AV. nWIJ’tg‘WI‘ \ .. ‘--.-.= .. '3'; . «3' ‘_ . ¢ 5’: , n:-.,.~ .. s. L w u .._.-.:. .u‘ . at. L. A.“ V"? . -4. . . u ‘v‘ 3‘ ~.‘~“ .,1..l ‘J ‘ v! \r ”A.” 3.: an. , I‘m ‘N‘V.’ .: I? 1:“ ‘1}. .. ‘nd'JA-u fixar am. an.» 3’ '2 ; z ? This is to certify that the thesis entitled Investigation of the molecular basis of Ml-a-mediated disease resistance in barley presented by Samuel Peter Simons has been accepted towards fulfillment of the requirements for MOS. Botany and Plant Pathology degree in I , I . AA (a (rt/mi» [-qu w‘ {v1 /C\. Major professor Date Q]- ’7’ / 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Mlchlgan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE J IL__] I; ________ ll l I — l! r—i i— l MSU Is An Affirmative ActiorVEqual Opponunlty Institution cmma-nt L INVESTIGATION OF THE MOLECULAR BASIS OF M—a-MEDIATED DISEASE RESISTANCE IN BARLEY By Samuel Peter Simons A THESIS . . Submitted to . . .MlChl an State Umversrty 1n partial ful ent of the requirements for the degree of ‘ MASTER OF SCIENCE Department of Botany and Plant Pathology 1991 .v 0/;- S I)” V aft-’3- ABSTRACT INVESTIGATION OF THE MOLECULAR BASIS OF M-a-MEDIATED DISEASE RESISTANCE IN BARLEY By Samuel P. Simons Three attempts were made to identify a molecular marker for the barley M-a locus, which encodes resistance to the powdery mildew pathogen, Erysiphe graminis f. sp. bordei. Progeny of crosses between barley isolines carrying alleles M—a6 or MI-313 were scored for powdery mildew resistance, ADHl, and waxy, in an effort to identify a reported unstable genetic element. No mutants were recovered, thus, we were unable to confirm the existence of an unstable element. In a second approach, polypeptide profiles of coleoptiles from resistant and susceptible isolines were examined by two-dimensional polyacrylamide gel electrophoresis. Two previously undescribed sets of polymorphic polypeptides were observed. However, no correlation with the MI-a locus was found. Polypeptides from resistant and susceptible isolines were analyzed over a 12 hour period following inoculation with the fungus. No induced differences were found. More sensitive methods are needed to identify polypeptides associated with resistance to E. g. hordei. DEDICATED to my wife LORRIE and to my children PETER, JEREMY, and KELLY ACKNOWLEDGMENTS My thanks and appreciation to Dr. Shauna Somervjlle for providing challenging problems and financ1al support. Ialso WlSh .to t ank Dr. SomerVille and the other members of my %iidanCe committee, Dr. Lee McIntosh, Dr. Jerry Dodgson, and Dr. Bar Sears for their support and suggestions while gu1dmg me through an important step in my development as a screnttst. . . The PRL and Genetics Pro am prowded an open environment for the dlSCUSSlon of seience. My 5 ay at MSU was made enjoyable by my fellow students andpthcr members of the PRL, espeCiallyin the case of my fellow BamHI/Pi dogs, I thank them for many fruitful discusSions and ood times at the enetics retreats, ASPP meetm s and trips to the l_oatcd Goat. MarCia Kieliszewski must be tha ed for rQViding scientific adv1ce and perspective over the course of many lunc times. Many peo 1e helped in the course of these studies. I espec1ally want to acknowle e Lisa Churgay for her techmcal assistance, Lyla Melkerson-;Wa on. for trainm and aSSistance on the Visage 110 and Kurt Stepmtz for his timel an excellent help With all the fi ures. Seeds for; this work were. prow ed by Dr. Mosemanand Dr. K¢ ster. Dr. R. Wise was of help in prov1ding suggestions which may account for the disparity of our results and his. Fmanc1al su port came from the DOE-BER (DE-AC02-76- ER01338), The . ichigan Agricultural E criment Station (#1648) and the National Institutes of Health GM4OO .4 . I also Wish to acknowledge the finanCial aSSistance of the .P . , especia y Hans Kende who did his best for the students during his time as director. . Finally, my friends outSide the smentific estabhshment supported me throughout this endeavor es ec1ally Nan Case, Dick Lorencen, The members of Dimondale ni ed Methodis Church, and of course my wgfe,.I.orrie. M . children, whose names I do remember provrde me With JO when t gs looked their worst. Contrary to popular behef, they allowe me to keep my samty, not lose it. iv TABLE OF CONTENTS LIST OF TABLES .................................... vii LIST OF FIGURES .................................. viii LIST OF ABBREVIATED TERMS ....................... ix INTRODUCTION ..................................... 1 LITERATURE REVIEW ............................... 3 Literature Cited ................................. 18 CHAPTER I. Investigation of a genetic instability in barley Abstract . ................ ' ............. 24 Introduction .......................... 26 Materials and Methods .................. 28 Results. .............................. 32 DiscuSSion -. ........................... 37 Literature Cited ........................ 39 CHAPTER II. Identifying polypeptides associated with disease resistance usmg near-isogenic lines Abstract . ............................. 41 Introduction .......................... 42 Materials and Methods .................. 45 Results. .............................. 48 DiscuSSion .. ........................... 57 Literature Cited ........................ 6O CHAPTER III. Examination of polypeptide patterns from coleoptiles of reSistant and susceptible barley lines following inoculation With Erysrphe grammis f. sp. border Abstract................. ............ 62 Introduction .......................... 63 Materials and Methods .................. 66 Results .............................. 68 Discussion ............................ 74 Literature Cited ........................ 77 SUMMARY AND FUTURE DIRECTION ................ 79 Literature Cited ........................ 83 APPENDIX A ...................................... 84 APPENDD( B ...................................... 87 LIST OF TABLES Table 1 Disease reaction phenotypes for specific host pathogen interactions according to the gene -for -gene hypotheSis ............. . .............. 6 Variants recovered at the Waxy, ADHI and MI-a loci . . . . 33 3 Genetic markers adjacent to the Ma locus in near-isogenic barley hues carrying various M—a alleles .................................... 51 4 Development stage of E: g. bordei race CR3 after mocula‘ ion onto coleoptiles of AlgR and AlgS .......... 69 5 Protein changes in AI R and AlgS at 24 hours after inoculation With E. g. order, race CR3 ............... 73 vii LIST OF FIGURES Figure 1 Physiological models of the gene -for-gene interaction ...................................... 8 2 ADHl isozymes from seeds of putative ADH] mutants, separated by native polyacrylamide gel electrophoreSis ................................. 34 3 Southern blot .of genomic DNA cut with BamHI . and probed With pM9 -15.1, which contains a genomic clone of ADHI ................................. 36 4 Autoradio am demonstratinsg constitutive differences in the pro em profile of Alg ....................... 49 5 Autoradio am demonstratin constitutive differences in the pro ein profile of Alg ...................... 50 6 Autoradiograms of 35S -labelled coleoptile polypeptides in the region of p6.7 ............................. 54 7 Linkage map of the short arm of barley chromosome 5 in the region of MI-a ............................. 55 8 Electrophoretic atterns of coleo tile. polype titles. from umnoculated bar e lines and bar ey lines 4 pi With raceCR30fE.g. order............‘., ............. 72 A Model of barley chromosome 5 showing the genetic interpretation of Table 3 .......................... 85 HRGP IEF kbp NEPHGE PCR PR RAPD LIST OF ABBREVIATED TERMS alcohol dehydrogenase Algerian R (CI-16137) Algerian S (CI-16138) complementary . deoxyribonucleic aCld centimorgan disintegrations per minute hours post inoculation h droxyproline -rich glycoprotem isoelectric focusing kilobase pairs kilodaltons nonequilibrium pH gel electrophoreSis near-isogenic lines polymerase chain reaction isoelectric point athogenesis-related fprotem) randomly am lified polymorphic NA SDS -PAGE sodium dodecyl sulfate polyacrylamide gel electrophorCSis 2MB ,B-mercaptoethanol INTRODUCTION Disease resistance genes are fundamental to plant survival in nature. At present however, no plant resistance gene has been isolated and our understanding of the role of such genes in directing plant responses to pathogen attack is incomplete. The isolation and molecular characterization of plant resistance genes would represent a significant advance in our knowledge of host-pathogen interactions. We have chosen to use the barley-barley powdery mildew system in our efforts to isolate and molecularly characterize a plant disease resistance gene. Two approaches were used in an attempt to isolate an allele of the M-a disease reaction locus of barley. Chapter I describes an effort, based on identification of a putative transposable element, to use transposon tagging as a method for isolating MI-a alleles. In Chapter H, I have examined the protein profiles of several near- isogenic barley lines carrying different alleles of M—a. We hoped to identify a constitutive polypeptide representing the MI-a gene product by this method. Analysis of a polymorphic polypeptide using the inherent genetic advantages of near-isogenic lines