Volume 7, Issue 12, Pages 1740-1755 (December 2014) The Stripe Rust Resistance Gene Yr10 Encodes an Evolutionary-Conserved and Unique CC–NBS–LRR Sequence in Wheat  Wei Liu, Michele Frick, Réné Huel, Cory L. Nykiforuk, Xiaomin Wang, Denis A. Gaudet, François Eudes, Robert L. Conner, Alan Kuzyk, Qin Chen, Zhensheng Kang, André Laroche  Molecular Plant  Volume 7, Issue 12, Pages 1740-1755 (December 2014) DOI: 10.1093/mp/ssu112 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 RAPD Amplification of a 1100-bp Polymorphic Fragment Using Primer OPE5 ‘5’-TCAGGGAGGT-3″. R, bulk of Cot fractionated DNA from 14 resistant lines; S, bulk of Cot fractionated DNA from four susceptible lines; r, Cot fractionated DNA from individual resistant lines; s, Cot fractionated DNA from individual susceptible lines; M, 100-bp ladder. Arrowhead indicates the 1100-bp polymorphic fragment. Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Fluorescent In Situ Hybridization of Probe 4B to Moro Metaphase Chromosomes. The hybridization signals are located at the distal ends of the short arm of chromosome 1B (white arrows). Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Alignment of Deduced Amino Acid Sequences of Genomic Clones 4B (Upper) and 4E (Lower). Conserved regions CC, P-loop, kinase 2a, kinase 3a, conserved domains 2 and 3, conserved domains Q/EGF and HD, and LRR are underlined. * indicates identical residues;: indicates conserved substitutions;. indicates semi-conserved substitutions. Sequences of fragments γK2a, γSLR, and γCLR used for gene silencing are underlined with the thick line. Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Characterization of Clones 4B and 4E. (A) Comparison of the consensus sequence of 14 amino acids and variable region of the 11 leucine-rich repeats between clones 4B and 4E. (B) Cartoon representation of the identified cis-acting elements of the promoter regions of clones 4B (upper) and 4E (lower). The predicted transcription start site (TSS) is indicated for clone 4B. (C) Southern blot of RT–PCR amplicons obtained with 4E specific and Δ4E primers. Δ4E primers amplified 359-bp and 302-bp fragments from clones 4B and 4E, respectively. 4E primers amplified a 524-bp fragment only in 4E. Wells 4B, 4E, and R represent amplification products from the 4B plasmid, the 4E plasmid, and genomic samples from four different stripe rust resistant lines. Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Comparison of Deduced Amino Acid Sequences including Insertions from Genomic Sequences for Yr10 and Homologs Using ClustalW2. Sequences are Triticum aestivum Yr10 (AF149112), Aegilops tauschii (AF509533), Dasypyrum breviaristatum (EU428764), Brachypodium distachyon (XM_003577421), Sorghum bicolor (SORBIDRAFT_05g018980), and Oryza sativa (LOC_Os11g37759.2). Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Phenotypic Reaction of Fielder, Moro (Yr10), and Transformed Fielder (Yr10) (T1) after Inoculation with Pst Races SRC-84 (A) and CDL-29 (B). Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Moro Wheat Leaves Inoculated with BSMV Constructs Used to Silence the Wheat Phytoene Desaturase (PDS). (A) Mock inoculation with sterile buffer. (B) Moro inoculated with the BSMV:γ00 construct. (C) Moro inoculated with the BSMV:γPDS-as construct. Photos were taken at 12 d after treatments. Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 8 Stripe Rust Reaction in Silenced Moro Leaves Inoculated with P. striiformis at 20 dpi. (A, B) Moro and Moro transfected with BSMV:γ00, incubated for 8 d, and subsequently inoculated with race CDL-29. (C, D) Moro and Moro transfected with BSMV:γ00 and subsequently inoculated with race SRC-84 (note necrosis caused by the hypersensitive reaction). (E–G) Moro transfected with the γK2a, γSLR, and γCLR, and inoculated with race SRC-84 (note presence of striping in (E), (F), and (G), and necrotic regions). Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 9 qRT–PCR of Yr10 in Moro Following Inoculation with P. striiformis. BSMV vectors carrying different Yr10 domain target fragments, γKa2, γSLR, and γCLR were transfected into Moro and after subsequently being inoculated with race SRC-84. Values were compared to the treatment receiving the BSMV vector without an insert (BSMV:γ00). Molecular Plant 2014 7, 1740-1755DOI: (10.1093/mp/ssu112) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions