Deciphering the molecular mechanisms of Lr34-mediated disease resistance in wheat

Dr. Dharmendra Singh

Introduction

Leaf rust (Puccinia triticina) Stripe rust (Puccinia striiformis)

Stem rust (Puccinia graminis)

Powdery mildew (Blumeria graminis)

Introduction

Lr34/Yr18/Sr57/Pm38/Ltn1 durable, race non-specific multi-resistance locus on 7DS

Emerge recently after hexaploid wheat 8000 years ago

Deployed in wheat breeding for >100 years in >50% of wheat cultivar worldwide

ABC transporter (24 exons; 11.8 kb; ABCG)

Homoeologs: Lr34-A (7A) and Lr34-B (4A)

Deletion of TTC in exon 11 at 546 bp - Phenylalanine

C/T SNP in exon 12 at 634 bpTyrosine Histidine

Lr34 sus-D

Source: Krattinger et al. 2011, Plant J, 65: 392-403

Lr34 evolution and orthologs

Source: Krattinger et al. 2011, Plant J, 65: 392-403

Lr34 orthologs present

Lr34 orthologs absent

Lr34 mediated resistance in maize

Source: Sucher et al. 2017, Plant Biotechnol J, 15: 489-496

Common rust 12 DAI Northern corn leaf blight 10 DAI

Lr34res provides partial resistance against common rust and NCLB in maize

Lr34 mediated resistance in sorghum

P. purpurea pustule development on control and transgenic sorghum leaves at 14 DPI.

Source: Schinippenkoetter et al. 2017, Plant Biotechnol J, doi:10.1111/pbi.12723

Lr34res provides partial resistance against sorghum rust

Lr34 mediated resistance in rice

Source: Krattinger et al. 2017, Plant Biotechnol J, 14: 1261-1268

Response to KJ105, KJ201 and CHE80861 strains of M. oryzae in rice transgenic and sib lines 7 dpi

Lr34 mediated seedling resistance in durum wheat

Phenotypic analysis of Lr34 durum seedlings for (a) leaf rust and (b) stripe rust

Source: Rinaldo et al. 2017, Plant Biotechnol J, 15: 894-905

Lr34 mediated resistance in barley

Source: Boni et al. 2017, Plant Biotechnol J, doi:10.1111/pbi.12765

Transgenic barley shows Ta-Lr34res-mediated disease resistance against leaf rust and powdery mildew

How Lr34 leads to APR?

• What associated genes involved?

How such genes interact ?

• What is overall mode of action?

Lr34 offers• Broad spectrum resistance

• Leaf rust• Stripe rust• Stem rust• Powdery mildew• Rice Blast• Corn leaf blight

• Cross species effectiveness• Maize• Rice• Barley• Sorghum• Durum

• Durability

• Variation in response under

different environment

Background and resources

Chara wild type (+ Lr34) Chara heavy ion irradiation mutant (HII), 1-263 (+ Lr34)

RIKEN-Japan RI-beam facility using a Neon ion beam at 50 Gy with LET at 63 KeV/mm2 (Fitzgerald et al. 2010)

Hypothesis

Mutation within Lr34 gene

Deletion of Lr34 gene

Irradiation leads to deletion of other significant gene or set of genes

Deleted genes or factors leads to susceptibility

Objective

Identification of gene or set of genes essential for Lr34 function

Puccinia triticina pathotype 76-1,3,5,7,9,10,12,13+Lr37

Chara (CE) 14 DAI Chara HII 1-263 (CM) 14 DAI

Histopathological studiesPlant 2 hpi 4 hpi 6 hpi

Chara HII 12 germinating spore /cm

258 with germ tube

153 with appresorium

Chara 4 germinating spore /cm

148 with germ tube

30 with appresorium

Chara HII

Chara

SSV

Appresorium

Appresorium

Germ tube

Spore

Spore

Germ tube

No SSV

Leaf samples collected in Farmer’s reagent at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 30, 36, 42, 48, 72, 96, 168 hpi

Dugyala et al. (2015) Plant Methods 11:54

Histopathological studies

Chara HII at 12 hpi Chara HII at 14 hpi Chara HII at 18 hpi Chara HII at 24 hpi

Chara at 12 hpi Chara at 14 hpi Chara at 18 hpi Chara at 24 hpi

HMC

SSV

No HMC

Selected 72 hours post inoculation (hpi) as sampling time-point

Four biological replicates were collected

RNA-seq (100 bp paired reads) performed on Illumina HiSeq 2500 at Beijing Genome Institute, Hong Kong

9.0 Gb sequences (2.25 Gb/ condition) from 16 libraries

Conditions Codes

Chara mock CE-M

Chara infected CE-I

Chara mutant mock CM-M

Chara mutant infected CM-I

Sample collection and sequencing

Sequence analysis workflow (Tuxedo analysis pipeline)

Filtering

• Removing low quality reads

• Trimming adapters

Alignment to Puccinia Ref.

• P. triticina transcriptome

• Broad Institute, MIT, USA

• Rust read abundance

Alignment to Wheat Ref.

