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Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
Agricultural Sciences in China
June 2011
2011, 10(6): 827-837
Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato SUN Wan-yu, ZHAO Wan-ying, WANG Yuan-yuan, PEI Cheng-cheng and YANG Wen-cai Department of Vegetable Science, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, P.R.China
Abstract The resistance in tomato plants to bacterial speck caused by Pseudomonas syringae pv. tomato is triggered by the interactions between the plant resistance protein Pto and the pathogen avirulence proteins AvrPto or AvrPtoB. Fen is a gene encoding closely related functional protein kinases as the Pto gene. To investigate the status of resistance to the pathogen and natural variation of Pto and Fen genes in tomato, 67 lines including 29 growing in China were subject to disease resistance evaluation and fenthion-sensitivity test. Alleles of Pto and Fen were amplified from genomic DNA of 25 tomato lines using polymerase chain reaction (PCR) and sequences were determined by sequencing the PCR products. The results indicated that none of the 29 cultivars/hybrids growing in China were resistant to bacterial speck race 0 strain DC3000. Seven of eight tomato lines resistant to DC3000 were also fenthion-sensitive. Analysis of deduced amino acid sequences identified three novel residue substitutions between Pto and pto, and one new substitution identified between Fen and fen. A PCR-based marker was developed and successfully used to select plants with resistance to DC3000. Key words: tomato, bacterial speck, Pto, Fen, natural variation, marker-assisted selection
INTRODUCTION Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato (Pst) with two reported races 0 and 1, is an economically important disease in cool and moist environmental conditions (Yang and Francis 2007). Although resistant sources have been identified in many wild species, a single gene Pto with additive to dominant action from the Solanum pimpinellifolium has been the only source of resistance widely bred into cultivated tomato (S. lycopersicum). Pto encodes a serine/threonine kinase conferring resistance specifically to Pst race 0 strains expressing the avirulence genes avrPto or avrPtoB (Martin et al. 1993a; Kim et al. 2002; Mur et al. 2008; Hofmann 2009). Tomato lines carrying the Pto locus are also sensitive to an
organophosphorous insecticide fenthion (Laterrot and Moretti 1989). Exposure of leaves to fenthion results in the rapid development of small necrotic lesions in fenthion-sensitive tomato lines (Martin et al. 1994; Loh and Martin 1995). The locus controlling this phenotype termed as Fen co-segregates with the Pto locus and is mapped to the same region of Pto on chromosome 5 (Carland and Staskawicz 1993; Martin et al. 1993a, 1994). The Fen gene is also originally discovered in the wild species S. pimpinellifolium and encodes a serine/threonine kinase responsible for sensitivity to fenthion (Martin et al. 1994; Loh and Martin 1995). Further study revealed that both Pto and Fen alleles exist in bacterial speck-susceptible and fenthioninsensitive tomato cultivars and encode active protein kinases (Jia et al. 1997). However, these alleles don’t act as resistance to bacterial speck or sensitive to
Received 19 July, 2010 Accepted 21 November, 2010 SUN Wan-yu, E-mail: [email protected]; Correspondence YANG Wen-cai, Professor, Tel: +86-10-62734136, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60068-0
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fenthion. Natural variation of Pto gene has been investigated in seven wild species of tomato including S. pimpinellifolium, S. pennellii, S. peruvianum, S. habrochaites, S. chilense, S. parviflorum, and S. chmielewskii (Rose et al. 2005, 2007). The work also identified 10 novel amino acid substitutions associated with the absence of AvrPto recognition and HR (Rose et al. 2005). Although both Pto and Fen were firstly discovered in S. pimpinellifolium and their origin in cultivated tomato could be traced back by pedigree analysis, it remains unclear whether all Pto and Fen alleles in cultivated tomato are from the same species. In addition, no work on natural variation of the Fen gene has been reported to date. The first occurrence of tomato bacterial speck in China was in 1997. The pathogen was initially identified in Heilongjiang, Jilin, and Liaoning provinces, China (Zhao et al. 1999, 2001a; Feng et al. 2000). However, it quickly spread to all provinces and is becoming a threat to tomato production in North China (Zhao et al. 2001b, 2004; Wang et al. 2006a; Deng et al. 2008). Resistance to the disease in cultivars/hybrids growing in China is limited. Zhao et al. (2000) identified 15 lines resistant to an isolate Pst27 from 76 cultivars/ hybrids and 106 breeding lines. But most of these resistant cultivars/hybrids/breeding lines are from few institutes and thus may be from the same resistant source. A recent study found that none of 18 processing tomato varieties tested was resistant to an isolate Pst12 (Wang et al. 2006b). There is no further study on evaluation of resistance to the disease in cultivars/ hybrids currently growing in China. The work described here was initiated to understand the status of resistance to tomato bacterial speck in some cultivars/ hybrids currently growing in China, to investigate natural variation of Pto and Fen alleles in tomato, and to develop an efficient approach to marker-assisted selection of resistance to bacterial speck in tomato.
MATERIALS AND METHODS Plant materials and experimental design A total of 67 tomato lines including 30 fresh-market cultivars/hybrids, 18 landraces from Latin America, nine processing varieties, six S. lycopersicum var. cerasiforme
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lines, and four S. pimpinellifolium lines were subject to disease resistance evaluation and fenthion-sensitivity test (Table 1). Lines ONT7710 containing the Pto locus (Pitblado and MacNeill 1983) and LA3343 were used as resistant and susceptible controls, respectively. Most fresh-market and processing cultivars/hybrids are currently growing in China. Seeds of all lines were germinated in 288 Square Plug Tray Deep (Taizhou Longji Gardening Materials Co., Ltd., Zhejiang, China) filled with a mixture of peat soils and vermiculite (3:1) in the greenhouse. One-month old seedlings were transplanted into 10 cm (diameter)×8 cm (height) pots filled with the same soil mixture in the growth room. Water and fertilizer were supplied as needed. Evaluations of lines for response to bacterial speck and fenthion were performed in three replicated experiments established using randomized complete block designs with two blocks containing five plants for each line.
Inoculum preparation and disease evaluation DC3000, a race 0 strain of Pst, was grown on King’s B agar medium (King et al. 1954) at 26°C for 48-72 h. Bacterial cells were removed from the agar plates and suspended in ddH2O. The inoculum (1×108 CFU mL-1) was infiltrated through the back of three fully expanded leaflets on the 3rd true-leaf when plants were at the 5th true-leaf stage using the method described by Yang and Francis (2005). The plants were misted with water one hour before infiltration and kept at 22-28°C in a humid environment after infiltration. Hypersensitive response (HR) was recorded 24-72 h after inoculation. Ten days after infiltration, all previous infiltrated leaves were removed and the second infiltration was conducted as described above.
Scoring plant reactions to fenthion Seedlings were exposed to the insecticide by spraying 0.2% fenthion (Taizhou Huangyan Weihuan Pesticide and Chemicals Manufactory, Zhejiang, China) and 0.05% L-77 Silwet (Beijing SINOPCR Bio&Tech Co., Beijing, China) dispersed in ddH2O. The development of necrotic lesions or entire leaf necrosis was monitored for a period of 10 d after treatment according to the description in Martin et al. (1994).
