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
RNA Silencing
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
TGS
Initiates and
maintains the
heterochromatic
state of certain
DNA regions
PTGS
Affects gene
expression by
degradation of
mRNA or
repression of RNA
translation
miRNA
ta-siRNA
NATsiRNA
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
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
– 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
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
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
Aims
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
Methods
•Novel
miRNAs and Conserved
miRNAs
TME3 and T200
plantlets
Apical developing
Total RNA
•Predicts
miRNAs
from
high-throughput
sRNA
sequencing
mock inoculated or
leaves collected
Extraction
data
without
requiring
a
putative
precursor
infected
SACMV at 4at 12, 32 and
(HMWPEG
miRProf
miRCat with•Input
criteria – miRNA*
6 leaf stagegenome and miRNA identification
67dpi
Protocol)
12dpi
Weighted
Count
•Raw count/Genome matches
32dpiWeighted
Kit
Genomics)
67dpi
•Raw count/Genome matches
Count
T200
susceptible
•(Weighted count/Total number of reads in this sample)*
Normalised
Count
Filtered
for
•Conserved
miRNAs
Illumina
sRNAs
using expression levels of sRNAs matching
•Determines
normalised
HiSeq2000 (LGC
known
miRNAs
in miRBase
Ambion
MirVana
000 000
1
•(Weighted count/Total number of reads in this sample) *
Normalised 1 000 000
Count
•Log2(Normalised SACMV/Normalised Mock)
•Log2(Normalised SACMV/Normalised Mock)
Fold Change
•Students t-test
Statistical
significance
TME3
Tolerant
Fold Change
Statistical
significance
•Students t-test
mir166
mir396
mir156
mir167
mir319
mir393
mir408
mir171
mir157
mir398
mir858
mir159
mir168
mir535
mir160
mir6445
mir169
mir162
mir170
mir2950
mir397
mir399
mir530
mir5538
mir164
mir172
mir390
mir395
mir2111
mir403
mir477
mir6478
mir827
mir1511
mir384
mir394
mir4995
mir5139
mir6300
mir8155
40
TME3
– Tolerant:
35
• Identified
290 individual conserved miRNAs belonging to 40 miRNA
families
30
Number of individual miRNAs belongig to each family
Results – Conserved miRNAs
25
20
15
10
5
0
miRNA families
The number of individual miRNA members belonging to each miRNA
family in TME3
Results
TME3 – Tolerant
12dpi
• All miRNA families had log2 fold
changes between 2 and -2
TME3 – Tolerant
32dpi
Flowering time,
Leaf
12% • 11 miRNA families (27%) had log
2
development,
Floral
organ
fold changes greater than 2 and
12%
identify, 50%
less than -2
• 8 upregulation
• 3 downregulation
Auxin signalling,
12%
Adaptive
responses to
stress, 25%
Role of conserved plant miRNAs
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
mir166
mir156
mir396
mir408
mir167
mir171
mir319
mir157
mir159
mir393
mir398
mir169
mir535
mir168
mir858
mir397
mir160
mir162
mir170
mir399
mir6445
mir172
mir390
mir395
mir164
mir2111
mir2950
mir477
mir530
mir6478
mir827
mir1507
mir1511
mir384
mir394
mir403
mir482
mir4995
mir5139
mir5538
mir6300
mir8155
T200 – susceptible:
• Identified
317 individual conserved miRNAs belonging to 42 miRNA
30
families
• Two miRNA
families present in T200 that were not observed in TME3
25
• miR1507 and miR482
The number of individula miRNA members belonging to each family
35
20
15
10
5
0
miRNA Famlies
The number of individual miRNA members belonging to each
miRNA family identified in T200
T200 – Susceptible
Regulation of
miRNAs and
siRNAs; 25%
12dpi
• 4 (10%) miRNAs had log2 fold
changes greater than 2
(upregulation)
Adaptive
responses to
stress, 75%
Role of conserved plant miRNAs
T200 – Susceptible
Floral organ
identify, 87%
32dpi
Flowering time,
• 23 miRNA families (55%) 13%
had
log2
Regulation
Adaptive
miRNAs
fold responses
changes
to greater than 2 ofand
and
siRNAs
less stress,
than 52%
-2
; 13%
• 18 upregulated
• 5 downregulated
Leaf polarity, 43%
Leaf development,
87%
Roles of conserved plant miRNAs
Auxin
signalling,
13%
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 Veinclearing virus:
• miR168 ; miR169
; miR398
T200 – Susceptible
Adaptive
responses to
stress, 50%
67dpi Auxin Signalling,
10% families (23%) had
• 10 miRNA
log2 fold changes greater than 2
and less than -2
Leaf development,
• 6 upregulated
20%
• 4 downregulated
Floral organ
identity, 20%
Flowering time,
10%
Role of conserved plant miRNAs
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)
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
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
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
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
Acknowledgements
•
•
•
•
NRF
Casquip Starch Manufacturing Co.
Cassava Biotech lab members
My supervisor Prof Rey for her
guidance and support