My talk given at Microbes in Norwich 2013

1. Diversity of Crinkler Effector Genes
in Phytophthora infestans isolates.
Graham Etherington
8 Feb 2013

2. Outline
• Phytophthora infestans
– Biology and evolution
– Effector genes
• Crinklers
– Gene architecture
– VLVVVP recombination motif
• Aims
• Methods
– Looking for the VLVVVP motif
– Making use of all the data
• Applying methods to data
• Confirming results
• Comments/To-do

3. Phytophthora infestans
• Oomycete
• Cause of Late Blight in Solanum species (e.g. Potato)
• Responsible for the Irish Potato Famine (1845)
• Global potato yield losses of 16%
• Economic losses of £4.3 billion annually

4. • Capable of sexual and asexual reproduction
• High evolutionary potential
- striking capacity for genetic change
- rapidly adapts to overcome resistant plants
• Phytophthora - from Greek (phytón) “plant” and
(phthorá) “destruction” - the plant-destroyer!
Phytophthora infestans

5. P. infestans effector genes
• Secreted from pathogen – alters host to enable infection
• High polymorphism and positive selection
• Forms a ‘haustorium’ which delivers effector molecules to
plant cells.
• Two main cytoplasmic effectors:
– RXLRs
– Crinklers
Beynon 2006

6. Crinkler effector genes
Haas 2009
Conserved
N-terminal
Diverse
C-terminal
Torto 2003

7. Crinklers
N-terminal C-terminal
Recombination hotspot.
Look at sequence diversity
around this site.
Roman Numerals:
V+L+V+V+V=70
=70P

8. Aims
• Do P.infestans isolates contain unique
Crinkler genes?
• What is the diversity of Crinklers within
P.infestans?
• How many Crinklers are shared between
different isolates?

9. The data
• 76nt PE Illumina genomic sequences of P.
infestans isolates
– EC3527 (51x)
– EC3626 (64x)
– UK 3928 (50x)
– US 22 (50x)
– NL 07434 (50x)
Test data (99% seq similarity)

10. Looking for VLVVVP in genome
• Take isolate reads, make 6-frame translations and
look for ‘VLVVVP’ recombination site?
• Control experiment – available resources:
– P. infestans reference sequence (T30-4)
– All annotated transcripts
• including Crinklers
• Make 6-frame translation of genome and look for
VLVVVP motif
– Where are the motif hits?

11. Looking for VLVVVP in genome
• 231 VLVVVP hits:
– 81 in annotated Crinklers
– 18 in other transcripts
– 132 outside any annotated feature
• Conclusion: Looking for the VLVVVP motif in 6-
frame translations results in a very high
number of false positives.

12. DNA sequence
• Extract Crinkler gene sequences using P.
infestans annotation.
• Align sequences
• Locate VLVVVP motif coding sequence
• Create ‘variable consensus’ DNA sequence
from alignment.

13. Consensus sequence
Consensus sequence logo
GTGCTGGTGG[TC]G[TG]T[TG]CC
V L V V V P

14. Testing the consensus sequence
• No. of consensus motifs found in P. infestans genome:
194
– in annotated Crinklers: 185
– in any other annotated genes: 0
– not found in any annotated region: 9
• Blast 300nt upstream region
– 8 Crinkler gene hits, 1 false positive
• 193/194 hits were Crinklers
• Conclusion: Looking for the ‘variable consensus’
sequence in reads should result in very low number of
false positives.

15. Methods
• Search reads for variable consensus.
• Truncate reads at consensus.
– ATCGGTTCATGTGCTGGTGGTGTTTCCGCCGTG becomes
GTGCTGGTGGTGTTTCCGCCGTG
• Count the occurrence of each sequence:
GTGCTGGTGGTGGTTCCGCCGTG 100
GTGCTGGTGGCGTTGCCATGTG 70
GTGCTGGTGGCGTTGCCA 5
GTGCTGGTGGCGTTGCCCGCGTG 1
GTGCTGGTGGTGGTTCCGCC 30
• Remove singletons

16. Making use of all the data
• In the datasets there are read-counts for short ‘sub-
sequences’ which are part of one or more longer
unique ‘super-sequences’.
GTGCTGGTGGTGGTTCCGCCGTG 2
GTGCTGGTGGTGGTTCC 8
GTGCTGGTGGT 55
GTGCTGGTGGTTAACGGT 40
GTGCTGGTGGTTAACGGTACGTAA 25
• Interested in the longest unique super-sequences,
but don’t want to throw away information in the
shorter sub-sequences.

