Diversity of Crinkler Effector Genes in Phytophthora infestans isolates.

Graham Etherington

8 Feb 2013

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

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

• 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

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

Crinkler effector genes

Haas 2009

ConservedN-terminal

DiverseC-terminal

Torto 2003

CrinklersN-terminal C-terminal

Recombination hotspot.Look at sequence diversity around this site.

Roman Numerals:V+L+V+V+V=70=70P

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?

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)

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?

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.

DNA sequence

• Extract Crinkler gene sequences using P. infestans annotation.

• Align sequences• Locate VLVVVP motif coding sequence• Create ‘variable consensus’ DNA sequence

from alignment.

Consensus sequence

Consensus sequence logo

GTGCTGGTGG[TC]G[TG]T[TG]CCV L V V V P

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.

Methods

• Search reads for variable consensus.• Truncate reads at consensus.– ATCGGTTCATGTGCTGGTGGTGTTTCCGCCGTG becomes

GTGCTGGTGGTGTTTCCGCCGTG• Count the occurrence of each sequence:

GTGCTGGTGGTGGTTCCGCCGTG100GTGCTGGTGGCGTTGCCATGTG 70GTGCTGGTGGCGTTGCCA 5GTGCTGGTGGCGTTGCCCGCGTG1GTGCTGGTGGTGGTTCCGCC 30

• Remove singletons

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 2GTGCTGGTGGTGGTTCC

8GTGCTGGTGGT

55GTGCTGGTGGTTAACGGT

40GTGCTGGTGGTTAACGGTACGTAA 25• Interested in the longest unique super-sequences,

but don’t want to throw away information in the shorter sub-sequences.

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

Proportional Representation

A ABCDE100

Bi ABCDEFGH144

Bii ABCDEFGA6

A ABCDE100

Bi ABCDEFGH48

Bii ABCDEFGA2

100

5048 96

100

502 4

sub-sequence A ABCDE 100super-sequence Bi ABCDEFGH 48super-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

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).

EC CrinklersAs amino acid sequences

Shared by both 59

ec3626 only 4

ec3527 only 1

Total 64

motif ec3527 ec3626VLVALPPGTSSAPISDGSDFWLS 56 22VLVALPPGTSSAPISDGTCRDFN 15 16VLVALPPGTSSAPISDGTDFWLSRF 216 106VLVALPPGTSSAPISDGTDLWLSRF 1103 899VLVALPPGTSSAQ*AREQVCSSTRH 11 13VLVALPPWTSSAPISDGTNLWLSRF 100 81VLVALPSGTSSAPISDGTDLWLSRF 58 40VLVVVPDEAGGAVASVEPS 11 18VLVVVPDEAGGAVASVV 11 7VLVVVPDGAGGSASDTSRIDR 2 8VLVVVPDGAGGSASDTSRMDRLFDK 322 413VLVVVPDVAGYAVASVELSAAPTT 22 21VLVVVPDVAGYAVASVEPSAAPTTI 13 25VLVVVPE*EQPVSPPQKN*SAVST 36 41VLVVVPEGAGGDLELASLLSTTIQ 19 32VLVVVPEGAVGSALSQPANSATIPN 20 15VLVVVPEGGKRHPSNGWFAEFFH 17 24VLVVVPEHDGAISNDMSAVTTP 49 66VLVVVPEHDGTISNDMSAVTTPLIV 99 49VLVVVPEHDGTISNDMSAVTTPLT 25 25VLVVVPEITTTV*VRERKDEVLMA 50 14VLVVVPEQAQGQPGLWLVTGSVD 19 25VLVVVPEQDGKIKRYVCSDNATDSR 147 95VLVVVPEQDGTISKEMFAATTPLT 91 55VLVVVPEQDGTISKEMSAATSPLTV 28 11VLVVVPEQDGTISKEMSAATTPLTL 57 55VLVVVPEQDGTISKEMSAATTPLTV 654 437VLVVVPEQDGTISNDLSAVTTPLTV 4 28VLVVVPEQDGTISNDMSAVTTPLTV 605 463VLVVVPEQGFSVPTVSQDGVFDHCI 36 41VLVVVPEQGSSVL*TVFSTTAVIHS 24 34VLVVVPEQGSSVPTVSLDGVFDHCS 58 96

