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TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11/00 c/CIHG’DateDuapeSvpu RESISTANCE AGAINST ER WINIA AMYLOVORA INDUCED IN APPLE TREES BY ACIBENZOLAR-S-METHYL By Kimberly Lynn Maxson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 2000 ABSTRACT RESISTANCE AGAINST ER WINIA AMYLOVORA INDUCED IN APPLE TREES BY ACIBENZOLAR-S-METHYL By Kimberly Lynn Maxson Acibenzolar-S-methyl (ASM), a synthetic inducer of pathogenesis—related (PR) protein expression and systemic acquired resistance (SAR), was evaluated for its control of fire blight in Malus x domestica (apple). PR protein gene expression was evaluated with real time PCR to determine an appropriate resistance induction interval. Expression of putative apple PR genes related to PR-l and 8 from Pyms pyrifolia and PR-2 from Cicer arietinum was elevated in ASM-treated apple seedlings. ASM induced a 10-fold increase in apple PR-l, and PR—8 RNA expression and a lOO-fold increase in apple PR-2 RNA expression. Elevated PR gene expression was observed 2 to 7 days after treatment. In Jonathan trees, treatment with ASM at 75 mg/l was initiated one week prior to infection and weekly applications thereafter were more effective than biweekly applications at protecting shoots and spurs from fire blight resulting from natural and inoculated infections. In Fuji trees, increasing rates of ASM (0 to 300 mg/l) demonstrated a dose response in fire blight severity, and ASM combined with streptomycin showed a synergistic response. ASM applications of 150-300 mg/l beginning at least one week prior to bloom and continuing weekly throughout the critical period are recommended for optimal fire blight control. ASM combined with streptomycin is recommended for further study. To my parents Ted and Lucille Maxson, who have always believed in me. iii ACKNOWLEDGMENTS I would like to thank Dr. Alan Jones for giving me the opportunity to work on the "Cutting edge" of fire blight research and learn under his direction. His patient guidance and teaching have been invaluable. Thanks also to Drs. Raymond Hammerschmidt and Sheng Yang He for the enlightening discussions, encouragement, and helpful suggestions. This project would not have been possible without the financial support of Novartis Crop Protection, Inc. I wish to thank Allison Tally, Ven Lengkeek, Charlie Pearson and others at Novartis for their interest in this research. Special thanks to Elise Schnabel, Gayle McGhee, and Qiaoling Jin who I am convinced know all of the secrets of genetics and molecular biology and were kind enough to share them with me; to Gail Ehret for help with applications and seedling germination; to Guido Schnabel and Katalin Kasa for helpful discussions, camaraderie, and ice cream breaks; to Luis Velasquez and Julie Greyerbiehl, the gurus of protein; and to Dave Almy for his persistence and skill at extractions and RT- PCR. I am also gratefiil to Drs. David Netzly, John Wise, and Martin Wiglesworth for sparking the interest and giving me the experience I needed to get here in the first place. iv TABLE OF CONTENTS LIST OF TABLES .............................................................................................................. vi LIST OF FIGURES ........................................................................................................... vii REVIEW OF LITERATURE Systemic Acquired Resistance ................................................................................. 1 Literature Cited ...................................................................................................... 10 RESISTANCE AGAINST ER WINIA AMYLOVORA INDUCED IN APPLE TREES BY ACIBENZOLAR—S-METHYL .......................................................................................... 1 5 Introduction ............................................................................................................ 15 Materials and Methods ........................................................................................... 17 Results .................................................................................................................... 25 Discussion .............................................................................................................. 34 Literature Cited ...................................................................................................... 42 APPENDIX A: PATHOGENESIS-RELATED PROTEIN AND ACTIN SEQUENCES ALIGNED FOR DEGENERATE PRIMER DESIGN ................................................................................................... 46 APPENDIX B: CHITINASE ACTIVITY IN APPLE SEEDLINGS TREATED WITH ACIBENZOLAR-S-METHYL (ASM) ........................................ 52 Literature Cited ........................................................................................ 