Molecular Plant 7, 1740–1755, December 2014
RESEARCH ARTICLE
The Stripe Rust Resistance Gene Yr10 Encodes an Evolutionary-Conserved and Unique CC–NBS–LRR Sequence in Wheat
Wei Liua,b, Michele Frickb, Réné Huelb,c, Cory L. Nykiforukb,d, Xiaomin Wanga,b, Denis A. Gaudetb, Fran?ois?Eudesb, Robert L.?Connerb,e, Alan?Kuzykb,f, Qin?Chenb,f, Zhensheng?Kanga,1, and André?Larocheb,1
a State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China b Agriculture and Agri-Food Canada, Lethbridge Research Centre, 5403 1st Avenue South, Lethbridge, Alberta T1J 4B1, Canada c Current address: The International Commission on Missing Persons, Alipasina 45A, Sarajevo 71000, Bosnia and Herzegovina d Current address: Emergent BioSolutions, 155 Innovation Drive, Winnipeg, MB R3T 5Y3, Canada e Current address: Agriculture and Agri-Food Canada, Morden Research Centre, Unit 100–101, Route 100, Morden, MB R6M 1Y5, Canada f Retired from Agriculture and Agri-Food Canada
ABSTRACT The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionaryconserved and unique CC–NBS–LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC–NBS–LRR sequence is unique among known CC–NBS–LRR R genes in wheat but highly conserved homologs (E?=?0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC–NBS–LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species. Key words: seedling or all-stage stripe rust resistance; gene functionality; transgenesis; gene silencing; homolog sequences; coiled-coil region; nucleotide-binding site; leucine-rich repeat domain. Liu W., Frick M., Huel R., Nykiforuk C.L., Wang X., Gaudet D.A., Eudes F., Conner R.L., Kuzyk A., Chen Q., Kang Z., and Laroche A. (2014). The stripe rust resistance gene Yr10 encodes an evolutionary-conserved and unique CC–NBS–IRR sequence in wheat. Mol. Plant. 7, 1740–1755.
INTRODUCTION
Stripe rust or yellow rust, caused by Puccinia striiformis Westend. f.?sp. tritici Eriksson (Pst), is one of the most damaging diseases of wheat (Triticum aestivum L.) worldwide, particularly in cooler and wetter regions (Chen, 2005). During severe epidemics, yield losses can exceed 75% and quality losses due to shriveling of kernels may be extensive (Conner and Kuzyk, 1988). The deployment of resistant cultivars is the most economical and preferred control method. Two major forms of resistance have been deployed to control stripe rust: seedling or all-stage resistance (R gene-mediated resistance) and adult plant resistance (APR)
(Chen, 2005). To date, over 50 stripe rust resistance genes (R genes and APR genes) have been catalogued but only a handful of these resistance genes have been deployed
To whom correspondence should be addressed. A.L. E-mail andre. laroche@agr.gc.ca, tel. 403-317-2224, fax 403-382-3156. Z.K. E-mail kangzs@https://www.doczj.com/doc/f913290363.html,, tel. 86-29-87082857, fax 86-29-87082892. ? Her Majesty the Queen in right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CIBS and IPPE, SIBS, CAS. doi:10.1093/mp/ssu112 Advance Access publication 20 October 2014 Received 23 July 2014; accepted 22 September 2014
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in commercial cultivars (McIntosh et?al., 2005; Kuraparthy et?al., 2007). To date, two stripe rust resistance APR genes, Yr36 (Kinase-START gene) and Lr34/Yr18 (ABC transporter gene), have been identified and characterized (Fu et al., 2009; Krattinger et?al., 2009). No stripe rust resistance seedling resistance genes have been characterized. According to current models of host–parasite interactions, R genes encode protein receptors that specifically interact with corresponding effectors produced by the pathogen and encoded by Avr genes, to induce effectortriggered immunity (ETI) (Jones and Dangl, 2006; Boller and He, 2009). Members of the largest class of R genes encode cytoplasmic proteins with a nucleotide-binding site (NBS) and several leucine-rich repeats (LRRs). However, in cereals, only a few R genes encoding NBS–LRR proteins have been characterized including NBS–LRR genes governing wheat leaf rust resistance (Lr1, Lr10, Lr21) (Feuillet et?al., 2003; Huang et al., 2003; Cloutier et al., 2007), stem rust resistance (Sr35, Sr33) (Periyannan et al., 2013; Saintenac et?al., 2013), and powdery mildew resistance (Pm3b, Pm8) (Yahiaoui et al., 2004; Hurni et al., 2013). NBS–LRR genes have not been identified to date among stripe rust-resistant genes in?wheat. The wheat cultivar Moro possesses the seedling resistance gene Yr10 located on the short arm of chromosome 1B (Metzger and Silbaugh, 1970). Alternative sources for the stripe rust resistance gene Yr10 were identified in T.?spelta accession 415 (Kemma and Lange, 1992) and in T.?vavilovii accession AUS22498 (Bariana et?al., 2002). The Yr10 gene continues to provide effective resistance to stripe rust in wheat in most wheat-growing areas but virulent Pst races have been reported (Chen et?al., 2010). The putative Yr10 sequence was first cloned in wheat and submitted to GenBank as accession # AF149112 in 2000. The NBS domain of Yr10 was used to identify putative NBS–LRR resistance gene family members associated with leaf and stripe rust resistance in the terminal regions of homoeologous chromosome group 1S of wheat (Spielmeyer et?al., 2000). Xpsp3000 and/or Gli-B1 can be used for markerassisted selection of Yr10 (Bariana et al., 2002). Previous studies focused on molecular markers linked to Yr10 in wheat but no functional analysis of Yr10 has been reported. Because of the complexity and size of the wheat genome, identification and cloning of genes are timeconsuming and difficult. Moreover, analyzing gene function is complicated by the difficulty of transforming wheat (Jones, 2005). Virus-induced gene silencing (VIGS) using barley stripe mosaic virus (BSMV) has been adapted for use in other cereals (Holzberg et?al., 2002) and employed for silencing rust candidate R genes in wheat (Scofield et?al., 2005; Cloutier et?al., 2007; Zhou et?al., 2007). In the present study, we demonstrated that an expressed genomic sequence from chromosome 1B (clone 4B) constitutes the stripe rust resistance gene Yr10. Clone 4B represents a novel CC–NBS–LRR sequence and its
Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
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presence in cultivars possessing Yr10 and in lines segregating for this gene was demonstrated. Functionality of Yr10 was assessed using stable plant transformation and BSMV– VIGS. Molecular analyses were supported by histological observations during infection. Highly conserved homologs were absent in wheat but were identified in the wheat progenitor Aegilops tauschii and in several host and nonhost monocots for stripe rust including Dasypyrum breviaristatum, Brachypodium distachyon, Oryza sativa, and Sorghum bicolor. Related sequences were also identified in genomic databases of maize (Zea mays) and sugarcane (Sacharrum spp.). The presence of CC–NBS–LRR homologs in related species that are non-hosts to stripe rust and their potential future benefit as sources of resistance genes are discussed.
