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Research performed by Andrew Tsai and Helen Oh in early 2011. Identifies potential genes that regulate miRNA activity, especially in regards to growth and development.
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miRNA activity in Arabidopsis thaliana plantsHelen Oh Andrew Tsai
Purpose
To identify potential genes that regulate miRNA activity, especially in regards to
growth and development.
Overview
To identify those potential genes:Mutagenize with T-DNA insertions Screen using BASTA Screen using ABA Screen for typical mutant miRNA phenotypes Find location of T-DNA insertion Determine whether miRNA target genes are
affected Germination assay BASTA assay
Mutagenize with T-DNA insertion
Overview
To identify those potential genes: Mutagenize with T-DNA insertions
Screen using BASTA Screen using ABA Screen for typical mutant miRNA phenotypes Find location of T-DNA insertion Determine whether miRNA target genes are
affected Germination assay BASTA assay
Screen using BASTA
Ten plants germinated on BASTA.
Overview
To identify those potential genes: Mutagenize with T-DNA insertions Screen using BASTA
Screen using ABA Screen for typical mutant miRNA phenotypes Find location of T-DNA insertion Determine whether miRNA target genes are
affected Germination assay BASTA assay
Screen using ABA
Six plants displayed mutated responses to ABA , specifically they displayed
hypersensitive root growth.
Overview
To identify those potential genes: Mutagenize with T-DNA insertions Screen using BASTA Screen using ABA
Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion Determine whether miRNA target genes are
affected Germination assay BASTA assay
Screen for typical mutant miRNA
phenotypeHOAT2-2
Screen for typical mutant miRNA
phenotypesHOAT3-2
Screen for typical mutant miRNA
phenotypeHOAT5-2
Overview
To identify those potential genes: Mutagenize with T-DNA insertions Screen using BASTA Screen using ABA Screen for typical mutant miRNA phenotypes
Find location of T-DNA insertion Determine whether miRNA target genes are
affected Germination assay BASTA assay
Find location of T-DNA insertion
TAIL PCR was performed.
This gel shows that AD primers are about 100 bps.
AD
2
AD
1/A
D2
Find location of T-DNA insertion
TAIL PCR product for HOAT2-2AD2 and HOAT3-2AD2 are about 750 bps.
Lower bands are AD primers.
HO
AT2-2
AD
2
HO
AT3-2
AD
2
HO
AT2-2
AD
1
HO
AT2-2
AD
3
HO
AT3-2
AD
1
HO
AT3-2
AD
3
HO
AT5-2
AD
1
Find location of T-DNA insertion
Heavy upper bands could be due to precipitated DNA.
Lower bands are AD primers.
HO
AT5-
2A
D2
HO
AT5-
2A
D3
Find location of T-DNA insertion
DNA sequence analysis performed.
HOAT3-2: Had greater or equal to
200 as alignment score. Expected value was 0.0.
Find location of T-DNA insertion
AT3G09720 codes for a protein with a p-loop. Binds nucleotides
and ATP Helicase functions
AT3G09730 has no known function.
upst
rea
m
dow
nst
ream
upst
rea
m
Overview
To identify those potential genes: Mutagenize with T-DNA insertions Screen using BASTA Screen using ABA Screen for typical mutant miRNA phenotypes Find location of T-DNA insertion
Determine whether miRNA target genes are affected
Germination assay BASTA assay
Determine whether miRNA target genes are affected
GRF3 is a known target of miRNA.
Look at amount of mRNA transcribed from GRF3 to determine if miRNA activity is
potentially altered.
Determine whether miRNA target genes are affected
Non-degraded RNA has the following bands: 28s rRNA and 18s rRNA.
Create cDNA for HOAT3-2 and HOAT5-2.
28s rRNA
18s rRNA
HO
AT3-2
HO
AT5-2
Col-
0
HO
AT2-2
Determine whether miRNA target genes are affected
sqPCR was performed.
26 cycles
HO
AT3-2
HO
AT5-2
DD
L
Col-
0
HO
AT3-2
HO
AT5-2
DD
L
Col-
0
Col-
0
26 cycles• Actin primers (above).
• GRF3 primers (below).
Determine whether miRNA target genes are affected
qPCR was performed.