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Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
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Table 1 Tomato lines and their responses to Pseudomonas syringae pv. tomato race 0 strain DC3000 and insecticide fenthion Genotype LA3343 73-45 DVRINTA IKRAM Menhir Prorita Shannon Baiguoqiangfeng Baili Baoguan Baoluota Beiying Daianna Fenqi Jiafen 15 Jiali Jiaren Jiabao Shanghai 908s Weihebaoguan Meifen 1 Nunhems 256 Nunhems 509 Qinfen Shijifeng Yingfen 8 Zhongshu 5 Zhongshu 6 Zhongza 101 Zhongza 105 LA2283 LA2285 LA0126 LA0358 LA0147 LA0473 LA1251 LA0134C LA1565 LA0468 LA1462 LA1162 LA0113 LA2307 LA2304 LA1460 LA0404 LA0477 ONT7710 FG02-7536 Gailiang VF Hongfanbuluo 1 Hongfanbuluo 2 Hongyu A Liger 87-5 Xinyin 98-1 OH88119 LA1310 PI114490 LA4133 LA1218 LA0172 LA2-473 PI128216 LA2804 LA1589 LA1269 1)
Species S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. pimpinellifolium S. pimpinellifolium S. pimpinellifolium S. pimpinellifolium
Origin China China China China China China China China China China China China China China China China China China China China China China China Peru Peru Ecuador Colombia Honduras Peru Ecuador Peru Mexico Chile Mexico Cuba Peru Peru Peru Guatemala Peru Peru Canada USA China China China China China China USA Peru USA Mexico Bolivia Peru Peru
Market type Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Processing Processing Processing Processing Processing Processing Processing Processing Processing Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild
Reaction to DC3000 1)
Fenthion
No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR HR No HR No HR No HR No HR No HR HR HR No HR No HR No HR No HR No HR No HR No HR No HR HR No HR No HR No HR No HR HR HR HR HR
Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Sensitive Insensitive Insensitive Insensitive Insensitive Insensitive Sensitive Sensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Sensitive Sensitive Sensitive Sensitive
HR, hypersensitive response.
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Pto and Fen genes amplification
Marker-assisted selection
Young leaves were collected from at least eight plants of each line. Genomic DNA was isolated using the modified CTAB method as described by Kabelka et al. (2002). Primers specific to Pto (forward: 5´GCAGCAAACTTCCTTTTTGC-3´, reverse: 5´TGGGAGGTGCCATAAGTTGT-3´) and Fen (forward: 5´-CTGACCTTGCCATCCCTATC-3´, reverse: 5´TGCCCTGTGAGTTTTCGATT-3´) genes were designed using primer 3 (Rozen and Skaletsky 2000) based on the sequence (AF220602) obtained from NCBI website (http://www.ncbi.nlm.nih.gov). PCR reactions were conducted in a 50 μL volume consisting of 10 mmol L-1 Tris-HCl (pH 9.0 at room temperature), 50 mmol L-1 KCl, 1.5 mmol L-1 MgCl2, 50 μmol L-1 of each dNTP (Vigorous Biotechnology Beijing Co., Beijing, China), 0.4 μmol L-1 primers, 30 ng genomic DNA template, and 2 U of Taq DNA polymerase (TaKaRa Biotechnology Dalian Co., Dalian, China). Reactions were heated at 94°C for 3 min followed by 36 cycles of 1 min denaturing at 94°C, 1 min annealing at 54°C, and 3 min extension at 72°C with the final extension reaction at 72°C for additional 5 min. Amplification was performed in a PTC-100TM programmable termal controller (MJ Research, Inc. Watertown, MA).
A pair of primers (forward: 5´-ATCTACCCACAATG AGCATGAGCTG-3´, reverse: 5´-GTGCATACTCCA GTTTCCAC-3´) for selecting materials with resistance to bacterial speck in Yang and Francis (2005) was used to genotype 1 216 individuals of an F2 population derived from a cross between OH88119 and PI128216. PI128216 is a wild species with HR to DC3000 and OH88119 is a processing tomato line susceptible to the pathogen. Disease resistance evaluation and genomic DNA isolation were conducted using the same approaches as described above. PCR was conducted according to Yang and Francis (2005). However, in this study, fragment size difference was observed (see Results) between resistant and susceptible lines. Therefore, length polymorphism instead of cleaved amplified polymorphism (CAP) described in Yang and Francis (2005) was used to distinguish resistant and susceptible lines. PCR products were separated using 6% denaturing gel and stained with silver staining approach as described in Chen et al. (2009).
DNA sequencing and sequence analysis PCR products were separated on 1% agarose gel, purified using Cycle-Pure Kit (Omega Bio-Tek, Inc., GA, USA), and sequenced for both forward and reverse directions using a ABI 3730 (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed and assembled using sequencer 4.0 (Gene Codes Corporation, Ann Arbor, MI, USA). All genomic DNA sequences were deposited into NCBI with GenBank accession numbers of HN341885 through HN341925. ORF finder from NCBI was used to deduce the amino acid sequence. Multiple-alignment was conducted using ClustalX 1.83 (Thompson et al. 1997). Nucleotide sequences for Pto homologs Fen, Pth2, Pth3, Pth4, and Pth5 from Rio Grande 76R (AF220602) and VFNT cherry (AF220603) along with all sequences obtained in this study were used to construct a parsimonious tree.
RESULTS Responses of 67 tomato lines to DC3000 and fenthion All lines had consistent reactions to DC3000 in two infiltration experiments. Seven lines, PI128216, FG027536, LA1589, LA1269, LA2804, LA0113, PI114490, and the resistant control ONT7710 showed HR to DC3000 within 24 h after infiltration, while the remaining 59 lines including the susceptible control LA3343 didn’t show any symptom till 72 h after infiltration (Table 1). Some of these lines had water-soaked symptom 72 h after infiltration. But most of them showed water-soaked symptom 5 d after infiltration. Of the eight lines showing HR, LA0113, FG02-7536, and ONT7710 are cultivated tomato. LA0113 is a landrace from Peru. The resistance in FG02-7536 is from PI128216 (Yang and Francis 2005), and the resistance in ONT7710 is from S. pimpinellifolium (Pitblado and MacNeill 1983). The other five are either S. lycopersicum var. cerasiforme lines or
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
S. pimpinellifolium. None of cultivars/hybrids growing in China showed HR to DC3000. At the 5th d after fenthion spray, lesions occurred on leaves of ONT7710, LA1589, LA0113, FG02-7536, PI128216, LA2804, and LA1269 plants. However, line PI114490 had HR to DC3000, but didn’t show any symptoms. There was no symptom on plants of other lines till 10th d after spray either (Table 1).
Variation of Pto gene All eight resistant lines and 17 susceptible lines were randomly selected for DNA variation analysis. The primers specific to Pto gene amplified a fragment from genomic DNA of 16 tomato lines. Nine tomato lines didn’t have any PCR product. There was no nucleotide sequence variation for Pto alleles in eight resistance lines. However, sequence polymorphisms were detected between Pto and pto alleles as well as within pto alleles. Nucleotide sequences were 966 bp in resistance lines (Pto allele) and 960 bp in susceptible lines (pto allele), respectively. A total of 41 single nucleotide polymorphisms (SNPs) and one 6 bp indel (AATGAG) were observed between resistance and susceptible lines (Table 2), of which 14 SNPs were identified between one susceptible line LA0468 and other seven susceptible lines. Indeed, 13 of the 14 nucleotides were same as those in resistance lines and only one SNP (A/C) was unique between LA0468 and other lines. Another SNP (G/A) existed between three sus-
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ceptible lines (LA4133, LA1460, and LA2283) and other lines. The indel occurred in the position of 399-404 bp and didn’t cause ORF shift. Of 41 SNPs, approximately 56% were transversion substitutions. There were two amino acid deletions in the deduced amino acid sequences of susceptible lines due to the 6 bp deletion in their nucleotide sequences (Fig. 1). Comparisons of deduced amino acid sequences revealed that eight nucleotide substitutions were synonymous, while 80.5% substitutions were non-synonymous (Table 2). These non-synonymous substitutions resulted in 26 variable amino acids (Fig. 1). Of the 26 amino acid substitutions, 23 have been previously described (Chang et al. 2002; Rose et al. 2005). The remaining three were novel, of which Val55 in resistant lines was substituted by Ala55 in susceptible lines. The other two substitutions, Gly151/ Ala151 and Thr288/Ala288, between resistant and susceptible tomato lines also existed in susceptible lines. In addition, eight more substitutions between resistant and susceptible lines were also observed within susceptible lines. Thus, only 16 substitutions existed between Pto and pto.
