expression profiles of the late blight resistance gene …...expression profiles of the late blight...
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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