1. TILLING, a non-GMO approach to engineer genetic variation for disease resistance © copyright BenchBio Source: Seattle TILLING project Manash CHATTERJEE Founder & Director

2. © copyright BenchBio Overview of the talk About BenchBio Our Technology-TILLING How it is done Some examples of disease resistance using TILLING

3. BENCH BIO LTD. (BBIO) Indian plant biotech company set up in 2007, strong links with EU. Science based contract research and service in plant biotechnology Focussed on discovering induced alleles from plants- mutant- allele discovery platform, M-TILLING/TILLING Focussed on discovering natural alleles from plants- natural allele discovery platform, N-TILLING/Eco-TILLING Focussed on genes for agricultural and consumer value Food, feed as well as non-food crops

4. © copyright BenchBio TILLING  A novel technology that allows rapid selection of mutants from any gene in any organism.  The initial development and testing used Arabidopsis.  TILLING now also applied to all crops and organisms such as mouse, fish, flies and worms.  Public labs worldwide now engaged in TILLING  Takes advantage of large amounts of sequence information available in public databases

5. © copyright BenchBio TILLING / ECOTILLING CONCEPT Product 1, tomato Product 2, rice Product 3, wheat Product 4, sorghum ………….etc Endo1 Colbert T et al Plant Physiol 2001

6. INRA-KSU - May 2011 M2 seeds 8 seeds 3 weeks Taking away Check on agarose gel (1%) Qiagen Dneasy 96 plant kit® gDNA extraction EMS POOL plates -600 bp -500 bp -370 bp -230 bp -100 bp LICOR Gel ENDO1 PCR PCR amplification And barcoding TILLING Pipeline

7. © copyright BenchBio Pools screening + WT Homozygote family identification 12345678P ? Family identification 4800 M2 plants 35 mutants: Product Crosses TILLING summary

8. © copyright BenchBio TILLING:Advantages Both natural and mutagenised populations in any organism can be screened. Mutagenised plants have a large number of randomly distributed mutations per plant genome. Plants heterozygous for a mutation can be detected (lethality is not a problem). Both nonsense (knockout) and missense mutations can be recovered. No transgenic manipulations are required (non- genetically modified product.

9. © copyright BenchBio Examples Potyvirus resistance Powdery Mildew

10. Potato virus Y on Tomato Infected Non-infected VPg : virus-encoded genome linked protein P1 HC-Pro P3 CI Nia Nib CP VPg AAAAAAA

11. Translation initiation factors: kingpin in plant resistance to RNA viruses Viruses require host factors to perform their infectious cycle Molecular cloning of recessive resistance genes Characterization of A. thaliana mutant lines Translation initiation factors eIF4E & eIF4G

12. Translation initiation factor eIF4E eIF4E plays an important role in initiation of translation CAP binding protein eIF4E eIF4G PABP 5´CAP 3´polyA AAAAA 40S eIF3 eIF4F Eukaryotic initiation complex P1 HC-Pro P3 CI Nia Nib CP VPg AA VPg Translation initiation factors: kingpin in plant resistance to RNA viruses mo1/ LMV/ Lettuce pvr2/ PVY/ Pepper sbm1/ PsBMV/ Pea rym/BaYMV/ Barley Nsv/MNSV/Melon Zym/ZYMV/Watermelon

13. Induced mutations in translation initiation factors: 283 induced mutations with 91 in exonic regions out of witch 33 missense mutations Courtesy: Dr A. Bendahmane, URGV

14. Induced mutations in translation initiation factors: Courtesy: Dr A. Bendahmane, URGV

15. TILLING Translation Initiation Factors eIF4E1 Splicing mutant JO55 WT e1858 Mutant eIF4Eiso eIF4E2 e597 Growth rescue of a yeast strain depleted for eIF4E The eIF4E1 splicing mutant is impaired in cap-binding activity & mRNA translation in yeast The splicing mutant is immune to PVY (Potato Virus Y) and PepMoV (Pepper Mottle Virus). PVY LYE 90V PepMoV ELISA test: Courtesy: Dr A. Bendahmane, URGV

16. S MVVEDSMKATSAEDLSNSIANQNPRGRGGDEDEELEEGEIVGDDDLDSSNLSASLVHQPH R MVVEDSMKATSAEDLSNSIANQNPRGRGGDEDEELEEGEIVGDDDLDSSNLSASLVHQPH ************************************************************ S PLEHSWTFWFDNPSAKSKQATWGASIRPIYTFSTVEEFWSVYNNIHHPSKLAMRADLYCF R PLEHSWTFWFDNPSAKSKQATWGASIRPIYTFSTVEEFWSVYNNIHHPSKLAMRADLYCF ************************************************************ S KHKIEPKWEDPVCANGGKWTVNFPRGKSDNGWLYTLLAMIGEQFDCGDEICGAVVNVRSG R KHKIEPKWEDPVCANGGKWTVNFPRGKSDNGWLYTLLAMIGEQFDCGDEICGAVVNVRSG ************************************************************ S QDKISIWTKNASNEAAQASIGKQWKEFLDYNESIGFIFHDDAKKFDRHAKNKYMV R QDKISIWTKNASNEAAQASIGKQWKEFLDYNESIGFIFHDDAKKFDRLAKNKYMV *********************************************** ******* TILLING eIF4E gives MNSV resistance eIF4E-Ved (S)eIF4E-PI (R) His228 Leu228 Single Nucleotide Polymorphism (SNP) eIF4E Resistance/ Susceptibility to MNSV Position 228 EcoTILLING of 100 melon accessions Courtesy: Dr A. Bendahmane, URGV Family: Carmovirus +ssRNA

17. Family: Caulimoviridae dsDNA-RT

18. © copyright BenchBio Virus induced gene silencing of SlMlo1 causes enhanced PM resistance Bai et al, 2008 Example- Powdery Mildew

19. Conclusions TILLING can be used to generate disease resistant plants Quality and size of the mutant population very important for the success Both reverse and forward genetic screens can be done using the mutant population Relatively low cost compared to GM Products are non-GM, better acceptance by the public © copyright BenchBio

20. Thanks www.benchbio.com Abdel BENDAHMANE URGV Lab, INRA Bench Bio Team