Tuesday theme 1 1220 1235 large briefing room mulusew

1. Recognition of Phytophthora infestans in Potato (Solanum tuberosum l.): Scr74 gene as an
example
Mulusew Kassa Bitew
Dr. Vivianne Vleeshouwers
Emmanouil Domazakis
10th Triennial Conference
October 11 , 2016

2. content
 Introduction
 Methods followed
 Result obtained
 What concluded & Recommended?

3. P. infestans
(Marshall, 1995; Judelson 1997; Vleeshouwers et al., 2011)
• Late blight of potato and tomato, cause of Irish famin
• Can destroy a potato crop within a few days due to:
 Elevated virulence as an R gene destroyer
 Capability to rapidly adapt to resistance plants
 High evolutionary potential

4. Cont’d
• Late blight is the most devastating disease of
potato that affect the entire plant, stem, leaves
and tubers; resulting in a yield loss of 16%
worldwide
(Vleeshouwers et al., 2006)

5. Possibilities to control P.infestans
 In many parts of the world, P. infestans is now resistant to
fungicides and
 It still poses a major threat, and it has evolved to overcome
most of the control measures that were introduced over the
years.
 So:
it is sensible to seek sources of genetic resistance to the
pathogen in the wild potato plants
And to do this understanding the recognition of
Phytophthora infestans in potato and using effector to
accelerate R gene is the root of the problem to figure it
out

6. What helps the pathogen to break the R gene?
A. Reproduction: Sexual ; A1+A2
(Haas et al ., 2009)
• Reproduce asexually
 Sporangia
 Zoospores
• Sexual reproduction can
also occur when both
mating types are present
 Oospores:
♀ oogonium &
♂antheridium

7. What helps the pathogen to break the R gene?
B. largest and the most complex genome sequence so far in
chromarveolates~240Mb.
 The fast evolving effector gene are localised to P. infestans genome.
 This complex effectors fails in to two categories;
 Apoplastic effectors that accumulate in the plant intercellular space
(apoplast) which includes secreted hydrolytic enzymes such as:
 proteases
 lipases and glycosylase, and
necrotizing toxins such as small cycteine-rich-proteins (SCRs).
 Cytoplasmic effector that are translocate directly in to the plant cell by
specialised infection structure called the haustorium belong to the RXLR
and CRN families
(Haas et al ., 2009)

8. How Plant respond to effectors of the pathogen?
(Jones and Dangl, 2006)
Plants have two branches of immune system mediated by:
 Apoplastic receptor = Pattern recognition receptors: Trans-membrane PRRs which
respond to microbial associated molecular pattern (MAMPS/PAMP)
 Cytoplasmic receptors = nucleotide-binding site leucine-rich repeat (NBS-LRR)
proteins recognizing cytoplasmic effectors
In general:
 Most PRR are identified as transmebrane receptor-like kinases (RLk/Ps)
 But there is no PRR in P. Infestans, as far as I know

9. What is he question then?
 There is a thought that P. infestans effectors are apoplastic
effectors and which is the cause of pathogenicity ever
 No Pattern Recognition Receptors (PRR) identified for
oomycetes
?

10. Objectives
 Study the sequence variation of the Scr74 gene family on 12 P.
infestans isolates & search for novel Scr74 variants = Polymorphism
of Scr74
 Evaluate the response of potato genotypes to the different Scr74
variants through effectoromics = recognition

11. Scr74 effector family of P. infestans
 Secreted cysteine-rich protein 74 amino acids = Scr74
 Candidate apoplastic effector gene
 Highly polymorphic and under diversifying selection
  Is this protein being recognized in Solanum sp.?
  Does this polymorphism affect the recognition by plant receptors?
(Orsomando et al., 2001; Bos et al. 2003 ; Liu et al., 2005)

12. Over view of the Method
Genomic DNA
isolation
PCR amplification
Cloning to pGEM-T
easy vector
Sequencing
Cloning to
pGR106 vector for
PVX
PVX assay
Scoring necrosis
Detached leaf
assay
Scoring P.
infestans growthSelection of Scr74
polymorphic genes
Selection of plant-pathogen
combination based on PVX
results

13. P. infestans isolates & Potato genotypes used in this study
 12 isolates of P. infestans used for
studying Scr74 diversity
EC 1 90128
H3OPO4 IPO-C
IPO-0 PIC99183
UK7824 PIC99189
PIC99177 UK3928-A
89148-09 Katshaar
Plant material
Species name genotype name
S. verrucosum (wild
diploid)
VER909-1
VER910-5
VER914-7
VER914-9
VER922-1
VER922-2
VER989-1
VER989-2
VER989-3
VER989-4
S. tuberosum (diploid) RH89-039-16
S. tuberosum (doubled
haploid)
DM1-3616R44
 12 Potato genotypes used for PVX assay