17. Proportional Representation
• Distribute read counts of smaller sub-
sequences to longer super-sequences
proportionately:
– assign more read counts to common super-
sequences
– assign less read counts to rarer super-sequences

18. Proportional Representation
A ABCDE 100
Bi ABCDEFGH 144
Bii ABCDEFGA 6
A ABCDE 100
Bi ABCDEFGH 48
Bii ABCDEFGA 2
100
50
48 96
100
50
2 4
sub-sequence A ABCDE 100
super-sequence Bi ABCDEFGH 48
super-sequence Bii ABCDEFGA 2
Sub-sequence A is shared between super-sequences Bi and Bii as follows:
50
Assign to Bi
Assign to Bii
+48
+2

19. Recap
• Identify the best way to find Crinkler
recombination motif (VLVVVP) in P. infestans =
variable consensus sequence.
• Search for the variable consensus in the isolate
reads.
• Create ‘proportionally representative’ longest
super-sequences.
• Produce Venn Diagram of shared and unique
sequences (both as DNA seqs and protein seqs).

20. EC Crinklers
As amino acid sequences
Shared by both 59
ec3626 only 4
ec3527 only 1
Total 64
motif ec3527 ec3626
VLVALPPGTSSAPISDGSDFWLS 56 22
VLVALPPGTSSAPISDGTCRDFN 15 16
VLVALPPGTSSAPISDGTDFWLSRF 216 106
VLVALPPGTSSAPISDGTDLWLSRF 1103 899
VLVALPPGTSSAQ*AREQVCSSTRH 11 13
VLVALPPWTSSAPISDGTNLWLSRF 100 81
VLVALPSGTSSAPISDGTDLWLSRF 58 40
VLVVVPDEAGGAVASVEPS 11 18
VLVVVPDEAGGAVASVV 11 7
VLVVVPDGAGGSASDTSRIDR 2 8
VLVVVPDGAGGSASDTSRMDRLFDK 322 413
VLVVVPDVAGYAVASVELSAAPTT 22 21
VLVVVPDVAGYAVASVEPSAAPTTI 13 25
VLVVVPE*EQPVSPPQKN*SAVST 36 41
VLVVVPEGAGGDLELASLLSTTIQ 19 32
VLVVVPEGAVGSALSQPANSATIPN 20 15
VLVVVPEGGKRHPSNGWFAEFFH 17 24
VLVVVPEHDGAISNDMSAVTTP 49 66
VLVVVPEHDGTISNDMSAVTTPLIV 99 49
VLVVVPEHDGTISNDMSAVTTPLT 25 25
VLVVVPEITTTV*VRERKDEVLMA 50 14
VLVVVPEQAQGQPGLWLVTGSVD 19 25
VLVVVPEQDGKIKRYVCSDNATDSR 147 95
VLVVVPEQDGTISKEMFAATTPLT 91 55
VLVVVPEQDGTISKEMSAATSPLTV 28 11
VLVVVPEQDGTISKEMSAATTPLTL 57 55
VLVVVPEQDGTISKEMSAATTPLTV 654 437
VLVVVPEQDGTISNDLSAVTTPLTV 4 28
VLVVVPEQDGTISNDMSAVTTPLTV 605 463
VLVVVPEQGFSVPTVSQDGVFDHCI 36 41
VLVVVPEQGSSVL*TVFSTTAVIHS 24 34
VLVVVPEQGSSVPTVSLDGVFDHCS 58 96
motif ec3527 ec3626
VLVVVPEQGSSVPTVSQDGVFDHCI 136 249
VLVVVPEQGSSVPTVSQDGVFDHCS 61 82
VLVVVPESFGVDSQLLQLQEALLQ 29 17
VLVVVPGIITTVEVRERKDDKLIMA 25 52
VLVVVPGITTTVEVRERKDEMLMA 45 10
VLVVVPGITTTVEVRERKDEVLMAE 173 251
VLVVVPGLVASTVTIVIEEAAGSKP 110 126
VLVVVPGQGSSVPTVSQDGVFDH 14 5
VLVVVPGQRLPIAATAIHEPHPA 14 34
VLVVVPKGKNDRSAAMAIGVAPSLP 227 379
VLVVVPKGRNDRSAAMAIGVAP 5 10
VLVVVPKQDGTISNDTSAATTPLT 39 28
VLVVVPKQGTSVPTVSQDGVFDHCN 12 48
VLVVVPMPSVGSKRSADEIADVQKR 21 29
VLVVVPPPSVGSKRSADEIADVQK 13 17
VLVVVPPQDDLRSPAMTLLEAILPY 22 25
VLVVVPRSRETMTHGAQWILR 20 8
VLVVVPRSRETTTHGALWILRFN 16 9
VLVVVPRSTGDDDAWSPKDLCTVD 27 40
VLVVVPRSTGDDDAWSPMDPKVQLN 695 699
VLVVVPRSTGDDDAWSPMDSK 3 44
VLVVVPRSTGDDYALSPNDLCTLDP 28 50
VLVVVPRSTGDGDAWSPMDPKVQLN 29 25
VLVVVPRVAPAPENKRKRKRMEDED 37 24
VLVVVPSDDVVVPVSVPVAVPTGPE 25 39
VLVVVPVGAGVGVGQDVSMDVPAA 83 141
VLVVVPVGAGVGVGQDVSMHVPAAV 7 8
VLVVVPGLVASTVTIVIEEAAES 0 43
VLVVVPRSAGDDDAWS 0 5
VLVVVPYGAG 0 3
VLVVVPYPEQ 0 9
VLVALPPGKS 2 0