55 LIST OF TABLES Table 1. Sequences of specific and degenerate primers for PCR amplification of genes in apple genomic DNA and cDNA .......................................................................... l8 Appendices: Table A1. List of amino acid sequences aligned for degenerate primer design ............... 47 vi LIST OF FIGURES Figure 1. Detection by agarose gel electrophoresis of pathogenesis-related (PR) and actin genes amplified from Gala apple DNA and Jonathan apple cDNA with degenerate primer pairs aj264-aj263 (PR-l), aj277-aj278A (PR-2), aj28lA-aj282A (PR-8), and aj665-aj666 (actin). Ladder lanes are l-kb Ladder Plus (Life Technologies) ............................... ' ....... 26 Figure 2. Amino acid alignment of apple sequences with their most similar matches from GenBank, and approximate primer placement. Arrows indicate direction of polymerization. “ * “ indicates amino acids that match the respective GenBank entry. “X” indicates an ambiguous reading in the nucleotide sequence for which a corresponding amino acid cannot be determined ............................................................... 28 Figure 3. Expression of PR-l, PR-2, and PR-8 in cDNA prepared from total RNA extracts from apple leaf samples collected 0, 2, 5 and 7 days after Jonathan seedlings were sprayed with ASM or water. cDNA concentrations were measured with real-time PCR and units reported are the base 10 logarithm of the PR-n to actin concentration ratio. Error bars denote the standard deviation for each mean .................................................... 30 Figure 4. Incidence of fire blight infected spurs on Jonathan apple trees after two flowers on each of 50 spurs were inoculated with strain Eal 10 of Erwinia amylovora 8 days after ASM applications were initiated and 4 days after streptomycin applications were initiated. Data, taken 20 days after inoculation, are from one experiment with four single tree replicates. Bars labeled with the same letter are not significantly different. A least significant difference (LSD) of 20% was determined according to an analysis of variance (ANOVA) with a = 0.05 .................................................................................................... 32 Figure 5. Fire blight strikes per tree (top) and the incidence (middle) and severity (bottom) of fire blight on Jonathan apple inoculated with Erwinia amylovora. The number of strikes (trauma blight) per tree was evaluated 16 (1999) and 12 (2000) days after a severe storm. Percent shoot infection is the average percent of inoculated shoots which became infected. Of the infected shoots, the severity of the infection was measured in the current year's growth as the length of the canker divided by the length of the shoot. Percent shoots infected and percent canker extension were evaluated 2 weeks after inoculations were made. Bars within a year labeled with the same letter are not significantly different. LSDs were determined within years according to an AN OVA with a = 0.05 .............................................................................................................................. 33 Figure 6. Relationship of canker extension in ‘Fuji’ apple trees inoculated with E. amylovora to the rate of ASM applied. Treatments were applied weekly for three weeks and inoculations were made 7 days after the first application. The data are pooled from vii two experiments in 2000 with three single tree replicates of 10 shoots per tree. Line depicts a linear trend (y = - 0.11x + 94; R2 = 0.93) ........................................................... 34 Figure 7. Effect of four sprays applied weekly on the percent of the current season growth that became necrotic following inoculation with Erwinia amylovora 7 days after the first applications. The data are pooled from two experiments in 2000 with three single tree replicates of 10 shoots per tree. Str = streptomycin. Bars labeled with the same letter are not significantly different. An LSD of 19% was determined according to an ANOVA with a = 0.05 ............................................................................................... . ....... 35 Appendices: Figure Ala. PR-l amino acid sequences from Nicotiana tabacum, Zea mays, Capsicum annuum and Vitis vinifera aligned for observation of conserved regions. Boxed regions denote areas used for degenerate primer design ................................................................ 48 Figure Alb. PR-2 amino acid sequences from Nicotiana tabacum, Zea mays and Prunus persica aligned for observation of conserved regions. Boxed regions denote areas used for degenerate primer design ............................................................................................. 49 Figure Alc. PR-8 amino acid sequences from Cucumis sativas, Hevea brasiliensis and Vitis vinifera aligned for observation of conserved regions. Boxed regions denote areas used for degenerate primer design ..................................................................................... 50 Figure Ald. Actin amino acid sequences from Homo sapiens, Neurospora crassa and Phytophthora infestans aligned for observation of conserved regions. Boxed regions denote areas used for degenerate primer design ................................................................ 51 Figure B1. Chitinase activity of total protein extracted from apple seedlings 7 days afier treatment with ASM at 1.5 g/l (lanes l-7 and 9) or with water (lanes 10-17). Lane 8 is empty .................................................................................................................................. 54 viii REVIEW OF LITERATURE SYSTEMIC ACQUIRED RESISTANCE INTRODUCTION The phenomenon of plant resistance is universally observed, however the _ mechanisms behind the resistance are relatively unknown. Many studies throughout the later part of the twentieth century have contributed to the limited understanding we have today. Intense investigation in plant resistance has revealed that there are a number of mechanisms involved. Plants employ vertical resistance, which is specialized in specific cultivars of species against a specific pathogen or group of pathogens. One example is gene-for-gene resistance, first studied by Flor (13), in which a single plant gene (R) confers resistance by interacting with a single pathogen gene (Avr). In response to the interaction of the two genes or their gene products, plants initiate rapid cell death surrounding the location of invasion. This is generally referred to as the hypersensitive response (HR) and is characterized by the appearance of small necrotic lesions in the host tissue (10, 21, 31). Plants also employ a horizontal resistance, which is the expression of constitutive resistance mechanisms such as lignified cell walls, thick cuticle, sequestered antimicrobial secondary metabolites, and inducible resistance mechanisms (23). Inducible plant resistance occurs locally with HR, phytoalexin accumulation, callose formation, increased cell wall lignification and cross-linking (19), and systemically with salicylic acid (SA) and peroxidase accumulation, expression of pathogenisis related (PR) proteins, and broad spectrum disease resistance (22, 34, 39). Systemic resistance can be induced by pathogen or insect attack, natural and synthesized chemical elicitors, or plant growth promoting rhizobacteria (PGPR) (37). Systemic resistance induced by PGPR typically does not affect PR- protein levels and is referred to as induced systemic resistance (ISR) (20, 30). There are exceptions, however, such as ISR induced in tobacco and bean by fluorescent bacteria in which increased PR- protein levels are observed (28, 49). Systemic resistance induced by pathogen or insect attack, or by chemical elicitors typically results in the induction of PR proteins and is referred to as systemic acquired resistance (SAR) (34). SAR induced by pathogen invasion is usually the result of a salicylic acid-dependant pathway, and the PR-proteins induced typically belong to different families than those induced by insects. This review will focus on the history, induction mechanisms, and agricultural applications of the salicylic acid-dependent systemic acquired resistance induced in plants by plant pathogens and chemical elicitors. HISTORY OF SAR Initial Discoveries Observations of acquired "pysiological immunity" in plants were first reviewed by Chester in 1933 (5), and are generally considered to be the first evidence of SAR. In 1961, Ross (33) showed that tobacco (var. Xanthi nc) resistance to Tobacco Mosaic Virus (TMV) could be enhanced by a previous inoculation with the virus a few days prior to a challenge inoculation. An earlier publication by Cruickshank and Mandryk in 1960 (7) indicated that blue mold (Peronospora tabacina) stem infection conferred resistance to foliar pathogens in tobacco. Peronospora-induced SAR in tobacco was further studied by Cohen and Kuc (6, 25) who reported that it required 3 weeks for development, and that heat-killed conidia were not able to induce SAR, indicating an induction interval requirement and an active pathogen role in SAR induction. In 1970, Gianinazzi et a1. (16) discovered that TMV-inoculated tobacco exhibited a marked increase in certain proteins, later termed pathogenesis-related (PR) proteins (PRs). They were described as acid-soluble, protease-resistant, acidic proteins located primarily in the extracellular space. Basic homologues were described by van Loon and van Kammen (43). Ward et a1. performed a detailed study of these proteins in tobacco and described 9 gene families induced locally and systemically in response to TMV inoculation (46). Presently, recognized PRs belong to 14 gene families (12, 15, 38, 44). Proteins are classified as "pathogenesis-related" if they are (i) induced by a pathogen in plant tissues that don't normally express the protein and (ii) induced in response to two different plant-pathogen interactions or are shown to be induced by two or more independent laboratories in the same interaction (44). In 1979, White (48) reported that exogenous applications of acetyl salicylic acid (aspirin) and other benzoic acid derivatives induced PR proteins and protection against TMV in tobacco. In 1983, van Loon (41) proposed a possible link between SA and SAR. Others reported salicylic acid (SA) accumulation in cucumber and tobacco in response to SAR induction by pathogen infection (11, 26, 29). Further studies in transgenic tobacco confirmed a relationship between SA and SAR. Tobacco transformed with a naphthalene hydroxylase G (nahG) gene (catalyzes the degradation of SA to the non-inducer, catechol) from Pseudomonas putida did not respond to SAR induction with SA accumulation and never exhibited SAR (14). This suggested that the SAR response to pathogen infection was SA-dependant. SAR models The tobacco-TMV combination is the classic model for SAR study, however Tobacco necrosis virus (TNV), Thielaviopsis basicola, Peronospora tabacina, Pseudomonas syringae and Pseudomonasfluorescens strain CHAO also induce SAR in tobacco against a variety of fungal, bacterial, and viral pathogens (25, 28, 46). Cucumber is also used as a model for studying SAR with induction by TNV, Pseudomonas Iachrymans, P. syringae, and Colletotrichum lagenarium (25). SAR in monocots has been demonstrated with P.s. pv. syringae protection of rice against Pyricularia oryzae (36). Since its discovery in 1960, SAR has been demonstrated in a variety of species including: green bean, cotton, potato, soybean, tomato, alfalfa, asparagas, bean, carnation, cucumber, Arabidopsis, muskmelon, oilseed rape, pearl millet, radish, red clover, rice, sicklepod and Stylosanthes guianensis (37). Perhaps the most useful model for dissection of the SAR mechanism is Arabidopsis thaliana. SAR can be induced in A. thaliana by Turnip crinkle virus (TCV), P.s. pv. tomato and F usarium oxysporum (1, 27, 34, 40). A. thaliana mutants exhibiting constitutive lesion formation, termed lesion simulating disease (Isd1-7) and accelerated cell death (acd2), constitutively express high levels of PRs and disease resistance (9, 18, 47). Constitutive immunity (cim2-3) mutants constitutively express PRs in the absence of cell death and still exhibit increased disease resistance (35). nahG mutants are transformed with the NahG gene from Pseudomonas putida, are not able to accumulate or respond to SA treatment and do not exhibit SAR in response to pathogen infection (24). Non-immunity(nim1) and non-expressor of PR genes (nprI) mutants show an absence of PR accumulation afier SA treatment and decreased resistance to Peronospora parasitica compared to the wild type (2, 3, 8). Non-race-specific disease resistance (ndrI) mutants are susceptible to many P. parasitica and P. syringae DC3000 strains irrespective of virulence, however the HR response in ndrI does not differ from that observed in the wild-type (4). These mutants are generally classified as either SAR constitutive (lsd1-7, acd2, cim2-3,) or SAR compromised (nahG, nimI, nprI, ndrI) and their implications will be discussed in the SAR mechanisms section. SAR MECHANISMS The physiological process resulting in SAR is largely unknown, however elegant experiments with Arabidopsis mutants, transgenic tobacco, and a wide range of host- pathogen combinations have attempted to further define the roles of SA and PR-proteins in SAR. Studies with transgenic plants focused on the role of SA in SAR, and the relationship between lesion formation and SA accumulation. Host-pathogen combinations demonstrate the role of PRs in SAR. Although further study is needed, general trends and pathways in SAR are emerging. Role of SA in SAR In the early 1990’s studies in SAR indicated systemic and local accumulation of SA in cucumber and tobacco (11, 26, 29). Malamy (26) observed that SA induced PR genes, and Ward et. a1. (46) confirmed that the PRs induced by SA were the same as those induced biologically. Working independently, Metraux et. al. and Malamy et. a1. (26, 29) both suggested SA as a putative endogenous signal for SAR. Additional studies indicated that the signal for SAR is translocated in phloem (25). Rasmussen et al. (32) found evidence contradicting SA as the translocated SAR signal. Cucumber was inoculated with P. syringae pv syringae and the inoculated leaf was removed before SA accumulation occured in leaf 1. However, SAR expression and systemic SA accumulation was not affected. Studies with nahG tobacco confirmed that SA was not the translocated signal. NahG shoots were grafted onto wild type Xanthi nc rootstocks and vice versa. After SAR induction in the rootstock, the wild type rootstocks exhibited SA accumulation, but no SAR was evident in the nahG shoots, as expected. NahG rootstocks failed to accumulate SA in response to induction, however the wild type shoot exhibited the SAR induced phenotype (45). Transgenic Arabidopsis studies have attempted to further define the role of SA in SAR. When the NahG gene is expressed in constitutive immunity (cim3) mutants, the