1. Using artificial microRNAs to induce Cassava Brown Streak Disease resistance in cassava
Henry Wagaba

2. Outline
Introduction
RNA interference (RNAi) using UCBSV and CBSV coat protein sequences
Use of artificial microRNAs (amiRNAs) to induce CBSD resistance
Conclusions

3. Genome structure of CBSD causing viruses
UCBSV genome
CBSV genome
70% nucleotide identity
UCBSV isolates share % nucleotide identity
CBSV isolates share % nucleotide identity
Family: Potyviridae
Genus:
Ipomoviridae
Encodes the P1 serine proteinase that suppresses RNA silencing
Contains a Maf/HamHI-like gene-pyrophosphatase
Lacks the helper component proteinase (HC-pro)
It is perhaps interesting to speculate that this
could be a reflection of how relatively few viruses are able
to infect Euphorbiaceae hosts
ICTV’s species demarcation is <75% nucleotide identity or difference in polyprotein cleavage sites

4. RNAi strategy by expression of dsRNA targeting CBSV coat protein
6K1 C1
6K2
Vpg
NiaPro
NIb
Ham1H CP
5’UTR
3’UTR
FL-CP UCBSV (1101 bp)
Δ FL-CP UCBSV (896 bp)
NT-CP UCBSV (400bp)
CT-CP UCBSV (500 bp)
Cloned in sense and antisense orientation
pCsVMV
UCBSV-S
UCBSV-AS
Pdk Intron
TNos

5. Virus challenge with different homologous UCBSV isolates using sap inoculation
UCBSV-AS
Tnos
UCBSV-S
Pdk intron
pCsVMV
UCBSV isolates

6. Virus challenge with different non-homologous CBSV isolates using sap inoculation
UCBSV-AS
Tnos
UCBSV-S
Pdk intron
pCsVMV
CBSV isolates

7. RT-PCR to detect UCBSV and CBSV in challenged transgenic in selected N
RT-PCR to detect UCBSV and CBSV in challenged transgenic in selected N. benthamiana lines
Controls FL CT NT
UCBSV-[UG:Kab:07] (1101nt)
CBSV-[TZ:Zan:07] (1134nt)
Patil et al., Molecular Plant Pathology (2011) 12(1), 31–41

8. Screening for resistance to CBSD in N
Screening for resistance to CBSD in N. benthamiana RNAi transgenic lines
Transgenic FL17 line challenged with UCBSV (homologous virus)
[Isolate: Kenya:Mwalumba:07]

9. Grafting challenge of transgenic cassava Cv. 60444 with UCBSV
Transgenic resistant root-stock
Transgenic resistant
CBSD Infected scion
UCBSV source
2-3 months after grafting

10. siRNA expression and UCBSV challenge of transgenic cassava lines
Expt1
Expt2
Wild type 60444
8/8 (0%)
10/10 (0%)
0/8 (100%)
0/7 (100%)
0/3 (100%)
0/9 (100%)
0/6 (100%)
Not tested
0/2 (100%)
0/31 (100%)
Yadav et al., Molecular Plant Pathology (2011) 12(7), 677–687

11. Cloning of N-Terminal and ΔFL hairpin constructs using coat protein sequences from UCBSV and CBSV
pCsVMV
ΔCP-FL CBSV
Pdk intron
ΔFL CBSV
T-Nos
CBSV NT CP
ΔCP-FL
CBSV
ΔCP-FL UCBSV
ΔCP-FL-UCBSV

12. Transient siRNA expression and protection in N. benthamiana
Transformed into N. benthamiana and cassava
Next-> Challenge transgenic T0 lines to test for resistance

13. The use of Artificial microRNAs in virus protection
Virus targeted
Virus
Genes targeted
Resistance acquired?
Plants tested
Precursor used
Niu et al., (2006)
Turnip Yellow mosiac virus (TYMV)
P69 (silencing suppressor)
Yes
A. thaliana
miRNA159a
(A. thaliana)
Turnip mosaic virus (TuMV)
Hc-pro (suppressor)
Qu et al., 2007
Cucumber mosaic virus (CMV)
2b (silencing suppressor)
N. benthamiana
miRNA171a
(A. thalaiana)
Zhang et al., 2010
2a and 2b
Tomato
3’UTR
Fahim et al., 2011
Wheat streak mosaic virus (WSMV)
Conserved regions
Wheat
miR395 (rice) polycistronic
Ai et al., 2011
Potato virus Y
Coat protein
Potato

