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

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 ?

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

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

Construction of infectius cassava begomovirus clones

Inoculation of cassava for begomovirus infection

Inoculation of cassava genotypes with cloned Cassava mosaic viruses

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

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

Differential response of Cassava breeding lines to CMD recovery non recovery

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

Proof of VIGS principle by interfering with chlorophyll biosynthesis Magnesium chelatase MgCHlI 501 600 N. bnthamiana_ChlI (501) TAGGTGCTACTGAGGACAGGGTATGTGGCACAATCGACATTGAGAAAGCTCTTACTGAGGGTGTGAAGGCTTTCGAGCCTGGTCTTCTTGCTAAAGCTAA Cassava_ChlI (1) ----------------------------------------------------------------------------------CCTTCTTGCTAAAGCTAA 601 700 N. benthamiana_ChlI(601) CAGAGGAATACTTTACGTCGATGAGGTTAATCTTTTGGACGACCATTTAGTAGATGTTCTTTTGGATTCTGCAGCATCAGGATGGAACACTGTTGAAAGA Cassava_ChlI (19) TAGAGGGATTCTTTATGTGGATGAAGTTAACCTTTTAGATGACCACTTAGTGGATGTTCTATTGGATTCTGCTGCCTCAGGATGGAACACTGTGGAGAGA 701 800 N. benthamiana_ChlI(701) GAGGGGATTTCAATCTCACATCCGGCCCGATTTATCCTAATTGGTTCGGGTAATCCTGAAGAAGGAGAACTTAGGCCACAACTTCTTGATCGATTTGGAA Cassava_ChlI (119) GAGGGTATTTCTATTTCACATCCTGCACGGTTCATTTTGATTGGCTCTGGCAACCCTGAAGAAGGGGAGCTCAGGCCACAGCTACTTGATAGATTTGGAA 801 900 N. benthamiana_ChlI(801) TGCATGCCCAAGTGGGGACCGTGAGAGATGCAGAGCTGAGAGTAAAGATCGTTGAGGAAAGAGCTCGTTTTGATAAGAACCCCAAGGAATTCCGGGAGTC Cassava_ChlI (219) TGCATGCACAAGTAGGAACTGTTAAAGATGCGGAGCTGAGAGTGAAGATAGTTGAAGAAAGAGGCCGTTTTGATAAAAACCCAAAAGAATTTCGTGATTC 901 1000 N. benthamiana_ChlI(901) ATACAAGGCAGAGCAAGAAAAGCTCCAGAATCAAATCGACTCAGCTAGGAACGCTCTTTCTGCTGTTACAACTGCTCATGATCTTCGAGTTAAAATCTCC Cassava_ChlI (319) TTACAAGGCAGAGCAAGAGAAGCTACAACAACAAATTGCCTCAGCTAGAACGTCTCTTTCTTCTGTGCAGATAGATCATGACCTCAAAGTAAAAATATCC 1001 1100 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.

VIGS DNA A expression of cassava ChlI Gene silencing Symptome sind schon 10-14 Tage früher als Virussymptome sichtbar » blocks chlorophyll biosynthesis in cassava und also degrades the orthologous gene in N. benthamina

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

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

» ChlI silencing is bound to efficient virus replication

Specificity of „gene silencing“ 501 600 Benthamiana_ChlI (501) TAGGTGCTACTGAGGACAGGGTATGTGGCACAATCGACATTGAGAAAGCTCTTACTGAGGGTGTGAAGGCTTTCGAGCCTGGTCTTCTTGCTAAAGCTAA Cassava_ChlI (1) ----------------------------------------------------------------------------------CCTTCTTGCTAAAGCTAA 601 700 Benthamiana_ChlI (601) CAGAGGAATACTTTACGTCGATGAGGTTAATCTTTTGGACGACCATTTAGTAGATGTTCTTTTGGATTCTGCAGCATCAGGATGGAACACTGTTGAAAGA Cassava_ChlI (19) TAGAGGGATTCTTTATGTGGATGAAGTTAACCTTTTAGATGACCACTTAGTGGATGTTCTATTGGATTCTGCTGCCTCAGGATGGAACACTGTGGAGAGA 701 800 Benthamiana_ChlI (701) GAGGGGATTTCAATCTCACATCCGGCCCGATTTATCCTAATTGGTTCGGGTAATCCTGAAGAAGGAGAACTTAGGCCACAACTTCTTGATCGATTTGGAA Cassava_ChlI (119) GAGGGTATTTCTATTTCACATCCTGCACGGTTCATTTTGATTGGCTCTGGCAACCCTGAAGAAGGGGAGCTCAGGCCACAGCTACTTGATAGATTTGGAA 801 900 Benthamiana_ChlI (801) TGCATGCCCAAGTGGGGACCGTGAGAGATGCAGAGCTGAGAGTAAAGATCGTTGAGGAAAGAGCTCGTTTTGATAAGAACCCCAAGGAATTCCGGGAGTC Cassava_ChlI (219) TGCATGCACAAGTAGGAACTGTTAAAGATGCGGAGCTGAGAGTGAAGATAGTTGAAGAAAGAGGCCGTTTTGATAAAAACCCAAAAGAATTTCGTGATTC 901 1000 Benthamiana_ChlI (901) ATACAAGGCAGAGCAAGAAAAGCTCCAGAATCAAATCGACTCAGCTAGGAACGCTCTTTCTGCTGTTACAACTGCTCATGATCTTCGAGTTAAAATCTCC Cassava_ChlI (319) TTACAAGGCAGAGCAAGAGAAGCTACAACAACAAATTGCCTCAGCTAGAACGTCTCTTTCTTCTGTGCAGATAGATCATGACCTCAAAGTAAAAATATCC 1001 1100 Benthamiana_ChlI (1001) AAGGTCTGTGCAGAACTAAATGTCGATGGACTTAGAGGTGATATAGTCACTAACAGGGCAGCACGAGCGTTGGCTGCACTAAAAGGAAGAGATAAGGTAA Cassava_ChlI (419) AGGGTTTGTGCTGAGCTGAATGTG---------------------------------------------------------------------------- Ca Chl149 Ca ChlI 161 Ca CHl 126 Cassava ChlI fragment 442

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

Specifity of „gene silencing“ - amiRNA

EACMV VIGS – to induce resistance with virus derived genes

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