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

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. 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. GENETIC ANALYSIS OF PLANT SUSCEPTIBILITY TO
TOBACCO ETCH VIRUS

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. 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

7. 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.
—

8. TEV-bar positive selection

9. 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

10. 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)

11. 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

12. 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

13. 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)

14. 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)

15. 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

16. 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)

18. TEV-P450/R7402 selection
TEV-P450
Mock
+ R7402
Ecotype: C24

19. High-throughput inoculation

20. EMS-mutagenized A. thaliana

21. An early view of screen flat
Confirm putants by testing with TEV-GUS
in M3 generation

22. TEV-P450/R7402 selection
B149
TEV-P450
C24
Mock
TEV-P450
+ R7402
Mock
+ R7402
24 days post R7402

24. Dynamics of infection of TEV GUS in C24 plants
1 dpi
16 dpi
3 dpi
Mock
TEV GUS
2 dpi
8 dpi
18 dpi

25. 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.

26. Development of infection foci of TEV-GUS 3 dpi
C24 - Mock
C24 - TEV GUS
B149 - Mock
B149 - TEV GUS

27. Development of infection foci of TEV-GUS 4 dpi
C24 - TEV GUS C24 - Mock
B149 - Mock
B149 - TEV GUS

28. 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

29. Development of infection foci of TEV-GUS 8 dpi
C24 - Mock
C24 - TEV GUS
B149 - Mock
B149 - TEV GUS

30. Development of infection foci of TEV-GUS 16 dpi
C24 - Mock
C24 - TEV GUS
B149 - Mock
B149 - TEV GUS

31. Development of infection foci on inoculated leaves of C24 and B149
C24
B149
3 dpi
8 dpi
16 dpi

32. 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

33. Susceptibility of B149 to TuMV - 10 dpi
C24
B149
A
B
Mock
TuMV
Mock
TuMV

34. Susceptibility of B149 to TCV - 9 dpi
B149
C24
Mock
TCV

35. Susceptibility of B149 to TCV - 11 dpi
B149 Mock
C24 Mock
B149 TCV
C24 TCV

36. Development of infection foci on inoculated leaves of C24 and B1
attempt to map phenotype
C24
B149
3 dpi
8 dpi
16 dpi

37. Exceptions:
When foci in contact with midrib or at the edges of leaves
(pictorial)

38. 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)

39. Is the defect leaf specific?

40. Is the defect leaf specific?
C24
Cover and fire – TEV-GUS
B149
Few days later

42. 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

43. 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

44. THE FUN BEGINS HERE !!!!!!!

45. Suppressed leaf infectivity phenotype of B149
conferred by a novel genetic mechanism

46. THE FUN DOESN’T STOP THERE !!!!!

47. 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

48. WHAT DO YOU SAY FOR THAT ?????

49. (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