is described. Examination of the early biochemical events of the barley-barley powdery mildew interaction is of interest for determining the developmental processes involved in activating host defenses. Additionally, polypeptides identified by their involvement in the 2 earliest events of pathogenesis can be used as the basis of experiments to isolate the resistance gene. Methods for obtaining synchronized development of the pathogen over the early time course of attempted infection (Masri and Ellingboe, 1966) allows for such examination. Chapter III is devoted to efforts to identify protein changes in resistant and susceptible barley plants during the early stages of pathogenesis. LITERATURE REVIEW The first report of a plant resistance gene that followed a Mendelian inheritance pattern was published in 1905 (Ellingboe, 1976). In the 85 intervening years a great deal has been learned about the genetic, physiological and biochemical aspects of pathogenesis. Despite this intensive study, we still have an incomplete mechanistic understanding of plant disease resistance. Pathogen invasion elicits a variety of responses in plants (Slusarenko and Longland, 1986). These responses may include, singly or in combination, cell wall modification, phytoalexin accumulation, the hypersensitive necrosis response, or synthesis. of pathogenesis- related (PR) proteins (Halverson and Stacey, 1986). These metabolic changes are mediated by two different types of resistance. Non-host resistance is defined as resistance expressed at a species level. For example, an organism that is pathogenic on pea may not be pathogenic on bean. Once a pathogen overcomes non-host resistance, it must establish a basic compatibility at the species level (Ellingboe, 1976). Race-specific resistance overlies this basic compatibility by allowing specific host lines to be resistant to specific races of a normally pathogenic organism. It has recently been suggested that non-host resistance in bacteria is an extension of race-specific resistance to all lines of a specific host 4 (Whalen et al., 1988; Kobayashi et al., 1989). Bacterial pathogens used in this study are separated taxonomically into different pathovars of the same bacterial species based on their ability to grow on certain host plant species. The bacteria grow on specific species, but elicit the hypersensitive response, a programmed cell death, in other plant species. This interaction, though eliciting a response similar to that which occurs in race-specific resistance, is generally defined as non- host resistance. Clones of race-specific avirulence genes (Avr) have been isolated from Pseudom onas syringae pv. tomato. When the clones are transferred into P. s. egcinea, a soybean pathogen, these bacteria elicit a race-specific resistance response in soybean. One of these clones, Aer, confers on Escherichia coli the ability to elicit a race-specific hypersensitive necrosis response in specific cultivars of soybean. Taken together, the three Avr clones isolated from P. s. tomato elicit resistance on all lines of soybean tested suggesting that a battery of race-specific avirulence genes is sufficient to explain non- host resistance. Heath (1991) has countered these arguments with a vigorous defense of the distinction between race-specific and non-host resistances. Pointing out differences in timing that characterize race- specific and non-host responses in fungi, species crossover of race- specificity is explained as the result of evolution of biotypes or pathovars subsequent to the development of race-specificity. Also, Gabriel and Rolfe (1990) argue that pathovars represent different races and, therefore, what are called non-host resistance reactions are actually race-specific resistance reactions. It is most likely that non- host resistance can be separated from race-specific resistance. 5 Non-host resistance does not necessarily depend upon host specific responses but rather it may simply result from the inability of a potential pathogen to recognize a particular plant species as a host. For instance, a fungal spore may fail to germinate or, once germinated, the germ tube may not recognize the host surface (Callow, 1984; Hoch et al., 1987; Allen et al., 1991). However, most forms of non-host resistance to biotrophic pathogens are active (Heath, 1981). Basic compatibility may result from the pathogen overcoming the plant non-host associated defense responses by suppressing or avoiding their activation or by inactivating or becoming insensitive to (bio)chemical defenses. Where basic compatibility exists, race-specific resistance may evolve. Race-specific resistance is often conditioned