• IWGSC wheat reference 1.0

• Putative deletion regions

• Differential gene expression with Cufflinks WT mock WT infected mutant mock mutant infected

0

5000

10000

15000

20000

25000

30000

35000

P. triticina abundance based on read alignment

Differential expression with Cufflinks

CE-M vs CE-I

CM-M vs CM-I

CE-M vs CM-M

CE-I vs CM-I

Gene Enrichment Analysis

Putative genes and pathways of interest

Experiment Upregulated Downregulated

CE-M vs CE-I 2135 1901

CM-M vs CM-I 291 276

CE-M vs CM-M 436 1268

CE-I vs CM-I 486 616

Upregulated distribution Downregulated distribution

Key findings

Putative deletion regions in Chara HII 1-263

6 deletion region identified based on collinearity of deleted genes

Located on 1A, 2D, 5A, 5B, 5D and 7D chromosomes

Genes present in deleted regions

1A

TraesCS1A01G431400 Retrovirus-related Pol polyprotein LINE-1

TraesCS1A01G431600 Endonuclease/exonuclease/phosphatase family protein

TraesCS1A01G433000 DNA-(apurinic or apyrimidinic site) lyase

TraesCS1A01G433300 Bifunctional lycopene cyclase/phytoene synthase

2D

TraesCS2D01G387800 AT hook motif-containing protein, putative

TraesCS2D01G387900 PIF1 helicase

TraesCS2D01G389900 RNA-directed DNA polymerase related family protein

TraesCS2D01G390500 DNA-directed RNA polymerase II subunit RPB1

TraesCS2D01G393400 Retrovirus-related Pol polyprotein transposon TNT 1-94

TraesCS2D01G393600 LINE-1 reverse transcriptase like

TraesCS2D01G393900 Retrotransposon protein, putative, Ty1-copia subclass

5A

TraesCS5A01G164400 Retrotransposon protein, putative, unclassified

TraesCS5A01G164700 Retrotransposon protein, putative, Ty3-gypsy subclass

TraesCS5A01G165600 ATP-dependent DNA helicase pif1

TraesCS5A01G166000 Protein FAR1-RELATED SEQUENCE 5

TraesCS5A01G166100 Protein FAR1-RELATED SEQUENCE 5

TraesCS5A01G167200 Protein phosphatase 5.2

TraesCS5A01G168400 Collagen alpha-1(VIII) chain

TraesCS5A01G168500 LINE-1 reverse transcriptase

TraesCS5A01G168600 Pol polyprotein

TraesCS5A01G168700 Transposon Ty3-I Gag-Pol polyprotein

5D

TraesCS5D01G488800 Retrotransposon protein, putative, unclassified

TraesCS5D01G488900 Retrotransposon protein, putative, Ty1-copia subclass

TraesCS5D01G491600 PP2A regulatory subunit TAP46

7D

TraesCS7D01G076300 DHHC-type zinc finger family protein

TraesCS7D01G076400 Transposon protein, putative, Pong sub-class

TraesCS7D01G077100 Glutamate receptor 2.9

TraesCS7D01G078100 Disease resistance RPP8-like protein 3

TraesCS7D01G080200Retrotransposon protein, putative, unclassified, expressed

TraesCS7D01G080400 Serine/threonine-protein kinase

5B

TraesCS5B01G340000 Polynucleotidyl transferase, ribonuclease H-like superfamily

TraesCS5B01G340100 Glucan endo-1,3-beta-glucosidase

TraesCS5B01G340900 Gamma-aminobutyric acid receptor subunit rho-2

TraesCS5B01G348900 Transposon Ty3-I Gag-Pol polyprotein

TraesCS5B01G349000 Gag-Pol polyprotein

TraesCS5B01G349300 phototropin 2

TraesCS5B01G350100 UvrB/UvrC domain protein (DUF3506)

TraesCS5B01G490500 Retrovirus-related Pol polyprotein from transposon TNT 1-94

TraesCS5B01G490800 Endoglucanase

TraesCS5B01G490900 Retrotransposon protein, putative, unclassified

• Stilbene biosynthetic and metabolic processes• Coumarin biosynthetic and metabolic processes• Lignin biosynthetic process (cell wall fortification)• Phenyl propanoid pathways (PAL, Flavonoids)

Key biological processes

• Serine O-acyltransferase activity (gene regulation)• Cinnamyl-alcohol dehydrogenase activity (Lignin synthesis)• Sinapyl alcohol dehydrogenase activity (Lignin synthesis)• Licheninase activity, cation binding (Hydrolase activity)

Key molecular function

Work in progress

To detect significant deletion, bulk segregant genotyping of Chara × Chara HII F2 crosses performed

Analysis leads to key genomic region associated with Lr34 resistance

The genes present in that region will be identified and validated using qPCR and knockout studies

CE CM

Pooled DNA of susceptible lines

Pooled DNA of resistant lines

Genotyping and analysis

Acknowledgements

Dr. Lee HickeyQAAFI, The University of Queensland

Dr. Ian GodwinSAFS, The University of Queensland

Hickey Lab The University of Queensland

Dr. Jonathan PowellCSIRO, Brisbane

Dr. Kemal KazanCSIRO, Brisbane

Brisbane Canberra

Dr. Evans LagudahCSIRO, Canberra