Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Molecular Genetics of Host-Virus Interactions

This is one of my past work on host-virus interactions. If you want a clean copy, contact me to get one.

  • Login to see the comments

  • Be the first to like this

Molecular Genetics of Host-Virus Interactions

  1. 1. MOLECULAR GENETIC ANALYSIS OF HOST-VIRUS INTERACTIONS Suresh Gopalan, Ph.D (work done mid 1998 – early 2001) Institute of Biological Chemistry Washington State University Pullman, WA Based on last presentation at: Prof. Frederick M. Ausubel Lab, Department of Molecular Biology, MGH & Harvard Medical School March, 2006
  2. 2. Significance Accomplishments: 1. Identified novel themes host susceptibility and immunity to viral pathogens, using a single stranded RNA virus. 2. Identified novel mutants using high-throughput screening and molecular genetic analysis. 3. Demonstrated mutli-factorial interactions (and genetic loci) affecting local and systemic responses of host to viral pathogens. 4. Developed several hypothesis using above, later proved correct. Practical Significance: Engineering/manipulating disease and resistance mechanisms applicable to a variety of host-pathogen interactions. Tools for the study of multi-pathogen infections, and interactions between different immune responses.
  3. 3. GENETIC ANALYSIS OF PLANT SUSCEPTIBILITY TO TOBACCO ETCH VIRUS
  4. 4. TOBACCO ETCH VIRUS (TEV) (a positive strand RNA virus of picoRNA virus family) P1 HCPro P3 CI 6 NIa NIb Cap Translation and proteolysis P1 55 121 88 HC-Pro 6 +NIa P3 NIa +NIa CI NIb Cap
  5. 5. Recombinant TEV Genomes SP6 P1 HCPro P3 CI 6 NIa NIb Cap TEV vector NcoI ClaI MluI KpnI NIa site (E N L Y F Q S) Reporter Viruses GFP, GUS TEV-GFP, GUS Selectable Viruses bar, P450 TEV-bar, P450
  6. 6. Properties of restricted TEV movement phenotype • • • • • • Restricts TEV to inoculated leaves • C24/Col difference due to a single, dominant locus,RTM1 • Additional mutants revealed restriction mediated by multi-component system Specific to TEV Does not affect cell -cell movement -to No hypersensitive response No induction of systemic acquired resistance C24 Col-3 Non-inoculated tissue 16 days p.i. — Not compromised in mutants deficient in HR/SAR type resistance pathways C24 Col-3 Inoculated leaves 3 days p.i. —
  7. 7. TEV-bar positive selection
  8. 8. Location of RTM1, RTM2 and RTM3 Loci in the Arabidopsis Genome ATEAT1 AtGST2B RTM1 frohc mi390 g4523 C425 CTR1 RTM2 m518 ypm255 CDPK9 AIG1 mi260 AT.LOX2A CZSOD2 CDR1 agp66 nga707 m366 g4026 RTM3 nlp mi462 II sah4 III IV GSA1 I One locus V
  9. 9. RTM1: Similar to lectin jacalin and related proteins with one/more jacalin repeats RTM2: N-terminal region with similarity to small HSPs, a-crystalline domain. C-terminal extension no similarity to known protein/domains RTM3: Cloned (contributor on that project)
  10. 10. Advantages of TEV-Arabidopsis pathosystem 1. Ability of TEV to tolerate insertion still retain infectivity (i.e., Availability of reporter viruses (GUS, GFP etc.) and selectable viruses (bar, P450) 2. Lack of any obvious infection phenotype 3. High throughput inoculation technique 4. Tools available and being developed for Arabidopsis research
  11. 11. GENETIC ANALYSIS OF PLANT SUSCEPTIBILITY TO TEV Punch line title: A multidirectional non-cell autonomous control conferred by a novel genetic mechanism restricts Tobacco Etch Virus susceptibility in Arabidopsis
