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1 Genetic Engineering for Virus Resistance in Plants: Different Viruses Demand Different Strategies Roger Beachy Donald Danforth Plant Science Center St. Louis, MO Improving the Human Condition through Plant Science Genetic Engineering for Virus Resistance in Plants: Different Viruses Demand Different Strategies Lecture will include: 1. Review of strategies used for genetic modification for resistance to virus diseases 2. Tobacco mosaic virus: a simple RNA virus 3. Tobacco streak virus in peanuts: a relevant application of (2) 4. Rice tungro bacilliform pararetrovirus 5. Cassava mosaic disease, a geminivirus Improving the Human Condition through Plant Science First Field Trial of a Food Crop in 1987, TMV-Resistant Tomatoes Coat Protein Mediated Resistance first described in December, To date, a single public sector transgenic crop has reached market. Why not more success? What steps can be taken to enhance contribution to food production in the future? In 1987 Dr. Roger Beachy, foreground at right, joins scientists to test the first genetically modified plants in a field trial in Jerseyville, Il. Improving the Human Condition through Plant Science The American Society for Cell Biology 1

2 Strategies for Deriving Virus Disease Resistance Pathogen Derived Resistance Protein Mediated Resistance Coat Protein Mediated Resistance Replicase Mediated Resistance Movement Protein Mediated Resistance RNA-mediated resistance srnas that target vrnas for degradation Non-Pathogen, Protein Mediated Resistance Transcription regulators: TFs, and szfps DNA binding proteins Interferon-like strategies; ds-rna degrading enzymes Translation initiation factors/co-factors Improving the Human Condition through Plant Science Protein-Based Strategies for Virus Disease Resistance The Model System: Coat Protein Mediated Resistance to TMV. Q: why/how does it work? And, can it be made more effective? TSV in peanuts for India: real world application of CP-MR Rice Tungro Disease: Novel approaches to control disease caused by plant pararetrovirus G5, a protein from an Inovirus to control geminivirus replication Improving the Human Condition through Plant Science The American Society for Cell Biology 2

3 A Primer on TMV and CP-MR RdRp MP CP Replication from a molecular view Replication from a cellular view CP-MR to TMV in tomato Model for CP-MR in blocking infection Mutations in CP Sequence and the role of Structure in CP-MR Mutant CPs have Different Effects on CP-MR, including VH Resistance The American Society for Cell Biology 3

4 ,5 5,5 4,5 3,5 2,5 1,5 0,5-0,5 ibioseminars: Roger Beachy, April 2008 Effects of CP Mutants on TMV Gene Expression BY BY-CP BY-CP T42W BY-CP D77K BY-CP D77R CP Level Replicase level Level BY-CP E50R BY-CP E50D Hpi Hpi. MP Level Hpi. Mutant CPs have different impacts virus gene expression, and replication, and as a consequence impact the efficacy of CP-MR. How? BY-2 Protoplast infected by TMV, 21 hpi Localization of Viral Proteins with Specific Ab: Red = rep; Green = MP; Blue = CP Early Stage Puncta Effects of CPs on Formation of VRCs, by Class Protoplasts infected with TMV MP:GFP-BY2 group Mid Stage Small bodies Large bodies 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Late Stage Mid Stage Early Stage by2 2842w 28w 77a 77e 77n Protoplasts infected with TMV MP:GFP-WTCP group Late Stage Bodies and fibers Bodies,fibers,punct Long fibers 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% wtcp 89w 50k 50m 50r Late Stage Mid Stage Early Stage Protoplasts infected with TMV MP:GFP-42W group 100% 80% 60% Late Stage Mid Stage 40% 20% Early Stage Puncta and Fibers Hairs 0% w 50d 50q 77r CP and mutants can act as (+) or ( ) regulators of replication, included by limiting formation of VRCs (virus replication complexes The American Society for Cell Biology 4

5 Views of infected protoplasts at ~ 22 HPI CP T42W, Transgenic cell Non-Transgenic cell Comparing Structures of w.t. CP and CP-T42W EM analyses of w.t. and CP-T42W Structural Model for w.t. and CP-T42W grna (+) RdRp MP CP grna (-) Replicase complex MP SgRNA CP aggregates CP SgRNA Mutant CP aggregates of late in the infection. The American Society for Cell Biology 5

6 Proposed Model of Cell to Cell Spread of TMV infection Reducing MP production limits Cell-cell spread and increases resistance Summary of Study of Mutant CPs Mutants of CP that do not form elongated (quaternary) structures do not confer CP-MR Mutants that assemble like w.t. CP restrict infection per se, but do not restrict replication, resulting in good to modest levels of CP-MR Mutants that have aberrant assembly can restrict infection and reduce virus replication by reducing formation of virus replication complexes (VRCs), thereby enhancing CP-MR Production of transgenic groundnut with resistance to tobacco streak ilarvirus A collaborative Project: R.N. Beachy/DVR Reddy K.K. Sharma, ICRISAT, Hyderabad, India Hindu; SEPT, 8,2000 Deccan Chronicle: Aug, 12, 2001 Deccan Chronicle: May, 16, 2001 The New Indian Express: Sept, 14,2000 The American Society for Cell Biology 6

7 History of TSV in India First observed in 1996 in sunflower. A disease epidemic occurred in groundnut during Kharif 2004 in Ananthapur, AP; >10 mil farmers affected Symptoms were confused to those caused by Tospoviruses (PBNV) The occurrence of TSV confirmed in the year 2000 from 3 independent laboratories (ICRISAT, MAHYCO & IARI) TSV on Other Crops sunflower Marigold GENOME STRUCTURE of TSV an Ilarvirus The American Society for Cell Biology 7