RESULTS
Identification and Characterization of a Polymorphic Fragment Linked to Yr10
Using RAPD, a polymorphic DNA fragment of about 1100 bp was identified in the resistant bulk of Cot fractionated DNA from BC4F5 lines from a Moro x Fielder cross using the OPE5 primer (Figure 1). Stripe rust responses were scored using Pst race SRC-84 that is avirulent on lines carrying Yr10. The 1100-bp fragment was present in all the 14 resistant BC4F5 lines, and absent in all susceptible lines tested. Following initial sequencing results, specific PCR primers based on extending the original decamer sequence (SCARs) were designed (Supplemental Table?1). The SCAR primers amplified a specific DNA fragment on both fractionated and total genomic DNA originating from resistant lines. This fragment, designated E51100, was also present in
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.
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the parental wheat cultivar Moro and accession PI 178383, the source of stripe rust resistance in Moro. Analysis of a segregating population of 874 lines showed that E51100 was perfectly linked to the phenotype (422 R: 452 S). The length of the fragment was 1085 bp after complete sequencing and similar to the 1100-bp deduced from agarose gel sizing (Supplemental Table?1). BLAST analysis of E51100 at the time when no monocot NBS gene sequences were available showed that a portion of this fragment had a small but significant level of homology at the amino acid level (score 94, P < 1.4 × 10–4) with the P-loop region of the L6 rust resistance gene in flax (Lawrence et?al., 1995).
The Specific 1100-bp DNA Fragment Is Detected in All Wheat Lines Carrying Yr10
A very wide hexaploid wheat germplasm collection, which included Jacmar (Yr10), an Avocet + Yr10 near-isogenic line (NIL), Avocet S, 16 other Avocet NILs carrying different Yr genes, Corrigin (S), Corrigin + Yr10, QT3960 (S), QT3960 + Yr10, 79W93 (S), 79W93 + Yr10, Cappelle Desprez (Yr3a), Compair (Yr8), T. spelta (Yr5), and T. spelta accession 415 (Yr10), was screened using the SCAR primers derived from the 1100-bp fragment on DNA isolated from leaves of seedlings. The 1100-bp fragment was amplified only in lines known to carry Yr10 and its identity was verified by Southern blot hybridization (data not shown). No fragment was observed in gels and Southern blots of PCR amplicon gels for DNA samples isolated from susceptible lines or expressing other Yr resistance genes. Upon hybridization of a biotinylated clone 4B probe, a strong and specific signal was observed only on the distal end of the short arm of chromosome 1B (Figure?2), the reported genetic location of Yr10 (Metzger and Silbaugh, 1970; McIntosh et al., 2005). The identification of chromosome 1B was facilitated by the presence of a nucleolar organizer region on this chromosome. 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).
Isolation and Characterization of Clones 4B and?4E
Seven of 350 000 genomic clones from a Moro sub-genomic library were hybridized with the E51100 fragment. Of these, five gave a weak signal whereas two others, clones 4B and 4E, exhibited a strong signal and were selected. Clones 4B and 4E were estimated to be 6.3 and 7.0 kb in size, respectively, from restriction digests and their sequences comprised ORFs of 3630 and 3662 nucleotides, respectively (from the ATG translation start site to the termination codon TGA) (GenBank accessions # AF149112 and AF149113). These two clones are 84% identical and a similar level of conservation was observed between intron and exons for the two clones. Both clones contain a single intron (1155 bp for 4B and 1129 bp for 4E). The presence and exact location of the
intron in the kinase-2A domain within the NBS region of clone 4B were verified using cDNA clones obtained by RT– PCR. The intron includes nucleotides 834 to 1988 of Yr10 genomic sequence accession # AF149112 and is inserted between the second and third codon of amino acid # 278 arginine (R) (Figure?3). The intron in clone 4E was assigned by consensus homology with clone 4B. Nine deletions and four insertions ranging from 1 to 123 nucleotides located in the intron and LRR region were observed in clone 4E. The two most important deletions were located in the LRR region (57 bases after nucleotide 2740)?and in the intron (14 bases after nucleotide 1911)?while an insertion of 123 nucleotides was observed at the 3’ end of the coding sequence (nucleotide 3540). The deduced amino acid sequences from clones 4B and 4E encode predicted open reading frames of 824 and 843 amino acids, respectively (Figure 3). Both clones exhibit features of NBS-type disease-resistance genes. These characteristics include a putative coiled-coil (CC) region at the 5’ end, a central NBS, and a LRR domain at the 3’ end (Figure?3). In both clones, P-loop, kinase-2a and kinase-3a domains, conserved domains 2 and 3, as well as other conserved domains were identified (Figure?3). Eleven imperfect LRR motifs were deduced in the 3’ end of clone 4B, based on the identification of the 14-amino acid (aa) consensus sequence xxLxxLxxLxxLxL/I. The length of each unit varied from 21 to 31 aa (Figure?4A). When compared to clone 4B, one additional LRR motif was identified at the end of the sequence in clone 4E. However, due to the deletion of 19 aa, 11 LRR repeats were also identified in this clone (Figure?4A). Using primers specifically designed to amplify the 4E sequence (Supplemental Table?1), clone 4E was found to be closely linked to clone 4B, as only one recombination event
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Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
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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 ?K2a, ?SLR, and ?CLR used for gene silencing are underlined with the thick line.
was observed in a random subset of 55 BC2F3 and BC3F3 segregating lines. This suggested that clones 4B and 4E were members of the same gene cluster. Approximately 1300 bp were sequenced upstream of the translation start codon for each genomic clone. Using RACE–PCR of cDNA, it was possible to identify the position of a transcription start site (TSS) 159 bp upstream from the ATG only in clone 4B. Furthermore, a putative TATA-box and a CAAT-box were detected 191 bp and 202 bp upstream of the translation start site in clone 4B (Figure?4B). No evidence for these two boxes could be found in clone 4E. However, numerous light-responsive elements and a few stress (ABRE and Myb) and plant defense (EIRE (elicitor), WUN (fungal), ER (ethylene), MeJA (methyljasmonate), TCA (salicylic acid))responsive elements were identified within the 1300-bp upstream of ATG in both clones. Only a few of these elements were in similar positions within the two clones. The absence of a TSS and putative TATA-box and CAAT-box in clone 4E suggests that this clone is a non-expressed pseudogene. mRNA corresponding to clone 4B was present in very low abundance and at least 4 ?g of poly-A+ RNA from Moro were needed to detect an mRNA of approximately
2.5 kb in size using Northern blot analysis (data not shown). Using RT–PCR with different primers, two overlapping cDNA clones corresponding to clone 4B were obtained and assembled (GenBank accession # AF149114). There was near perfect identity (99.9%) between the cDNA sequence and genomic clone 4B, as only two nucleotide substitutions were observed with the obvious exception of the intron. Using two pairs of specific primers with RT–PCR, the sequence corresponding to clone 4B was fully expressed in wheat lines carrying Yr10 whereas the 4E sequence seems to represent a non-expressed sequence (Figure?4C). These results demonstrate that the sequence corresponding to clone 4B is expressed in Moro wheat carrying the Yr10 resistance gene and that 4E represents a pseudogene.
