Identification and characterization of effector genes from wheat stripe rust

Discovering the effector genes of Puccinia striiformis f.sp. tri.ci

John Rathjen The Australian Na;onal University

Stripe rust and Australian wheat produc;on

Annual losses

Control cost

GM Murray & JP Brennan 2009. Grains Research & Development Corpora?on. Australian Government

hAp://www.apsnet.org/edcenter/intropp/lessons/fungi/Basidiomycetes/Pages/StemRust.aspx

Meiosis

Urediniospores (2n)

Teliospores (2n)

Basidiospores (1n)

Pycniospores (1n)

Aeciospores (2n)

Wheat

Barberry

alternate host

Sexual host insignificant in Australia

Dikaryo?c – Two haploid nuclei

Puccinia striiformis f.sp. tri0ci Barley grass yellow rust Psd– grows on Dactylis glomerata (Cocksfoot) Psp – grows on Poa pratensis (Kentucky blue grass) Stripe rust of Phalaris spp., Bromus spp., “wheat grass”, etc, etc

P. striiformis in Australia

Pst-‐1979

Pst-‐WA (2002)

BGYR (2000)

(~20 strains)

(~6 strains)

Psd

Psp

How can we define effector genes?

•  Generally, effectors are thought to be small secreted proteins.

•  This is sufficient to build a list of such proteins if genomic sequence is available.

•  In some cases, amino acid mo?fs such as RxLR or YxC are present…but don’t seem to be diagnos?c.

•  Another important criterion is expression of candidate effector genes in planta, where that informa?on is available.

Puccinia genomics

•  Pgt (stem rust) genome (Duplessis et al. 2011) is about 90 Mb, encoding about 17,000 genes – Pgt expected to be similar.

•  This was assembled with a lot of “last-generation sequencing” which helps with scaffolding and sequence assembly.

•  Transposable elements account for about 45% of the genome.

•  Calling genes from NGS assemblies can be problematic, and can be difficult to detect expression of fungal genes in infected tissue (but these are the most interesting genes).

•  There are ongoing unresolved problems with the dikaryotic nature of rusts.

•  Broad Institute (Cuomo) has a good Pst assembly in the pipeline.

Perils and pi`alls of next-‐genera?on sequencing (NGS).

•  NGS – boAom up or ‘shotgun’ assembly of millions of small sequence reads, using high-‐performance compu?ng. Technologies include:

•  Illumina – millions of very short reads (~100 bp). •  Roche-‐454 – fewer numbers of longer reads (~500 bp).

•  Tradi?onal (Sanger) sequencing – long reads 800-‐1000 bp.

DNA sequencing; the impossible triangle

Tradi?onal Sanger sequencing of physical con?gs

NGS

Perils and pi`alls of next-‐genera?on sequencing (NGS).

AATATAAAACCAAAGATACTGATATCTTAGCGGCTTTCCGAATGACCCCACAACCTGGAG

Nucleus 1 Nucleus 2

Detec?ons of sequence polymorphisms in small-‐read assemblies


X

X

X

X

C/G

Detec?ons of sequence polymorphisms in small-‐read assemblies -‐ II


X

X

X

X

C/G

X

X

X

X

T/A

Detec?ons of sequence polymorphisms in small-‐read assemblies -‐ II


X

X

X

X

C/G

X

X

X

X

T/A T C A C T A

G G

The “phase” problem

Repeats and mul?copy genes are difficult to assemble from small reads

Repeats (transposons…effectors?) assemble poorly or not at all. This is obvious in NGS genome assemblies. It’s a considerable problem for genomics of Puccinia spp.