by the presence of a single, usually dominant, resistance allele in the plant and a single pathogen avirulence allele, which is also usually dominant. Thus, race- specific resistance is only expressed when the host resistance gene is paired with the appropriate, complementary, avirulence gene. This general rule is known as the gene-for-gene hypothesis (Table 1). Flor first devised this hypothesis to describe the genetic relationships that he observed in the flax-flax rust interaction (Flori, 1946; Flor, 1947; Flor, 1955). A The gene-for—gene hypothesis implies that resistance is an active process involving gene products of the host resistance allele and the pathogen avirulence allele. A number of working physiological models have been developed to explain the gene-for-gene hypothesis (reviewed by Gabriel and Rolfe, 1990). Table 1: Disease reaction phenotypes for specific host-pathogen interactions according to t e gene-for-gene hypotheSis. Host Geno e Pathogen typ Genotype R- rr A- Incompatible Compatible aa Compatible Compatible Incompatible: resistant host, avirulent pathogen Compatible: susceptible host, virulent pathogen 7 Albersheim and Anderson—Prouty (1975) proposed the "elicitor- receptor" model (Figure 1a) in which the resistance gene product is postulated to be a cell surface receptor that binds an avirulence gene product expressed on the pathogen surface. Other variations of this model postulate that the avirulence gene product is either an extracellular metabolite (Keen, 1982) or an activator of an endogenous host elicitor (Davis et al., 1984). Binding of the avirulence gene product or a host-derived elicitor by the surface receptor is thought to activate a signal transduction pathway leading to active host defense. The "dimer" model (Ellingboe, 1982) is used to describe a direct interaction of a resistance gene product and avirulence gene product, which, rather than activating defense responses, inactivate basic compatibility (Figure 1b). The "ion-channel" model (Gabriel et al., 1988) is similar to the "elicitor-receptor" model with the resistance gene product represented as a cell surface ion channel that is opened in the presence of the avirulence gene product (Figure 1c). Rather than activating cell defenses via a signal transduction pathway, the ion channel would directly initiate hypersensitive cell death through ion efflux. In both the "dimer" and the "ion-channel" models, the physiological responses, such as phytoalexin and PR protein accumulation, are considered consequences, rather than causes, of resistance. All of the above models suggest three methods of examining gene- for-gene interactions. One may attempt to gain insight by analyzing the resistance gene product, the avirulence gene product, or the biochemical changes that occur subsequent to the interaction of these two factors. Involvement of a protein product of a resistance (a) I' . . I II : III (C) The ion channel delense model Pathogen W SM“ 'mmfiwm E Co v were"; ... ............... "283132“ mm" "mm” sunflouou at 9000'“ (9.16031. “an“ “flue" ! rocognition at not.“ UDHNO'MI ‘ ' end/orolhoelwrtlu 00 U D A l Specialized imam. ouch ee Swim Wales 0 r> 4 \ weeeomm, Musical etc 0 x e ___,_,.I 2 it t z ,,,,, v v v v I . ””r I fl Ceu nu PAS 1’ ...... ”I mm .0000 \ A‘ .' MIIDNIIKII ‘ \ ” ‘~-.°.‘3‘.”.‘!- Nucleus ..... ’ 3 reuneo enzyme a 9.": Plant Cell Ooenmo oi the «on cam {} m in plume vacuum :9' :flaq‘fc'_ o" (0) Wm mo'm' '3... ”‘33.?" Basic _ompatibiliiy Spealic Incompatibility g as in _I) lo, regulatory or an oodm r oiieci etlects 3 38mm: t protein W —> m: "ovum" r \ fix] ! |, Collapse ol enern-uo sure a , \\ \\ Host cell death inco) V] o. oiuma “MW." and Once! A/ \ \] onset cl hco ."OCID HCD I A m»"”“”" at in 9' . direct effects L R 9"” Plant Cell um men-is Fi gu.re1 Ph siolo cal d 15 " _ _ ..- . Gabriel and kRolfeg119 9810 e 0f the gene for gene interaction. From 9 gene is suggested by the temperature sensitive behavior of the Sr6 allele in wheat (Loegering, 1966). This allele confers race-specific resistance to wheat rust (Puccinia graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn.), which is impaired at the non-permissive temperature of 25°C. Temperature sensitivity is commonly interpreted as resulting from an unstable tertiary structure in the protein product of the affected gene. Therefore, the Sr6 allele is likely to encode a protein product actively involved in resistance. However, no race- specific resistance gene or gene product has been isolated. Several avirulence (Avr) alleles have been cloned from bacteria (Staskawitz et al., 1984; Gabriel et al., 1986; Kobayashi et al., 1989). Although no biochemical