Intracellular highways in the plants: the role of the cytoskeleton in CaMV infections.

James Schoelz

42 Division of Plant Sciences Faculty 101 Graduate Students

34 MS67 PhD

Five Program Areas Crop, Soil and Pest ManagementEntomologyHorticulturePlant Stress BiologyPlant Breeding, Genetics and Genomics

Plant Sciences Biological SciencesBiochemistryComputer ScienceForestry ChemistryUSDA-ARS Plant Genetics Unit

The IPG --- who we are…• Established in 1981 to develop a program of excellence in

plant science• 58 faculty teams; recognized for interactive and cooperative

research and education community• 2 National Academy of Science members• 18 Fellows of the American Association for the Advancement

of Science (AAAS)

In 2010, MU was ranked 14th in the world among universities for plant science research (Times Higher Education)

Building on a strong history in plant genetics

INTERDISCIPLINARY PLANT GROUP

U N I V E R S I T Y O F M I S S O U R I

A RABIDOPSISC O L U M B I A W I L D T Y P E

PROF. GEORGE RÉDEI (1921-2008)

Older view of Inclusion Bodies:

P6 inclusion bodies are garbage dumps; they form after the protein has finished its specific task.

CaMV P6 Protein

Older view of Inclusion Bodies:

P6 inclusion bodies are garbage dumps; they form after the protein has finished its specific task.

New Interpretation:

In the space of a few days, the P6 protein is able to assemble into a “virion factory”. Cells initially contain many, small P6 inclusion bodies that utilize the cell’s cytoskeleton to move throughout the interior of the cell. They will eventually coalesce into a single inclusion the size of a chloroplast.

Central hypothesis – P6 inclusion bodies function to deliver virions to the plasmodemata for transport to adjacent cells

CaMV P6 Protein

What Every Plant Virus Must Do to Survive

R

(-)3'

(+)5'

5 ' (+)

cp cp cp

cp cp

(+ )5'

(- )

(+ )5'R

RR R R R

RR

5' (+)

Replicate Progeny

Genomes

Move to Other Plants

Uncoat the

Viral Genome

Express Proteins

needed for genome

Replication.

Enter the Cell

Produce proteins needed

for Virion formation and

Cell to cell MovementMove through

the plant

Assemble Virions

Cauliflower Mosaic virus

Source of the the 35S promoter

First plant virus genome to be sequenced – 1981

First plant virus to be cloned in infectious form – 1981

First viral avirulence gene i.e. a pathogen gene that triggers a plant defense response) to be characterized by recombinant DNA techniques – P6 - 1984

Genome Structure of Cauliflower Mosaic virus

Gene I - cell-to-cell movement

Gene II - aphid

transmission

Gene III - DNA binding;

aphid transmission

Gene IV - coat protein

V - reverse

transcriptase

VI - Translational

transactivator/Forms

inclusion bodies.

CaMV

8.0 kb

(Modified from Haas et al., 2002)

Turnip leaf epidermal

strip stained with

phloxine B illustrates

CaMV inclusions

Electron micrograph of a

turnip leaf cell infected with

CaMV

Viruses have developed Movement Proteins (MPs) to facilitate transport through plasmodesmata

Class I

Class IIVirions

Major Movement Classes : MP:RNA complexes & Virions

Tubules mediate cell-to-cell movement of virions of some

virusesTubule-like structures (aprox. 35 nm dia) containing a single row of virions are present in plants infected by CPMV.

See Pouwels et al., Mol Plant Path. 3:411 (2002).

Tubules Mediate Cell-to-cell Movement of Virions

Tubule-like structures (aprox. 35 nm dia) containing a single row of virions are present in plants infected by CPMV.

Movement proteins are responsible for tubule formation

See Pouwels et al., Mol Plant Path. 3:411 (2002).

Tubules Mediate Cell-to-cell Movement of Virions

Tubule-like structures (aprox. 35 nm dia) containing a single row of virions are present in plants infected by CPMV.

Movement proteins are responsible for tubule formation

Tubules containing virions have also been observed for:NepovirusesTospovirusesCaulimoviruses

See Pouwels et al., Mol Plant Path. 3:411 (2002).

Where are CaMV P6 inclusion bodies

found within the cell?

The Agroinfiltration Technique

1. Grow Agrobacterium tumefaciens

carrying the desired binary vector in 1.0

ml of broth overnight at 28 C in a

shaker/incubator.

