Deciphering the molecular mechanisms of Lr34 …...Deciphering the molecular mechanisms of...
Transcript of Deciphering the molecular mechanisms of Lr34 …...Deciphering the molecular mechanisms of...
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