function has been identified for these clones, active function has been inferred by the dominance of the cloned genes when transferred into other pathovars and races. A low molecular weight factor, which elicited a hypersensitive response on specific cultivars of soybean, was secreted by E. coli cells transformed with an Aer clone. These results suggest that the Aer gene product acts on a common bacterial metabolite (Keen et al., 1989). However, since the Aer clone was isolated from a tomato pathogen and acts on soybean, it may be argued that the Aer product is activating non- host resistance. A protein that specifically elicits chlorosis and necrosis on tomato cultivars with the Ct? resistance gene has been purified from the fungal pathogen CIadosporium fulvum Cooke [syn. FuIVia fuIva (Cooke) Cif.] (De Wit et al., 1989). This peptide appears to be extracellular and its biochemical function has likewise not yet been deduced. Clearly more work is necessary, but we can infer that the avirulence gene product acts extracellularly. 10 A number of biological molecules have been correlated with the events of resistance, but no cause-effect relationship has been demonstrated. Among the more intensely studied molecules are the secondary metabolites of low mass known as phytoalexins (Dixon and Harrison, 1990). Phytoalexins exhibit anti-microbial activity and have been shown to accumulate preferentially in some incompatible interactions (Cramer et al., 1985; Habereder et al., 1989). Phenylalanine ammonia lyase (PAL), the first enzyme in the phytoalexin pathway in bean, is induced in suspension culture cells within 3 minutes following application of elicitor preparations from Colletotn'chum Iindemuthianum (Sacc. & Magnus) Lams.-Scrib. (Lawton and Lamb, 1987). PAL transcripts increase in relative abundance within 5 hours post inoculation (hpi) of potato with Pbytopbtbora mfestans (Mont) de Bary (Fritzemeier et al., 1987) or soybean infected with root rot (Phytophthora megasperma Drechs. f. sp. glycinea T. Kuan & D.C. Erwin) (Habereder et al., 1989). PAL has also been observed to increase in wheat (Moerschbacher et al., 1989) and barley (Shiriashi et al., 1989) following infection, although lignin, not phytoalexin, is thought to be the final product. Chalcone synthase (CHS), the first committed step in isoflavonoid phytoalexin biosynthesis, has also been observed to increase in inoculated potato and bean plants (Dixon and Harrison, 1990). Although phytoalexins may accumulate preferentially in incompatible interactions, their importance in race-specific resistance is questionable. In one study of the interaction between soybean and P. m. glycmea, phytoalexin accumulation did not precede hypersensitive cell death (Yoshikawa et al., 1978). Thus, in this interaction, the hypersensitive necrosis 11 response may play a more important role than phytoalexin accumulation in the expression of resistance. Cell wall modifications also occur in plants during disease development. Hydroxyproline-rich glycoproteins (HRGPs) have been shown to increase following infection (Mazau and Esquerré-Tugayé, 1986). Presumably, these proteins help provide a structural barrier to infection. However, the induction of HRGPs is slow and occurs equally in compatible and incompatible interactions, though individual HRGP genes may respond differentially (Dixon and Harrison, 1990). In addition to their role in cross-linking toxic phenolics into the plant cell wall, peroxidases are also thought to be involved in strengthening the wall (Bolwell et al., 1985). Specific intercellular peroxidases were induced in inoculated barley, however, the peroxidases accumulated equally in resistant and susceptible cultivars (Kerby and Somerville, 1989). While gene products which can modify cell walls are clearly induced following pathogen challenge, a role for the cell wall in excluding potential pathogens has not been shown. Infection of several plant species results in the accumulation of PR proteins, which are believed to act to provide induced resistance to viruses, bacteria, and fungi (Gianinazzi, 1984). These proteins are divided into a number of classes and include chitinases, glucanases, protease inhibitor/a-amylase, and many proteins with no identified functions (Van Loon, 1985). PR proteins are found both in the extracellular space and in the vacuole. Accumulation of PR proteins occurs over a time course of days rather than hours and, thus, PR proteins are most likely not involved in the relatively rapid race- specific resistance response. Thionins are another class of small 12 molecules that are induced in infection and are toxic to fungi (Bohlmann et