14. Scr74 amplification
PCR using Scr74 specific primers:
 forward primer Scr74-FCla (59-GGAAATCGATCCGGTCATCGTCACTACTCAACAGCTCG–39)
and
 reverse primer Scr74-RNot (59- GGAAGCGGCCGCTTCATTCATTTGATTATCACTGTATCTC-39)
 Bands were excised from gel and cloned into pGEM-T easy
pGEM-T

15. Colony development and PCR confirmation
• Plating & blue/white screening
• Colony PCR confirmation
• Isolation of plasmid DNA and sequencing
 8 colonies per transformation to E. coli

16. Distribution of Scr74 variants on the tested P. infestans isolates
P. infestans
isolates
Scr74 variants
C3b
B11a
C4_1
C10
B11_1a
E6_a1c
B3a_1ab
E6_1
C4c
B10_1b
B3a_2b
B3a_1b
C4_2c
C4_3c
B3a_1bb
D5_1ad
E5
C4_4c
H1
C3a
D5_2ad
D5_3ad
E6_a2c
B10_2b
D6
D5_1d
D5_2d
90128 x
89148-09 x
EC 1 x
H3OPO4 x
IPO-0 x x x
IPO-C x x x x
Katshaar x x x
PIC99177 x x
PIC99183 x x
PIC99189 x x
UK3928-A x x x x x x
UK7824 x
Reference
Liuetal.,2005
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Liuetal.,2005
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Liuetal.,2005
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Liuetal.,2005
Liuetal.,2005
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Liuetal.,2005
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Liuetal.,2005
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Liuetal.,2005
Liuetal.,2005
Liuetal.,2005
Liuetal.,2005
 One isolate can have one or more Scr74 variants

17. Cloning of novel Scr74
 27 different Scr74 variants were identified
 Obtained Scr74 variants were cross checked with Liu et al. (2005)
 4 novel variants identified
• Scr74-B11 →H3OPO4 , Scr74-E6_1 → PIC99177
• Scr74-H1 → UK3928-A , Scr74-C4_1 → IPO-0
Colony PCR of 4 novel Scr74
variants in PVX vector

18. Amino acid variation of Scr74 variants
Identity
Mature proteinsSignal peptides
 8 conserved cysteine a.a. residues → Scr74 gene family is conserved in P.
infestans
 7 variants have stop codon = pseudo gene
 A total of 21 variablea.a. sites found and 20 are found in the mature protein
 Highlight sites under diversifying selection

19. Amino acid sites under diversifying selection
 13 amino acid sites were under positive diversifying selection (p > 95%)
 Positions 3L, 28R, 30D, 58Y, 38L, 41K, 43T, 48K, 52A, 55I, 36H, 62S, 69S
 Analysis was done using PAML package
Model Estimate of parameters InL Diversifying selection sites Model
comparison
2L p- values
M0: One
ratio
w = 1.95 -643 not allowed
M1: neutral P0 = 0.56, P1 = 0.44 -635 not allowed
M2:
Selection
P0 = 0.74, P1 = 0, P2 = 0.26, w =
8.27
-620 3L, 30D, 58Y, 38L,41K, 52A,62S M1 VS. M2 32 1.13E-07
M3:
discrete
P0 = 0.048, P1 = 0.69, P3 = 0.26, w1
= 0.19, w2 = 0.19, w3 = 8.27
-620 3L, 28R, 30D, 58Y, 38L, 41K, 43T,
48K, 52A, 55I, 36H, 62S, 69S
M0 VS. M3 46 2.46E-09
M7: b P = 0.005, q = 0.007 -636 not allowed
M8:
b+w
P0 = 0.074, P =23.3,
q = 99, P1 = 0.26, w = 8.27
-620 3L, 30D, 58Y, 38L, 41K,48K, 52A, 55I, 62S,
69S
M7 VS. M8 32 1.13E-07
InL = log likelihood value, 2L is likelihood ration test= 2(InL alternative hypothesis – InL null hypothesis)
 Diversifying selection analysis (based on Liu et al., 2005)

20. Amino acid sites under diversifying selection
Identity
52A
48K
55I
58Y
62S
69S
28R
30D
3L
Mature proteinsSignal peptides
36H
38L
41K
43T

21. Evaluation of the response of potato genotypes to the
different Scr74 variants
Experimental set up
 5 plants per genotype
 2 spots per effector, per leaf
 3 leaves per plant
 Controls:
 pGR106 empty vector as
negative
 CRN2 as positive
control
 PVX assay to study the recognition of 17 Scr74 variants
 Screen on 12 selected Solanum sp.
17 Scr74 variants were screened with PVX
• 13 variants from Liu et al., 2005
• 4 cloned (own new one)

