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
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