Clone 4B Encodes an Evolutionary-Conserved CC–NBS–LRR Sequence
The deduced aa sequence of genomic clone 4B exhibits the highest homology (E-value = 0.0) to CC–NBS–LRR sequences from wheat lines identified as carrying the Yr10 resistance in Chinese cultivars (GenBank accessions
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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 ?4E primers. ?4E 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.
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# HM461975, HM231239) as well as to highly conserved homologs from D. breviaristatum (EU428764), A. tauschii (AF509533), B. distachyon (XM_003577421), S. bicolor
Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
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(SORBIDRAFT_05g018980), and O.?sativa (LOC_Os11g37759). Protein alignment among the genomic sequences for these ORFs and the interspersed introns indicated a high level of
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G EG F I H E EQGK S L Y EV G ED Y I A E L I N K S G E G L V R E E Q G MS L Y E L G E D Y I A E L I N K S G EG F I H V EQGK S L Y EV G ED Y I A E L I N K S G EG F V Q E EH GR S L Y EV G ED Y F H E L I N K S G EG I I QK QH GQ T F Y EA G ED C F ED L I N R S C E G F V R A E H MK T L H V V G M E Y L N E L WN K S
590 . . . . | . . . . |
L L L L I L
. . . . |
510 520 530 540 550 560 570 580 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
600 . . . . |
Triticum aestivum Dasypyrum breviaristatum Aegilops tauschii Brachypodium distachyon Sorghum bicolor Oryza sativa
484 V Q P MY I N I A N K A S S V R V H D MV L D L I 482 I Q P MD T S I A D K A S S V R V H D MV L D L I 485 V Q P MD I N I A N K A S S V R V H D MV L D L I 479 I Q P V D I K S G N K A S A C R V H D MV R D L V 497 I Q P M I L T MG T MY E P T R - - - - - - - - 480 I Q P I S N - C D N M PWD Y C L H D MV L D L I
T S L S N E EN F L A T T S L S N E EN F V A T T S L S N E EN F L V T T S L S S E EN F L T I - - - - - - - - - - - T F L S N E EQ F MT S
L GGQQ T R S L PR K L GGQQ T R S L PS K L GGQH T R S L PG K L GD L Q PV S A S S K - - - - - - - - - - - K L GD QQ PML V PH K
I I I I I I
R R L S L Q S S N E E D V Q P M P T MS S L S H V R S R R L S I Q T S N E E D V K Q M P T MR S S S H V R S R R L S L Q T S N E E D V R P M P T MS S L S H V R S H R L S V Q K I N E D D F K Q L A T MS - L F H A R S R R L F L Q N MK V E D V K K V V T MN - L S RWR S R R L S L QS I K Q EY F K L I S N A D - L S H V R S
L L L L L L
TV FSKD LS L TV FSKD LR L TV FSKD LS L F V F G Q D MN L T V S S EA F T L I V S K QA F S L
LSA LSGF LS ELSGF LSA LSGF L PA L S S F L PTLSSF L PN L S S F
LV LV LV PV P I PV
L RA L CV L RA L RA I RV L RV
L L L L L L
. . . . |
610 620 630 640 650 660 670 680 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
690 . . . . | . . . . |
700 . . . . |
Triticum aestivum Dasypyrum breviaristatum Aegilops tauschii Brachypodium distachyon Sorghum bicolor Oryza sativa
584 582 585 578 564 578
D L S G C E E V G N H H MK D D L T GCK EV CDH H L K D D L S G C Q E V G N H H MMD D L S CC L NV DNY HV K I N L Y GC T QV D N S H C K H D L S GC D QV D N R H C R D
I I I I I I
CN L FH L RY CK L FH L RY CN L FH L RY CS MFH L RY CN L CH L RY CNML H L RY
L S L EG T S LS LKGTS L S L QG T S L S L CN TS LK LS L TS L N L HR TS
I I I I I I
TE I TE I T EV TK I TE I SE I
PK E I S N L R L L Q L P K E MS N L Q L L Q L P K E MS N L Q L L Q V PV E I GN L Q F L K V PN E I GN L Q L L Q F P E E I GN L Q F L L V
L V I R S T K MK K F P S T F V Q L G Q L V F - - I D MG N R E V S - - - R L L L K S L D I R S T K MK K L P S T F V Q L R Q L V F - - V D MG N K MV S - - - T L L L K A L D I R S I R I K K L P S T F V L L R Q L V S - - A D MG T R MV S - - - T L L L K S L D I S Q T G I E V L P S E F V Q L T Q L V Y L H I D MS V R L P E G L WN L K S L W L D L N M T N I K A L P P T F V Q L R K L E F L C V D N R T R L P EC L GN L I S L QK L D I T K T R L R E V P S T F V Q L Q Q L V D L C V G P G MR L P D D F G N L K T L Q S
- - - - - - - - - - - - L S PR I WPH
I I
- - - - - - - - - - - - T I SS Y V MS
. . . . |
710 720 730 740 750 760 770 780 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
790 . . . . | . . . . |
800 . . . . |
Triticum aestivum Dasypyrum breviaristatum Aegilops tauschii Brachypodium distachyon Sorghum bicolor Oryza sativa
668 666 669 667 664 678
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P TML C E L S ML P T M L R N MG G L
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T E L R R L M L R F D DWD D E S Y E E T F V Q C L S N L V N L E S L Q I T K L R H L S I R F H EWD E S Y Q K S F E L C L S N - - - - L V N L R S
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - F D C H N G L G S N S N I G M L L T T G P Q Q L R S MN I G P G T V R C V I T V R V Y E G V MD S K C E N L S P G P Q Q L E D I D MN R S V A N S V
- - - MS T - - - MS T - - - MS T - - - MS S P RW L P S P I WMS S
L PS L S S L PS L S S L PS L S S LSCLS F L FA L SA LS FLSS
LA LA LA LV LD LD
I I I I I I
G G T H T K
. . . . |
810 820 830 840 850 860 870 880 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |
890 . . . . | . . . . |
900 . . . . |
Triticum aestivum Dasypyrum breviaristatum Aegilops tauschii Brachypodium distachyon Sorghum bicolor Oryza sativa
681 - I G E L R E E D L Q I 679 - L K E P R E D D L Q I 682 - L R E L R E E D L Q I 681 G L K T L R V E D L Q V 763 - L L T L Q E E D L Q V 773 - L K T L G H K D L Q I
L GS M PS L GS M PS L GS M PS L GS I PS L GS I PS L GN M PS
L H D L S I D V GYWER GR D K R L V L RD L S I Y V PY V GD F T NK R L V L H D L S L F V V Y QGK V T Y K R L V L C D L D I WV V E P T Q E R H S R L L L R S L Y I WV K E H R K D R C K R L A L S D L T L WV N E P T Q D R H E R L V
I DS G - S P FRS L T D S G - C P F ES L I GN G - C P F QS L I D S S - Y P F QC L I GS D D C P F GC L I DNC - Y P FHC L
T R F S I K GC G F I D F M F A QG T L QK L Q I L E L S I F GK A I K D R F GD F Q F G L T R F R I K S WS A I G F M F A Q G T F I N L L K E I I S L V - - - - - - - - - - - - - - T R F S I K S I EA T V F M F A QG T L QK L Q I L E L D L Y V EK T K H Q F GD F Q F G L T S L K I A S R - V M E L K F A Q G A MQ K L Q T L K I R L S V R Q T WD Q F G N L D F G L T K I R I G P G - A M E V Q F A A G A MR K L R T A R L D L H V R H T L D Q F G D F H C G L T F L K L MA N - N M E V A F A Q G S MQ K L Q K L R F G F G V R K T V D Q F G N F D F G L
EN L S S - - - - EN L S S ENV S S ES L S S EK I S -
. . . . |
910 920 930 940 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .
Triticum aestivum Dasypyrum breviaristatum Aegilops tauschii Brachypodium distachyon Sorghum bicolor Oryza sativa
780 L E H V Y V D A R G R G I I P S Q E A E L S G A 757 - - - - - - - - - - - - - - - - - - - - - - - 781 V E H V Y V V A R S G - - - - G S E D E L T N A 779 L K H V Y V G RWS K P D - P G E V E A A E A I 862 L E R V I V H MN C Y R A E L E E V E A A E A S 870 V E H L D V C V N Y S D A N RW E V N A A E A A
L EK E L D I N PNK P T L T V K - V T PR - - - - - - - - - - - - - - - - - - - - - - L EK E L D I N PN K P I L T V K QV T PC I RK A L DV N PS K P T L V F S K V V RHV I R K A L D L N PNR PS F E L EK V V - - I R E A V N MN P N N P T L K L T T L F V L L
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).
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Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
Molecular Plant
Rust sporulation was not affected using the BSMV:?00 vector and the same pathogenic race (Figure?8B). In the incompatible interaction between Moro and race SRC-84, HR on Moro was observed on untreated and BSMV:?00-treated Moro leaves (Figure?8C and 8D). Symptoms were similar to those on SRC-84-inoculated Moro, demonstrating that the BSMV:?00-vector did not alter the normal compatible and incompatible interactions. Successful silencing of the Yr10 gene was observed following transfection with BSMV carrying three independent antisense DNA fragments as evidenced by the formation of pustules and infection levels ranging between 47% and 63% of cells at the microscopic level, in treatments inoculated with race SRC-84 that is avirulent on Moro (Table?1 and Figure?8E–8G). The incompatible interaction was only partially inhibited, since sectors lacking pustules were observed (Figure?8E–8G). Restriction of pustule formation in leaf sectors suggested that transfection had occurred only for part of the leaves with the BSMV vector carrying the silencing sequences. In these non-sporulating sectors, typical HR flecking was observed (Figure?8E and 8G). The highest level of silencing was observed with ZKHUHE\ Vymptoms were not significantly different in severity from Moro plants inoculated with the compatible CDL-29 race (Table?1). In all three silencing treatments, infection percentages were significantly higher (P ≤ 0.05) than those in Moro inoculated with race SRC-84 (Table?1). Microscopically, hyphal development in all treatments was similar up to 10 dpi. At 10 dpi, hyphae in fully compatible interactions started to spread and ramify surrounding mesophyll cells, frequently encircling them (Supplemental Figure 1). There were similar hypersensitive responses in Moro and BSMV:?00-treated Moro when inoculations were performed with the avirulent race SRC84. There were also no differences in the detailed infection events between Moro-Yr10 inoculated with the virulent race CDL-29 and Moro-Yr10 silenced with any of the three BSMV constructs when inoculated with the avirulent race SRC-84. These results demonstrated that silencing of Yr10
conservation considering the evolutionary distance among these species (Figure?5). The intron located in the kinase-2a domain was common to all species. An additional intron was present in the LRR domains of B. distachyon and S.?bicolor, but in two different locations.
Clone 4B Corresponds to Yr10 and Plays a Key Role in Moro Resistance to Pst
The stripe rust-susceptible cultivar Fielder was transformed with clone 4B under control of its native promoter. Fortysix plants were regenerated and produced seeds from a total of 40 bombarded scutella. These putative transgenic seedlings were screened directly with Pst race SRC-84 (virulent on Fielder). One transgenic line developed no pustules and displayed the characteristic hypersensitive response observed for the resistant cultivar Moro (Figure?6A). When the same transgenic line was inoculated with Pst race CDL29 (virulent on Moro), abundant rust development was observed (Figure?6B). These results demonstrate that clone 4B exhibits the same functional properties as Yr10 when transformed into the susceptible variety Fielder. Our initial tests using the BSMV–VIGS system in Moro demonstrated that Moro was susceptible to BSMV as mosaic symptoms could clearly be identified on the inoculated leaves, demonstrating systemic infection of BSMV in this cultivar. Eight days post inoculation (dpi) of the first and second leaves with the test vector BSMV:?PDS-as which silences the wheat phytoene desaturase gene, evident photobleaching was apparent in the second or third leaves of Moro and bleaching was clearly seen at 12 dpi. Simultaneously, no evidence of photobleaching was observed in plants infected with the native virus (BSMV:?00) (Figure?7). Three different fragments located in two domains of Yr10 (clone 4B) (Figure?3) were selected for silencing using BSMV–VIGS. Chlorotic spots first observed on Moro leaves 12 dpi with the virulent race CDL-29 was followed by extensive pustule formation at 20 dpi (Figure?8A).
