Rpp5 Asian Soybean Rust Resistance Genes

Cloning, Mapping and Identification of the Rpp1-Rpp5 Rpp5 Asian Soybean Rust Resistance Genes Jenelle Meyer 1, Danielle Silva 2, Martijn van de Mortel 3, Chunling Yang 3, Chris Zhang 3, Kerry Pedley 4, John Hill 3, Randy Shoemaker 1,3, Ricardo Abdelnoor 2, Steven Whitham 3 and Michelle Graham 1,3 1 USDA-ARS, ARS, Corn Insects and Crop Genetics Research Unit, 2 Embrapa Soja, 3 Dept. of Plant Pathology, Iowa State University 4 USDA-ARS ARS Foreign Disease-Weed Science Research Unit, www.swicofil.com, cropwatch.unl.edu/ archives/25/crop5-6.htm, http://www.uoguelph.ca/research/news/articles/25/june/aphid_biocontrol.shtml, Soybean plant and flower. Credit: Jamie O'Rourke, ISU,

Outline Introduction to Asian Soybean Rust Analyses of the Rpp4 locus in susceptible genotype Wm82 and the resistant genotype PI45925B Status of Rpp1-3 3 and Rpp5

Asian Soybean Rust (ASR) Caused by the fungus Phakopsora pachyrhizi Infects many legume species (soybean, Phaseolus, kudzu) First identified in the Eastern Hemisphere early 19 s Now in all major soybean producing countries Yield losses range from 1-8% U.S. commercial cultivars lack resistance Images: Unknown source, DTN

Asian Soybean Rust Resistance Resistance is very rare, over 16, germplasm accessions screened Breeding efforts rely on germplasm with partial resistance or tolerance Five resistance loci have been identified: Rpp1, Rpp2, Rpp3, Rpp4, Rpp5 Our objective: To clone the Rpp4 ASR resistance gene.

Microscopic Studies of Rpp2, Rpp3 and Rpp4 Resistant Interaction Susceptible Interaction Resistance to ASR mediated by Rpp4 leads to the formation of red/brown lesions and localized cell death, indicative of a hypersensitive response. Images: D. da Silva, M. van de Mortel

Microarray Studies of Rpp2 van de Mortel et al. (27) used microarray analyses to examine changes in gene expression in response to ASR 2 1 2 Chorismate mutase Susceptible and resistant genotypes both had: - early induction of basal defense genes followed by return to normal levels. - a second defense response However, the 2nd defense response occurred 24 hours earlier in the resistant line A similar response was seen for Rpp3 and Rpp4 (van de Mortel et al., in preparation). 2 1 6 4 2 3 2 1 3 2 Chalcone isomerase Isoflavone 2'-hydroxylase 2'-hydroxydihydrodaidzein reductase We hypothesize Rpp4 is a classical disease resistance genes. 1 Isoflavone-7- O-methytransferase 48 96 144 192 24 288 Time (hai) van de Mortel et al. 27. Molecular Plant-Microbe Interactions

Research Objectives: 1. Identify markers linked to Rpp4. 2. Develop BAC contig corresponding to the Rpp4 locus. 3. Sequence candidate BACs. 4. Identify candidate resistance genes. 5. Silence candidate genes using VIGS (virus induced gene silencing).

PI45925 BRS184 F 2 population (8) Soybean Consensus Map (linkage group G) 76.76 cm Silva et al. 28. Theoretical and Applied Genetics 89.94 cm

The Wm82 Rpp4 BAC Contig M7A12 M176I1 M4E17 I16I22 M171H4 I16K21 M18K13 I45O14 I51A11 M92N2 I75D11 M158G13 I13L24 I79L4 M9G19 M3L17 I63J17 I4P17 I42K8 I57P16 I45C4 M33J17 I52J5 I16L9 M27E18 I1C5 I4P17R Sat_143 I4P17F I52J5R I75D11R I75D11F I57P16F M158G13F I57P16R I13L24R 79L4F Satt288 79L4R I13L24F M9G19R M171H4R I45O14F M171H4F A885_1 73.4 cm 76.8 cm 79.2 cm Williams82 is susceptible to ASR.

