molecular genetics of host-virus interactions
DESCRIPTION
This is one of my past work on host-virus interactions. If you want a clean copy, contact me to get one.TRANSCRIPT
MOLECULAR GENETIC ANALYSIS OF
HOST-VIRUS INTERACTIONS
(work done mid 1998 – early 2001)
Institute of Biological Chemistry
Washington State University
Pullman, WA
Suresh Gopalan, Ph.D
Based on last presentation at:
Prof. Frederick M. Ausubel Lab,
Department of Molecular Biology, MGH & Harvard Medical School
March, 2006
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.
GENETIC ANALYSIS OF PLANT SUSCEPTIBILITY TO
TOBACCO ETCH VIRUS
6
Translation and
proteolysis
HC-Pro NIa
121 88
P3 NIb Cap CI
P1 55
+NIa +NIa
P1 Pro P3 CI 6 NIa NIb Cap HC-
TOBACCO ETCH VIRUS (TEV)
(a positive strand RNA virus of picoRNA virus family)
SP6 HC- Pro P1 P3 CI 6 NIa NIb Cap
TEV vector
GFP, GUS
TEV-GFP, GUS
TEV-bar, P450
(E N L Y F Q S)
Recombinant TEV Genomes
bar, P450
NcoI ClaI MluI KpnI NIa site
Reporter Viruses
Selectable Viruses
C24 Col - 3 Non - inoculated tissue — 16 days p.i.
C24 Col - 3 Inoculated leaves — 3 days p.i.
Properties of restricted TEV movement phenotype
• Restricts TEV to inoculated leaves
• Specific to TEV
• Does not affect cell - to - cell movement
• No hypersensitive response
• No induction of systemic acquired resistance
• Not compromised in mutants deficient in
HR/SAR type resistance pathways
• C24/Col difference due to a single, dominant
locus, RTM1
• Additional mutants revealed restriction
mediated by multi-component system
TEV-bar positive selection
Location of RTM1, RTM2 and RTM3 Loci in the Arabidopsis Genome
II III
IV
V
AtGST2B
C425
CZSOD2
m366
mi390
m518
mi260
agp66
sah4
CTR1
CDPK9
CDR1
nlp
GSA1
g4523
ypm255
AT.LOX2A
nga707
ATEAT1
frohc
AIG1
g4026
mi462
I
RTM3
RTM1RTM2
One locus
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)
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
GENETIC ANALYSIS OF PLANT SUSCEPTIBILITY TO TEV
A multidirectional non-cell autonomous control conferred
by a novel genetic mechanism restricts Tobacco Etch Virus
susceptibility in Arabidopsis
Punch line title:
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)
HCPro
dsRNA
siRNA
dicer
RISC
systemic silencing
Adapted from Matzke et. al. Science (August 2001)
An
An
viral RdRp
RNA virus
cellular RdRp
aberrant RNA
PTGS/RNAi
degradation
p25
avrB
Selectable/Reporter Virus – to elicit HR
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
C24 ------------> R7402 -------------> Dead plants
EMS mutagenized --------------> R7402 ------> Surviving plants
C24/M2 (altered susceptibility
to herbicide/virus,
or escapes)
TEV-P450
TEV-P450
Schematic of TEV-P450 selection
Ecotype: C24
TEV-P450 Mock
+ R7402
TEV-P450/R7402 selection
High-throughput inoculation
EMS-mutagenized A. thaliana
Confirm putants by testing with TEV-GUS
in M3 generation
An early view of screen flat
B149 C24
TEV-P450/R7402 selection
TEV-P450 Mock TEV-P450 Mock
+ R7402 + R7402
24 days post R7402
1 dpi
2 dpi
3 dpi
8 dpi
16 dpi
18 dpi
Mock
TEV GUS
Dynamics of infection of TEV GUS in C24 plants
Rate of cell-cell movement of TEV-GUS in
inoculated leaves of B149 and C24
F
oci
dia
me
ter
(nu
mb
er
of
epid
erm
al c
ells
)
h
Data are from atleast 39 foci. P value for variation within each data
set was less than 0.001.