21. Proportional representation of all DNA
super-sequences.
Shared and unique motifs
Number in brackets refers to percent of dataset.
…as amino acid sequences

22. UK/US/NL isolate Crinklers
motif uk us nl
VLVALPPGTSSAPISDGSDFWLSR 12 26 37
VLVALPPGTSSAPISDGTCRDFNI 28 72 47
VLVALPPGTSSAPISDGTDFWLSRF 119 259 198
VLVALPPGTSSAPISDGTDLWLSRF 860 1352 1336
VLVALPPWTSSAPISDGTNLWLSRF 101 143 232
VLVALPSGTSSAPISDGTDLWLSRF 10 105 17
VLVVVPDEAGGAVASVEPSAAPT 5 26 145
VLVVVPDGAGGSASDTSRMDRLFDK 147 347 399
VLVVVPE*EQPVSPPQKN*S 3 16 17
VLVVVPEGAGGDLELASLLSTTIQE 12 18 50
VLVVVPEGAVGSALSQPANSATI 15 49 37
VLVVVPEGGKRHPSNGWFAEFFHP 12 55 79
VLVVVPEHDGAISNDMSAVTTPLTV 18 55 53
VLVVVPEHDGTISNDMSAVTTPLIV 42 116 157
VLVVVPEITTTV*VRERKDEVLM 3 94 29
VLVVVPEQAQGQPGLWLVTG 6 18 34
VLVVVPEQDGKIKRYVCSDNATDSR 78 150 154
VLVVVPEQDGTISKEMFAATTPLTL 16 165 49
VLVVVPEQDGTISKEMSAATSPLT 63 34 18
VLVVVPEQDGTISKEMSAATTPLTV 252 895 793
VLVVVPEQDGTISNDLSAVTTPLTV 16 6 57
VLVVVPEQDGTISNDMSAVTTPLTV 307 902 619
VLVVVPEQGFSVPTVSQDGVFDHCI 15 28 17
VLVVVPEQGSSVL*TVFSTTAVIHS 18 8 38
VLVVVPEQGSSVPTVSQDGVFDHCI 71 255 283
VLVVVPEQGSSVPTVSQDGVFDHCS 71 106 14
VLVVVPESFGVDSQLLQLQEALLQ 10 52 26
VLVVVPGITTTVEVRERKDEVLMAE 60 453 353
VLVVVPGLVASTVTIVIEEAAG 34 85 103
motif uk us nl
VLVVVPKGKNDRSAAMAIGVAPSLP 147 333 173
VLVVVPKQDGTISNDTSAATTPLT 7 24 77
VLVVVPKQGTSVPTVSQDGVFDHC 2 44 25
VLVVVPMPSVGSKRSADEIADVQ 3 10 21
VLVVVPPPSVGSKR 9 22 15
VLVVVPPQDDLRSPAMTLLEAILPY 21 27 20
VLVVVPRSAGDDDAWSPMD 3 8 17
VLVVVPRSRETMTHGAQWILRFN 9 17 62
VLVVVPRSRETTTHGAL 4 9 4
VLVVVPRSTGDDDAWSPKDLCTVDP 12 104 133
VLVVVPRSTGDDDAWSPMDPD 12 52 11
VLVVVPRSTGDDDAWSPMDPKVQLN 101 364 262
VLVVVPRSTGDDYALSPNDLCTLD 33 18 25
VLVVVPRSTGDGDAWSPMDPK 14 11 6
VLVVVPRVAPAPENKRKRKRME 4 19 21
VLVVVPSDDVVVPVSVPVAVPT 9 11 20
VLVVVPVGAGVGVGQDVSMDVPAAV 82 211 130
VLVVVPDQTEDANLSQRFSDL 3 42 0
VLVVVPDVAGYAVASVEP 0 20 18
VLVVVPEQGSSVPTVSLDGVFDHCS 0 81 72
VLVVVPGIITTVEVRERKDDKLIMA 0 30 36
VLVVVPGITTTVEVRERKDEMLMA 0 49 20
VLVVVPGQRLPIAATAIHEPH 0 27 22
VLVVVPDVDGYAV 8 0 0
VLVVVPGQGSSVP 5 0 0
VLVVVPEQDGEIKRYVCSDSAT 0 3 0
VLVVVPGIKTTVEVRERKDEVLMA 0 9 0
VLVALPPGTSKIGR 0 0 3
VLVVVPSPSVGS 0 0 10
VLVVVPYPEQAQVDMVHE 0 0 16
Shared by all 46
UK/US 1
US/NL 5
UK only 2
US only 2
NL only 3
Total 59