constitutive SAR phenotype is lost (35), indicating the dependance of SAR on SA accumulation. Studies crossing lsd], 2, 4, 6, and 7 with nahG mutants indicate that a feedback loop involving SA may be present in the SAR pathway (35). Expectedly, progeny from lst, 4, 6 and 7 crosses with nahG lost the PR-l expression and resistance to P. parasitica observed in the lsd parents. While lesion formation was retained in the crosses with lst and 4 parents, it was suppressed in crosses with lsd], 6 or 7 parents. When lsd] and 6 mutants expressing NahG were treated with an elicitor which works downstream of SA, lesion formation was regained. These studies indicate the possibility of two types of lesion formation: (i) lesion formation involved in the initiation of SA accumulation which would result in PR expression and SAR and (ii) lesion formation which'results from SA accumulation in a feedback loop. Role of PR-proteins in SAR PR—proteins were defined by van Loon et a1. (42) as plant proteins that accumulate after pathogen attack or in related situations (such as after elicitor treatment). In tobacco, basic PRs are located in the vacuole and acidic PRs accumulate in the extracellular space, however this trend does not hold in all plants (3 7). PR-proteins have been characterized by their functions either in enzyme bioassays or by sequence homology. Known PRs are beta-1,3-glucanases, chitinases, thaumatin-like, proteinase-inhibitors, endoproteinases, peroxidases, ribonuclease-like, defensin, thionin, and lipid-transfer proteins (44). Despite these classifications, the actual role of PR-proteins in planta is uncertain. In 1997, Cao et a1. (3) discovered that the gene controlling the SAR-repressed phenotype in nprI Arabidopsis mutants, NPRl , is a positive regulator of PR protein expression and SAR. A single mutation in the nprI -1 mutant, which causes suppression of PR protein expression in response to infection, occurs on an ankyrin-repeat which are typically involved in protein-protein interactions. Some well studied proteins with ankyrin-repeats are transcription regulators, which suggests a possible direct role for the NPRl gene product in PR-protein expression. It has subsequently been found in other crops. AGRICULTURAL APPLICATIONS OF SAR SAR research has primarily been driven by the possibility that a greater understanding of plant resistance may enable growers to harness this phenomenon for increased crop protection. The many mechanisms involved in SAR suggest that the possibility of resistance development in pathogens would be extremely low. Exogenous applications of SA require careful optimization to prevent phytoxicity (37) which is impractical on a field scale, and which initiated the search for a SAR inducing chemical for agricultural use. To be considered an SAR-inducer, the chemical must meet the following criteria (37): (i) show no direct antimicrobial effect in vitro, (ii) protect against a range of pathogens (iii) activate a host response which can be demonstrated as similar to the biologically-induced response. Many have been studied such as 2,6- dichloroisonicotinic acid (INA) and acibenzolar-S-methyl (ASM) which have both been shown to work within the SA-induced SAR pathway, directly downstream of SA (24). INA was developed by Novartis Crop Protection Inc. (formerly Ciba-Geigy) for field scale applications. It has little or no antifungal effect in vitro and has been shown to protect dicots as well as monocots from a variety of fungal, bacterial and viral pathogens (3 7). Its usefulness in crop protection is limited however, by its phytotoxic effects in many crop species. It is still widely used as a research tool in SAR. ASM, a benzothiadiazole, was also developed by Novartis and is quite similar to INA with the exception of phytotoxicity. ASM is generally well tolerated by most crops, like INA has no antifimgal effect in vitro, and has demonstrated protection of monocots and dicots against a variety of pathogens (37). ASM’s success in field studies has I propelled its commercialization (17). It is now registered under the name Bion (countries other than USA), or Actigard (USA). It is continually being evaluated on new crops such as fruit trees. CONCLUSIONS Since its discovery in the 1960’s, SAR has continually provided us with clues about the physiology and genetics of plant metabolism. However variable it seems to be between different plant species, general trends in SAR include its dependence on SA, its characteristic PR-protein expression, and its broad spectrum protection against pathogen attack. 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Weymann, K., Hunt, M., Uknes, S., Neuenschwander, U., Lawton, K., Steiner, H. Y., and Ryals, J. 1995. Suppression and restoration of lesion formation in Arabidopsis lsd mutants. The Plant Cell 7:2013-2022. 48. White, R. F. 1979. Acetylsalicylic acid (Aspirin) induces resistance to Tobacco Mosaic Virus in tobacco. Virology 99:410-412. 