Variation of Fen gene Nucleotide sequences for Fen gene were obtained from the 25 tomato lines used for Pto allele variation analysis. The sequences were 963 bp in fenthion-insensitive lines (fen allele) and 957 bp in fenthion-sensitive lines (Fen
Table 2 Occurrence of base substitutions and their association with amino acid alternation in tomato Pto and Fen genes Substitution type Transition
Subtotal Transversion
Subtotal Insertion/Deletion Total
Base substitution A/G G/A C/T T/C A/T A/C C/A C/G G/C G/T T/G T/A T/C/A AATGAG TATGAG
No. of occurrence 4 2 6 6 18 3 8 2 3 1 1 3 2 0 23 1 0 42
Pto Non-synonymous 4 2 3 2 11 3 7 2 3 1 1 3 2 0 22 1 0 34
Synonymous
No. of occurrence
Fen Non-synonymous
Synonymous
0 0 3 4 7 0 1 0 0 0 0 0 0 0 1 0 0 8
2 3 1 4 10 0 2 0 2 2 3 2 3 1 15 0 1 25
2 2 1 2 7 0 1 0 2 1 2 2 3 0 11 0 1 18
0 1 0 2 3 0 1 0 0 1 1 0 0 1 4 0 0 7
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Fig. 1 Variable amino acids in the Pto gene in tomato. The numbers at the top indicate the corresponding codon positions in the predicted amino acid sequence of the Pto allele (AF220602) from Solanum pimpinellifolium. Number in bold indicates the novel residue substitution detected in this study. Invariable amino acids are removed. Amino acids in susceptible (No HR) tomato lines different from those in resistant (HR) tomato lines are highlighted in grey. A dash (-) indicates a gap. The same as below.
allele). Sequence variation was observed in both sensitive and non-sensitive lines. A total of 25 SNPs with 60% transversion substitutions and one 6 bp indel (TATGAG) were observed between fen and Fen alleles (Table 2). Five SNPs were line specific. One SNP (T/G) was identified between LA1462 and other lines, two SNPs (A/G) were detected between LA4133 and others, and two SNPs (T/C) were discovered between LA1269 and others. Particularly, one SNP had three nucleotides, one T in fenthion-sensitive line LA1269, one C in other fenthion-sensitive lines, and one A in fenthion-insensitive lines. The indel occurred in the position of 399-404 bp, which was at the same location of the indel in Pto gene. The indel in nucleotide sequence didn’t cause ORF shift but two amino acids deletion in insensitive lines (Fig. 2). Of the 25 nucleotide substitutions, seven were synonymous, while 72% were non-synonymous (Table 2). Sixteen variable amino acids were identified due to the SNPs and indel in nucleotide sequences (Fig. 2). No variation was observed within fenthion-sensitive lines at amino acid sequence level. All 16 residue substitutions occurred between fenthion-insensitive and fenthion-sensitive lines, and 15 of them were described in Chang et al. (2002). The only novel substitution was at the position of 163 aa. However, this substitution only occurred in one insensitive line LA1462 with Arg substituted Leu. Thus, this substitution might not be associated with fenthion-sensitivity.
Fig. 2 Variable amino acids in the Fen gene in tomato. The numbers at the top indicate the corresponding codon positions in the predicted amino acid sequence of the fen allele (LA4133) from Solanum lycopersicum var. cerasiforme. Amino acids in insensitive tomato lines different from those in sensitive tomato lines are highlighted in grey.
Comparison between Pto and Fen Pto and Fen had high homology at both nucleotide and amino acid sequence levels. Pairwise comparisons between these two genes obtained from resistance and susceptible lines revealed that Pto and Fen genes had 88% similarity at nucleotide sequence level and 78% identities at deduced amino acid sequence level. However, there was no significant similarity at nucleotide sequence level outside the coding regions. All Pto alleles and pto alleles formed Pto clade, while all Fen and fen alleles formed Fen clade (Fig. 3). Pth3 and Pth5 were closest to the Pto clade, followed by Pth4, and then Pth2. All Pto alleles were in one cluster and pto alleles were in another cluster. Similarly, all Fen alleles were in one cluster and fen alleles were in another cluster.
Marker-assisted selection for resistance to DC3000 As described above, there was one 6 bp indel in both
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Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
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Fig. 3 Parsimonious tree of the nucleotide sequences of alleles of Pto and Fen. HR stands for hypersensitive response to Pseudomonas syringae pv. tomato race 0 strain DC3000.
pto and Fen alleles. The forward primer used for markerassisted selection in previous study (Yang and Francis 2005) spanned the sides of the indel region (Fig. 4). Sequence comparison suggested that the primers could amplify a 552-bp fragment from for the Pto allele and a 534-bp fragment for fen allele. No PCR product
could be obtained for pto and Fen alleles using the primers. The DNA sequences of Fen/fen alleles in this region were 18 bp shorter than those in Pto/pto alleles. The 18 bp size difference was then used as a marker to distinguish resistant and susceptible lines in this study.
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Fig. 4 Partial alignment of nucleotide sequences for pto, Pto, fen, and Fen indicates the primer regions.
As expected, a 552-bp fragment from PI128216 and a 534-bp fragment from OH88119 were amplified by the primers (Fig. 5). The F1 plant had both fragments. In the F2 population, all resistance lines had the 552-bp fragment or both 552- and 534-bp fragments, while all susceptible lines only had the 534-bp fragment. This result indicated that the marker co-segregated with phenotypes and suggested that the marker was suitable for marker-assisted selection in breeding for resistance to bacterial speck race 0 using the Pto gene as the resistance source.
DISCUSSION
Fig. 5 Image of polyacrylamide gel for separating PCR fragments amplified from two parents, F1, and part individuals of F2 population derived from a cross between OH88119 and PI128216 using the length polymorphism marker. M, 100 bp standard marker; R, individuals with hypersensitive response to Pseudomonas syringae pv. tomato race 0 strain DC3000; S, individuals without hypersensitive response to Pseudomonas syringae pv. tomato race 0 strain DC3000.
PCR amplification of Pto and Fen genes depends on the primer specificity. Using a pair of primers, Rose et al. (2005) obtained PCR products from 49 of 61 individuals though only two individuals lacking the Pto alleles revealed by Southern blot. The failure of amplifying Pto from specific individuals isn’t always correlated with the absence of the gene (Rose et al. 2005). In this study, the primers specific to Fen gene could easily amplify the Fen/fen alleles from all 25 tomato lines, while the primers specific to Pto gene could only amplify Pto/pto alleles from 16 of 25 tomato lines. A careful inspection of the sequence (AF220602) found that the forward primer located at 101-120 bp upstream
and the reverse primer located at 165-184 bp downstream of the gene. Sequence comparison revealed that there was no similarity between the Pto (AF220602) and pto (AF220603) alleles in upstream region. Thus the lack of amplification of a Pto allele could be due to the sequence divergence at the upstream region. Natural variation in the reaction to strains of Pst expressing AvrPto can be largely attributed to the sequence variation in the Pto gene (Rose et al. 2005). More than 10 amino acid substitutions associated with AvrPto recognition and HR have been identified in Pto sequences (Sessa et al. 2000; Rose et al. 2005). In the amino acid sequence of Pto, residues 182-209 is believed to be the
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Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
activation segment containing a determinant K202xTLxxxD209 (x indicates an invariant amino acid residue) responsible for AvrPto binding. Substitutions at Thr204 and Tyr207 result in lack of AvrPto binding activity (Rathjen et al. 1999). In this study, there was no sequence variation in Pto alleles in eight resistant lines suggesting that the Pto gene in these lines might come from the same ancestor. However, great sequence variations were identified between Pto and pto. Among the three novel substitutions, only one Val55/Ala55 might be associated with AvrPto recognition. Two substitutions were in the determinant. Leu205 and Ile208 in resistance lines were substituted by Phe205 and Met208 in susceptible lines, respectively. These two substitutions in susceptible lines might be related to the failure of AvrPto binding. Previous study identified two deletion regions (residues 19-23 and 196-198) in pto obtained from VFNT Cherry (Jia et al. 1997). But these two deletions didn’t occur in this study. Indeed, there are more residue substitutions in pto from VFNT cherry than from other varieties. This suggested that residue substitutions and deletions in VFNT cherry might be variety-specific. Fenthion-sensitivity has widely been used to select plants with resistance to bacterial speck. The Fen gene has 88% nucleotide identity to Pto and is separated from Pto by approximately 23 kb (Martin et al. 1994). It is unlikely that recombination will separate fenthion-sensitivity and bacterial speck resistance in most breeding applications. In this study, lines had variations for Pto also showed variation in Fen. However, one line PI114490 showing HR to DC3000 of Pst race 0 was not sensitive to fenthion. Sequence analysis found that PI114490 had homolog of Pto and fen alleles. Therefore, the resistance to bacterial speck race 0 in this line might be controlled by Ptoh, an allele of Pto, described by Tanksley et al. (1996). Marker-assisted selection has the potential of accelerate breeding progress. Molecular markers linked to Pto have been available for over 10 years, e.g., Martin et al. 1993b. Recently, a PCR-based DNA marker has been developed based on the cloned gene, with polymorphisms detected as CAP using restriction enzyme Rsa I (Coaker and Francis 2004). Using this CAP marker can easily pyramid resistance to bacterial speck and bacterial spot in one variety (Yang and Francis 2005).