Rpp4 Candidate Genes Encode NBS/LRR Proteins 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 55 kb 75 kb 1 kb 125 kb 15 kb 175 kb 2 kb Rpp4 candidate R-genes 1. Q9ZT68 Resistance protein candidate RGC2K 1E -113 2. Q1HF9 uncharacterized protein 5E -53 3. A5BCQ9 uncharacterized protein 1E -93 4. Unknown 5. Q9ZT68 Resistance protein candidate RGC2K 1E -11 6. Unknown 7. Q6UUN7 Putative gag-pol polyprotein* 1E -14 8. Q4315 Cellulase 1E -5 9. Q9LFC1 Uncharacterized protein 2E -46 1. Q9M2D1 Copia-type polyprotein* 4E -47 11. Q9M2D1 Copia-type polyprotein. 12. Q2R3T4 Retrotranposon protein 1E -15 13. Q9ZR3T4 Retrotranposon Tto1 1E -22 14. Unknown 15. Q9ZT68 Resistance protein candidate RGC2K 1E -111

The Rpp4 Contig Can be Linked to the Soybean Whole Genome Assembly. M7A12 M4E17 M176I1 I16I22 M171H4 I16K21 M18K13 I45O14 I51A11 M92N2 I75D11 M158G13 I13L24 I79L4 M9G19 M3L17 I63J17 I4P17 I42K8 I57P16 I45C4 M33J17 I52J5 I16L9 M27E18 I1C5 Scaffold_21 Sat_143 Satt288 A885_1 73.4 cm 76.8 cm 79.2 cm No other R-genes R candidates can be identified in this region. www.phytozome.net

Soybean Rpp4 Candidate Genes Three Rpp4 candidate genes identified, belonging to the CC-NBS NBS-LRR family of resistance genes. Most closely related to the lettuce RGC2 genes which confer resistance to downy mildew and root aphid 1. One additional homolog (RLG) of the Rpp4 candidate genes was located in the genome on LG D1A. Genes are large, spanning 15-25kb. Genes are not represented by ESTs nor any public microarray platform. 1 Meyers et al. 1998. Plant Cell.

Structure of the Rpp4 Candidate Genes RLG (LGD1A) NBS- required for ATP binding, signaling molecule LRR- provide binding surface for pathogen avr protein or plant target

Can we use the Wm82 Rpp4 candidate genes to silence Rpp4 in the resistant genotype? Assumption 1: Rpp4 is a member of this gene cluster. Assumption 2: Rpp4 shares >85% nucleotide identity with the genes in this cluster.

Virus-Induced Gene Silencing (VIGS) Bean pod mottle virus (BPMV) genome organization: RNA-1 VPg VPg Co-Pro Helicase Pro RdRp Poly(A) RNA-2 insert VPg 468 (775) CR 3522* / MP L-CP S-CP Poly(A) dsrna forms during virus replication & triggers soybean defenses leading to post-transcriptional transcriptional gene silencing Virus engineered by and image provided by Chunquan Chris Zhang (Iowa( State University) Zhang et al. 29. Molecular Plant-Microbe Interactions

Can we use the Wm82 Rpp4 candidate genes to silence Rpp4 in the resistant genotype? Assumption 1: Rpp4 is a member of this gene cluster. Assumption 2: Rpp4 shares >85% nucleotide identity with the genes in this cluster.

Silencing Rpp4 with VIGS Empty VIGS BPMV 1. Infect Rpp4 genotype with VIGS construct Rpp4 Rpp4 Rpp4 VIGS BPMV No silencing of Rpp4 R genotype R phenotype 2. Infect VIGS plants with P. pachyrhizi Silencing of Rpp4 R genotype S phenotype Control: Mock VIGS Control: Empty VIGS Exp: NBS VIGS Exp: LRR VIGS

Are there differences in gene number or gene expression between the R and S parents? Gene2 Gene3 Gene1 Rpp4_NBD_F/R: Amplify and differentiate the NBD of all Rpp4 candidate genes Gene2 Gene3 Wm82 (S):, and (Wm82) Gene1 PI45925B (R):,,, Rpp4C4 and Rpp4C5 (PI45925B) Gene2 Gene3 candidate genes Gene1 Rpp4_F/R: Amplify the NBD of all Rpp4 candidate genes.