49. Zdor, R. E., and Anderson, A. J. 1992. Influence of root colonizing bacteria on the, defense responses of bean. Plant and Soil 140:99—107. l4 INTRODUCTION Fire blight, caused by Erwinia amylovora (Burrill) Winslow et al., is a devastating disease of apple (Malus x domestica Borkh.) which causes extensive destruction of bearing tissues, and frequently results in tree death. In 2000, a fire blight epidemic in southwest Michigan cost apple growers an estimated 42 million in losses. E. amylovora initially infects apple blossoms and actively growing shoots then spreads through the vascular system to infect susceptible rootstocks (14). Wind-driven rain and pollinating insects spread the bacteria once they are established in blossoms; therefore, fire blight control during the bloom period is critical to prevent severe losses. Control measures include copper compounds sprayed at bud break followed by two to four applications of the antibiotics streptomycin and oxytetracycline throughout the bloom period. Copper ions are toxic and can adversely affect fruit finish. Limiting the use of copper to bud break can reduce the risk of hit injury. Antibiotic applications can be made without risk to the fruit; however, E. amylovora has developed resistance to streptomycin in some areas (4, 5, 19, 20) and its use in some countries is prohibited. Oxytetracycline is substituted or mixed with streptomycin in areas of the United States where streptomycin resistance has been identified. Antibiotic treatments are only locally systemic, and their efficacy is often enhanced by infection period forecasts with a prediction model (2). In the United States, antibiotics and copper compounds have been used to control fire blight for 50-70 years and are the best bacteriocides available, but their efficacy is often inadequate for fire blight control in high-density plantings of susceptible varieties. There is also concern that increased use of antibiotics for plant 15 health could impact resistance in human bacterial pathogens. Apple growers need new fire blight control methods with increased efficacy and low environmental impact. Novel approaches to fire blight control include utilizing the plant resistance phenomenon called "systemic acquired resistance" (SAR). SAR occurs systemically in the host plant in response to pathogens which cause a rapid, local development of necrosis. SAR induction is associated with an increase in peroxidase activity, accumulation of salicylic acid (SA), and increased expression of several pathogenesis- related proteins (PRs) (11, 22). The SAR response can also be elicited independant of infection by exogenous applications of SA or other synthetic elicitors (24). One of these SAR elicitors is acibenzolar—S-methyl (ASM), which is marketed as Actigard and Bion (Novartis Crop Protection, Inc., Greensboro, NC). ASM has demonstrated systemic activity and broad spectrum efficacy in a variety of plant species against bacterial, fungal, and viral diseases (8, 9, 12, 13, 23). In pome fruit crops Ishii et al. (10) reported that ASM provided protection against scab (Venturia nashicola) and rust (Gymnosporagium asiaticum) in Japanese pear (Pyrus pyrifolia) and Brisset et al. (3) reported that ASM protected apple trees from fire blight. These studies indicate that resistance can be induced in fruit trees with ASM treatment and is effective in apples against E. amylovora, however, a practical treatment strategy for ASM on apple trees is still needed. What remains unknown is the level of resistance attained by ASM treatment, the pre-infection interval necessary to reach this level, and the application interval necessary for maintenance of resistance. The intention of this study was to determine the post- application interval needed for the highest level of PR-protein induction, correlate 16 expression of PR genes with disease resistance, and develop an application schedule that would optimize ASM's mode of action in apple trees to prevent fire blight. MATERIALS & METHODS PCR of target genes from apple: Amino acid sequences of PR-proteins and actin found in GenBank were aligned with the aid of DNASTAR's Lasergene software (DNASTAR Inc., Madison, WI) to identify conserved regions for which PCR primers could be designed. Sequences of PR-l were Nicotiana tabacum, GenBank accession number D90196; Zea mays, U82200; Capsicum annuum, AF 053343; and Vitis vinifera, AJ003113; those of PR-2 were N. - tabacum, M60460; Z. mays, M95407; and Prunus persica U49454; the PR-8 sequences were Cucumis sativas, M84214; Vitis vinifera, 1705812; and Hevea brasiliensis, 116359; and the actin sequences were Homo sapiens, NP_001605; Neurospora crassa, P78711; and Phytophthora infestans, JE0414. Degenerate primer pairs aj263-aj264, aj277- aj278A, aj281A-aj282A, and aj665-aj666 were designed for the amplification of PR-l , PR-2, PR-8, and actin genes, respectively (Table 1). DNA was isolated fi'om 0.1g fresh weight of Gala and McIntosh apple leaves with a DNeasy