al., 1988). These molecules accumulate in the secondary cell wall appositions (papillae) of barley and are preferentially accumulated in incompatible interactions (Ebrahim-Nesbat et al., 1989). Accumulation of these molecules occurred after 32-44 hpi, long after resistance is expressed (Ellingboe, 1972; Johnson et al., 1979). Therefore, thionin induction is also too late to be involved in race- specific resistance. Efforts have also been devoted to identifying proteins or RNA species associated with the disease response through the use of two- dimensional gel electrophoresis (2D-PAGE) and in vitro translation. Hadwiger identified approximately 21 mRNA species that are induced in both resistant and susceptible host lines of pea during the early interactions with the fungus Fusarium solani (Mart.) Sacc. when growth of the fungus is suppressed in both lines (HadWiger and Wagoner, 1983). Interestingly, the level of induced mRNAs declined in the compatible interaction at 18 hpi, coincident with resumption of fungal growth in the susceptible host. Davidson et a1. (1987) identified six infection-related cDNAs which increased in abundance in barley (Hordeum vulgare L.) following inoculation with Eiysz'phe grami'm's DC. f. sp. hordei Em. Marchal. The accumulation of the infection- related mRNA was similar in the compatible and incompatible interactions until after the expression of race-specific resistance. Similar results have been obtained in non-host interactions of wheat (Schweizer et al., 1989), turnip (Collinge et al., 1987), and potato (Marineau et al., 1987). These infection-related mRNAs are likely associated with general plant defenses rather than race-specific 13 responses. In most cases, no function has been assigned to the induced mRNAs. It is obvious from this discussion that a great amount of work has been devoted to analyzing race-specific disease resistance as described by the gene-for-gene hypothesis. Nevertheless, much of the process is unexplained and no biochemical molecule has been identified which can be exploited in isolating clones of resistance genes by standard molecular techniques. A well-defined genetic system of resistant and susceptible plant lines with a homogenous background would be of use for defining events and molecules which are correlated with race- specific resistance. Further, the ability to synchronize the infection process of the pathogen, combined with specific knowledge of the cytological events of disease progression in both compatible and incompatible interactions, would be a powerful tool for defining cause- effect relationships between identified molecules and resistance. The barley-powdery mildew interaction provides all these advantages. The interaction of the barley powdery mildew with barley follows the gene-for-gene relationship (Moseman, 1966). Greater than ten loci (designated MI) have been identified as conferring race-specific resistance to powdery mildew (Sogaard and Jorgensen, 1982). The M1- a disease reaction locus is of special interest due to its complex nature. More than 30 alleles, expressing a range of resistant phenotypes, have been identified at this locus (Giese, 1981). Molecular characterization of these alleles would be of interest in determining the functional basis of the different disease reaction phenotypes. Additionally, as a complex locus, examination of cloned alleles may yield insight into the evolution of new resistance alleles. 14 Near-isogenic pairs differing only in the presence or absence of specific M-a alleles for resistance to E. g. bordei' have been developed (Moseman, 1972; Kolster et al., 1986; Kolster and Stolen, 1987). Cytological investigation of the events leading to resistance or susceptibility is relatively easy as the powdery mildew disease is localized to the epidermal surface of aerial portions of the plant. Quantitation of these events is made possible by the development of methods for the synchronous development of E. g. bordei (Masri and Ellingboe, 1966). ' A two step process of general non-host specific responses overlaid by race-specific resistance can be argued in the barley-powdery mildew interaction (Thordal-Christensen et al., 1987). Non-host resistance may be triggered by attachment of the primary germ tube, which is unique to E. g. border, to the host surface (Thordal-Christensen et al., 1987; Carver and Bushnell, 1983). The cessation of primary germ tube growth is correlated with papillae formation at 4»6 hpi. This early papillae response results in the termination of infection by wheat or pea powdery mildew pathogens on barley and, thus, is a non-host defense response. Race-specific resistance can