8 Evaluating transgenic groundnut with TSV Coat Protein for CP-Mediated resistance Control TSV+ve Delayed symptoms Resistant plant Resistant plant Developing a model for TSV using AlMV Science 306: The American Society for Cell Biology 8

9 Rice Tungro Disease From IRRI Virion Life Cycle of Plant Pararetrovirus DNA Mini- Chromosome Nucleus mrna pregenome RNA Translation trna Cytoplasm Assembly The American Society for Cell Biology 9

10 RTBV Genome and Promoter GATA ASL Box II Box I TATA +45 Rice Transcription Factors Bind to Box II of RTBV Promoter to Control Gene Expression RLP1 N P BTB S bzip C bzip Effects of RF2a, RF2b and RLP1 on Rice Development Ck Ubi::RF2a(-) Tungro Disease CK Cs::RLP1 Cs::GUS Cs::RF2b(-) The American Society for Cell Biology 10

11 Dominant Negative Effects Caused by Expression of the bzip Domain of RF2a in Transgenic Rice Plants B C D E F C C C C C Greenhouse Trial of Transgenic Rice Lines with Elevated RF2a and RF2b Expression Vector: GLH Virus: RTBV RTSV , Wild type (TP309); 2, Susceptible control(lpo345); 3, Resistant control (MT5); 4, Ubi::RF2a-1; 5, Ubi::RF2a-2; 6, Cs::RF2b-1; 7, Cs::RF2b-2 Impact of Elevated RF2a and RF2b on Tungro Disease Symptom Development RTBV inoculation Mock Ubi:RF2a TP309 RTBV inoculation Mock Ubi:RF2a TP309 PhilRice The American Society for Cell Biology 11

12 Impact of Elevated RF2a and RF2b on RTBV Replication and Accumulation RT PCR, rice protoplasts RT PCR, rice protoplasts The American Society for Cell Biology 12

13 % 50% 40% 30% 20% 10% 0% con AC1-31 Y44 Y100 Y85 ibioseminars: Roger Beachy, April 2008 Control of the Cassava Geminiviruses by sirna Strategy Virus Clones Virus Genomes p35s AC1 1087nts Gene Constructs Cassava Viral Disease Cassava Transformation Cassava Virus Resistant Cassava Challenge Cassava Regeneration Control of the Cassava Geminiviruses by the G5 Strategy Inovirus Genome ssdna binding G5 Protein pcsvmv G5 original NOS WT-G5 Improved G5 Super G5 Gene Constructs Inovirus Cassava Mosaic Disease (CMD) Cassava Transformation Assessment of G5 SLCMV Resistance % Virus-Free Plants Control 2300 TMS30572 CsVMVg5n1/1 35Somegag5n1/1 35Somegag5n9/1 CsVMVldg5n24/1 Resistance to CMD Cassava Challenge Cassava Regeneration SLCMV G5 mediated resistance to CMV- Sri Lanka Strain The American Society for Cell Biology 13

14 G5 mediated resistance to CMV-Uganda G5 Current Status of Cassava/Geminivirus Projects Pre-commercial processes being implemented in all experiments Applications for confined field trial under review by IBCs in Uganda and Kenya (cv 60444) Transformation of local, farmer preferred cvs in progress using best gene constructs PROJECT DIRECTORS: Claude Fauquet and Nigel Taylor; with cast of many Research & Development of Transgenic Crops requires a long term commitment Research in the Lab RESEARCH Research in the Greenhouse Initial Field Trials PRODUCT DEVELOPMENT Additional Field & Regulatory Work COMMERCIAL- IZATION Breeding, Multiplication & Distribution 5 10 yrs 1 3 yrs 3-5 yrs 4 6 yrs 5 10 yrs BioCassava Plus Phase I Mycotoxin Control Project Phase I VIRCA Phase I Phase II VIRCA Phase II The American Society for Cell Biology 14

15 Strategies for Virus Disease Resistance: What will be effective in disease control in Africa, Asia and elsewhere? Pathogen Derived Resistance Protein Mediated Resistance Coat Protein Mediated Resistance Replicase Mediated Resistance Movement Protein Mediated Resistance RNA-mediated resistance srnas that target vrnas for degradation Non-Pathogen, Protein Mediated Resistance Transcription regulators: TFs, and szfps DNA binding proteins Interferon-like strategies; ds-rna degrading enzymes Translation initiation factors/co-factors Improving the Human Condition through Plant Science RTBV Project - Credits Danforth Center Shunhong Dai* Yi Liu Isabel Ordiz* Liping Pei* Jane Symington Xiaoping Wei* Zhihong Zhang Sarah Youngstrom TSRI Carlos Barbas III* Laurent Magenant PhilRice Antonio Alphonso TMV Project - Credits S. Asurmendi M. Soto-Aguilar M. Bendhamane T. Smith (collaborator) A. Bazzini T. Woodford-Thomas H. Berg (collaborator) T. Moravec J. Bick Support Provided by: M. Fujiki NIH, NSF, DDPSC S. Kawakami Fellowships from Gov ts M. Fujiki of Japan, Argentina The American Society for Cell Biology 15

16 Genetic Engineering for Virus Resistance in Plants The American Society for Cell Biology 16