motif ec3527 ec3626VLVVVPEQGSSVPTVSQDGVFDHCI 136 249VLVVVPEQGSSVPTVSQDGVFDHCS 61 82VLVVVPESFGVDSQLLQLQEALLQ 29 17VLVVVPGIITTVEVRERKDDKLIMA 25 52VLVVVPGITTTVEVRERKDEMLMA 45 10VLVVVPGITTTVEVRERKDEVLMAE 173 251VLVVVPGLVASTVTIVIEEAAGSKP 110 126VLVVVPGQGSSVPTVSQDGVFDH 14 5VLVVVPGQRLPIAATAIHEPHPA 14 34VLVVVPKGKNDRSAAMAIGVAPSLP 227 379VLVVVPKGRNDRSAAMAIGVAP 5 10VLVVVPKQDGTISNDTSAATTPLT 39 28VLVVVPKQGTSVPTVSQDGVFDHCN 12 48VLVVVPMPSVGSKRSADEIADVQKR 21 29VLVVVPPPSVGSKRSADEIADVQK 13 17VLVVVPPQDDLRSPAMTLLEAILPY 22 25VLVVVPRSRETMTHGAQWILR 20 8VLVVVPRSRETTTHGALWILRFN 16 9VLVVVPRSTGDDDAWSPKDLCTVD 27 40VLVVVPRSTGDDDAWSPMDPKVQLN 695 699VLVVVPRSTGDDDAWSPMDSK 3 44VLVVVPRSTGDDYALSPNDLCTLDP 28 50VLVVVPRSTGDGDAWSPMDPKVQLN 29 25VLVVVPRVAPAPENKRKRKRMEDED 37 24VLVVVPSDDVVVPVSVPVAVPTGPE 25 39VLVVVPVGAGVGVGQDVSMDVPAA 83 141VLVVVPVGAGVGVGQDVSMHVPAAV 7 8VLVVVPGLVASTVTIVIEEAAES 0 43VLVVVPRSAGDDDAWS 0 5VLVVVPYGAG 0 3VLVVVPYPEQ 0 9VLVALPPGKS 2 0

Proportional representation of all DNA super-sequences.

Shared and unique motifs

Number in brackets refers to percent of dataset.

…as amino acid sequences

UK/US/NL isolate Crinklersmotif uk us nlVLVALPPGTSSAPISDGSDFWLSR 12 26 37VLVALPPGTSSAPISDGTCRDFNI 28 72 47VLVALPPGTSSAPISDGTDFWLSRF 119 259 198VLVALPPGTSSAPISDGTDLWLSRF 860 1352 1336VLVALPPWTSSAPISDGTNLWLSRF 101 143 232VLVALPSGTSSAPISDGTDLWLSRF 10 105 17VLVVVPDEAGGAVASVEPSAAPT 5 26 145VLVVVPDGAGGSASDTSRMDRLFDK 147 347 399VLVVVPE*EQPVSPPQKN*S 3 16 17VLVVVPEGAGGDLELASLLSTTIQE 12 18 50VLVVVPEGAVGSALSQPANSATI 15 49 37VLVVVPEGGKRHPSNGWFAEFFHP 12 55 79VLVVVPEHDGAISNDMSAVTTPLTV 18 55 53VLVVVPEHDGTISNDMSAVTTPLIV 42 116 157VLVVVPEITTTV*VRERKDEVLM 3 94 29VLVVVPEQAQGQPGLWLVTG 6 18 34VLVVVPEQDGKIKRYVCSDNATDSR 78 150 154VLVVVPEQDGTISKEMFAATTPLTL 16 165 49VLVVVPEQDGTISKEMSAATSPLT 63 34 18VLVVVPEQDGTISKEMSAATTPLTV 252 895 793VLVVVPEQDGTISNDLSAVTTPLTV 16 6 57VLVVVPEQDGTISNDMSAVTTPLTV 307 902 619VLVVVPEQGFSVPTVSQDGVFDHCI 15 28 17VLVVVPEQGSSVL*TVFSTTAVIHS 18 8 38VLVVVPEQGSSVPTVSQDGVFDHCI 71 255 283VLVVVPEQGSSVPTVSQDGVFDHCS 71 106 14VLVVVPESFGVDSQLLQLQEALLQ 10 52 26VLVVVPGITTTVEVRERKDEVLMAE 60 453 353VLVVVPGLVASTVTIVIEEAAG 34 85 103