first be observed at 10- 12 hpi when penetration by the appressorial germ tube is preferentially halted in cultivars carrying resistance alleles (Thordal- Christensen et al., 1987; Ellingboe, 1972; Johnson et al., 1979). Resistance at this stage is characterized by the appearance of cytoplasmic aggregates and fluorescent papillae at the site of attempted penetration (Kita et al., 1981; Koga et al., 1980; Johnson et al., 1979). Any fungal propagules that successfully penetrate resistant cells are halted at 22-24 hpi by disruption of normal haustoria 15 formation and the hypersensitive necrosis response (Kita et al., 1981; Johnson et al., 1979; Ellingboe, 1972). The paired near-isogenic lines of barley, combined with a detailed understanding of the sequence of events that occur in the interaction with E. g. bordei provide a unique tool for the isolation of a resistance gene or gene product. Several methods are available that allow us to take advantage of this excellent genetic system for the isolation of a factor associated With MI-a mediated resistance. Transposon tagging has been used to isolate several genes in maize (Federoff et al., 1984; Hake et. a1, 1989). This technique has two advantages for cloning a race-specific resistance gene. The primary advantage is that no knowledge of the biochemical nature of the resistance gene is necessary. Transposon tagging requires only a phenotypic screen. The second useful property is that mobile elements insert into genes at random at a high rate. Genomic libraries generated from mutant plants harboring the element can be screened using the cloned element to isolate the gene of interest. Two groups have attempted to tag the Rp] locus of maize, which encodes race- specific resistance to Puccinia sorghi Schwein. (Pryor, 1987; Bennetzen et al., 1988). These attempts have been unsuccessful due to a high rate of spontaneous mutation at Rp], which makes it extremely difficult to identify mutants caused by insertion of a transposable element. No transposon has been isolated from barley; however, Wise and Ellingboe (1985) have reported a genetic instability in progeny of crosses between lines carrying the MI-a6 and M1-a13 alleles. We have examined progeny of this cross in an effort to isolate a barley transposon for the purpose of molecularly tagging and cloning the 16 MI-a disease reaction locus (see. Chapter I). We were unable to repeat the results of Wise and Ellingboe. In fact, no transposon has been identified in barley. However, the potential exists in other plant species such as maize for isolating resistance genes if the loci are not subject to high mutation rates. Transposon tagging will also be useful in other organisms once an appropriate transposon is cloned from the chosen species or introduced on a plasmid. The Ac element of maize, for instance, has been shown to be active in tomato (Yoder, 1988). Another approach to the problem of isolating a resistance gene is to identify the gene product. As a result of purification and partial sequencing of the gene product, clones of the resistance gene could be isolated by standard cloning techniques. Alternatively, the resistance gene can be identified by "backtracking" through host molecules induced specifically in the early phase of the resistant response. This method involves the use of gel retardation assays to identify polypeptides that bind to cis-acting regulatory sequences of genes encoding induced host molecules. Both methods require identification of either a constitutively produced or induced host polypeptide. Two- dimensional gel electrophoresis has been used to identify polypeptides induced in response to various treatments (Hadwiger and Wagoner, 1983; Mansfield and Key, 1987; Ramagopal, 1987; Sachs et al., 1980). Gabriel and Ellingboe (1982) also used this approach in an unsuccessful attempt to identify the gene product, presumed to be constitutively produced, of the P112 (powdery mildew) resistance gene of wheat. Gels of leaf extracts from near-isogenic Wheat lines were compared following silver staining. No polymorphic polypeptides were identified in lines carrying different alleles of the Pm locus. Their 17 method was deemed to lack sufficient sensitivity to identify such a product. Davidson et a1. (1987) also used leaf material in an examination of barley isolines differing for single resistance alleles. Several cDNA clones were found to be induced following inoculation. These infection-related genes did not differ in expression between resistant and susceptible lines until after the expression of resistance. The use of near-isogenic lines, 35S-methionine for autoradiographic detection, and