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However, this marker requires PCR amplification and enzyme restriction-digestion. It adds time and money to selection. In this study, comparision between sequences of Pto/pto and Fen/fen alleles identified a fragment size difference (18 bp) between resistant and susceptible lines. The marker was successfully used to select plants with resistance to Pst race 0. Breeding for resistance to bacterial speck has been successful because the resistance conferred by Pto is simply inherited and efficient and accurate evaluation techniques are available (Emmatty et al. 1982). Varieties with resistance to Pst race 0 have been developed and deployed in many countries (Yang and Francis 2007). However, this is not the case in China. Since the pathogen didn’t occur before 1997, there was no effort on breeding for resistance to this disease till the beginning of this century. Subsequently, the disease spread quickly throughout the country in the past decade. In addition to race 0, race 1 has been reported in several countries (Lawton and MacNeill 1986; Buonaurio et al. 1996; Donner and Barker 1996; Arredondo and Davis 2000). The wide introduction of cultivars containing the Pto gene for resistance is possibly driving a change in the pathogen population structure. Thus, there is a vital need in developing varieties with resistance to bacterial speck in a short period in China. In conclusion, there was no natural variation in Pto and Fen but great variations were found in pto and fen. These variations might be associated with specific protein binding/recognition. A lack of resistance to bacterial speck in tomato cultivars/hybrids currently growing in China might be the cause of quick expansion of the disease. Thus breeders need pay more attention to the disease and may develop varieties with resistance to the disease using the length polymorphism marker developed in this work.
Acknowledgements We would like to thank Tomato Genetics Resource Center at University of California (Davis) in the USA, Dr. David M. Francis at the Ohio State University, Ohio, USA, and Mr. Li Jishuo from Nunhems Beijing Seeds Company, Shandong, China, for providing tomato germplasms. The study was supported by the National High Technology Research and Development Program
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of China (2006AA10Z1A6) and the Program for New Century Excellent Talents in University, China (NCET08-0531).
References Arredondo C R, Davis R M. 2000. First report of Pseudomonas syringae pv. tomato race 1 on tomato in California. Plant Disease, 84, 371. Buonaurio R, Stravato V M, Cappelli C. 1996. Occurrence of Pseudomonas syringae pv. tomato race 1 in Italy on Pto gene-bearing tomato plants. Journal of Phytopathology, 144, 437-440. Carland F M, Staskawicz B J. 1993. Genetic characterization of the Pro locus of tomato: semi-dominance and cosegregation of resistance to Pseudomonas syringae pathovar tomato and sensitivity to the insecticide Fenthion. Molecular and General Genetics, 239, 17-27. Chang J H, Tai Y S, Bernal A J, Lavelle D T, Staskawicz B J, Michelmore R W. 2002. Functional analyses of the Pto resistance gene family in tomato and the identification of a minor resistance determinant in a susceptible haplotype. Molecular Plant-Microbe Interactions, 15, 281-291. Chen J, Wang H, Shen H L, Chai M, Li J S, Qi M F, Yang W C. 2009. Genetic variation in tomato populations from four breeding programs revealed by single nucleotide polymorphism and simple sequence repeat markers. Scientia Horticulturae, 122, 6-16. Coaker G L, Francis D M. 2004. Mapping, genetic effects, and epistatic interaction of two bacterial canker resistance QTLs from Lycopersicon hirsutum. Theoretical and Applied Genetics, 108, 1047-1055. Deng G, Qu X, Chen X R, Yang C D, Xue L. 2008. Pathogen identification of bacterial leaf speck of tomato based on 16S rDNA, physiology and biochemistry in Gansu Province. Plant Protection, 34, 47-51. (in Chinese) Donner S C, Barker S J. 1996. Pto resistance will not be effective in Australia (Abstr.). In: Proceedings of the 8th International Congress on Molecular Plant-Microbe Interactions. Knoxville, TN, USA. Emmatty D A, Scott M D, George B F. 1982. Inoculation technique to screen for bacterial speck resistance of tomatoes. Plant Disease, 66, 993-994. Feng L Y, Zhao T C, Sun F Z, Liu Q, Wang Y S. 2000. Occurrence of tomato bacterial speck in Liaoning Province. Liaoning Agricultural Sciences, 1, 53-54. (in Chinese) Hofmann N R. 2009. The tomato Pto kinase uses shared and unique surfaces to recognize divergent avirulence proteins. The Plant Cell, 21, 1623. Jia Y L, Loh Y T, Zhou J M, Martin G B. 1997. Alleles of Pto and
Fen occur in bacterial speck-susceptible and fenthioninsensitive tomato cultivars and encode active protein kinases. The Plant Cell, 9, 61-73. Kabelka E, Franchino B, Francis D M. 2002. Two loci from Lycopersicon hirsutum LA407 confer resistance to strains of Clavibacter michiganensis subsp. michiganensis. Phytopathology, 92, 504-510. Kim Y J, Lin N C, Martin G B. 2002. Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity. Cell, 109, 589-598. King E O, Ward M K, Raney D E. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. The Journal of Laboratory and Clinical Medicine, 44, 301-307. Laterrot H, Moretti A. 1989. Linkage between Pto and susceptibility to fenthion. Report of Tomato Genetics Cooperative, 39, 21-22. Lawton M B, MacNeill B H. 1986. Occurrence of race 1 of Pseudomonas syringae pv. tomato on field tomato in southwestern Ontario. Candian Journal of Plant Pathology, 8, 85-88. Loh Y T, Martin G B. 1995. The disease-resistance gene Pto and the fenthion-sensitivity gene Fen encode closely related functional protein kinases. Proceedings of National Academy of Sciences of the USA, 92, 4181-4184. Martin G B, Brommonschenkel S H, Chunwongse J, Frary A, Ganal M W, Spivey R, Wu T, Earle E D, Tanksley S D. 1993a. Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science, 262, 1432-1436. Martin G B, Carmen de Vicente M, Tanksley S D. 1993b. Highresolution linkage analysis and physical characterization of the Pto bacterial resistance locus in tomato. Molecular PlantMicrobe Interactions, 6, 26-34. Martin G B, Frary A, Wu T, Brommonschenkel S, Chunwongse J, Earle E D, Tanksley S D. 1994. A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death. The Plant Cell, 6, 1543-1552. Mur L A J, Kenton P, Lloyd A J, Ougham H, Prats E. 2008. The hypersensitive response; the centenary is upon us but how much do we know? Journal of Experimental Botany, 59, 501-520. Pitblado R E, MacNeill B H. 1983. Genetic basis of resistance to Pseudomonas syringae pv. tomato in field tomatoes. Canadian Journal of Plant Pathology, 5, 251-255. Rathjen J P, Chang J H, Staskawicz B J, Michelmore R W. 1999. Constitutively active Pto induces a Prf-dependent hypersensitive response in the absence of avrPto. EMBO Journal, 18, 3232-3240. Rose L E, Langley C H, Bernal A J, Michelmore R W. 2005. Natural variation in the Pto pathogen resistance gene within species of wild tomato (Lycopersicon). I. Functional analysis