Col-0 DDL HOAT3-2 HOAT5-20
200
400
600
800
1000
1200
qPCR
Plant Name
Perc
enta
ges
Determine whether miRNA target genes are affected
26 cycles 30 cycles
26 c
ycl
es
30 c
ycl
es
HO
AT3-2
HO
AT5-2
DD
L
Col-
0
HO
AT3-2
HO
AT3-2
, 30H
OAT5-2
DD
L
Col-
0
26 c
ycl
es
26 c
ycl
es
26 c
ycl
es
30 c
ycl
es
30 c
ycl
es
30 c
ycl
es
HO
AT3-2
, 30 C
ol-
0,
30
Col-
0,
30
HO
AT3-2
, 20
HO
AT3-2
, 20 C
ol-
0,
20
Col-
0,
20
Actin primers
ATG09730 and ATG09720 primers
Overview
To identify those potential genes: Mutagenize with T-DNA insertions Screen using BASTA Screen using ABA Screen for typical mutant miRNA phenotypes Find location of T-DNA insertion Determine whether miRNA target genes are
affected
Germination assay BASTA assay
Germination Assay
plated onto ABA plates of concentrations:
0 μM
0.5 μM
1 μM
1.5 μM
Germination Assay
wt 2-2 3-2 5-20
10
20
30
40
50
60
70
80
90
100
0 μM ABA
Experimental
Percent Germi-nation
• Baseline
• note 3-2 low
germination
Germination Assay
wt 2-2 3-2 5-20
10
20
30
40
50
60
70
80
90
100
0.5 μM ABA
Experimental
Percent Germi-nation
• Mimics 0 ABA control
• T-value of 0.5275
• Don’t see too much
from this plate
Germination Assay
wt 2-2 3-2 5-20
10
20
30
40
50
60
70
80
90
100
1 μM ABA
Experimental
Percent Germi-nation
• Start seeing trends
• 3-2, 5-2
hypersensitive
• 2-2 ambiguous
Germination Assay
wt 2-2 3-2 5-20
102030405060708090
100
1.5 μM ABA
Experimental
Percent Germi-nation
• Similar to 1.0 μM
ABA
• 3-2, 5-2
Germination Assay
Seeds from HOAT 2-2 may have a T-DNA
insertion but are not hyper/hyposensitive to
ABA!
Overview
To identify those potential genes: Mutagenize with T-DNA insertions Screen using BASTA Screen using ABA Screen for typical mutant miRNA phenotypes Find location of T-DNA insertion Determine whether miRNA target genes are
affected Germination assay
BASTA assay
BASTA Assay - Theory
GIVEN:
T-DNA insertion confers BASTA resistance
ABA-hypersensitive plants on BASTA – should all have T-DNA insertion
Plated surviving plants from ABA treatment onto BASTA plates
HYPOTHESIS: ABA hyper/hyposensitivity caused by T-DNA
insertion.
BASTA Assay
0.5 1 1.50
20
40
60
80
100
Survival on BASTA
Col-0HOAT 2-2HOAT 3-2HOAT 5-2
[ABA] in μM
Percent Survival
• Data does not confirm
theory
• Most surviving plants
have BASTA resistance
BASTA Assay - Conclusions
Seeds from HOAT 2-2 HAVE a T-DNA
insertion but are not hyper/hyposensitive to
ABA!Contrary to original screen
2-2 originally identified as hypersensitive
Possible Explanations
Chance from first hyper/hyposensitivity assay (n = 1)
NOT chance from this experimentn = large (despite contamination)
Possibly growth conditions
BOTTOM LINE: We have a lot of factors at
work here that we don’t know about!
Possible Explanations
0.5 1 1.50
20
40
60
80
100
Survival on BASTA
Col-0HOAT 2-2HOAT 3-2HOAT 5-2
[ABA] in μM
Percent Survival
Possible interaction
between ABA and
BASTA
Conclusions
HOAT 2-2, HOAT 3-2, HOAT 5-2
Root growth assay potential to have miRNA mutations
Observable “miRNA phenotypes”
Conclusions
HOAT 3-2, HOAT 5-2 Increase in GRF3 expression – KNOWN
miR396-REGULATED GENE
Decrease in ATG09720 gene expression T-DNA insertion inside gene.
Future work: How do changes in these gene expressions explain original phenotype?
References
Earley, et al., An endogenous F-box protein regulates ARGONAUTE1 in Arabidopsis thaliana Silence 2010, 1:15
Rodriguez RE, Mecchia MA, Debernardi JM, Schommer C, Weigel D, Palatnik JF. 2010. Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development 137, 103–112.
Dr. John Wagner, personal communication.
Allison Lesher, personal communication.
NCBI BLAST database: gene identification
TAIR database: gene information