14. Advantages of using amiRNAs
Use of the artificial microRNA (amiRNA) strategy against UCBSV and CBSV
Advantages of using amiRNAs
Multiple targeting is possible
Very specific with fewer off-target effects to complementary host-targets
microRNAs also active at low temperature
Sablok et al., 2011
Degradation of viral RNA
Process of plant genetic transformation using amiRNAs

15. Cloning of UCBSV and CBSV amiRNA using the A. thaliana miR159a backbone
CAGTTTGCTTATGTCAGATCCATAATATATTTGACAAGATACTTTGTTTTTCGATAGATCTTGATCTGACGATGGAAGTAGAGCTCCTTAAAGTTCAAACATGAGTTGAGCAGGGTAAAGAAAAGCTGCTAAGCTATGGATCCCATAAGCCCTAATCCTTGTAAAGTAAAAAAGGATTTGGTTATATGGATTGCATATCTCAGGAGCTTTAACTTGCCCTTTAATGGCTTTTACTCTTCTTTGGATTGAAGGGAGCTCTACAT
A. thaliana miR159a bp predicted folded structure
Target
XbaI site
21-nt target
miR159a-P1[CBSV]
TCATCTAGATGATCTGACGATGGAAGATGGTGAGACCTGGTTGGAGTCATGAGTTGAGCAGGGTAAAG
miR159a-P1[UCBSV]
TCATCTAGATGATCTGACGATGGAAGATGATTCGTCCTGGATGGAGTCATGAGTTGAGCAGGGTAAAG
miR159a-Nib[UCBSV]
TCATCTAGATGATCTGACGATGGAAGGATTGTGATGGTAGTAGGTTTCATGAGTTGAGCAGGGTAAAG
miR159a-CP [UCBSV]
TCATCTAGATGATCTGACGATGGAAGCTGGCAGCGAATGTTGGTAGACATGAGTTGAGCAGGGTAAAG
tNos
A. thaliana miR159a bp
Sense
Anti-sense
pCsVMV

16. Transient expression and protection from amiRNAs against CBSV

17. Aligned sequence regions of P1, Nib and CP targeted genes of UCBSV and CBSV
miR159a-P1[UCBSV]
miR159a-CP[UCBSV]
miR159a-P1[CBSV]
miR159a-NIb[UCBSV]
UCBSV
CBSV

18. Summary of selected CBSV and UCBSV genes targeted
amiRNA
Targeted
UCBSV or CBSV
Transient miRNA expression
Transient protection
Stable expression in N. benthamiana
miR159a-P1[CBSV]
CBSV
Positive
+++
Yes
miR159a-P1[UCBSV]
UCBSV ++
miR159a-Nib[UCBSV]
Both
miR159a-CP [UCBSV]

19. Stable protection against CBSV and UCBSV in N. benthamiana T2 lines
There is a variation in versus differential expression pattern of amiRNAs versus resistance
There is possibly differential uptake of dsRNAs by AGO proteins
AGO proteins may bind to the target RNA and amplification of target miRNAs
P1-CBSV

20. Virus resistant line miR159a-CP[UCBSV] challenged with UCBSV
LINE 10-4 plants
Control plants

21. Future activities on artificial microRNAs
Characterize T3 N. benthamiana plants transformed with amiRNA constructs;
map cleavage sites using 5’ RACE
amplification of the signal
Concatemer 21nt miRNAs to target different genes of both UCBSV and CBSV
Transform them into cassava for CBSV and UCBSV protection
The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediated transient expression in Nicotiana benthamiana confirms that the siRNA-initiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.

22. Conclusions
The delta-FL-UCBSV CP sequence protects against both UCBSV and CBSV in N. benthamiana, and cassava
Artificial microRNAs targeting P1, Nib and CP genes of UCBSV and CBSV have a potential to protect against UCBSV and CBSV in N. benthamiana, when fused, may lead to more durable resistance

23. Acknowledgement VIRCA project Academic advisors
Millennium Science Initiative (MSI)
ILTAB members
Greenhouse staff
Makerere University
Academic advisors
Dr. Mukasa Ssettumba
Dr. Nigel Taylor
Dr. Yona Baguma
Dr. Titus Alicai
Dr. Claude M. Fauquet
Donald Danforth
Plant Science Center

24. VIRCA acknowledges support by