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of Pto alleles. Genetics, 171, 345-357. Rose L E, Michelmore R W, Langley C H. 2007. Natural variation in the Pto disease resistance gene within species of wild tomato (Lycopersicon). II. Population genetics of Pto. Genetics, 175, 1307-1319. Rozen S, Skaletsky H. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Molecular Biology, 132, 365-386. Sessa G, D’Ascenzo M, Martin G B. 2000. Thr-38 and Ser-198 are Pto autophosphorylation sites required for the AvrPtoPtomediated hypersensitive response. EMBO Journal, 19, 2257-2269. Tanksley S D, Brommonschenkel S, Martin G. 1996. Ptoh, an allele of Pto conferring resistance to Pseudomonas syringae pv. tomato (race 0) that is not associated with fenthion sensitivity. Report of Tomato Genetics Cooperative, 46, 2829. Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, Higgins D G. 1997. The CLUSTER_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876-4882. Wang X H, Li G Y, Ren Y Z, Xue X W, Huang S F. 2006a. Pathogen identification of bacterial spot of processing tomato in Xinjiang. Acta Agriculturae Boreali-occidentalis Sinica, 15,72-74. (in Chinese) Wang X H, Li G Y, Ren Y Z, Xue X W, Huang S F. 2006b. Identified the resistance of processing tomato varieties to bacterial spot. Northern Horticulture, 3, 5-6. (in Chinese)
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patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase. The Plant Cell, 16, 2809-2821. Yang W C, Francis D M. 2005. Marker-assisted selection for combining resistance to bacterial spot and bacterial speck in tomato. Journal of the American Society for Horticultural Science, 130, 716-721. Yang W C, Francis D M. 2007. Genetics and breeding for resistance to bacterial diseases in tomato: prospects for marker assisted selection. In: Razdan M K, Mattoo A K, eds., Genetic Improvement of Solanaceous Crops. vol. 2: Tomato. Science Publishers, USA. pp. 379-420. Zhao T C, Sun F Z, Feng L Y, Han W H, Xu W K. 2000. Evaluation of resistance to bacterial speck in tomato. Plant Protection, 26, 49-50. (in Chinese) Zhao T C, Sun F Z, Li M Y, Zhang G F, Dai C Z, Cui Y Y, Yang H, Wang W L. 2004. Occurrence and control of bacterial speck in tomato. China Vegetables, 4, 64. (in Chinese) Zhao T C, Sun F Z, Song W S. 2001a. Pathogen identification of bacterial speck of tomato. Acta Phytopathologica Sinica, 31, 37-42. (in Chinese) Zhao T C, Yu L, Sun F Z, Feng L Y. 1999. Occurrence and control of tomato bacterial speck. Plant Protection, 25, 56. (in Chinese) Zhao Z J, Lin Z M, Zhao X J, Zhao T C, Sun F Z. 2001b. Occurrence of tomato bacterial speck in Shanxi Province. Plant Protection Technology and Extension, 21, 37. (in Chinese)
Wu A J, Andriotis V M E, Durrant M C, Rathjen J P. 2004. A (Managing editor ZHANG Yi-min)
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June 2011
2011, 10(6): 827-837
Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato SUN Wan-yu, ZHAO Wan-ying, WANG Yuan-yuan, PEI Cheng-cheng and YANG Wen-cai Department of Vegetable Science, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, P.R.China
Abstract The resistance in tomato plants to bacterial speck caused by Pseudomonas syringae pv. tomato is triggered by the interactions between the plant resistance protein Pto and the pathogen avirulence proteins AvrPto or AvrPtoB. Fen is a gene encoding closely related functional protein kinases as the Pto gene. To investigate the status of resistance to the pathogen and natural variation of Pto and Fen genes in tomato, 67 lines including 29 growing in China were subject to disease resistance evaluation and fenthion-sensitivity test. Alleles of Pto and Fen were amplified from genomic DNA of 25 tomato lines using polymerase chain reaction (PCR) and sequences were determined by sequencing the PCR products. The results indicated that none of the 29 cultivars/hybrids growing in China were resistant to bacterial speck race 0 strain DC3000. Seven of eight tomato lines resistant to DC3000 were also fenthion-sensitive. Analysis of deduced amino acid sequences identified three novel residue substitutions between Pto and pto, and one new substitution identified between Fen and fen. A PCR-based marker was developed and successfully used to select plants with resistance to DC3000. Key words: tomato, bacterial speck, Pto, Fen, natural variation, marker-assisted selection
INTRODUCTION Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato (Pst) with two reported races 0 and 1, is an economically important disease in cool and moist environmental conditions (Yang and Francis 2007). Although resistant sources have been identified in many wild species, a single gene Pto with additive to dominant action from the Solanum pimpinellifolium has been the only source of resistance widely bred into cultivated tomato (S. lycopersicum). Pto encodes a serine/threonine kinase conferring resistance specifically to Pst race 0 strains expressing the avirulence genes avrPto or avrPtoB (Martin et al. 1993a; Kim et al. 2002; Mur et al. 2008; Hofmann 2009). Tomato lines carrying the Pto locus are also sensitive to an
organophosphorous insecticide fenthion (Laterrot and Moretti 1989). Exposure of leaves to fenthion results in the rapid development of small necrotic lesions in fenthion-sensitive tomato lines (Martin et al. 1994; Loh and Martin 1995). The locus controlling this phenotype termed as Fen co-segregates with the Pto locus and is mapped to the same region of Pto on chromosome 5 (Carland and Staskawicz 1993; Martin et al. 1993a, 1994). The Fen gene is also originally discovered in the wild species S. pimpinellifolium and encodes a serine/threonine kinase responsible for sensitivity to fenthion (Martin et al. 1994; Loh and Martin 1995). Further study revealed that both Pto and Fen alleles exist in bacterial speck-susceptible and fenthioninsensitive tomato cultivars and encode active protein kinases (Jia et al. 1997). However, these alleles don’t act as resistance to bacterial speck or sensitive to
Received 19 July, 2010 Accepted 21 November, 2010 SUN Wan-yu, E-mail: [email protected]; Correspondence YANG Wen-cai, Professor, Tel: +86-10-62734136, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60068-0
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fenthion. Natural variation of Pto gene has been investigated in seven wild species of tomato including S. pimpinellifolium, S. pennellii, S. peruvianum, S. habrochaites, S. chilense, S. parviflorum, and S. chmielewskii (Rose et al. 2005, 2007). The work also identified 10 novel amino acid substitutions associated with the absence of AvrPto recognition and HR (Rose et al. 2005). Although both Pto and Fen were firstly discovered in S. pimpinellifolium and their origin in cultivated tomato could be traced back by pedigree analysis, it remains unclear whether all Pto and Fen alleles in cultivated tomato are from the same species. In addition, no work on natural variation of the Fen gene has been reported to date. The first occurrence of tomato bacterial speck in China was in 1997. The pathogen was initially identified in Heilongjiang, Jilin, and Liaoning provinces, China (Zhao et al. 1999, 2001a; Feng et al. 2000). However, it quickly spread to all provinces and is becoming a threat to tomato production in North China (Zhao et al. 2001b, 2004; Wang et al. 2006a; Deng et al. 2008). Resistance to the disease in cultivars/hybrids growing in China is limited. Zhao et al. (2000) identified 15 lines resistant to an isolate Pst27 from 76 cultivars/ hybrids and 106 breeding lines. But most of these resistant cultivars/hybrids/breeding lines are from few institutes and thus may be from the same resistant source. A recent study found that none of 18 processing tomato varieties tested was resistant to an isolate Pst12 (Wang et al. 2006b). There is no further study on evaluation of resistance to the disease in cultivars/ hybrids currently growing in China. The work described here was initiated to understand the status of resistance to tomato bacterial speck in some cultivars/ hybrids currently growing in China, to investigate natural variation of Pto and Fen alleles in tomato, and to develop an efficient approach to marker-assisted selection of resistance to bacterial speck in tomato.
MATERIALS AND METHODS Plant materials and experimental design A total of 67 tomato lines including 30 fresh-market cultivars/hybrids, 18 landraces from Latin America, nine processing varieties, six S. lycopersicum var. cerasiforme
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lines, and four S. pimpinellifolium lines were subject to disease resistance evaluation and fenthion-sensitivity test (Table 1). Lines ONT7710 containing the Pto locus (Pitblado and MacNeill 1983) and LA3343 were used as resistant and susceptible controls, respectively. Most fresh-market and processing cultivars/hybrids are currently growing in China. Seeds of all lines were germinated in 288 Square Plug Tray Deep (Taizhou Longji Gardening Materials Co., Ltd., Zhejiang, China) filled with a mixture of peat soils and vermiculite (3:1) in the greenhouse. One-month old seedlings were transplanted into 10 cm (diameter)×8 cm (height) pots filled with the same soil mixture in the growth room. Water and fertilizer were supplied as needed. Evaluations of lines for response to bacterial speck and fenthion were performed in three replicated experiments established using randomized complete block designs with two blocks containing five plants for each line.
Inoculum preparation and disease evaluation DC3000, a race 0 strain of Pst, was grown on King’s B agar medium (King et al. 1954) at 26°C for 48-72 h. Bacterial cells were removed from the agar plates and suspended in ddH2O. The inoculum (1×108 CFU mL-1) was infiltrated through the back of three fully expanded leaflets on the 3rd true-leaf when plants were at the 5th true-leaf stage using the method described by Yang and Francis (2005). The plants were misted with water one hour before infiltration and kept at 22-28°C in a humid environment after infiltration. Hypersensitive response (HR) was recorded 24-72 h after inoculation. Ten days after infiltration, all previous infiltrated leaves were removed and the second infiltration was conducted as described above.
Scoring plant reactions to fenthion Seedlings were exposed to the insecticide by spraying 0.2% fenthion (Taizhou Huangyan Weihuan Pesticide and Chemicals Manufactory, Zhejiang, China) and 0.05% L-77 Silwet (Beijing SINOPCR Bio&Tech Co., Beijing, China) dispersed in ddH2O. The development of necrotic lesions or entire leaf necrosis was monitored for a period of 10 d after treatment according to the description in Martin et al. (1994).
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Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
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Table 1 Tomato lines and their responses to Pseudomonas syringae pv. tomato race 0 strain DC3000 and insecticide fenthion Genotype LA3343 73-45 DVRINTA IKRAM Menhir Prorita Shannon Baiguoqiangfeng Baili Baoguan Baoluota Beiying Daianna Fenqi Jiafen 15 Jiali Jiaren Jiabao Shanghai 908s Weihebaoguan Meifen 1 Nunhems 256 Nunhems 509 Qinfen Shijifeng Yingfen 8 Zhongshu 5 Zhongshu 6 Zhongza 101 Zhongza 105 LA2283 LA2285 LA0126 LA0358 LA0147 LA0473 LA1251 LA0134C LA1565 LA0468 LA1462 LA1162 LA0113 LA2307 LA2304 LA1460 LA0404 LA0477 ONT7710 FG02-7536 Gailiang VF Hongfanbuluo 1 Hongfanbuluo 2 Hongyu A Liger 87-5 Xinyin 98-1 OH88119 LA1310 PI114490 LA4133 LA1218 LA0172 LA2-473 PI128216 LA2804 LA1589 LA1269 1)
Species S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. lycopersicum var. cerasiforme S. pimpinellifolium S. pimpinellifolium S. pimpinellifolium S. pimpinellifolium
Origin China China China China China China China China China China China China China China China China China China China China China China China Peru Peru Ecuador Colombia Honduras Peru Ecuador Peru Mexico Chile Mexico Cuba Peru Peru Peru Guatemala Peru Peru Canada USA China China China China China China USA Peru USA Mexico Bolivia Peru Peru
Market type Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Fresh market Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Landraces Processing Processing Processing Processing Processing Processing Processing Processing Processing Wild Wild Wild Wild Wild Wild Wild Wild Wild Wild
Reaction to DC3000 1)
Fenthion
No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR No HR HR No HR No HR No HR No HR No HR HR HR No HR No HR No HR No HR No HR No HR No HR No HR HR No HR No HR No HR No HR HR HR HR HR
Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Sensitive Insensitive Insensitive Insensitive Insensitive Insensitive Sensitive Sensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Insensitive Sensitive Sensitive Sensitive Sensitive
HR, hypersensitive response.
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Pto and Fen genes amplification
Marker-assisted selection
Young leaves were collected from at least eight plants of each line. Genomic DNA was isolated using the modified CTAB method as described by Kabelka et al. (2002). Primers specific to Pto (forward: 5´GCAGCAAACTTCCTTTTTGC-3´, reverse: 5´TGGGAGGTGCCATAAGTTGT-3´) and Fen (forward: 5´-CTGACCTTGCCATCCCTATC-3´, reverse: 5´TGCCCTGTGAGTTTTCGATT-3´) genes were designed using primer 3 (Rozen and Skaletsky 2000) based on the sequence (AF220602) obtained from NCBI website (http://www.ncbi.nlm.nih.gov). PCR reactions were conducted in a 50 μL volume consisting of 10 mmol L-1 Tris-HCl (pH 9.0 at room temperature), 50 mmol L-1 KCl, 1.5 mmol L-1 MgCl2, 50 μmol L-1 of each dNTP (Vigorous Biotechnology Beijing Co., Beijing, China), 0.4 μmol L-1 primers, 30 ng genomic DNA template, and 2 U of Taq DNA polymerase (TaKaRa Biotechnology Dalian Co., Dalian, China). Reactions were heated at 94°C for 3 min followed by 36 cycles of 1 min denaturing at 94°C, 1 min annealing at 54°C, and 3 min extension at 72°C with the final extension reaction at 72°C for additional 5 min. Amplification was performed in a PTC-100TM programmable termal controller (MJ Research, Inc. Watertown, MA).
A pair of primers (forward: 5´-ATCTACCCACAATG AGCATGAGCTG-3´, reverse: 5´-GTGCATACTCCA GTTTCCAC-3´) for selecting materials with resistance to bacterial speck in Yang and Francis (2005) was used to genotype 1 216 individuals of an F2 population derived from a cross between OH88119 and PI128216. PI128216 is a wild species with HR to DC3000 and OH88119 is a processing tomato line susceptible to the pathogen. Disease resistance evaluation and genomic DNA isolation were conducted using the same approaches as described above. PCR was conducted according to Yang and Francis (2005). However, in this study, fragment size difference was observed (see Results) between resistant and susceptible lines. Therefore, length polymorphism instead of cleaved amplified polymorphism (CAP) described in Yang and Francis (2005) was used to distinguish resistant and susceptible lines. PCR products were separated using 6% denaturing gel and stained with silver staining approach as described in Chen et al. (2009).