Transcriptome

Transcriptome

Illumina mate-‐paired Illumina pair-‐end (2)

454 RNA-‐seq

454 RNA-‐seq

Genome

Illumina RNA-‐seq

Illumina RNA-‐seq

454 mate-‐pair

NGS datasets for stripe rust bioinformatics

16831 contigs

14682 contigs

1299 ORFs-SP

515 ORFs-SP

418 ORFs-SP

100 ORFs-SP

Contamina;on removal

Secreted proteins predic;on Non-‐transmembrane domains

Unique or non-‐overlapping ORFs

Protein length ≤ 300aa

High expression

454 sequencing of isolated haustoria

transcriptome

Lab tests

Illumina sequencing

Prediction of small secreted proteins (SSPs) from the haustorial transcriptome

433 ≤ 300 aa 98 > 300 aa

311 ≤ 4 Cysteines 220 > 4 Cysteines

91 have 1 mo?f , 18 in the ‘correct’ loca?on 42 have 2 mo?ves, 23 correct loca?on

Protein length

Cysteine content

9 have 3 or more mo?ves, 8 correct loca?on Y/F/WxC mo?f

Pgt hypothe?cal protein

Specific hit (most from Pgt)

Not available

e-‐val ≤ 10-‐25

e-‐val > 10-‐25

BLASTn BLASTx

74 211

29 38

291 419

Invertase 1,3-‐β-‐glucosidase Pepsin A Chi?n deacetylase Glucose-‐regulated protein Previous SP from Pst

No memes No clusters/tribes

Validation and investigation of effector candidates

100 sequenced and cloned in TOPO

75

24

Narayana Upadhyaya and Diana Garnica

Ø R-‐AvrR recogni?on assay Ø Inhibi?on of plant cell death Ø Localisa?on Ø Influence on host metabolism

AvrM type-‐III delivery/ P. fluorescens

AvrM

avrM

Agro/AvrM

PST-80 housekeeping genes are not single allele

Housekeeping Gene Copy Number

0

1

2

3

4

5

6

7

8

9

10

221 18 39 60 81 102

123

144

165

186

207

233

254

275

312

333

368

389

418

453

483

521

295

467

443

511

Copy num

ber

Boeva V, et al. (2011) Control-‐FREEC: Bioinforma?cs. 2011 Dec 6

PST-80 Effector genes are present with variable copy number

Effector Allele Number, 6

0

1

2

3

4

5

6

7

1 21

41

308 61

81

101

121

141

161

181

201

221

241

261

281

326

346

366

415

456

494

290

434

486

471

519

Allele Num

ber

PST_80 Effector Allele Number Effector gene copy number

Effector gene, nominal ranking

Copy num

ber

Effector copy number variations between Pst-80 and BGYR

0

1

2

3

4

5

6

7

1 51 101 151 201 251 301 351 401 451 501

Axis Title

Axis Title

Effector Allele Number Effector gene copy number

Effector rank (nominal)

Copy num

ber

Effector copy number variations between Pst-80 and Pst-130 (US)

0

2

4

6

8

10

12

1 13

25

37

49

61

73

85

97

109

121

133

145

157

169

181

193

205

217

229

241

253

265

277

289

301

313

325

337

349

361

373

385

397

409

421

433

445

457

469

481

493

505

517

Allele Num

ber

Effector Number

PST_130 Effector Allele Number

Effector Allele Number

Effector gene copy number

Copy num

ber

Effector number (nominal)

Cantu et al. PLOS One (2011)

Housekeeping genes do not show the same degree of varia?on in copy number

Conserved Gene Copy Number

01234567

1 51 101 151 201 251 301 351 401 451 501

Gene

Pred

icted

Cop

y Num

ber

PST_conBGYR_con

BGYR

Pst-‐80 Control-‐FREEC predic?on of CNVs


Copy number varia?on in Pst effectors

•  Copy number varia?ons are readily apparent in Pst effector genes, with many single copy.

•  Sequence polymorphisms are also apparent, but these are harder to annotate because of NGS assemblies.

•  Single-‐copy effectors may allow the pathogen to mutate rapidly to virulence.

Barley grass yellow rust (BGYR) – a stripe rust that jumped?

BGYR (2000)

Wheat stripe (1980)

wheat Barley grass

Stripe rust and BGYR 99+% iden?cal in effector genes so far sequenced

Sequencing summary

•  We amplified and sequenced the PCR products of 50 candidate effector genes from Pst-80 and BGYR and found 99 single nucleotide polymorphisms (SNPs).