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
in Moro resulted in a susceptible interaction that was macroscopically and microscopically similar to a fully compatible interaction. qRT–PCR on the fourth leaves sampled from 0 dpi to 14 dpi in these treatments demonstrated a generalized downregulation in the expression of the Yr10 gene in treatments silenced with either of the three partial sequences
Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
1747
of the Yr10 gene (Figure?9). The largest down-regulation was observed for BSMV:?CLR followed by BSMV:?Ka2 and BSMV:?SLR treatments in Moro. Compared with BSMV:?00, BSMV:?CLR, BSMV:?Ka2, and BSMV:?SLR showed reductions of 74.1%, 71.6%, and 46.3%, respectively (Table 1). Yr10 transcript levels (Figure?9) were consistent with stripe rust severities in the leaves (Table?1).
DISCUSSION
Despite the vast size and complexity of the wheat genome, we were able to identify and characterize the stripe rust resistance gene Yr10 by isolating a Cot DNA fraction and developing and screening a sub-genomic library. The first approach yielded a DNA fraction enriched for coding sequences (Eastwood et?al., 1994) which eventually led to identification of a DNA polymorphic fragment (E51100) that was perfectly linked to the resistance phenotype in a large segregating population of 874 lines. This approach has also been widely employed in other laboratories to reduce the complexity of large genomes in identifying different genes (Eastwood et al., 1994; Lamoureux et al., 2005; Paterson, 2006). The latter approach led to the identification of different candidate clones from which clones 4B and 4E were characterized. Sub-genomic libraries were also successfully employed to reduce genome complexity in identification of genes (Mandaokar and Koundal, 1996) and microsatellite markers (Farias et?al., 2006) in species without genomic reference. The original sub-genomic library approach was adapted to further accelerate gene discovery by using NGS
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:?00 construct. (C) Moro inoculated with the BSMV:?PDS-as construct. Photos were taken at 12 d after treatments.
Figure?8 Stripe Rust Reaction in Silenced Moro Leaves Inoculated with P.?striiformis at 20?dpi.
(A, B) Moro and Moro transfected with BSMV:?00, incubated for 8 d, and subsequently inoculated with race CDL-29. (C, D) Moro and Moro transfected with BSMV:?00 and subsequently inoculated with race SRC-84 (note necrosis caused by the hypersensitive reaction). (E–G) Moro transfected with the ?K2a, ?SLR, and ?CLR, and inoculated with race SRC-84 (note presence of striping in (E), (F), and (G), and necrotic regions).
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Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
Molecular Plant
et?al., 2003), and Lr21 (Huang et?al., 2003), two stem rust resistance genes (Sr35, Sr33) (Periyannan et al., 2013; Saintenac et al., 2013), and two powdery mildew resistance genes (Pm3b, Pm8) (Yahiaoui et?al., 2004; Hurni et?al., 2013). Based on the presence and location of intron(s), Lr10 appears to be the most closely related sequence to Yr10. However, identity between the two sequences at the amino acid level was only 40% while at the nucleotide level it was barely significant. This uniqueness also extends to the number of imperfect LRR units; Yr10 possesses 11 whereas Lr10 possesses 14. This would be expected due to the putative role of the LRR domain of plant R proteins in determining recognition specificity (Dodds et?al., 2006). Given the low level of homology between Yr10 and the other seven cloned wheat CC–NBS–LRR resistance genes (Feuillet et al., 2003; Huang et al., 2003; Yahiaoui et al., 2004; Cloutier et al., 2007; Hurni et al., 2013; Periyannan et?al., 2013; Saintenac et?al., 2013), Yr10 is clearly distinct from previously characterized wheat NBS–LRR genes. Yr10 exhibited high similarity to sequences from different cereal and grasses, demonstrating that Yr10 and its homologs represent a well-conserved gene family among monocots with the kinase-2a domain intron present in all species. To date, this is a two-member family represented by one expressed gene and one pseudogene in wheat. The identification of a few highly conserved homologs in A. tauschii, D. breviaristatum, B. distachyon, O. sativa, and S.?bicolor (Figure?5) suggests that the sequence encoding Yr10 originates from a theoretical progenitor more than 60 MYA (million years ago), prior to divergence of the Panicoideae (sorghum, maize) and the ancestor that further evolved into the Pooideae (wheat, rye, barley, and wild relatives) and the Ehrhartoideae (rice) about 50 MYA (Chalupska et al., 2008). We do not know at this point whether Yr10 represents a gain of function in wheat or whether that functionality was present in the ancestral progenitor leading to speciation of Aegilops, Dasypyrum, Triticum, and Brachypodium, rice, sorghum, maize, and
technology (Herman et al., 2009). We characterized two highly related genomic sequences (clones 4B and 4E) from Moro wheat and further demonstrated that clone 4B was the only expressed sequence corresponding to seedling resistance gene Yr10. Yr10 belongs to the cytoplasmic CC– NBS–LRR class of genes known to represent many different resistance genes in plants (McDowell and Simon, 2006). Yr10 is the first CC–NBS–LRR gene reported to provide resistance to stripe rust in cereals. The CC–NBS–LRR class of genes is well represented in monocots (Tarr and Alexander, 2009). Among the few cloned CC–NBS–LRR disease-resistance genes in wheat, three genes are related to leaf rust resistance, namely Lr1 (Cloutier et?al., 2007), Lr10 (Feuillet
Table?1 Silencing of Yr10 Transcripts and Stripe Rust Infection Levels in Treated Moro Wheat Leaves.
Treatment? Silencing efficiency at 8 dpi (%)* N/A 74.1 71.6 46.3 N/A
N/A
Rust infection (%)?,§ 16 dpi 58.3 ± 3.6a,§ 42.3 ± 16.5 32.0 ± 17.3a,b,c 31.0 ± 6.0a,b,c 6.8 ± 5.1d
2.3 ± 0.4d
a,b
20 dpi 73.7 ± 2.7a 63.2 ± 6.0a,b 51.3 ± 14.6b,c 47.3 ± 8.8b,c 2.8 ± 1.8d
4.3 ± 1.0d
CDL-29 ?CLR ?K2a ?SLR SRC-84
?00
? Treatments as follows: CDL-29 is virulent on Yr10; SRC-84 is avirulent on Yr10; BSMV–VIGS constructs ?K2a, ?SLR, and ?CLR target different regions of the mRNS (see the ‘Methods’ section and Figure?3); ?00 corresponds to the empty vector. Except for the treatment receiving the virulent strain CDL-29, all other treatments were inoculated with the avirulent strain SRC-84. * Silencing efficiency represents the percentage reduction in the Yr10 transcript. ? Infection values represent percentage of the inoculated leaf area exhibiting pustules. § Means that are followed by different letters are statistically different using Duncan’s Multiple Range test.
Figure?9 qRT–PCR of Yr10 in Moro Following Inoculation with P.?striiformis.
