1. Geminivirus induced gene silencing for functional characterisation of cassava genes
Stephan Winter & Marianne Koerbler
Leibniz-Institute DSMZ Plant Virus Department, Germany

2. can this be achieved through VIGS “Virus-induced Gene silencing”
Identification of genes for crop improvement requires functional validation
can this be achieved through VIGS
“Virus-induced Gene silencing”
in Cassava ?

3. Virus-induced gene silencing, VIGS
Virus infections activate silencing processes
Sequence specific RNA degradation as active „natural antiviral defence in plants (PTGS),
» dsRNA is degraded to “short-interfering“ siRNA (21-24 nt)
Recombinant viruses carrying endogenous plant genes induce PTGS - VIGS
» homologous host sequences are degraded
» gene function can be deduced from the altered phenotype!
Advantages
Fast results, VIGS ≠ stabile Transformation;
Functional analysis of essential genes where „knock-out“ mutants are lethal;
High throughput for function studies in vivo, - EST, cDNA libraries from gene expression studies

4. VIGS vector construction - Begomovirus
ssDNA viruses with bipartite genomes
replicate through rolling circle amplification of DNA intermediates in nuclei of infected cells
Virus genes are translated from RNA transcribed from strand and complementary strand

5. Construction of infectius cassava begomovirus clones

6. Inoculation of cassava for begomovirus infection

7. Inoculation of cassava genotypes with cloned Cassava mosaic viruses

8. Establishing mixed CM virus infections
EACMV Ca123
EACMV Ca123 / SLCMV Ca36

9. Introducing pseudo-recombinant viruses
Introducing pseudo-recombinant viruses through inoculation of virus genomic component DNA A and DNA B mixes
EACMV Ca123A/ EACMV-UG Ca055B
EACMV Ca123 A/ B
EACMV-UG Ca55A/ B

10. Differential response of Cassava breeding lines to CMD
recovery
non recovery

12. Begomovirus DNA A ∆CP replacement vector
ORI
AV2
3'CP end
CP N-terminus
TraP
REn
Rep
left border
right border
VIGS Fragment ~ 500bp
pGreen II
CHlI gene fragment (440bp) from cassava and N. benthamiana

13. Proof of VIGS principle by interfering with chlorophyll biosynthesis
Magnesium chelatase MgCHlI
N. bnthamiana_ChlI (501) TAGGTGCTACTGAGGACAGGGTATGTGGCACAATCGACATTGAGAAAGCTCTTACTGAGGGTGTGAAGGCTTTCGAGCCTGGTCTTCTTGCTAAAGCTAA
Cassava_ChlI (1) CCTTCTTGCTAAAGCTAA
N. benthamiana_ChlI(601) CAGAGGAATACTTTACGTCGATGAGGTTAATCTTTTGGACGACCATTTAGTAGATGTTCTTTTGGATTCTGCAGCATCAGGATGGAACACTGTTGAAAGA
Cassava_ChlI (19) TAGAGGGATTCTTTATGTGGATGAAGTTAACCTTTTAGATGACCACTTAGTGGATGTTCTATTGGATTCTGCTGCCTCAGGATGGAACACTGTGGAGAGA
N. benthamiana_ChlI(701) GAGGGGATTTCAATCTCACATCCGGCCCGATTTATCCTAATTGGTTCGGGTAATCCTGAAGAAGGAGAACTTAGGCCACAACTTCTTGATCGATTTGGAA
Cassava_ChlI (119) GAGGGTATTTCTATTTCACATCCTGCACGGTTCATTTTGATTGGCTCTGGCAACCCTGAAGAAGGGGAGCTCAGGCCACAGCTACTTGATAGATTTGGAA
N. benthamiana_ChlI(801) TGCATGCCCAAGTGGGGACCGTGAGAGATGCAGAGCTGAGAGTAAAGATCGTTGAGGAAAGAGCTCGTTTTGATAAGAACCCCAAGGAATTCCGGGAGTC
Cassava_ChlI (219) TGCATGCACAAGTAGGAACTGTTAAAGATGCGGAGCTGAGAGTGAAGATAGTTGAAGAAAGAGGCCGTTTTGATAAAAACCCAAAAGAATTTCGTGATTC
N. benthamiana_ChlI(901) ATACAAGGCAGAGCAAGAAAAGCTCCAGAATCAAATCGACTCAGCTAGGAACGCTCTTTCTGCTGTTACAACTGCTCATGATCTTCGAGTTAAAATCTCC
Cassava_ChlI (319) TTACAAGGCAGAGCAAGAGAAGCTACAACAACAAATTGCCTCAGCTAGAACGTCTCTTTCTTCTGTGCAGATAGATCATGACCTCAAAGTAAAAATATCC
N. benthamiana_ChlI1001) AAGGTCTGTGCAGAACTAAATGTCGATGGACTTAGAGGTGATATAGTCACTAACAGGGCAGCACGAGCGTTGGCTGCACTAAAAGGAAGAGATAAGGTAA
Cassava_ChlI (419) AGGGTTTGTGCTGAGCTGAATGTG
Mg CHlI catalyses the insertion of Mg2+ into protoporphyrin IX.
This is the first unique step in the synthesis of chlorophyll.

16. VIGS DNA A expression of cassava ChlI
Gene silencing Symptome sind schon Tage früher als Virussymptome sichtbar
» blocks chlorophyll biosynthesis in cassava und also degrades the orthologous gene in N. benthamina

17. Systemic spread of the silencing“ signal
» ChlI “silencing” advances virus movement and virus symptoms classical mosaic symptoms are masked

18. ......systemic spread of the silencing“ signal
» ChlI Silencing - signal is not independent from virus infection

19. » ChlI silencing is bound to efficient virus replication

20. Specificity of „gene silencing“
Benthamiana_ChlI (501) TAGGTGCTACTGAGGACAGGGTATGTGGCACAATCGACATTGAGAAAGCTCTTACTGAGGGTGTGAAGGCTTTCGAGCCTGGTCTTCTTGCTAAAGCTAA
Cassava_ChlI (1) CCTTCTTGCTAAAGCTAA
Benthamiana_ChlI (601) CAGAGGAATACTTTACGTCGATGAGGTTAATCTTTTGGACGACCATTTAGTAGATGTTCTTTTGGATTCTGCAGCATCAGGATGGAACACTGTTGAAAGA
Cassava_ChlI (19) TAGAGGGATTCTTTATGTGGATGAAGTTAACCTTTTAGATGACCACTTAGTGGATGTTCTATTGGATTCTGCTGCCTCAGGATGGAACACTGTGGAGAGA
Benthamiana_ChlI (701) GAGGGGATTTCAATCTCACATCCGGCCCGATTTATCCTAATTGGTTCGGGTAATCCTGAAGAAGGAGAACTTAGGCCACAACTTCTTGATCGATTTGGAA
Cassava_ChlI (119) GAGGGTATTTCTATTTCACATCCTGCACGGTTCATTTTGATTGGCTCTGGCAACCCTGAAGAAGGGGAGCTCAGGCCACAGCTACTTGATAGATTTGGAA
Benthamiana_ChlI (801) TGCATGCCCAAGTGGGGACCGTGAGAGATGCAGAGCTGAGAGTAAAGATCGTTGAGGAAAGAGCTCGTTTTGATAAGAACCCCAAGGAATTCCGGGAGTC
Cassava_ChlI (219) TGCATGCACAAGTAGGAACTGTTAAAGATGCGGAGCTGAGAGTGAAGATAGTTGAAGAAAGAGGCCGTTTTGATAAAAACCCAAAAGAATTTCGTGATTC
Benthamiana_ChlI (901) ATACAAGGCAGAGCAAGAAAAGCTCCAGAATCAAATCGACTCAGCTAGGAACGCTCTTTCTGCTGTTACAACTGCTCATGATCTTCGAGTTAAAATCTCC
Cassava_ChlI (319) TTACAAGGCAGAGCAAGAGAAGCTACAACAACAAATTGCCTCAGCTAGAACGTCTCTTTCTTCTGTGCAGATAGATCATGACCTCAAAGTAAAAATATCC
Benthamiana_ChlI (1001) AAGGTCTGTGCAGAACTAAATGTCGATGGACTTAGAGGTGATATAGTCACTAACAGGGCAGCACGAGCGTTGGCTGCACTAAAAGGAAGAGATAAGGTAA
Cassava_ChlI (419) AGGGTTTGTGCTGAGCTGAATGTG
Ca Chl149
Ca ChlI 161
Ca CHl 126
Cassava ChlI fragment 442

21. Specifity of „gene silencing“ – minimal sequence context
Orthologous N. benthamiana ChlI results in effectives “gene silencing” in Cassava
Cassava ChlI short 3’ Genfragmente do not induce “gene silencing” in N. benthamiana

22. Specifity of „gene silencing“ - amiRNA

23. EACMV VIGS – to induce resistance with virus derived genes

24. high rates of infection and replication
Summary
VIGS vector constructs based on EACMV ∆CP are available for gene function studies in cassava
high rates of infection and replication
size constraint - DNA A vector constructs are stable < 800 nt
position constraint – DNA B vector constructs stable <150 nt
DNA A vector constructs are versatile for protein expression GFP ?
Geminivirus induced „gene silencing“, VIGS
depends on virus replication – not systemic!
extends to orthologous genes
specifity depends on sequence position und composition
dose dependency ?
replicating virus itself is target of silencing