Quantitative RT-PCR of Rpp4 Candidate Genes Time Point ASR Fold Change (R:S) 12 hai ASR 12 hai 6.49 5.2 Mock Control Fold Change (R:S) 12 hai mock 24 hai ASR 24 hai 3.92 2.84 24 hai mock 72 hai ASR 72 hai 4.82 2.86 72 hai mock 216 hai 5.8 3.1 216 hai ASR 216 hai mock Rpp4_F/R primers (amplify all genes)

Which of the Rpp4 Candidate Genes is Expressed? Sequences of expression products from Rpp4_NBD_F/R primers: Theoretical: Similar gene expression across all genes Gene1 CCGT CAATA ATAGCTTATAA 2 Gene2 CCGT-- --ATAGCTTATAA 2 Gene3 CCG TCAATAGC-- --ATAA 2 Gene4 CCATCAATAGC TCAATAGC--ATAA 2 Gene5 CCGTCAATAC CTTAAAA 2

Sequencing of RT-PCR products Sample Williams82/ASR/12hai Williams82/ASR/72hai Williams82/Mock/12hai Williams82/Mock/72hai Williams82 Total Williams82 Genomic DNA Expected Expression 35 136 6 1 1 8 27 15 8 88 79 9 337 27 15 Sample PI45925B/ASR/12hai PI45925B/ASR/72hai PI45925B/Mock/12hai PI45925B/Mock/72hai PI45925B Total PI45925B Genomic DNA 9 2 2 7 6 Rpp4C4 93 9 87 95 365 27 Rpp4C5 22 Primers: Rpp4_NBD_F/R

Sequencing of RT-PCR products Sample Williams82/ASR/12hai Williams82/ASR/72hai Williams82/Mock/12hai Williams82/Mock/72hai Williams82 Total Williams82 Genomic DNA Expected Expression 35 136 6 1 1 8 27 15 8 88 79 9 337 27 15 Sample PI45925B/ASR/12hai PI45925B/ASR/72hai PI45925B/Mock/12hai PI45925B/Mock/72hai PI45925B Total PI45925B Genomic DNA Expected Expression 9 41 2 2 7 77 6 27 Rpp4C4 93 9 87 95 365 27 122 Rpp4C5 22 1 Primers: Rpp4_NBD_F/R

Sequencing of RT-PCR products Sample Williams82/ASR/12hai Williams82/ASR/72hai Williams82/Mock/12hai Williams82/Mock/72hai Williams82 Total Williams82 Genomic DNA Expected Expression 35 136 6 1 1 8 27 15 8 88 79 9 337 27 15 Sample PI45925B/ASR/12hai PI45925B/ASR/72hai PI45925B/Mock/12hai PI45925B/Mock/72hai PI45925B Total PI45925B Genomic DNA Expected Expression 9 41 2 2 7 77 6 27 Rpp4C4 93 9 87 95 365 27 122 Rpp4C5 22 1 Rpp4C4 is the candidate gene for Rpp4-mediated resistance.

Sequencing of the Rpp4 locus in PI45925B

Sequencing of the Rpp4 locus in PI45925B 1. Develop BAC library from PI45925B (TOFU) 1. Screen library with primers designed from the Rpp4 candidate genes from Wm82. 1. Sequence candidate BACs. 1. Identify candidate resistance genes.

The Rpp4 locus in PI45925B TOFU 81O7 TOFU 98D16 TOFU 51G21 TOFU 2H3 TOFU 86D1 TOFU 66N11 TOFU 3J15 TOFU 4O11 Glyma18g: 55,747,53..55,914,731 167,678 bp? Rpp4 homology Transposon or Retrotransposon homology

The Rpp4 locus in PI45925B TOFU 81O7 TOFU 98D16 TOFU 51G21 TOFU 2H3 TOFU 86D1 TOFU 66N11 TOFU 3J15 4O11 Glyma18g: 55,747,53..55,914,731 Rpp4 homology 167,678 bp? Transposon or Retrotransposon homology Initiated sequencing of eight Rpp4 candidate BACs.

The Rpp4 locus in PI45925B 81O7 98D16 51G21 2H3 86D1 3J15 66N11 4O11 5K 1K 15K 2K 25K 3K 35K 4K 45K 469K Rpp4R1 Rpp4R2* Rpp4R2* Rpp4R3 Rpp4R3 Rpp4R4* Rpp4R4* Rpp4R5 Rpp4R5 Rpp4R6 Rpp4R6 Rpp4R7 Rpp4R7 Rpp4R8 Rpp4R8

Rpp4C5 Rpp4C4 The good... Rpp4C4 is still our candidate for Rpp4. Other genes in this cluster could also provide ASR resistance. The bad... The genes in the cluster range from 25 to 45 kb. The ugly The transformation vector is only 9 kb.