BSMV vectors carrying different Yr10 domain target fragments, ?Ka2, ?SLR, and ?CLR 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:?00).
Molecular Plant
sugarcane (Chalupska et?al., 2008). Additional experimental work is needed to verify whether these homologs from these species could represent novel R genes against stripe rust in wheat. Whether Yr10 homologs in rice, maize, and sorghum would confer resistance to wheat stripe rust or other pathogens is currently unknown. Although an important fitness cost has been suggested to explain the absence of different R genes in Arabidopsis (Vogel et al., 2002; Tian et al., 2003), alternative options may explain the absence of Yr10 or Yr10 homologs in wheat germplasm. If there was a major fitness cost associated with any gene, it would not be possible to identify the gene or homologs after thousands of years of evolution based on fitness cost (Brown and Rant, 2013). It has been reported that mutation and intragenic recombination in the LRR domain of a R gene resulted in maize lines with necrosis levels ranging from mild to heavy necrotic spotting (Brown and Rant, 2013). It is then possible that potential variants of Yr10 with a deleterious impact on the plant might have been eliminated during evolution, thus explaining the absence of Yr10 homologs in?wheat. Successful transformation of the susceptible cultivar Fielder (Su et?al., 2003) with clone 4B to impart resistance to P. striiformis race SRC-84 demonstrated its functionality as a Yr gene. The Yr10 allele carries all the necessary machinery to convert a fully compatible to a non-compatible interaction involving a hypersensitive response. A?similar finding was reported when the functionality of the leaf rust resistance gene Lr1 was demonstrated (Cloutier et?al., 2007). Incompatibility and compatibility are governed by the combined genotypes of host and pathogen (Jones and Dangl, 2006). Our results demonstrate that Yr10 encodes a CC–NBS–LRR protein that recognizes a corresponding avirulence protein(s) and elicits a hypersensitive response. BSMV–VIGS proved to be a very efficient tool to further characterize functionality of Yr10 with a high silencing efficiency based on utilization of three independent fragments of the target gene. Similar results were observed with the same viral vector in other wheat-pathogen systems (Holzberg et?al., 2002; Scofield et?al., 2005; Cloutier et?al., 2007). Successful gene silencing depends upon interplay between viral spreading and pathogen invasion. Both can be influenced by environmental conditions, especially temperature. The efficiency also depends on the insert size and nature of target sequence (Bruun-Rasmussen et?al., 2007). There was no evidence that the BSMV vector interfered with macro- or microscopic resistance responses governed by Yr10 to an avirulent P.?striiformis race. The presence of leaf sectors without pustules suggests that systemic spread of BSMV was incomplete or, alternatively, virus expression was reduced or lacking in these sectors, thus enabling normal or reduced expression of the Yr10 protein in these sectors. This phenomenon was also reported by others (Scofield et?al., 2005; Cloutier et?al., 2007; Zhou et?al., 2007; Yin et?al., 2011).
Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
1749
Silencing of Yr10 was accompanied by a reduction in the level of Yr10 transcripts, with the greatest reduction observed between 4 and 10 dpi which coincides with haustorial formation in Moro inoculated with a virulent strain of P.?striiformis (Wang et?al., 2014). The results also demonstrated that the key mechanism(s) for incompatibility involving Yr10 at the cellular level was silenced by all three constructs. NBS–LRR proteins mediate pathogen recognition in both mammals and plants, but the mechanisms are still unclear. Clone 4E, a Yr10 pseudogene, exhibited a characteristic inability to encode mRNA. Pseudogenes are considered disfunctional genes and are of genetic interest because they tend to evolve much faster than functional genes and mechanisms such as recombination and gene conversion can move these new and novel sequences between the pseudogene and functional alleles (Ota and Nei, 1994; Michelmore and Meyers, 1998). Thus, pseudogenes represent a mechanism for more rapid evolution of new versions of genes. Pseudogenes may be advantageous over functional genes in contributing to gene evolution, particularly if there is a genetic cost to maintaining functional R genes in the population (Michelmore and Meyers, 1998). P. striiformis is a rapidly evolving pathogen that is constantly generating new races with novel virulence combinations and physiological types (Chen, 2005; Chen et?al., 2010). Therefore, the 4E pseudogene may represent an adaptive mechanism in wheat to counter the rapid virulence shifts in the pathogen by accelerating the evolution of specific resistance genes capable of recognizing new pathogen forms (Jones and Dangl, 2006). The Yr10 resistance gene continues to that provide effective resistance to stripe rust in many parts of the world. We have cloned and sequenced the Yr10 gene and shown its functionality using transgenic expression and gene silencing. Yr10 and its pseudogene appear to be unique, evolutionarily conserved CC–NBS–LRR alleles conferring seedling stripe rust resistance. Highly conserved homologs were identified in the wheat progenitor A. tauschii and several host and non-host monocots. Future work is being directed towards cloning and characterizing homologs from non-host monocot species and evaluating the potential for different domains to contribute to stripe rust resistance in wheat. These species could serve as a new reservoir for resistance genes to an ever-evolving pathogen that is rapidly exhausting current resistance sources.