  12. 12. Components that could be identified by an altered susceptibility screen Necessary/accessory host factors for: 1. Replication/translation/assembly 2. Cell-Cell movement in inoculated leaves 3. Long-distance movement a. Entry into/exit from vasculature b. Transport through phloem 4. Re-establishing infection in systemic tissue 5. Other compatibility factors Components of defense pathway(s): 1. Constitutive activation of defense responses 2. Target/accessory factors of viral encoded suppressors of silencing and other defense responses (e.g., HC-Pro has been demonstrated to suppress silencing in Nicotiana plants)
  13. 13. PTGS/RNAi RNA virus viral RdRp dsRNA HCPro dicer systemic silencing p25 siRNA RISC An cellular RdRp An aberrant RNA degradation Adapted from Matzke et. al. Science (August 2001)
  14. 14. Selectable/Reporter Virus – to elicit HR avrB AvrB is an effector from Pseudomonas syringae (delivered through the type III secretion system) that causes a rapid programmed death of host cell in plants that have the corresponding R gene and other signaling components
  15. 15. Schematic of TEV-P450 selection C24 TEV-P450 R7402 -------------> Dead plants ------------> TEV-P450 EMS mutagenized --------------> R7402 ------> Surviving plants C24/M2 (altered susceptibility to herbicide/virus, or escapes)
  16. 16. TEV-P450/R7402 selection TEV-P450 Mock + R7402 Ecotype: C24
  17. 17. High-throughput inoculation
  18. 18. EMS-mutagenized A. thaliana
  19. 19. An early view of screen flat Confirm putants by testing with TEV-GUS in M3 generation
  20. 20. TEV-P450/R7402 selection B149 TEV-P450 C24 Mock TEV-P450 + R7402 Mock + R7402 24 days post R7402
  21. 21. Dynamics of infection of TEV GUS in C24 plants 1 dpi 16 dpi 3 dpi Mock TEV GUS 2 dpi 8 dpi 18 dpi
  22. 22. Rate of cell-cell movement of TEV-GUS in inoculated leaves of B149 and C24 diameter FociFoci diame ter (number of epide rmal cells) (number of epidermal cells) 12 12 B149 B149 C24 C24 10 10 8 6 4 2 h 0 0 0 20 20 40 40 60 60 80 80 100 100 120 120 Time (h) Time (h) Data are from atleast 39 foci. P value for variation within each data set was less than 0.001.
  23. 23. Development of infection foci of TEV-GUS 3 dpi C24 - Mock C24 - TEV GUS B149 - Mock B149 - TEV GUS
  24. 24. Development of infection foci of TEV-GUS 4 dpi C24 - TEV GUS C24 - Mock B149 - Mock B149 - TEV GUS
  25. 25. Development of infection foci on C24 and B14-9 infected with TEV-GUS Foci/plant C24# B14-9# P Experiment 1 343.4 (5) 12.05 (18) 9e-11 Experiment 2 93.4 (5) 4.9 (10) 2.4e-6 #Average (number of samples) Experiment 1: 3dpi; Experiment 2: 4 dpi Average plant weight (29 day old plants) during Experiment 1: B149: 0.67; C24:1.17. P = 3.8e-6
  26. 26. Development of infection foci of TEV-GUS 8 dpi C24 - Mock C24 - TEV GUS B149 - Mock B149 - TEV GUS
  27. 27. Development of infection foci of TEV-GUS 16 dpi C24 - Mock C24 - TEV GUS B149 - Mock B149 - TEV GUS
  28. 28. Development of infection foci on inoculated leaves of C24 and B149 C24 B149 3 dpi 8 dpi 16 dpi
  29. 29. Systemic movement of TEV -GUS in B149, C24 and Col 12 dpi 18 dpi ( /min/mg) GUS activitypmol 100 10 1 0.1 C24 Col B149 C24 Col B149 Plant Genotype Data from analysis of 10 plants
  30. 30. Susceptibility of B149 to TuMV - 10 dpi C24 B149 A B Mock TuMV Mock TuMV
  31. 31. Susceptibility of B149 to TCV - 9 dpi B149 C24 Mock TCV
  32. 32. Susceptibility of B149 to TCV - 11 dpi B149 Mock C24 Mock B149 TCV C24 TCV
  33. 33. Development of infection foci on inoculated leaves of C24 and B1 attempt to map phenotype C24 B149 3 dpi 8 dpi 16 dpi