Time (h)
Foci
dia
mete
r
0
2
4
6
8
10
12
0 20 40 60 80 100 120
B149
C24
Time (h)
0
2
4
6
8
10
12
0 20 40 60 80 100 120
B149
C24
(nu
mber
of
epid
erm
al
cell
s)
Time (h)
Foci
dia
mete
r
0
2
4
6
8
10
12
0 20 40 60 80 100 120
B149
C24
0
2
4
6
8
10
12
0 20 40 60 80 100 120
B149
C24
Time (h)
0
2
4
6
8
10
12
0 20 40 60 80 100 120
B149
C24
(nu
mber
of
epid
erm
al
cell
s)
C24 - Mock
C24 - TEV GUS
B149 - Mock
B149 - TEV GUS
Development of infection foci of TEV-GUS 3 dpi
C24 - TEV GUS C24 - Mock
B149 - Mock
B149 - TEV GUS
Development of infection foci of TEV-GUS 4 dpi
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)
Development of infection foci on C24 and B14-9
infected with TEV-GUS
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
Development of infection foci of TEV-GUS 8 dpi
C24 - Mock
C24 - TEV GUS
B149 - Mock
B149 - TEV GUS
Development of infection foci of TEV-GUS 16 dpi
C24 - Mock
C24 - TEV GUS
B149 - Mock
B149 - TEV GUS
C24 B149
3 dpi
8 dpi
16 dpi
Development of infection foci on inoculated leaves of C24 and B149
Data from analysis of 10 plants
Systemic movement of TEV - GUS in B149, C24 and Col
0.1
1
10
100
12 dpi 18 dpi
Plant Genotype
GU
S a
cti
vit
y (
p
mo
l /min
/ m g
)
C24 Col B149 C24 Col B149
C24 B149
Susceptibility of B149 to TuMV - 10 dpi
Mock TuMV Mock TuMV
A BA B
Susceptibility of B149 to TCV - 9 dpi
C24 B149
Mock
TCV
Susceptibility of B149 to TCV - 11 dpi
B149 -
Mock
B149 -
TCV
C24 -
Mock
C24 -
TCV
C24 B149
3 dpi
8 dpi
16 dpi
Development of infection foci on inoculated leaves of C24 and B149 -
attempt to map phenotype
Exceptions:
When foci in contact with midrib or at the edges of leaves
(pictorial)
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)
Is the defect leaf specific?
Cover and fire – TEV-GUS Few days later
C24 B149
Is the defect leaf specific?
Genetic Background wt leaf movement
C24 10/10
B149 0/12
C1221 0/10
C1221 X B149 0/10
C15-8 0/10
C15-8 X B149 0/10
C13-3 5/6*
C13-3 X B149 10/10
C13-7 10/10
C13-7 X B149 9/9
C18-78 10/10
C18-78 X B149 10/10
C24 X B149 8/8
B149 X C24/F2#1 50/65
B149 X Ler/F2#2 71/96
C24 X Ler/F2 75/75
* the only plant with no foci did not have any good leaves at this stage, but had
GUS activity in systemic tissue confirming infection#1 2 = 0.042 for 3:1 seggregation#22 = 0.432 for 3:1 seggregation
Genetic analysis of complementation of lsp mutants by B149
* the only plant with no foci did not have any good leaves at this stage, but had
GUS activity in systemic tissue confirming infection#1 2 = 0.042 for 3:1 seggregation#22 = 0.432 for 3:1 seggregation
Genetic Background wt leaf movement
C24 10/10
B149 0/12
C1221 0/10
C1221 X B149 0/10
C15-8 0/10
C15-8 X B149 0/10
C13-3 5/6 *
C13-3 X B149 10/10
C13-7 10/10
C13-7 X B149 9/9
C18-78 10/10
C18-78 X B149 10/10
C24 X B149 8/8
B149 X C24/F2 #1 50/65
B149 X Ler/F2 #2 71/96
C24 X Ler/F2 75/75
lsp1
Impaired in
susceptibility
to TuMV and
TEV
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
0 63 120 183 STOP STOP
lsp1-1 lsp1-2 B149?
SPLICE SITE
219
EiF(iso)4E (protein)
THE FUN BEGINS HERE !!!!!!!
Suppressed leaf infectivity phenotype of B149
conferred by a novel genetic mechanism
THE FUN DOESN’T STOP THERE !!!!!
Genetic Background wt leaf movement
C24 10/10
B149 0/12
C1221 0/10
C1221 X B149 0/10
C15-8 0/10
C15-8 X B149 0/10
C13-3 5/6*
C13-3 X B149 10/10
C13-7 10/10
C13-7 X B149 9/9
C18-78 10/10
C18-78 X B149 10/10
C24 X B149 8/8
B149 X C24/F2#1 50/65
B149 X Ler/F2#2 71/96
C24 X Ler/F2 75/75
* the only plant with no foci did not have any good leaves at this stage, but had
GUS activity in systemic tissue confirming infection#1 2 = 0.042 for 3:1 seggregation#22 = 0.432 for 3:1 seggregation
Genetic analysis of complementation of lsp mutants by B149
* the only plant with no foci did not have any good leaves at this stage, but had
GUS activity in systemic tissue confirming infection#1 2 = 0.042 for 3:1 seggregation#22 = 0.432 for 3:1 seggregation
Genetic Background wt leaf movement
C24 10/10
B149 0/12
C1221 0/10
C1221 X B149 0/10
C15-8 0/10
C15-8 X B149 0/10
C13-3 5/6 *
C13-3 X B149 10/10
C13-7 10/10
C13-7 X B149 9/9
C18-78 10/10
C18-78 X B149 10/10
C24 X B149 8/8
B149 X C24/F2 #1 50/65
B149 X Ler/F2 #2 71/96
C24 X Ler/F2 75/75
lsp1
lsp1-3 – based on
TuMV phenotype
WHAT DO YOU SAY FOR THAT ?????
(work done at)
Dr. JAMES CARRINGTON Laboratory
Institute of Biological Chemistry
Washington State University
Pullman, WA
Andrew Lellis
Stephen Chisholm
Kristin Kasschau
Robert Anderberg
Greenhouse staff
Juliana Gothard
Craig Whitney
Susan Vogtman
STEVE WHITHAM
Sunita Mahajan
Other undergraduate
students of the laboratory