motif uk us nlVLVVVPKGKNDRSAAMAIGVAPSLP 147 333 173VLVVVPKQDGTISNDTSAATTPLT 7 24 77VLVVVPKQGTSVPTVSQDGVFDHC 2 44 25VLVVVPMPSVGSKRSADEIADVQ 3 10 21VLVVVPPPSVGSKR 9 22 15VLVVVPPQDDLRSPAMTLLEAILPY 21 27 20VLVVVPRSAGDDDAWSPMD 3 8 17VLVVVPRSRETMTHGAQWILRFN 9 17 62VLVVVPRSRETTTHGAL 4 9 4VLVVVPRSTGDDDAWSPKDLCTVDP 12 104 133VLVVVPRSTGDDDAWSPMDPD 12 52 11VLVVVPRSTGDDDAWSPMDPKVQLN 101 364 262VLVVVPRSTGDDYALSPNDLCTLD 33 18 25VLVVVPRSTGDGDAWSPMDPK 14 11 6VLVVVPRVAPAPENKRKRKRME 4 19 21VLVVVPSDDVVVPVSVPVAVPT 9 11 20VLVVVPVGAGVGVGQDVSMDVPAAV 82 211 130VLVVVPDQTEDANLSQRFSDL 3 42 0VLVVVPDVAGYAVASVEP 0 20 18VLVVVPEQGSSVPTVSLDGVFDHCS 0 81 72VLVVVPGIITTVEVRERKDDKLIMA 0 30 36VLVVVPGITTTVEVRERKDEMLMA 0 49 20VLVVVPGQRLPIAATAIHEPH 0 27 22VLVVVPDVDGYAV 8 0 0VLVVVPGQGSSVP 5 0 0VLVVVPEQDGEIKRYVCSDSAT 0 3 0VLVVVPGIKTTVEVRERKDEVLMA 0 9 0VLVALPPGTSKIGR 0 0 3VLVVVPSPSVGS 0 0 10VLVVVPYPEQAQVDMVHE 0 0 16

Shared by all 46UK/US 1US/NL 5UK only 2US only 2NL only 3Total 59

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

Shared and unique motifs

Are Crinklers being correctly identified?

Left paired-read = Left primer

Right paired-read = Right primer

Crinkler gene

• Create primers from reads for sequencing

sequenceVLVVVP

Shared by NL/US

Shared by UK/US

NLUSCRN PITG_12090CRN PITG_12094

UKUSCRN PITG_19373CRN11

Crinklers correctly identified?

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.

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.

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.

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 250VLVVVPEQDGTISNDMSAVTTPLTVHIJKLMN 250VLVVVPEQDGTISNDMSAVTTPLTVOPQRST 200VLVVVPEQDGTISNDMSAVTTPLTVUVWXYZ 300

• To-do:– Larger-scale PCR/sequencing to confirm

shared/unique Crinklers in isolates.– What would we find with longer (targeted?)

sequences?

Acknowledgments

• Kamoun Group– Sophien Kamoun– Kentaro Yoshida– Marina País– Liliana Cano

• Dan MacLean