Plant Minikit according to the manufacturer's instructions (Quiagen, Inc., Valencia, CA). The degenerate primer pairs listed above were used to amplify fragments of PR-l and PR-8 from Gala DNA, PR-l, PR-2, and PR-8 from McIntosh DNA, and actin from Jonathan DNA. Reactions were preformed in a FTC-100 Programmable Thermal Cycler (MJ Research, Inc., Watertown, MA). PCR mixtures (50 pl) for the amplification of PR-proteins consisted of IX PCR Buffer (10 mM Tris-HCl, pH 8.3), 1.5 17 WW \I Table 1 Sequences of specific and degenerate primers for PCR amplification of genes in apple genomic DNA and cDNA. Target geneat (protein Primer family) ID Primer sequenceb PR-l aj264 GGNCAYTAYACNCARGTNGTNTGG PR-l aj263 TTNCCNGGNGGRTCRTARTTRCA PR-l aj 629 ACCCTACACGAGCCGAGTTRCG PR-l aj630 CATGCCATGGCCAAACIACYTGIGTRTARTGIC PR-l aj 708 GTAGGCGTTGGTCCCTTGAC PR-l aj 709 GATTGCAGTCGCCAACATGT PR-2 aj277 TAYATHGCNGTNGGNAAYGA PR-2 aj278A AACATNGCRAANAGRTANGT PR-2 aj636 TCAGAGGGAGGGAAGGGATTGG PR-2 aj637 AATTGCACTGTGGATGTTTTGGATGG PR-2 aj778 TCCGATGCCATTGCTTTTG PR-2 aj779 TTATGGACGAAACGGCAACA PR-8 aj281A TAYTGGGGNCARAAYGGNAA PR-8 aj 282A TARAAYTGNACCCANACRTA PR-8 aj638 CGGGTGGTGCGCAGTTCTAYGATGAG PR-8 aj639 CTCAACGGACACAACGGACAGGCAAAAA PR-8 aj780 CTCTTTTGAGCAGTTGGAACCA PR-8 aj 78 1 TGCCGGTAACCCCATGAA Actin aj665 AAYTGGGAYGAYATGGARAA Actin aj666 ATCCACATYTGYTGRAAN GT Actin aj 748 AACTTCGTGTTGCTCCTGAAGAG Actin aj 749 CAGTAGTACGACCACTGGCATAGAG ---- APl GGATCCTAATACGACTCACTATAGGGC ---- AP2 AATAGGGCTCGAGCGGC a Target gene family is putative based on most similar GenBank entry. Primers are listed 5' to 3'. Degeneracy is indicated with the standard degenerate code and " I " indicates an inosine nucleotide. 18 mM MgC12, 160 pM dNTPs, 25 pM of each primer, 1.25 U of Taq DNA polymerase (Life Technologies, Gaithersburg, MD), and 0015-0120 pM of apple DNA. Reaction mixtures were covered with a 40 pl layer of light mineral oil prior to amplification. Cycling parameters were 94°C for 5 min followed by 30 cycles of 94°C for 1 min; 47 °C (PR-1 and PR-8) or 53 °C (PR-2) for 2 min; and 72°C for 1 min. PCR mixtures (50 pl) for the amplification of actin consisted of 1X Expand Long Template PCR System Buffer (Roche Molecular Biochemicals, Indianapolis, IN), 10 mM dNTPs, 40 pM of each primer, 2.5 U taq DNA polymerase (Life Technologies), and 0015-0. 120 pM of apple DNA. Cycling parameters were 94°C for 2 min followed by 30 cycles of 94°C for 30 sec; 50°C for 1 min; and 72°C for 1 min. PCR products (8 pl) were separated on a 2% agarose gel, run in 0.5X Tris-borate-EDTA (TBE) buffer, stained with ethidium bromide, and visualized with UV light. The bands were purified with Wizard PCR Preps DNA Purification System (Promega Corp., Madison, WI), and quantified with a Genequant DNA/RNA Calculator. The amplified fiagments were sequenced and their deduced amino acid sequences were compared with the previously mentioned amino acid alignments according to protein family. All sequencing was preformed at Michigan State University Department of Energy Plant Research Laboratory sequencing facility using ABI dye-terminator chemistry (Applied Biosystems, Foster City, CA). To determine if the PR and actin genes were expressed in ASM-treated apple tissue, total RNA was isolated from leaves harvested 3 days after 28-year-old Jonathan trees were sprayed with ASM at 75 mg/l active ingredient (a.i.). An apple ZAP Express Custom cDNA library was constructed from total RNA by Stratagene (La J olla, CA). 19 Degenerate primers (Table 1) were then used to amplify fragments of PR-l, PR-2, PR-8 and actin from the cDNA library. An apple DNA library was used as a template to amplify longer regions of each gene for comparison with related genes in GenBank. McIntosh DNA was digested with the restriction enzyme, ScaI. Adapters (5'-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG CAG GT-3') and (5'-P-AC CTG CCC—NH2-3') were ligated onto the ends of the digested DNA according to the method in Seibert et al. (21). The 5' end of the apple PR—l gene was amplified fi'om the Seal library using nested PCR. Adapter primers, API and AP2 (Table 1), were paired with aj629, and then with aj630, respectively. Reaction mixtures (50 pl) for nested PCR were as described for actin amplification except Expand Long Template System Taq DNA Polymerase (Roche Molecular Biochemicals) was used. Cycling parameters were 94°C for 2 min followed by 30 cycles of 94°C for 30 sec; 65 °C for 30 sec; and 68°C for 5.5 min. The process was repeated with the PR-2 and PR-8 genes to amplify 5' and 3', respectively, out of the previously sequenced apple DNA. Primers API and AP2 were paired with aj636 and aj637 for PR-2 amplification in the 5' direction, and with aj638 and aj639 for PR-8 amplification in the 3' direction. PCR products were separated on 2% agarose gels, purified, and sequenced. Isolation of RNA and cDNA synthesis: Twenty-four 4-week-old Jonathan apple seedlings from open-pollinated seeds were sprayed to run-off