METHODS
Germplasm
Resistant F2 plants from a cross between stripe rust resistant cultivar Moro and susceptible cultivar Fielder were backcrossed to Fielder. Following backcrossing, thereafter all lines that were either homozygous resistant or
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Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
Molecular Plant
DNA fragment, DNA was isolated and fractionated (Eastwood et al., 1994) to obtain a single/low copy fraction for each line. Bulk segregants samples (Michelmore et?al., 1991) made from 14 resistant lines and four susceptible lines from the BC4F5 of Fielder x Moro were screened by RAPD–PCR with approximately 120 primers (Operon Technologies Inc., Alameda, CA, USA, Kits B–H) (Williams et?al., 1990; Demeke et?al., 1996). Polymorphic fragments were cloned in the pGEM–T vector (Promega, Madison, WI, USA), transfected into Escherichia coli DH5? cells (Invitrogen Canada Inc., Burlington, ON, Canada) and double-strand sequenced using ABI Prism or BigDye sequencing kits (Applied Biosystems, Foster City, CA, USA). Sequences were obtained using an automated sequencer (ABI 373A). Sequence analyses were performed using SeqEd (ver. 1.0.3 ABI) and MacDNASIS (ver. 3.6 Hitachi). SCAR primers were designed based on extension of decamers and nested primers using Oligo (ver. 5.0, National Biosciences) and synthesized on a Beckman 1000 DNA synthesizer. Southern blot analysis was carried out on restricted genomic DNA as described (Ausubel et?al., 1997) using the ≈1100-bp polymorphic fragment as a probe. Blots were hybridized at 55°C and washed four or five times for 45 min in pre/post-hybridization solution at 55°C (Laroche et al., 1995). Southern blot analyses of PCR amplified products were carried out using the same probe, only hybridizing at 65°C, washing once for 20 min in pre/post-hybridization solution at 65°C, and 2 × 20 min in 0.1 X SSPE/ 0.1% Sarkosyl at 65°C. Given the size of the wheat genome (>16 Gb) and the inherent difficulty in isolating the full-length sequence of the putative gene, sub-genomic library was made using EcoRI fragments from DNA isolated from three BC4F5 resistant wheat lines, based on the previous detection of an approximately 9.0-kb fragment by Southern blot using the ≈1100-bp polymorphic DNA fragment as the probe. Fragments of this approximate size were excised from low-melting-point agarose using the heat and freeze and squeeze method followed by phenol-chloroform extraction. The presence of the ≈1100-bp polymorphic fragment was verified by PCR using SCAR primers before cloning the sub-genomic DNA fraction into the ? Zap vector (Stratagene, La Jolla, CA,?USA). A cDNA library was constructed from mRNA isolated from leaves of 17-day-old Moro seedlings which were exposed to P. striiformis isolate SRC-84 for 48 h. Leaves were frozen and homogenized in liquid nitrogen before total RNA was obtained using TRIzol (Canadian Life Technologies). Hybond-mAP paper (Amersham) or MessageMaker (Invitrogen Canada Inc.) was used to obtain poly-A+ mRNA. Messenger RNA was reverse-transcribed using Superscript II RNase H- reverse-transcriptase (Invitrogen Canada Inc.) and adapters were ligated to double-strand cDNA before XhoI/EcoRI-restricted
homozygous susceptible were retained for use in identification of molecular markers associated with stripe rust resistance. A?total of 874 BC2F3 and BC3F3 lines were used. Eighteen BC4 F5 lines were used for identification of the initial polymorphic fragment. Resistant (R) and susceptible (S) lines of BC Fielder x Moro, Moro (R), Fielder (S), Cappelle Desprez (S), Compair (S), PI 178383 (R), Jacmar (R), Avocet, Avocet + Yr10 (R), 16 Avocet NILs carrying different Yr genes, Corrigin (S), Corrigin + Yr10 (R), QT3960 (S), QT3960 + Yr10 (R), 79W93 (S), 79W93 + Yr10 (R), T.?spelta (Yr5), and T.?spelta 415 (R) were grown in Cornell mix for 10–14 d under conditions reported elsewhere (Conner et?al., 1993). T.?spelta 415 was kindly provided by Dr. G.H.J. Kema, IPO-DLO, Wageningen, The Netherlands. Accession 415 was described as carrying the Yr10 gene, based on testing with different stripe rust strains, whereas most other stripe rust resistant T. spelta accessions carry the Yr5 resistance gene (Kemma and Lange, 1992). Cultivar Jacmar was kindly supplied by Dr. R.F. Line, USDA-ARS, Washington State University, Pullman, Washington, USA, and the susceptible and resistant (Yr10) NILs of Avocet, Corrigin, QT3960, and 79W93 were supplied by Drs. H.S. Bariana and R.A. McIntosh, Plant Breeding Institute, University of Sydney, Cobbitty, NSW, Australia.
Inoculation and Disease Assessment
BC2F3 and BC3F3 populations of 874 lines and parents were tested for reaction to the Pst race 44E14 (SRC-84) (Johnson et al., 1972) that is virulent to 19 genes but avirulent to Yr10 (Su et?al., 2003). The presence of Yr10 in wheat lines, including T. spelta 415, and transgenic lines was further verified by challenge with Pst races SRC-84, CDL-45, and CDL-29. The last race was reported to be virulent for Yr10 (Beaver and Powelson, 1969) and isolates SRC-84 and CDL45 are avirulent with a HR response on wheat cv. Moro (Chen and Line, 1992). Races CDL-29 and CDL-45 were supplied by Dr. R.L.?Line. Plants were grown in growth cabinets under a diurnal regime of 16 h of light (250 ?mol m–2 s–1) at 22°C and 8 h of darkness at 20°C until inoculation. After inoculation, seedlings were placed in the dark at 10°C for 2 d and then returned to a 16-h photoperiod in a high-humidity (95%) growth cabinet set at 12°C. Infection types were rated 17 dpi as described (Conner et?al., 1988).
DNA and RNA Isolation and Library Construction and Screening
DNA was isolated from all wheat lines as reported previously (Conner et al., 1993). In a few cases, a modified FastPrep DNA isolation method was used (Bio-101, La Jolla, CA, USA). For the identification of the initial polymorphic
Molecular Plant
fragments were unidirectionally cloned into the ? Zap vector (Stratagene). Approximately 350 000 PFU (80%–90% recombinants at 50 000 PFU per 150-mm Petri dish (Ausubel et?al., 1997)) from both cDNA and sub-genomic libraries were plated, lifted in duplicate, and hybridized with the ≈1100-bp fragment using the conditions described for genomic Southern blot analysis. Positive plaques were purified using secondary and tertiary screening before being double-strand sequenced. Northern blot analysis was performed with 4??g of poly-A+ RNA and hybridized and washed as described above for genomic Southern blot analysis. RT–PCR reactions were primed with either oligo-dT or specific primers derived from the genomic sequence. Molecular techniques were based on standard protocols (Sambrook et?al., 1989; Ausubel et?al., 1997). The sequence for the promoter region of clones 4B and 4E was obtained by primer walking.
Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
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after bombardment. Although the embryogenesis was slightly delayed and less frequent during the selection procedure with glufosinate, there was no impact on the quality of primary and secondary embryos produced. Totals of 46 and 54 T0 green plantlets for the 4B and 4E constructs, respectively, were recovered after selection with 5 mg L–1 of glufosinate ammonium salt (# C140300, Crescent Chemical, Hauppauge, NY, USA) in the different media starting 16 h after bombardment. Plantlets at the three-leaf stages were screened directly with P. striiformis SRC-84 as described above.
Chromosome Localization of Yr10 Using?FISH
The 6.3-kb 4B clone was labeled using the tyramide signal amplification for fluorescence in situ hybridization (TSAFISH, PerkinElmer), and hybridized to fixed root tip preparations of Moro wheat. After washing, chromosomes were counterstained with fluorescein as described in detail by Li et?al. (2003).
Sequence Analysis
Computer searches were carried out against non-redundant nucleotides and protein sequence databases registered in GenBank and SwissProt using the BLAST algorithm (Altschul et al., 1997). The promoter sequence was analyzed with BLASTN (https://www.doczj.com/doc/f913290363.html,/Blast.cgi), PLACE (www.dna.affrc.go.jp), Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) databases, Repbase (https://www.doczj.com/doc/f913290363.html,/repbase/index.html) and TIGR repeats database (http://plantrepeats.plantbiology.msu. edu/search.html).