  34. 34. Exceptions: When foci in contact with midrib or at the edges of leaves (pictorial)
  35. 35. 1. RESTRICTION IS NOT UNIFORM IN ALL CELL TYPES 2. A MULTI-DIRECTIONAL NON-CELL AUTONOMOUS CONTROL • Emanating from the infected cell and moving outside (i.e., prime-ahead mechanism) 2. Converging from many layers of outer cells towards foci (the strength of restriction proportional to layers contributing to restriction)
  36. 36. Is the defect leaf specific?
  37. 37. Is the defect leaf specific? C24 Cover and fire – TEV-GUS B149 Few days later
  38. 38. Genetic analysis of complementation of lsp mutants by B149 Genetic Background Genetic Background wt leaf movement C24 C24 10/10 B149 B149 0/12 C1221 C1221 0/10 C1221 X B149 0/10 C1221 X B149 C15-8 C15-8 0/10 C15-8 X C15-8 X B149B149 0/10 C13-3 C13-3 5/6* C13-3 X B149B149 10/10 C13-3 X C13-7 10/10 C13-7 C13-7 X B149B149 9/9 C13-7 X C18-78 10/10 C18-78 C18-78 X B149B149 10/10 C18-78 X C24 C24 X B149 X B149 8/8 wt leaf movement 10/10 0/12 0/10 0/10 0/10 0/10 5/6 * 10/10 10/10 9/9 10/10 10/10 8/8 B149 X C24/F2#1 B149 X C24/F2 50/65 #1 B149 X Ler/F2#2 B149 X Ler/F2 71/96#2 C24 C24 X Ler/F2 75/75 X Ler/F2 50/65 71/96 75/75 * lsp1 Impaired in susceptibility to TuMV and TEV the only plant with no foci did not have any good leaves at this stage, but had the only plant with no foci did not have any good leaves at this stage, but had GUS activity in systemic tissue confirming infection GUS=activityfor 3:1 seggregation confirming infection #1  2 0.042 in systemic tissue #1 2 #2 2 = 0.042 for 3:1 seggregation  = 0.432 for 3:1 seggregation *  2 = 0.432 for 3:1 seggregation #2
  39. 39. 1. B149 IS A PERFECT PHENOTYPIC ALLELE OF lsp1 MUTANT IMPAIRED IN SUSCEPTIBILITY TO TuMV AND TEV 2. B149 PHENOTYPE IS CONFERRED BY A MONOGENIC RECESSIVE LOCUS 3. B149 HAS A LESION IN THE SAME GENE CONFERRING lsp1 PHENOTYPE lsp1-1 STOP 0 lsp1-2 STOP 63 120 B149? 183 219 EiF(iso)4E (protein) SPLICE SITE
  40. 40. THE FUN BEGINS HERE !!!!!!!
  41. 41. Suppressed leaf infectivity phenotype of B149 conferred by a novel genetic mechanism
  42. 42. THE FUN DOESN’T STOP THERE !!!!!
  43. 43. Genetic analysis of complementation of lsp mutants by B149 Genetic Background Genetic Background wt leaf movement C24 C24 10/10 B149 B149 0/12 C1221 C1221 0/10 C1221 X B149 0/10 C1221 X B149 C15-8 C15-8 0/10 C15-8 X C15-8 X B149B149 0/10 C13-3 C13-3 5/6* C13-3 X B149B149 10/10 C13-3 X C13-7 10/10 C13-7 C13-7 X B149B149 9/9 C13-7 X C18-78 10/10 C18-78 C18-78 X B149B149 10/10 C18-78 X C24 C24 X B149 X B149 8/8 wt leaf movement 10/10 0/12 0/10 0/10 0/10 0/10 5/6 * 10/10 10/10 9/9 10/10 10/10 8/8 B149 X C24/F2#1 B149 X C24/F2 50/65 #1 B149 X Ler/F2#2 B149 X Ler/F2 71/96#2 C24 C24 X Ler/F2 75/75 X Ler/F2 50/65 71/96 75/75 * lsp1 lsp1-3 – based on TuMV phenotype the only plant with no foci did not have any good leaves at this stage, but had the only plant with no foci did not have any good leaves at this stage, but had GUS activity in systemic tissue confirming infection GUS=activityfor 3:1 seggregation confirming infection #1  2 0.042 in systemic tissue #1 2 #2 2 = 0.042 for 3:1 seggregation  = 0.432 for 3:1 seggregation *  2 = 0.432 for 3:1 seggregation #2
  44. 44. WHAT DO YOU SAY FOR THAT ?????
  45. 45. (work done at) Dr. JAMES CARRINGTON Laboratory Institute of Biological Chemistry Washington State University Pullman, WA STEVE WHITHAM Sunita Mahajan Andrew Lellis Stephen Chisholm Other undergraduate Kristin Kasschau students of the laboratory Robert Anderberg Greenhouse staff Juliana Gothard Craig Whitney Susan Vogtman

×