•  These were ALWAYS of a particular pattern – twin peak ‘dimorphisms’, rather than clear SNPs (dSNPs).

•  50 of these were'informative' dSNPs - 34 from BGYR, and 16 from Pst-80.

•  We amplified and sequenced these alleles from BGYR and Pst-80.

•  When we did this, we found that BGYR ALWAYS shared an allele with Pst-80, and the alternative allele was divergent.

•  We think that this is related to the dikaryotypic nature of P. striiformis.

1 8 6

2

4 7

5 3

1 8 6

2

4 7

5 3

Pst-‐80

BGYR

1 8 6

2

4 7

5 3

1 8 6

2

4 7

5 3

1 8 6

2

4 7

5 3

BGYR 1

8 6 2

4 7

5 3

Model for the origins of BGYR

Pst

BGYR unknown ancestor Anastamosis + Heterokaryosis

BGYR

Where did BGYR come from?

•  One line of evidence suggests that heterokaryosis is an underlying mechanism for the host jump – but we need to address the phase problem.

•  In the 1950’s, this was proposed as a mechanism to explain frequent mutation to virulence of stem rust on wheat.

•  We have detected four deleted effector genes, and will test these for recognition on barley grass by bacterial delivery.

•  Heterokaryosis potentially increases effector hemizygosity, which could both increase the effective effector compliment (for virulence) and allow rapid deletion of recognised effectors.

Acknowledgments •  Diana Garnica •  William Jackson

•  CSIRO Black Mountain •  Narayana Upadhyaya •  Peter Dodds •  Jeff Ellis

•  Univ Sydney CobbiAy •  Colin Wellings

Robert Park

•  Univ Exeter, UK •  David Studholme

Ø Take nutrients (sugars and aminoacids) from host Ø Generate precursors of metabolites and energy Ø Biosynthesise compounds necessary for the ul?mate produc?on of spores Ø Secrete pathogenicity factors (effectors)

Ø Use lipid reserves to generate energy Ø Grow (DNA replica?on, cell division) Ø Modify chi?n to avoid recogni?on

Haustoria:

Germinated spores:

0

10

20

30

40

50

60

70

80

0

2

4

6

8

10

12

14

1 23

45

398 69

91

113

135

157

179

201

223

245

267

314

336

358

409

431

492

290

436

517

502

Copy, A

llele and

SNP Num

ber

Effector Number

PST_80 Effector Copy Number, Allele Number and SNP Number

Effector Candidate Copy Number

Many effector genes are single copy

PST_80 effector genes in PST_130 have undergone significant modification

0

20

40

60

80

100

120

0

2

4

6

8

10

12

14

16

1 13

25

38

50

62

74

86

98

110

122

134

146

159

172

185

198

211

223

235

247

259

271

283

295

307

320

332

344

356

368

380

392

404

416

428

440

452

464

476

488

500

512

Copy, A

llele and

SNP Num

ber

Effector Number

PST_130 Effector Gene Variability

Effector Copy Number Effector Allele Number Effector SNP Number

Mapping BGYR genomic reads against 500 ‘conserved’ Pst genes

Conserved Gene Copy Number

01234567

1 51 101 151 201 251 301 351 401 451 501

Gene

Pred

icted

Cop

y Num

ber

PST_conBGYR_con

BGYR



Mapping BGYR genomic reads against 500 Pst effector candidates Effector Candidate Copy Number

0

2

4

6

8

1 51 101 151 201 251 301 351 401 451 501

Gene

Pred

icted

Cop

y Num

ber PST_e

ffBGYR_eff

BGYR



ToxA cell death dependent on Tsn1 is suppressed by stripe rust infec;on

+ToxA +H2O

+ToxA + stripe rust stripe rust

Diana Garnica with help from the Solomon lab

PST_79 effector gene Pstv_4835_1 has one copy and two alleles

Identification and characterization of effector genes from wheat stripe rust

Technology

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