Expression profiles of the late blight resistance gene Rpi-phu1

Śliwka J, Świątek M, Tomczyńska I, Stefańczyk E, Chmielarz M & Zimnoch-Guzowska E

Plant Breeding and Acclimatization Institute – National Research Institute, Młochów, Poland

Rpi-mcq1

Rpi-edn2

Rpi-dlc1

R8

I

VIII VII

VI V IV III II

XII XI X IX

R1

R3a R3b R5

R6 R7 R9 R10

R11 Rpi-sto2

Rpi-pcs1

Rpi-vnt1.1-1.3

R2 R2-like

Rpi-blb3

Rpi-abpt

Rpi-snk1.1-1.2

Rpi-edn1.1

Rpi-hjt1.1-1.3

Rpi-mcd1

Rpi-dmsf1

Rpi-bst1

Rpi-blb2

Rpi-ver1

Rber/Rpi-ber/Rpi-

ber1

Rpi-ber2

Rpi-blb1/RB

Rpi-bt1

Rpi-sto1

Rpi-pta1

Rpi-ver1-8

Rpi1

Rpi-avl1

Rpi-cap1

Rpi-qum1

Rpi-edn3

Rpi-pur1

Rpi-vnt2

Hot spots of R genes for resistance to P. infestans on potato genetic map

Solanum phureja

Solanum stenotomum

http://www.cgn.wageningen-ur.nl/pgr/collections/crops/potato/species.htm

Diploid , 2 EBN

Identification of the Rpi-phu1 gene on chromosome IX of potato genome

Rpi-phu1

GP129***

CP135***

GP39***

B7***

GP94***

S1D11*

2,7 cM

12,3 cM

6,4 cM

3,4 cM

4,9 cM

21,7 cM

IX

PR PS resistant susceptible progeny

Markers significantly linked with the resistance: * - p <0.05;*** - p<0.001

(U Mann-Whitney test)

okaNBS-Hae

Rpi-oka1.1

GP39

Rpi-phu1

2.7 cM

2.7 cM

2.7 cM

13.4 cM

TG551

okaNBS-Hae

4.0 cM

8.7 cM

2.7 cM

GP94

GP129

CP135

B7

S1D11

Rpi-phu1

(n=148)

1.3 cM

2.0 cM

T0156

TG591b

5.4 cM

TG186 [R]

TG35

S2h4 [R]

8.7 cM

1.3 cM

3.3 cM

0.16 cM

Rpi-oka1.1

(n=1213)

SokaM2.9L

TG186

C2_At2g47590

S. okadae = S. venturii

73

6 R

74

7 S

A6

18

+ -

Rpi-phu1 = Rpi-vnt1.1

Why are we interested in expression?

LB symptoms on Rpi-phu1 plants late in the season

Isolation of P. infestans

The isolate is not virulent on Rpi-phu1 plants in the lab

Hypotheses: 1.Isolate lost virulence in vitro 2.Rpi-phu1 gene is not (sufficiently) expressed due to:

•Genetic background •Plant age •Day length

?

Material

P. infestans

• MP324 avirulent on Rpi-phu1 plants

• MP1162 isolated from Rpi-phu1 plants but avirulent in lab tests

• EC1 described as virulent on Rpi-vnt1.1 plants

Plant material

diploid (2x)

tetraploid (4x)

Śliw

ka e

t a

l., 2

01

2

Methods

• Plants grown in climatic chambers • High relative humidity75-90% • temperature

– 16˚C night – 18˚C day

• Sampled on 0, 1, 3 and 5th day • Spray inoculation

Methods

• RNA isolation • Spectrum Plant Total RNA Kit, Sigma

• DNase I, Sigma

• RT-PCR • Maxima First Strand cDNA Synthesis Kit for RT-qPCR, Fermentas

• real-time PCR

• SYBR Green

• Relative expression

• α-tubulin

Relative expression level of Rpi-phu1 after inoculation with different P. infestans isolates

• P. infestans • MP324

• MP1162

• EC1

• Plants

• test: 04-IX-21 with Rpi-phu1 gene

• control: Craigs Royal

MP324

MP1162

EC1

Infection

04-IX-21 Craigs Royal

None of the P. infestans isolates was able to infect Rpi-phu1 plants efficiently

Potato genetic background

Potato genetic background day by day

Plant material

diploid (2x)

tetraploid (4x)

Śliw

ka e

t a

l., 2

01

2

Donor genome

12.5% 6.25% 3.12%

Plant age and Rpi-phu1 gene expression

• Gene Rpi-phu1 was expressed in all plants

• All plants were resistant

phu6 (Rpi-phu1) 3 6 9 12 14 weeks

Plant age

Plant age, day by day

Daylegth and Rpi-phu1 gene expression

• P. infestans: MP324

• Plants

• test: 04-IX-21 with Rpi-phu1

• control: Craigs Royal

• Warunki

• Short day (8h)

• Long day (16h)

Day length and Rpi-phu1 gene expression

Short day Long day

Re

lati

ve e

xpre

ssio

n

Summary

• Compatible interaction is a challenge in case of Rpi-phu1 • Rpi-phu1 expression is different in 2x and 4x potatoes

(donor genome?) and upregulated 1 day post inoculation (2x)

• In 3-week-old plant Rpi-phu1 expression is lower but it is enhanced after contact with pathogen

• No good explanation for the field observation so far • Day length? • Interaction of the above factors? • Avr-vnt1 may be switched off

Acknowledgements

J. Jones

The Sainsbury Laboratory, UK

Financed by:

LIDER/06/82/L-1/09/NCBiR/2010