Construction of BSMV-Derived Vector and Inoculation of?Virus
The genome of BSMV is made up of three RNA fragments: ?, ?, and ?, as previously reported (Holzberg et?al., 2002). A?modified ? fragment reported to enhance infection (Holzberg et al., 2002) was employed. The ? vector was engineered to either express in the antisense orientation a fragment of the wheat PDS gene or three DNA fragments corresponding to two different domains of the Yr10 gene: the kinase 2a (387 bp; Figure?3) and the LRR (S, 359 bp, proximal; C, 420 bp, distal; Figure 3). These fragments in their antisense orientation are represented as BSMV:?PDS-as, ?K2a, ?SLR, and ?CLR. The primers used for amplification of the different fragments are described in Supplemental Table?1. Amplicons with appropriate restriction sites (PacI and NotI) located outside the different domains were cloned in their antisense orientation in the ? cDNA fragment. Capped in vitro transcripts were prepared from three linearized plasmids (?, ?, and ?) using the mMESSAGE mMACHINE T7 in vitro transcription kit (Ambion, Austin, TX, USA), following the manufacturer’s protocol. Transcripts of each of the BSMV genomes (1.25??g ?l–1) were mixed in a 1:1:1 ratio (2.5??l of each for a total of 7.5??l per plant to be inoculated) and then combined with 45 ?l FES buffer (Pogue et?al., 1998). Two-week-old wheat seedlings were then inoculated by applying the mixture to the entire leaf surfaces of leaves 1 and 2 by twice sliding the gently pinched forefinger and thumb from the base to the tip of each leaf (Holzberg et al., 2002; Scofield et al., 2005). To study the efficiency of BSMV-induced silencing of the PDS gene, leaves were sampled at 2-d intervals from 0 to 16
Transformation of Fielder
DNA delivery to the tissues was carried out with the Helios Gene Gun System (Bio-Rad Laboratories (Canada) Ltd., Mississauga, ON, Canada). The plasmid pCOR113 carrying the marker gene bar, under control of the rice actin promoter (McElroy et?al., 1991) and a second vector pCOR113 carrying the candidate Yr10 resistance genes 4B and 4E under control of their native promoter were used. Equal masses of these two plasmids (25??g each) were mixed and coated on 25 mg of 1??m gold particles and distributed on the inside wall of 60-cm GoldCoat tubing according to the manufacturer’s instructions. Discharge pressure was set at 120–140 psi and a diffusion screen (# 165–2475) was used to ensure an even distribution of the gold particles carrying the plasmids. Forty scutella isolated from immature embryos (12 d post anthesis) from the genotype AC Fielder were bombarded with the gold particles. AC Fielder is susceptible to most common races of the stripe rust pathogen. The tissue culture protocol was essentially as reported (Eudes et?al., 2003) with the exception that excised scutella were placed onto a modified DSEM medium supplemented with 0.3 M mannitol, from 4 h before bombardment to 16 h
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Wheat Yr10 Encodes a Unique CC–NBS–LRR Sequence
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Scientific, Edmonton, AB, Canada). Leaf sections were examined using a Zeiss Universal research microscope using the 330–385-nm and 460–490-nm excitation and emission filters, respectively, and a HBO103W/2 light source. At least 40 infection sites were examined in each independent biological replication. Each treatment has three biological replications. The percentage of infection sites in mesophyll cells was also recorded. Statistical analysis was performed by SAS software.
d after BSMV transfection for RNA extraction and qPCR. Eight days following BSMV inoculation, plants were inoculated with Pst according to Conner et?al. (1988). Pst races SRC-84 and CDL-29, avirulent and virulent, respectively, on Moro were employed. Following inoculation, plants were misted with water and covered with a clear plastic bag and incubated overnight in the dark at 10°C. Plants were then transferred to a high-humidity (95%) greenhouse set at 16°C constant temperature. From the time of inoculation, plants were sampled every 2 d from day 0 to day 20. Inoculated seedling leaves were visually rated for stripe rust response, split along the midrib into two pieces, one half sampled for RNA isolation and qPCR, the other half was sampled for microscopic studies.
Accession Numbers
Sequence data from this article can be found in the EMBL/ GenBank data libraries under accession numbers AF149112, AF149113, and AF149114.
SYBR Green Quantitative Real-Time?PCR
Total RNA was extracted from 200 mg flash-frozen leaves using the RNeasy Plant Mini kit (QIAGEN Inc., Toronto, ON, Canada). First-strand cDNA was synthesized using the SuperscriptTM M-MLV reverse-transcriptase kit (Invitrogen) following the manufacturer’s protocol. An Applied Biosystems 7500 Fast Real-time PCR system was used to perform quantitative real-time PCR (qPCR) analyses using a QuantiTect SYBR Green PCR Kit (QIAGEN) according to the manufacturer’s instructions. Threshold (Ct) values for qPCR results were collected from each sample. Each value represents an average of three technical replications and results are based on three independent biological replications. The relative expression for each target gene was analyzed with REST software (Pfaffl et al., 2002) using the translation elongation factor 1 alpha subunit (TEF1; GenBank accession # M90077) as the reference gene. The expression of TEF1 was very stable during plant development under the different inoculation treatments. In all cases, expression of the target gene is presented as the expression level in the silenced plant relative to expression of the same gene in plants infected with BSMV:?00. Primers are listed in Supplemental Table?1.
SUPPLEMENTARY?DATA
Supplementary Data are available at Molecular Plant Online.
FUNDING
This study was financially supported by grants from Alberta Agricultural Research Institute and AAFC A-base projects to A.L. and National Basic Research Program of China (2013CB127700), 111 Project from the Ministry of Education of China (B07049) to Z.K.
ACKNOWLEDGMENTS
We are grateful to Gregory P.?Pogue, Large Scale Biology Corporation, Vacaville, CA, for providing the BSMV vectors and to members of André Laroche’s laboratory for assistance in the work. No conflict of interest declared.
Microscopic Observations and Host-Response Assessments
For whole mounts, leaves were cut into 1-cm pieces, decolorized in 100% ethanol for 24 h, stained in a boiling aqueous lactophenol cotton blue solution (one drop of LCB concentrate was mixed with 1.5 ml distilled water in a 2-ml tube; Pro-Lab Diagnostics, Richmond Hill, ON, Canada) for 30 min. In parallel, corresponding leaf sections were vacuum infiltrated with formalin-acetic acid-ethanol (Johansen, 1940), embedded in paraffin, sectioned with a rotary microtome, mounted on glass slides in Haupt’s adhesive, stained following Johansen’s quadruple stain method (Johansen, 1940), and mounted in Permount (Fisher
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