DNA sequencing and sequence analysis PCR products were separated on 1% agarose gel, purified using Cycle-Pure Kit (Omega Bio-Tek, Inc., GA, USA), and sequenced for both forward and reverse directions using a ABI 3730 (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed and assembled using sequencer 4.0 (Gene Codes Corporation, Ann Arbor, MI, USA). All genomic DNA sequences were deposited into NCBI with GenBank accession numbers of HN341885 through HN341925. ORF finder from NCBI was used to deduce the amino acid sequence. Multiple-alignment was conducted using ClustalX 1.83 (Thompson et al. 1997). Nucleotide sequences for Pto homologs Fen, Pth2, Pth3, Pth4, and Pth5 from Rio Grande 76R (AF220602) and VFNT cherry (AF220603) along with all sequences obtained in this study were used to construct a parsimonious tree.
RESULTS Responses of 67 tomato lines to DC3000 and fenthion All lines had consistent reactions to DC3000 in two infiltration experiments. Seven lines, PI128216, FG027536, LA1589, LA1269, LA2804, LA0113, PI114490, and the resistant control ONT7710 showed HR to DC3000 within 24 h after infiltration, while the remaining 59 lines including the susceptible control LA3343 didn’t show any symptom till 72 h after infiltration (Table 1). Some of these lines had water-soaked symptom 72 h after infiltration. But most of them showed water-soaked symptom 5 d after infiltration. Of the eight lines showing HR, LA0113, FG02-7536, and ONT7710 are cultivated tomato. LA0113 is a landrace from Peru. The resistance in FG02-7536 is from PI128216 (Yang and Francis 2005), and the resistance in ONT7710 is from S. pimpinellifolium (Pitblado and MacNeill 1983). The other five are either S. lycopersicum var. cerasiforme lines or
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Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
S. pimpinellifolium. None of cultivars/hybrids growing in China showed HR to DC3000. At the 5th d after fenthion spray, lesions occurred on leaves of ONT7710, LA1589, LA0113, FG02-7536, PI128216, LA2804, and LA1269 plants. However, line PI114490 had HR to DC3000, but didn’t show any symptoms. There was no symptom on plants of other lines till 10th d after spray either (Table 1).
Variation of Pto gene All eight resistant lines and 17 susceptible lines were randomly selected for DNA variation analysis. The primers specific to Pto gene amplified a fragment from genomic DNA of 16 tomato lines. Nine tomato lines didn’t have any PCR product. There was no nucleotide sequence variation for Pto alleles in eight resistance lines. However, sequence polymorphisms were detected between Pto and pto alleles as well as within pto alleles. Nucleotide sequences were 966 bp in resistance lines (Pto allele) and 960 bp in susceptible lines (pto allele), respectively. A total of 41 single nucleotide polymorphisms (SNPs) and one 6 bp indel (AATGAG) were observed between resistance and susceptible lines (Table 2), of which 14 SNPs were identified between one susceptible line LA0468 and other seven susceptible lines. Indeed, 13 of the 14 nucleotides were same as those in resistance lines and only one SNP (A/C) was unique between LA0468 and other lines. Another SNP (G/A) existed between three sus-
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ceptible lines (LA4133, LA1460, and LA2283) and other lines. The indel occurred in the position of 399-404 bp and didn’t cause ORF shift. Of 41 SNPs, approximately 56% were transversion substitutions. There were two amino acid deletions in the deduced amino acid sequences of susceptible lines due to the 6 bp deletion in their nucleotide sequences (Fig. 1). Comparisons of deduced amino acid sequences revealed that eight nucleotide substitutions were synonymous, while 80.5% substitutions were non-synonymous (Table 2). These non-synonymous substitutions resulted in 26 variable amino acids (Fig. 1). Of the 26 amino acid substitutions, 23 have been previously described (Chang et al. 2002; Rose et al. 2005). The remaining three were novel, of which Val55 in resistant lines was substituted by Ala55 in susceptible lines. The other two substitutions, Gly151/ Ala151 and Thr288/Ala288, between resistant and susceptible tomato lines also existed in susceptible lines. In addition, eight more substitutions between resistant and susceptible lines were also observed within susceptible lines. Thus, only 16 substitutions existed between Pto and pto.
Variation of Fen gene Nucleotide sequences for Fen gene were obtained from the 25 tomato lines used for Pto allele variation analysis. The sequences were 963 bp in fenthion-insensitive lines (fen allele) and 957 bp in fenthion-sensitive lines (Fen
Table 2 Occurrence of base substitutions and their association with amino acid alternation in tomato Pto and Fen genes Substitution type Transition
Subtotal Transversion
Subtotal Insertion/Deletion Total
Base substitution A/G G/A C/T T/C A/T A/C C/A C/G G/C G/T T/G T/A T/C/A AATGAG TATGAG
No. of occurrence 4 2 6 6 18 3 8 2 3 1 1 3 2 0 23 1 0 42
Pto Non-synonymous 4 2 3 2 11 3 7 2 3 1 1 3 2 0 22 1 0 34
Synonymous
No. of occurrence
Fen Non-synonymous
Synonymous
0 0 3 4 7 0 1 0 0 0 0 0 0 0 1 0 0 8
2 3 1 4 10 0 2 0 2 2 3 2 3 1 15 0 1 25
2 2 1 2 7 0 1 0 2 1 2 2 3 0 11 0 1 18
0 1 0 2 3 0 1 0 0 1 1 0 0 1 4 0 0 7
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Fig. 1 Variable amino acids in the Pto gene in tomato. The numbers at the top indicate the corresponding codon positions in the predicted amino acid sequence of the Pto allele (AF220602) from Solanum pimpinellifolium. Number in bold indicates the novel residue substitution detected in this study. Invariable amino acids are removed. Amino acids in susceptible (No HR) tomato lines different from those in resistant (HR) tomato lines are highlighted in grey. A dash (-) indicates a gap. The same as below.
allele). Sequence variation was observed in both sensitive and non-sensitive lines. A total of 25 SNPs with 60% transversion substitutions and one 6 bp indel (TATGAG) were observed between fen and Fen alleles (Table 2). Five SNPs were line specific. One SNP (T/G) was identified between LA1462 and other lines, two SNPs (A/G) were detected between LA4133 and others, and two SNPs (T/C) were discovered between LA1269 and others. Particularly, one SNP had three nucleotides, one T in fenthion-sensitive line LA1269, one C in other fenthion-sensitive lines, and one A in fenthion-insensitive lines. The indel occurred in the position of 399-404 bp, which was at the same location of the indel in Pto gene. The indel in nucleotide sequence didn’t cause ORF shift but two amino acids deletion in insensitive lines (Fig. 2). Of the 25 nucleotide substitutions, seven were synonymous, while 72% were non-synonymous (Table 2). Sixteen variable amino acids were identified due to the SNPs and indel in nucleotide sequences (Fig. 2). No variation was observed within fenthion-sensitive lines at amino acid sequence level. All 16 residue substitutions occurred between fenthion-insensitive and fenthion-sensitive lines, and 15 of them were described in Chang et al. (2002). The only novel substitution was at the position of 163 aa. However, this substitution only occurred in one insensitive line LA1462 with Arg substituted Leu. Thus, this substitution might not be associated with fenthion-sensitivity.
Fig. 2 Variable amino acids in the Fen gene in tomato. The numbers at the top indicate the corresponding codon positions in the predicted amino acid sequence of the fen allele (LA4133) from Solanum lycopersicum var. cerasiforme. Amino acids in insensitive tomato lines different from those in sensitive tomato lines are highlighted in grey.