2. Add acetosyringone, a signal produced by wounded plants that mobilizes the

transfer of the T-DNA of the binary vector into plant cells. Incubate overnight.

3. Fill a 5.0 ml syringe with Agrobacterium cells, apply the syringe to the

surface of a leaf, and gently infiltrate the solution into plant tissue.

Agroinfiltration of Gene VI of from different

CaMV strains

W260 Gene VI

D4 Gene VI

pW260VI rbcs35S

rbcs35SpD4VI

P6 inclusions are associated with actin

microfilaments, as well as ER and

microtubules.

P6-GFP

dsRed2-talin (labels thick

actin cables)

Co-agroinfiltrationof P6-GFP and

dsRed2-talin into N. benthamiana

and visualization of P6/actin

association.

Harries et al. (2009) The Cauliflower mosaic virus protein P6 forms motile inclusions that traffic

along actin microfilaments and stabilize microtubules. Plant Physiology 149, 1005-1016.

Phil Harries

P6 inclusion bodies can traffic along actin filaments.

Harries P et al. Plantphysiol 2009;149:1005-1016

©2009 by American Society of Plant Biologists

CaMV P6 inclusion bodies move on microfilaments

The actin inhibitor LatB inhibits CaMV infection.

Virions are assembled in the P6 inclusion bodies and most of the CaMV virions remain in the inclusion bodies.

P6 Inclusion Bodies as “Virion Factories”

Consequently, for CaMV virions to move from cell-to-cell there must be some mechanism for transfer of virions from the P6 inclusion body, the site for viral synthesis and virion assembly, to the plasmodesmata.

Nucleus

Model for P6 motility function

during CaMV infection

35S RNA

P6 protein

Microtubule

Microfilament

Virion

CaMV/P6

Inclusion

What host proteins interact with the P6

protein of CaMV?

What host proteins might potentially

facilitate intracellular movement to

plasmodesmata?

Previous yeast two-hybrid screens focused on

the role of P6 in translational transactivation and

silencing suppression

Eukaryotic translation factor 3 subunit g (eIF3g)(Park et al. 2001)

Ribosomal subunits L13, L18, and L24 (Leh et al., 2000)

RNA silencing protein DRB4 (Haas et al. 2008)

Interactions of P6 with virus and host proteins – Results of a Y2H Screen

Eighteen proteins identified in a total of 85 clones

Ribosomal initiation factor eIF3 ( represented in 17 independent clones)

Cytoskeleton-related

CHUP1 – interacts with actin for movement of chloroplasts on

microfilaments (represented in one clone)

Membrane Associations

AtSRC2.2 – C2 calcium-dependent membrane targeting protein

kinase C (represented in 17 independent clones)

CHUP1 is responsible for chloroplast movement within the cell in response to light intensity

Accumulation Avoidance

Low Light High Light

Redrawn from Wada (2013). Chloroplast movement. Plant Science 210, 177-182

Col-0 T-DNA line

129128C

White Band Assay

Analysis of Arabidopsis thaliana CHUP1 T-

DNA knockouts

Localization of CHUP1-CFP to the outer membrane of chloroplasts in N. benthamiana leaves

CHUP1-eCFP Chloroplasts

Merged

10 µm

Carlos Angel

Angel et al. 2013. The P6 protein of Cauliflower mosaic virus interacts with CHUP1, a plant protein which moves chloroplasts on actin microfilaments. Virology 443, 363-374.

Co-localization of CHUP1 with P6-Venus(YFP)

10 µm

MergedChloroplasts

CHUP1-eCFP P6-Venus(YFP)

Sample

CHUP1-GFP

P6-GFP

P6-RFP

+

-

-

-

+

-

-

-

+

+

-

+

-

+

+

72kDa

95kDa

72kDa

95kDa

TotalBlot: αGFP

TotalBlot: αRFP

72kDa

95kDa

Co-IP: αGFPBlot: αRFP

1 2 3 4 5

Sample

CHUP1-GFP

P6-GFP

P6-RFP

+

-

-

-

+

-

-

-

+

+

-

+

-

+

+

1 2 3 4 5

Co-Immunoprecipitation of P6-RFP with the truncated CHUP1-GFPCo-Immunopreciptation of P6-RFP with P6-GFP

Dominant Negative inhibition of chloroplast movement

Expression of a truncated CHUP1 protein consisting of

the first 500 amino acids, abolished the movement of

chloroplasts in Arabidopsis plants, acting as a

dominant negative inhibitor (Oikawa et al., 2008).