23. • UK 3928, US 22, NL 07434
Proportional representation of all
DNA super-sequences ..as amino acid sequences
Shared and unique motifs

24. Are Crinklers being correctly
identified?
Left paired-read = Left primer
Right paired-read = Right primer
• Create primers from reads for sequencing
VLVVVP

25. Shared by NL/US
Shared by UK/US
NL
US
CRN PITG_12090
CRN PITG_12094
UK
US
CRN PITG_19373
CRN11
Crinklers correctly identified?

26. Results
• EC data
– reflects the similarity of the two isolates (99%
identical at amino acid level).
– from the 64 longest unique sequences, 59 are found in
both isolates.
– at least one isolate has unique Crinklers.
• UK/US/NL data
– Most Crinklers are shared
– US genotype shares Crinklers with UK and NL, but very
few shared between UK and NL
– each isolate has 2-3 unique Crinklers.

27. Results
• EC v UK/US/NL
– 64 Crinklers in EC, 59 in UK/US/NL
• 52 shared by both
• 12 exclusive to EC isolates,
• 7 exclusive to UK/US/NL
• ‘VLVVVP’ not the only recombination motif.
– ‘VLVALP’ is also found in 8/64 in EC super-
sequences and 7/59 in UK/US/NL data.

28. Conclusions
• Variable consensus sequence
• ‘Proportional representation’
– identifies Crinkler diversity
– estimates abundance.
• P.infestans isolates do contain ‘unique’ Crinklers.
– Present in a few or totally unique?
• Application of methods on UK/US/NL data
reflects the greater sequence diversity between
the data.
• Crinklers prediction verified through sequencing.

29. Comments
• Method could bias the isolate with the most
reads – normalisation.
• Method shows diversity at the start of the
recombination hotspot
– can’t say much about the rest of the gene.
VLVVVPEQDGTISNDMSAVTTPLTV 1000
VLVVVPEQDGTISNDMSAVTTPLTVABCDEFG 250
VLVVVPEQDGTISNDMSAVTTPLTVHIJKLMN 250
VLVVVPEQDGTISNDMSAVTTPLTVOPQRST 200
VLVVVPEQDGTISNDMSAVTTPLTVUVWXYZ 300

30. • To-do:
– Larger-scale PCR/sequencing to confirm
shared/unique Crinklers in isolates.
– What would we find with longer (targeted?)
sequences?

31. Acknowledgments
• Kamoun Group
– Sophien Kamoun
– Kentaro Yoshida
– Marina País
– Liliana Cano
• Dan MacLean