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