Question:

Since our CHUP1-CFP construct consists of the 436 first

amino acids, can we block the movement of P6 –

Venus YFP inclusion bodies?

Time lapse imaging of P6- Venus YFP inclusion bodies

Time lapse imaging of AtCHUP-eCFP and P6- Venus YFP inclusion bodies

(Angel et al., 2012, submitted)

Question

What effect does the loss of CHUP1 have on CaMV

infections?

Experimental Design for Chup1 VIGS in N. edwardsonii

and inoculation of CaMV virions

CaMV virions inoculation21 days

5-6 week old plants

CaMV HRlocal lesions development

The Hypersensitive Response (HR – programmed cell death) in N. edwardsonii.

Necrotic local lesions induced by CaMV strain W260 in inoculated leaves. This virus is

unable to move systemically because N. edwardsonii mounts an effective defense.

Question:

Will lesion number or size be affected in

N. edwardsonii CHUP1 silenced plants?

Percentage of necrotic lesion development in N. edwardsonii : lesions

develop at a slower rate in CHUP1- silenced plants than in controls

(ANOVA, p=0.01).

The Association of CHUP1 with CaMV P6

Confirmed with Y2H, co-IP and co-

localization in vivo

Silencing of CHUP1 in N. benthamiana

significantly slows the rate of CaMV lesion

development.

Transient expression of a truncated CHUP1

blocks the movement of chloroplasts (Oikawa

et al. 2003) and P6 inclusion bodies (Angel

et al. 2013).

Nucleus

P6 protein

Microtubule

Microfilament

Virion

CaMV/P6

Inclusion

CHUP1 – May explain the interaction of P6 inclusion bodies with microfilaments, and participates in movement of P6 inclusion bodies on microfilaments.

CHUP1

Angel et al. 2013. The P6 protein of Cauliflower mosaic virus interacts with CHUP1, a plant protein which moves chloroplasts on actin microfilaments. Virology 443, 363-374.

Question

Are CaMV P6 inclusion bodies associated with

plasmodesmata?

Andres Rodriguez

Interactions of P6 with virus and host proteins – Results of a Y2H Screen

Eighteen proteins identified in a total of 85 clones

Ribosomal initiation factor eIF3 ( represented in 17 independent clones)

Cytoskeleton-related proteins

CHUP1 – interacts with actin for movement of chloroplasts on

microfilaments (represented in one clone)

Membrane Associations

AtSRC2.2 – C2 calcium-dependent membrane targeting protein

kinase C (represented in 17 independent clones)

SRC2.2 = Soybean Response to Cold

Three experimental techniques indicate an association

of AtSRC2.2 with CaMV P6

Y2H

Co-IP

Co-localization in vivo

Where is AtSRC2.2 found in the cell?

AtSCR2.2-RFP PDLP1-GFP

Cell wall

Plasma membrane

Overlay

Plasmolyzed cell

A portion of AtSRC2.2-RFP co-localizes with the plasmodesmal protein PDLP1

Co-agroinfiltration of PDLP1-GFP with AtSCR2.2-RFP into N. benthamiana leaves

AtSCR2.2-RFPP1-GFP

Overlay

A portion of AtSRC2.2-RFP co-localizes with CaMV P1-GFP

CaMV P1 (the CaMV movement protein) is localized to plasmodesmata

Co-agroinfiltration of P1-GFP with AtSRC2.2-RFP into N. benthamiana leaves

P1-GFP P1-GFP + untagged P1

Formation of tubules is likely carried out by untagged P1. P1-GFP allows for the visualization of the tubules.

Conti G.G., Vegetti G., Bassi M., Favali M.A. 1972. Some Ultrastructural and CytochemicalObservations on Chinese Cabbage Leaves Infected with Cauliflower Mosaic Virus. Virology 47, 697-700.

Longitudinal view

Transversal view

Conti G.G., Vegetti G., Bassi M., Favali M.A. 1972. Some Ultrastructural and CytochemicalObservations on Chinese Cabbage Leaves Infected with Cauliflower Mosaic Virus. Virology 47, 697-700.

AtSRC2.2 was co-localized with 43.1% of P1 tubules, with C2CDMT found at the base (80%) or within the tubule (20%).

AtSRC2.2-RFP MergedP1-GFP + Untagged P1

Question

Are P6 inclusion bodies associated with

plasmodesmata?