22. Plant material
Species name genotype name
S. verrucosum
(wild diploid)
VER909-1
VER910-5
VER914-7
VER914-9
VER922-1
VER922-2
VER989-1
VER989-2
VER989-3
VER989-4
S. tuberosum
(diploid)
RH89-039-16
S. tuberosum
(doubled haploid)
DM1-3616R44
Potato genotypes tested for response to Scr74
Selected highly homozygous wild potato accessions used for the PVX assay
 Grown in vitro for 1-2
weeks till strong roots
develop
 Transplanted in the
greenhouse in jiffy pots for
3-4 weeks
 Transplanted in big pots for
2-3 weeks
 PVX agro-infection scoring
after 2 weeks

23. Effectors VER909-1 VER910-5 VER914-7 VER914-9 VER922-1 VER922-3 VER989-1 VER989-2 VER989-3 VER989-4 RH89-039-16 DM1-3616R44
Scr74-A10 4 5 6 5 2 2 6 5 7 8 4 2
Scr74-D5-1 3 4 6 6 1 3 3 6 6 7 4 1
Scr74-B10-1 1 2 1 3 1 3 2 3 3 3 4 1
Scr74-D1 3 4 4 4 1 2 2 4 4 5 4 1
Scr74-D4 2 4 1 3 2 2 1 3 1 3 4 1
Scr74-B3a_1 3 0 1 3 2 1 1 1 1 0 5 1
Scr74-B7 4 1 2 4 2 1 1 2 1 0 5 1
Scr74-D6 4 1 2 4 2 0 1 2 0 0 5 1
Scr74-D2 3 1 2 4 3 0 0 2 0 0 5 1
Scr74-A11 2 1 1 2 0 0 3 3 4 5 5 1
Scr74-C3a 1 0 0 2 0 1 1 1 0 1 5 1
Scr74-C4 1 0 1 2 0 2 1 1 1 2 5 2
Scr74-C10 2 0 1 2 0 2 4 2 3 2 5 1
Scr74-B11 1 0 2 2 2 2 2 3 2 2 3 1
Scr74-E6_1 1 0 2 3 2 2 2 2 2 2 3 1
Scr74-C4_1 2 0 2 3 3 2 3 3 2 1 3 1
Scr74-H_1 1 0 2 3 3 3 2 3 2 2 3 1
pGR106 (neg) 1 0 1 1 2 1 0 1 1 1 4 1
CRN2 (Pos) 8 5 5 5 4 5 4 5 5 5 6 3
Some genotypes strongly respond to specific Scr74 variants with cell-death
 Plant cell-death was scored as 0, 1 or 2 and was transformed in 0-10 scale using
Excel
 Response matrix of 17 Scr74 effectors on 12 potato genotypes

24. Scr74-A10 and Scr74-D5-1 gave the strongest
cell-death in 4 S. verrucosum genotypes
 One-way ANOVA analysis between effector and genotype
 Letters indicate significant difference between effectors in each
genotype at P<0.01

25. Evaluation of P. infestans virulence on our selected
Solanum sp.
(Vleeshouwers et al., 2006)
 1st experiment was performed with 6 isolates
 All isolates except Katshaar were not aggressive enough
 2nd experiment was done with only isolate Katshaar
 6 leaves were spot inoculated (4 spots per leaf) per genotype
 DM and RH were used as susceptible controls
 Leaves were spot-inoculated by pipetting 10 μl
droplets of the diluted spore suspension on the
abaxial side of the leaf
 Pilot study to investigate if there is correlation between Scr74 variants
present in the isolate and its virulence on Scr74-responding genotype

26. P. infestans isolate Katshaar infection on 12 Solanum sp.
 The lesion size (length and width) was scored & Lesion area calculated
using 𝐴𝐴 =
1
4
𝜋𝜋(𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐿 ∗ 𝑊𝑊𝑊𝑊𝑊𝑊 𝑊𝑊𝑊)
 All S. verrucosum genotypes, except VER922-1, VER22-2 and VER909-1
has < 16mm2
 Effectors, i.e.,Scr74-D6, Scr74-D5_1 & Scr74-D5_2 were from Katshaar
isolate
 Largest lesion area scored in RH & DM genotypes

27. Conclusions
Scr74 is a highly polymorphic gene family in P. infestans (>27
variants exist)
 From the 12 wild potato genotypes screened, two S. verrucosum
(VER989-3 & VER989-4) genotypes gave the strongest cell-
death response to two Scr74 effectors (Scr74-A10 & Scr74-
D5_1)

28.  There is a potential involvement of Scr74 recognition in plant
defence against P. infestans.
 Strongly responding S. verrucosum genotypes have potentially
an immune receptor that might be an RLK or RLP which are part of
PRR since there is clear recognition of the Scr74 effector
Conclusions

29. Recommendation
As our result showed there is resistance wild
genotypes to P. infestans effector & would give
best production if they breed with different local
cultivars
So the identification and characterization of other
pathogen elicitor–plant receptor interactions could
also lead to novel strategies for engineering or
breeding for disease resistance.