Bacterium-induced basal resistance inhibits viral infection

in tobacco plants

László ZSIROS1,2, Ágnes SZATMÁRI1,

László PALKOVICS2, Zoltán KLEMENT1 Zoltán BOZSÓ1

1Plant Protection Institute of the HAS2 Corvinus University of Budapest Dept. of Plant Pathology

Main types of resistance in plants

Preformed Microorganism-induced

Hypersensitive response(HR)

Basal resistance(BR)

Early basal resistance(EBR)

Late basal resistance (LBR)

Comparison of basal and hypersensitive defense

Type of resistance Hypersensitive Basal

Plant-bacterium interaction

Specific Non-specific

Inducer bacterium Metabolically active pathogen

Pathogen, saprotroph, heat killed, etc.

Symptom Cell necrosis Symptomless

(detection with superinfection)

Induction of basal resistance (BR) and hypersensitive response (HR)

Plant cell

Basal resistance

Flagellin, LPSAvr proteins

Hypersensitive response

Intercellular space

Tobacco mosaic virus (TMV)

• Resistance in tobacco• HR – 78 hours• N gene

• Plant-TMV interaction• incompatible (HR)• compatible (mosaic leaves)

Changes at cell level during basal resistance (BR)

• change in pH level

• cell wall fortification

• papilla formation

• active oxygen forms (eg. hydrogen peroxide)

• bacterium envelopment

Aims of research

• Does a bacterial infection impede the symptoms/HR of a viral superinfection?

• Is symptom/HR inhibition attached to bated virus replication?

• How do some defense-related genes change in their transcription activity?

Materials and methods

• Plants– N. tabacum L. ‘Samsun’ nn (compatible)– N. tabacum L. ‘Xanthi’ NN (incompatible)

• Bacterial inoculation– Pseudomonas syringae pv. syringae hrcC mutant

• Viral inoculation– TMV

• Virus quantity and gene activity assay– Real-Time PCR

Inoculation method to assay early basal resistance (EBR)

Bacterial inoculationWater infiltration (mock) Viral inoculation

6 hours

TMV

HR-inhibition assay(‘Xanthi’ NN plant, incompatible tobacco-TMV interaction)

Bacterium (hrcC) + TMV

Water + TMV

Positive control (TMV only)

HR-inhibition assay(‘Xanthi’ NN plant, incombatible tobacco – TMV interaction)

Plant Pre-treatment Number of lesions

Size of lesions

Positive control

(TMV only)

- 228 2-3 mm

Early basal resistance

Water 188 2-3 mm

Bacterium 67 1 mm

Average results of repeated treatments

Inhibition of virus replication(‘Xanthi’ NN plant, incombatible tobacco – TMV interaction)

Representative results of repeated treatments

hrcC = Pseudomonas syringae pv. syringae hrcC mutáns

0.01

0.1

1

10

100

1000

0 1 2 3 4

days post TMV inoculation

Vir

us

qu

anti

ty a

s co

mp

ared

to

to

bac

co

acti

n e

xpre

ssio

n l

evel

TMV

Water + TMV

hrcC + TMV

Defense-related genes (‘Xanthi’ NN plant, incompatible tobacco – TMV interaction)

• Epoxide hydrolase – epoxide neutralization

• Ortho-methyl trasferase – cell wall synthesis

• Superoxide dismutase – superoxide degradation

Defense-related genes 1 (‘Xanthi’ NN plant, incompatible tobacco – TMV interaction)

Epoxide hydrolase

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4

days post TMV inoculation

Gen

e ex

pre

ssio

n a

s co

mp

ared

to

u

ntr

eate

d c

on

tro

l

TMV

Water + TMV

hrcC + TMV

Representative results of repeated treatments

hrcC = Pseudomonas syringae pv. syringae hrcC mutant

Defense-related genes 2 (‘Xanthi’ NN plant, incompatible tobacco – TMV interaction)

Representative results of repeated treatments

hrcC = Pseudomonas syringae pv. syringae hrcC mutant

Ortho-methyl transferase

0

5

10

15

20

25

30

0 1 2 3 4

days post TMV inoculation

Gen

e ex

pre

ssio

n a

s co

mp

ared

to

u

ntr

eate

d c

on

tro

l

TMV

Water + TMV

hrcC + TMV

Defense-related genes 3 (‘Xanthi’ NN plant, incompatible tobacco – TMV interaction)

Representative results of repeated treatments

hrcC = Pseudomonas syringae pv. syringae hrcC mutant

Superoxide dismutase

0

1

2

3

4

5

6

7

8

0 1 2 3 4

days post TMV inoculation

Gen

e ex

pre

ssio

n a

s co

mp

ared

to

u

ntr

eate

d c

on

tro

l

TMV

Water + TMV

hrcC + TMV

Symptom inhibition of EBR(‘Samsun’ nn plant, compatible tobacco – TMV interaction)

Water + TMV Bacterium + TMV TMV

Inhibition of virus replication(‘Samsun’ nn plant, compatible tobacco – TMV interaction)

Representative results of repeated treatments

hrcC = Pseudomonas syringae pv. syringae hrcC mutant

0.001

0.01

0.1

1

10

100

1000

10000

100000

1000000

0 1 2 3 4

days post TMV inoculation

Vir

us

qu

anti

ty a

s co

mp

ared

to

to

bac

co

acti

n e

xpre

ssio

n l

evel

TMV

Water + TMV

hrcC + TMV

Summary

• Related to EBR, there was inhibition observed in

– mosaic leaf symptom development;

– virus-induced HR development;

– the rate of virus replication;

– change in activity of some defense-related genes

Conclusions

• Possible reasons for HR or mosaic symptom inhibition during basal resistance:

– blocking of virus translocation in plant

– inhibition of virus replication (RNA silencing? priming?)

– non-specific defense

– hydrogen peroxide symptoms