75%

Regula-tion of

miRNAs and

siRNAs; 25%

Role of conserved plant miRNAs

32dpi• 23 miRNA families (55%) had log2

fold changes greater than 2 and less than -2

• 18 upregulated• 5 downregulated

T200 – Susceptible

Adaptive responses to stress;

52%Flowering time;

13%

Regulation of miRNAs and siRNAs ; 13%

Auxin sig-nalling; 13%

Leaf polarity; 43%

Leaf devel-opment; 87%

Floral organ identify; 87%

Roles of conserved plant miRNAs

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

log2 fold changes greater than 2 and less than -2

• 6 upregulated• 4 downregulated

T200 – Susceptible

Auxin Signalling; 10%

Leaf development; 20%

Floral organ

identity; 20%Flowering time; 10%

Adaptive responses to stress; 50%

Role of conserved plant 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)

Results – Novel miRNAs

TME3 - Tolerant

12dpi• 7 (21%) of the miRNAs had log2 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 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

T200 – susceptible 27 miRNA members belonging to 26 miRNA families

• 15 landrace specific12dpi• 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 267dpi• 2 (7%) of the miRNAs had log2 fold

changes greater than 2 and less than -2 • 1 upregulated• 1 downregulated

• 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

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