PDLP1-GFP P6-RFP MERGED

Some P6 inclusion bodies are found adjacent to plasmodesmal markers

Co-agroinfiltration of P6-RFP with PDLP1-GFP into N. benthamiana leaves

PDLP1-GFP P6-RFP MERGED

Aniline Blue P6-RFP MERGED

Thomas et al, 2008. Use of Aniline Blue as plasmodesmal marker

Some P6 inclusion bodies are found adjacent to plasmodesmal markers

P6

P1AtSRC2.2

Y2HCo-localization

in vivoCo-IP

Co-localization in vivo

Y2H (Hapiak et al, 2008)Co-localizationin vivo

Nucleus

P6 protein

Microtubule

Microfilament

Virion

CaMV/P6

Inclusion

CaMV P6 is capable of forming a complex with CaMV P1 protein, AtSRC2.2, PDLP1 at

plasmodesmata

CaMV MP (P1)

AtSRC2.2

PDLP1

Plasmodesma modified

into tubule by CaMV P1

Protein

A new function for CaMV P6: delivery of virions to

plasmodesmata

P6 inclusion bodies are considered the site for virion assembly and

accumulation

P6 inclusion bodies associate with and move on microfilaments

P6 inclusion body movement on microfilaments may be facilitated through an

interaction with CHUP1 and one or more myosins

At least a portion of P6 inclusion bodies are associated with

plasmodesmata

P6 appears to be capable to form a complex at the plasmodesmata with

CaMV P1 (the cell-to-cell movement protein), AtSRC2.2, and PDLP1.

Exactly what is the function of the CaMV P6 protein?

1. Is the matrix protein for the vacuolated, amorphous inclusion bodies: forms “virion

factories”

2. Avirulence gene product and symptom determinant

3. Translational transactivator: facilitates the translation of viral genes on the

polycistronic 35S RNA

4. A shuttle protein, moving between the cytoplasm and nucleus

5. A silencing suppressor

6. Regulates SA-mediated defenses and cell death pathways

7. Delivery of virions to plasmodesmata

Exactly what is the function of the CaMV P6 protein?

The “function” of P6 may be its capacity to interact with a broad range of host proteins.

The capacity to form indiscriminate associations with host proteins might be considered a survival mechanism to take advantage of whatever a host has to offer.

In this hypothesis, the composition of host proteins in the P6 inclusion bodies might be dynamic, constantly changing as the infection within the cell matures.

In

AcknowledgmentsSchoelz Lab (current and former members) Nelson Lab (current and former members)

Dr. Carlos Angel Dr. Philip Harries

Dr. Boovaraghan Balaji Dr. Xiaohua Yang

Dr. Andres Rodriguez Dr. Xin S. Ding

Yu Zhang Dr. Malay Saha

Sandra Valdes Bethany Bishop

Mohammad Fereidouni

Mustafa Adhab

Adam Adair

Dr. Scott M. Leisner and Lindy Lutz. (The University of Toledo, Toledo OH).

Dr. Michael Goodin and Kathleen Martin. (University of Kentucky, Lexington KY)

Dr. Aleksandr Jurkevic. (MU Molecular Cytology and Microscopy Core)

Dr. Howard Berg. (Cytology Core, The Donald W. Danforth Center, St. Louis MO).

Genome structures of plant viruses are

deceptively simple

Gene I - cell-to-cell movement

Gene II - aphid

transmission

Gene III - DNA binding;

aphid transmission

Gene IV - coat protein

V - reverse

transcriptase

VI – Translational

transactivator/Forms

inclusion bodies.

CaMV

8.0 kb

(Modified from Haas et al., 2002)

D1 D4D3D2

Host Range,

Symptoms & P6

Self AssociationMini TAV

RNA

Binding

Zinc

Finger

P6 Self Association

Chloroplast

Interaction

Leucine

zipper

Proline-rich,

interacts with

Profilin/Actin Leucine zipper

Coiled-coil Actin binding

CaMV P6 binding

1004 aa

CHUP1 structure

P6 structure CHUP1

binding

What domains are responsible for the interaction of

CHUP1 and CaMV P6?

(Modified from Wada and Suetsugu 2004)

P6

Is there any relationship between CaMV P6 and

chloroplast movement?

P6-interacting region10 218

360 aa

C2 Domain

6 112 189

Pro-Rich region

320

Cho & Stahelin, 2006

C2 domain is the second most abundant lipid binding domain.

C2-domain-calcium dependent proteins are involved in signal transduction and membrane trafficking.