Why is ASR Resistance Rare? The Rpp4 candidate genes are members of the most common class of R-genes. R There are >3 NBS/LRR R-genes R in Arabidopsis and >5 in Rice 1. NBS/LRR R-genes R are often clustered in the genome leading to duplication, recombination, and the evolution of new pathogen specificities 1. However, BLASTN searches (E<1-4 ) failed to identify additional genes with homology to the Rpp4 candidate genes. 1 Meyers et al., 25

Is the rarity of Rpp4 homologs linked to cost? Its possible that the cost of maintaining the Rpp4 homologs genes in the absence of ASR is greater that the benefit derived from resistance during pathogen attack. In the absence of P. syringae, RPM1 reduces seed number in Arabidopsis by 9% (Tian( et al. 23). Given the agronomic importance of soybean, a 9% reduction in yield would have been immediately selected against. If so, wild legumes may be a better source of resistance genes.

Status of Rpp1, Rpp2, Rpp3 and Rpp5

Status of Rpp1 Research Rpp1 (immune response) and Rpp1-b (hypersensitive response) map to the same region on soybean LG G (Hyten et al. 27, Chakraborty et al. 29). Rpp1 and Rpp1-b are either tightly linked genes or are alleles of the same gene. Microarray analyses of Rpp1 complete (Fredrick, USDA/ARS) Rpp1 and Rpp1-b are located between SSR markers Sct_187 and Sat_64. LG G (18) Rpp1 Rpp1-b Marker information can be used to browse genome sequence for candidate genes.

http://soybase.org/gbrowse/cgi-bin/gbrowse/gmax1.1/

Position on C18 SSR markers

Gene models: 7 NBS/LRR resistance genes, 3 protein kinases

Status of Rpp2 Research Rpp2 mapped to LG J by Silva et al. and Garcia et al.(27). rpp2? mapped to same region by Garcia et al. Rpp2 rpp2? Comparison with genome sequence identified 19 NBS/LRRs,, 15 protein kinases and 3 Mlo-like like genes. Fine mapping under way (Diers( and Hudson) Sequencing underway in PI 2397 (Rpp2,( Graham) VIGS of candidate genes underway (Graham, Whitham and Pedley) LG J Microarray analyses of Rpp2 complete.

Status of Rpp3 and Rpp?? (Hyuuga( Hyuuga) ) Research Rpp?(Hyuuga) mapped to LG C2 by Monteros et al. 27 (Hyuuga x Dillon). Rpp3 Rpp? Rpp3 mapped to same region by Hyten et al. 29 (PI 462312 x Wm82). Rpp? and Rpp3 are either allelic or tightly linked genes. Comparison with genome sequence identified 6 NBS/LRR genes and 26 receptor-like kinases in a 9 kb region. Microarray analyses of Rpp3 complete (Fredrick and Whitham) LG C2 (6)

Status of Rpp5 Research Rpp5 rpp5 Rpp5 (D, PI 2526), Rpp5 (ID, PI 47194), rpp5 (R, PI2456) mapped to LG N by Garcia et al. 28. Genes are either allelic or tightly linked. Comparison with genome sequence identified no obvious candidate resistance genes. Fine mapping currently underway (Diers) LG N (3)

Summary Release of the soybean genome sequence and improvements in genotyping have facilitated the mapping of ASR resistance genes. In all cases but one (Rpp5)( these efforts have identified a cluster of candidate resistance genes. Microarray analyses have been essential in understanding the mechanisms behind resistance. However, identifying a single candidate gene remains difficult.

Summary Our analyses of Rpp4 suggest that resistance loci are much more complex then expected. By combining molecular approaches, we have been able to identify a single candidate resistance gene. Continued analyses of the Rpp4 locus in soybean and Phaseolus will help us understand how novel resistance specificities are generated and may shed light on ASR s s ability to overcome resistance so quickly.

Acknowledgements Michelle Graham Jenelle Meyer Lori Lincoln Catherine Ford Randy Shoemaker Jamie O RourkeO Greg Peiffer Johanna Dobbs Kerry Pedley Rex Nelson Steven Cannon David Grant Steve Whitham Martijn van de Mortel Chunling Yang John Hill Chris Zhang Al Eggenberger Thanks to David Hyten,, USDA-ARS, ARS, Rpp1, Rpp1-b Brian Diers,, Univ. of Illinois, Rpp2, Rpp5 Roger Boerma,, Univ. of Georgia, Rpp3 Ricardo Abdelnoor Danielle Silva