with either ASM at 1.5 g/l a.i. or water. Six of the upper-most leaves from each of three replicates were harvested at days 0, 2, 5, and 7 post-application, quick-frozen in liquid nitrogen, and stored at -80 °C. The frozen leaves were ground in 20 liquid nitrogen and then added to a preheated (100 °C ) combination of 3 ml extraction buffer (100 mM Tris HCl (pH 8), 100 mM LiCl, 10 mM EDTA, 1% w/v SDS) and 3 ml Tris-saturated phenol. The mixture was vortexed for 1 min, 3 ml of chloroform were added, and the solution was vortexed again. The mixture was centrifiiged at 10,000 rpm for 20 min, the supernatant was collected, and the phenol/chloroform extraction was repeated. The supernatant from the second phenol/chloroform extraction was added to 1 volume of 4 M LiCl and precipitated at 4°C for 2 t018 hr. The mixture was centrifuged as above, and the pellet was resuspended in 400 pl water. All water used for RNA extraction was deionized and autoclaved with 0.1% v/v diethyl pyrocarbonate. Total RNA was precipitated with 2.5 volumes of 200 proof ethanol and 0.1 volume of 3M sodium acetate, pH 5.2, at -20°C for 1 tol 8 hr. RNA was pelleted by centrifugation at 10,000 rpm for 15 min, resuspended in water, quantified, and stored at -80°C in 5 pl aliquots at 1 pg/pl total RNA. Each 5 pl aliquot of total RNA was treated with RQl RNase-free DNase (Promega Corporation, Madison, WI) according to the manufacturer's protocol. RNA was precipitated with 2.5 volumes of 200 proof ethanol and 0.1 volumes of 3M sodium acetate, pH 5.2, at -20°C for 1 to 18 hr. Samples were centrifuged at 10,000 rpm for 15 min, the supernatant was discarded and the RNA pellet was allowed to dry. The RNA was then reverse-transcribed following the SuperScript II RNase H' Reverse Transcriptase (Gibco BRL Life Technologies, Grand Island, NY) protocol for first strand cDNA synthesis using random primers. The reaction was diluted 1 to 10 and used for expression analysis. 21 Expression of PR genes using real-time PCR: Expression of PR-l, PR-2, PR-—8, and actin in apple cDNA samples was quantified by real-time PCR. Primer pairs aj708—aj 709, aj778-aj779, aj780-aj781, and aj 748-aj 749 specific for putative apple PR-l, PR-2, PR-8 and actin genes, respectively, were designed with Primer Express software from Applied Biosystems (Table 1). Parameters for primer design were 20-80% CG content, Tm = 58-60 °C, and amplicon length of 50 tolSO bp. The PR gene fragments were amplified with an ABI Prism 7000 sequence detection system (Applied Biosystems) which measures the fluorescense of the double-stranded DNA binding dye, SYBR Green, as it binds to increasing amounts of the target sequence. PCR mixtures (30 pl reactions) consisted of IX SYBR PCR buffer, 3 mM MgC12, 1 mM dNTP Blend, 0.025 U/pl AmpliTaq Gold, and 0.01 U/pl AmpErase UNG from Applied Biosystems. To each mixture, 0.125 pg of cDNA fiom each sample was added. Cycling conditions were 2 min at 50°C, 10 min at 95 °C, and 40 cycles of 15 sec at 95 °C and 1 min at 59°C. Controls for each run included no-template controls, no-amplification controls in which 0.07% (wt/vol) sodium dodecyl sulfate (SDS) was added, and dilution series of target template previously described. Actin was amplified from each sample within a nm for a relative constitutive control. For each set. of primers, individual sample amplifications (PR-n and actin) were replicated three times and controls were replicated twice. The concentration of target sequence in each sample was determined from the dilution series of target template for both PR-n and actin. The expression of PR-proteins is reported as a concentration ratio of PR-n to actin. 22 ASM efficacy for fire blight control: The experiment was conducted in 1999 and 2000 on 28-yr-old Jonathan apple trees located at the Michigan State University Botany F arm in East Lansing. The experimental area consisted of a single outside row of trees. Each treatment (including the untreated check) was replicated three times in 1999 and four times in 2000. The experimental design was a randomized complete block with single-tree plots. Two ASM (Actigard 50WG) treatments at 75 mg/l were initiated 1 week pre-bloom, and successive sprays were applied on a 7- or 14-day interval. Streptomycin (Agrimycin 17, Novartis) at 100 mg/l was applied twice during bloom and then on a 7-day interval. All applications were applied to runoff. At full bloom, two blossoms per each of 50 spurs per tree (100 blossoms/tree) were inoculated with 1><106 cfu/ml of E. amylovora strain Eal 10 (17) by touching the stigmatic surface of the pistils with a cotton swab soaked with inoculum. The inoculum was prepared by suspending in sterile water bacteria from l8-hr-old cultures in Luria-Bertani (LB) broth (18). Percent of inoculated spurs infected was evaluated 20 days later. After three successive weekly ASM sprays and two biweekly ASM sprays, 25 shoots per tree were inoculated by bisecting two terminal leaves with scissors dipped in 1>