P6-GFP C2CDMT-RFP Overlay

P6-GFP - - + +

Initial ExtractBlot: αRFP

Co-IP: αGFPBlot: αRFP

Co-IP: αGFPBlot: αGFP

C2CDMT-RFP - + - +

Initial ExtractBlot: αGFP

70KDa

90KDa

70 kDa

90 kDa

1 2 3 4

Sample

B

A

C

D

C2CDMT-RFP is co-immunoprecipitated with P6-GFP

Three experimental techniques indicate an association

of C2CDMT with CaMV P6

Y2H

Co-IP

Co-localization in vivo

Where is C2CDMT found in the cell?

Question

Do myosins contribute to intracellular movement of

CaMV?

Andres Rodriguez

Carlos Angel

Yu Zhang

(Peremyslov et al., 2011)

Arabidopsis Myosins implicated in plant virus

movement through T-DNA knockouts or silencing

Development of CaMV infections in an A. thaliana chup1 T-

DNA knockout line versus wild type Col-0

Symptoms in the AtCHUP1 T-DNA knockout line were delayed, but CaMV

systemic symptom development was not abolished.

Days Post Inoculation (dpi)P

erc

en

tage o

f P

lan

ts w

ith

Syste

mic

Sym

pto

ms

0

10

20

30

40

50

60

70

80

9 10 11 12 13

Col-0

CHUP1

0

10

20

30

40

50

60

70

80

90

100

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Col-0

CHUP

Days Post Inoculation (dpi)

Pe

rcen

tage o

f P

lan

ts w

ith

Lo

ca

l L

esio

ns

0

10

20

30

40

50

60

70

80

90

100

7 8 9 10 11 12 13

Col-0

XI-20

20

40

60

80

100

120

10 11 12 13 14 15 16 17 18

Col

XI-2

Development of CaMV infections in an A. thaliana myosin XI-

2 T-DNA knockout line versus wild type Col-0

Pe

rcen

tage o

f P

lan

ts w

ith

Lo

ca

l L

esio

ns

Days Post Inoculation (dpi)Days Post Inoculation (dpi)P

erc

en

tage o

f P

lan

ts w

ith

Syste

mic

Sym

pto

ms

Primary lesion development in the myosin XI-2 T-DNA knockout line was

consistently delayed by one day, but CaMV symptom development was not

abolished.

Development of CaMV infections in the A. thaliana XI-2/chup1 double T-

DNA knockout line versus wild type Col-0

Pe

rcen

tage o

f P

lan

ts w

ith

Lo

ca

l L

esio

ns

Days Post Inoculation (dpi)Days Post Inoculation (dpi)P

erc

en

tage o

f P

lan

ts w

ith

Syste

mic

Sym

pto

ms

Primary lesion development in the double T-DNA knockout line was consistently

delayed by one day, and CaMV systemic symptom development was delayed by

one to three days.

0

10

20

30

40

50

60

70

80

90

100

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Col-0

CHUP/XI-2

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11

Col-0

CHUP/XI-2

PDLP1-GFP - - + +

Initial ExtractBlot: αRFP

Co-IP: αGFPBlot: αRFP

Co-IP: αGFPBlot: αGFP

P6-RFP - + - +

Initial ExtractBlot: αGFP

60KDa

90KDa

90 kDa

60 kDa

1 2 3 4

Sample

B

A

C

D

P6-RFP is co-immunoprecipitated with PDLP1-GFP

Heavy chain IgG

Development of CaMV infections in a Atsrc2.2 A. thaliana T-DNA

knockout line and in the triple T-DNA knockout pdlp123

Two tests were completed to compare the susceptibility of a Atsrc2.2 T-

DNA knockout line to wild type Col-0. There was no difference in the

timing of local or systemic symptoms.

By Contrast

A delay in CaMV infections was observed in the triple T-DNA knockout

pdlp123. However, even with the triple knockout line, CaMV

infections were not abolished (Amari et al., 2010).

Amari et al. 2010. A family of plasmodesmal proteins with receptor-like properties for plant viral

movement proteins. PLoS Pathogens 6 (9) e1001119 1-10.

100

A

Percentage of necrotic lesion development in N. edwardsonii : lesions

develop at a slower rate in CHUP1- silenced plants than in controls

(ANOVA, p=0.01).

Development of Local Lesions by CaMV in N. edwardsoniiand in CHUP1-Silenced Plants

Number of HR lesions per leaf was not statistically different (ANOVA, p=0.05)

Lesi

on

Nu

mb

ers

per

Lea

f