Comparison between Pto and Fen Pto and Fen had high homology at both nucleotide and amino acid sequence levels. Pairwise comparisons between these two genes obtained from resistance and susceptible lines revealed that Pto and Fen genes had 88% similarity at nucleotide sequence level and 78% identities at deduced amino acid sequence level. However, there was no significant similarity at nucleotide sequence level outside the coding regions. All Pto alleles and pto alleles formed Pto clade, while all Fen and fen alleles formed Fen clade (Fig. 3). Pth3 and Pth5 were closest to the Pto clade, followed by Pth4, and then Pth2. All Pto alleles were in one cluster and pto alleles were in another cluster. Similarly, all Fen alleles were in one cluster and fen alleles were in another cluster.
Marker-assisted selection for resistance to DC3000 As described above, there was one 6 bp indel in both
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Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
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Fig. 3 Parsimonious tree of the nucleotide sequences of alleles of Pto and Fen. HR stands for hypersensitive response to Pseudomonas syringae pv. tomato race 0 strain DC3000.
pto and Fen alleles. The forward primer used for markerassisted selection in previous study (Yang and Francis 2005) spanned the sides of the indel region (Fig. 4). Sequence comparison suggested that the primers could amplify a 552-bp fragment from for the Pto allele and a 534-bp fragment for fen allele. No PCR product
could be obtained for pto and Fen alleles using the primers. The DNA sequences of Fen/fen alleles in this region were 18 bp shorter than those in Pto/pto alleles. The 18 bp size difference was then used as a marker to distinguish resistant and susceptible lines in this study.
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Fig. 4 Partial alignment of nucleotide sequences for pto, Pto, fen, and Fen indicates the primer regions.
As expected, a 552-bp fragment from PI128216 and a 534-bp fragment from OH88119 were amplified by the primers (Fig. 5). The F1 plant had both fragments. In the F2 population, all resistance lines had the 552-bp fragment or both 552- and 534-bp fragments, while all susceptible lines only had the 534-bp fragment. This result indicated that the marker co-segregated with phenotypes and suggested that the marker was suitable for marker-assisted selection in breeding for resistance to bacterial speck race 0 using the Pto gene as the resistance source.
DISCUSSION
Fig. 5 Image of polyacrylamide gel for separating PCR fragments amplified from two parents, F1, and part individuals of F2 population derived from a cross between OH88119 and PI128216 using the length polymorphism marker. M, 100 bp standard marker; R, individuals with hypersensitive response to Pseudomonas syringae pv. tomato race 0 strain DC3000; S, individuals without hypersensitive response to Pseudomonas syringae pv. tomato race 0 strain DC3000.
PCR amplification of Pto and Fen genes depends on the primer specificity. Using a pair of primers, Rose et al. (2005) obtained PCR products from 49 of 61 individuals though only two individuals lacking the Pto alleles revealed by Southern blot. The failure of amplifying Pto from specific individuals isn’t always correlated with the absence of the gene (Rose et al. 2005). In this study, the primers specific to Fen gene could easily amplify the Fen/fen alleles from all 25 tomato lines, while the primers specific to Pto gene could only amplify Pto/pto alleles from 16 of 25 tomato lines. A careful inspection of the sequence (AF220602) found that the forward primer located at 101-120 bp upstream
and the reverse primer located at 165-184 bp downstream of the gene. Sequence comparison revealed that there was no similarity between the Pto (AF220602) and pto (AF220603) alleles in upstream region. Thus the lack of amplification of a Pto allele could be due to the sequence divergence at the upstream region. Natural variation in the reaction to strains of Pst expressing AvrPto can be largely attributed to the sequence variation in the Pto gene (Rose et al. 2005). More than 10 amino acid substitutions associated with AvrPto recognition and HR have been identified in Pto sequences (Sessa et al. 2000; Rose et al. 2005). In the amino acid sequence of Pto, residues 182-209 is believed to be the
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Natural Variation of Pto and Fen Genes and Marker-Assisted Selection for Resistance to Bacterial Speck in Tomato
activation segment containing a determinant K202xTLxxxD209 (x indicates an invariant amino acid residue) responsible for AvrPto binding. Substitutions at Thr204 and Tyr207 result in lack of AvrPto binding activity (Rathjen et al. 1999). In this study, there was no sequence variation in Pto alleles in eight resistant lines suggesting that the Pto gene in these lines might come from the same ancestor. However, great sequence variations were identified between Pto and pto. Among the three novel substitutions, only one Val55/Ala55 might be associated with AvrPto recognition. Two substitutions were in the determinant. Leu205 and Ile208 in resistance lines were substituted by Phe205 and Met208 in susceptible lines, respectively. These two substitutions in susceptible lines might be related to the failure of AvrPto binding. Previous study identified two deletion regions (residues 19-23 and 196-198) in pto obtained from VFNT Cherry (Jia et al. 1997). But these two deletions didn’t occur in this study. Indeed, there are more residue substitutions in pto from VFNT cherry than from other varieties. This suggested that residue substitutions and deletions in VFNT cherry might be variety-specific. Fenthion-sensitivity has widely been used to select plants with resistance to bacterial speck. The Fen gene has 88% nucleotide identity to Pto and is separated from Pto by approximately 23 kb (Martin et al. 1994). It is unlikely that recombination will separate fenthion-sensitivity and bacterial speck resistance in most breeding applications. In this study, lines had variations for Pto also showed variation in Fen. However, one line PI114490 showing HR to DC3000 of Pst race 0 was not sensitive to fenthion. Sequence analysis found that PI114490 had homolog of Pto and fen alleles. Therefore, the resistance to bacterial speck race 0 in this line might be controlled by Ptoh, an allele of Pto, described by Tanksley et al. (1996). Marker-assisted selection has the potential of accelerate breeding progress. Molecular markers linked to Pto have been available for over 10 years, e.g., Martin et al. 1993b. Recently, a PCR-based DNA marker has been developed based on the cloned gene, with polymorphisms detected as CAP using restriction enzyme Rsa I (Coaker and Francis 2004). Using this CAP marker can easily pyramid resistance to bacterial speck and bacterial spot in one variety (Yang and Francis 2005).
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However, this marker requires PCR amplification and enzyme restriction-digestion. It adds time and money to selection. In this study, comparision between sequences of Pto/pto and Fen/fen alleles identified a fragment size difference (18 bp) between resistant and susceptible lines. The marker was successfully used to select plants with resistance to Pst race 0. Breeding for resistance to bacterial speck has been successful because the resistance conferred by Pto is simply inherited and efficient and accurate evaluation techniques are available (Emmatty et al. 1982). Varieties with resistance to Pst race 0 have been developed and deployed in many countries (Yang and Francis 2007). However, this is not the case in China. Since the pathogen didn’t occur before 1997, there was no effort on breeding for resistance to this disease till the beginning of this century. Subsequently, the disease spread quickly throughout the country in the past decade. In addition to race 0, race 1 has been reported in several countries (Lawton and MacNeill 1986; Buonaurio et al. 1996; Donner and Barker 1996; Arredondo and Davis 2000). The wide introduction of cultivars containing the Pto gene for resistance is possibly driving a change in the pathogen population structure. Thus, there is a vital need in developing varieties with resistance to bacterial speck in a short period in China. In conclusion, there was no natural variation in Pto and Fen but great variations were found in pto and fen. These variations might be associated with specific protein binding/recognition. A lack of resistance to bacterial speck in tomato cultivars/hybrids currently growing in China might be the cause of quick expansion of the disease. Thus breeders need pay more attention to the disease and may develop varieties with resistance to the disease using the length polymorphism marker developed in this work.
Acknowledgements We would like to thank Tomato Genetics Resource Center at University of California (Davis) in the USA, Dr. David M. Francis at the Ohio State University, Ohio, USA, and Mr. Li Jishuo from Nunhems Beijing Seeds Company, Shandong, China, for providing tomato germplasms. The study was supported by the National High Technology Research and Development Program
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of China (2006AA10Z1A6) and the Program for New Century Excellent Talents in University, China (NCET08-0531).
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