coleoptile tissue significantly increases the sensitivity of this approach. The synchronized development of the pathogen also allows for examination, by the same method, of induced polypeptides at specific times during the disease progression. We have used these methods in an effort to identify proteins that are related to race- specific resistance or that represent the [WI-a gene product. Only through the molecular characterization of an isolated resistance gene can we gain insight into the cause-effect relationships of race-specific resistance. Presently, no resistance gene has been cloned nor has any resistance gene product been identified. As a result, we know nothing about the physiological mechanism of race- specific resistance in plants. This thesis describes the results of three approaches to isolating a resistance gene or gene product. 18 ' ratu ited Albersheim, P., and Anderson-Prouty AJ. (1975). Carbohydrates, proteins cell-surfaces, and the biochemistry of pathogeneSis. Annu. ev. Plant PhySlOl. 26:31-52. Allen, E.A., Hazen, B.E., Hoch, H.C., Kwon, Y., Leinhos, G.M.E., Staples, R.C., Stumpf, M.A., and Terhune, B.T. (1991).. A pressorium formation in response to topographical Signals by 2 rust species. Phytopathology 81:323-331. Bennetzen, J .L., Qin, M-M., Ingels, S., .and Ellin boe, AH. (1988 . Allele-speCific and mutator-assoc1ated insta 111 at the Rp disease resistance locus of maize. Nature 332:3 9-370. Bohlmann, H. Clausen, S., Behnke, S. Giese, H., Hiller C. Reimann- Phillip, U., Schrader, G., Barkholt V., and A e1, K. (1988). Leaf spec1 1c thionins of barley- a nove class of ce 1 wall roteins toxic to plant-pathogenic fungi and Bossibl involve in the defence mechanism of plants. EMB J. 7: 559-1565. Bolwell, G.P., Robbins, MP, and Dixon RA. (1985). Metabolic changes in eliCitor-treated bean cells. Enzymic responses in relation to ra id changes in cell wall composition. Eur. J. Biochem. 14 :571-578. Callow, J .A. (1984). Cellular and molecular recognition between higher plants and furligal flhogens. : Encyclopedia of Plant Physiolo Vol. 17, .F. skens an J. Heslop-Harrison, ed. Springer- erlag, Berlin. pp. 212-237. Carver, T.L.W., and Bushnell, W.R. (1983). The probable role of . primary germ tubes in water uptake before infection by Ery51phe grammis. PhySiol. Plant Pathol. 23:229-240. Collin e D.B. Milligan, D.E., Dow, J.M., Scofield, G., and Daniels, M. 1987). Gene expressmn in Brassrca cam estris showmg a ggpersensmve response to the tncompati 1e athogen anthomonas campestris pv. Vitians. lant 0]. Biol. 8:405-414. Cramer, C.L., Bell, J.N., Ryder, T.B., Bailey J .A, Schuch, W. Bolwell, G.P., Robbins, MP, Dixon, RA, and Lamb, C.J. (1985). C0- ordinated syntheSis of Rhytoalexm biosynthetic enzymes in oiczlc§%i§azll§I9-stressed ce 3 of bean (Phaseolus vulgaris L.). EMBO Davidson, A.D., Manners J .M., Simpson, RS, and Scott, K.J. (1987). cDNA clonin of in As induced in resistant barle durin igntectiSOp by lysiphe gramim's f. sp. bordei. Plant 01. Bio. 19 Davis, K.R., L on, G.D., Daryill,A.G. and Albersheim, P. (1984).. Host- a 0 en mteractions . , Endo olygalacturomc aeid lyase rom rwrma cartovora elic1ts h oa extn accumulation by releasing plant cell wall fragments. ant PhySIOI. 74:52-60. DeWit, P.J.G.M., van den Ackerveken, G.F,J.M., J oosten, M.H.AJ., and van Kan, J .A.L. (1989). A oplastic proteins involved in communication between toma o and the fungal athogen . Cladosporittm fulvumt In: NATO ASI Ser. 81 a Molecules in Plants and in Plant-Microbe Interactions. B ugtenberg, ed. Springer Verlag, Berhn. pp. 273-281. Dixon, RA, and Harrison, MJ. (1990). Activation structure, and or amzation of enes involved in microbial defence in plants. A v. Genet. 28: 65-234. Ebrahim-Nesbat, F., Behnke, S., Kleinhofs, A., and A e1, K. (1989). Cultivar-related differences in the distribution 0 cell-wall-bound thionins in compatibleand incom atible interactions between barley and powdery mildew. Plan 179:203-210. Ellin boe AH. 1972 . Genetics and h iolo of rima infection gby Erysr'ph(e grai)ninis. Phytopathglgsgy 6%01- 06. ry Ellingboe, AH. 1976 . Genetics of host-parasite interactions. : En clope 13 0 Plant Peysmlo , Vol. 4, R. Heitefuss an P.H. W ams, eds. Springer erlag, erhn. pp. 761-778. Ellingboe A.H.N(1982). Genetical aspects of active defence. In: Active Defence echanisms in Plants. Proc. NATO Conf. Cape Sgpmon, Greece, R.K.S. Wood, ed. Plenum, New York. pp. 179- Federoff, N.V., Furtek,.D.B.,.and Nelson, O.E., Jr. (1984). Cloning of the bronze locus in maize by a Simple and generalizable rocedure using the transposable controllin element Activator Ac). Proc. Nat . Acad. Sc1., USA 81:3825-3 29. Flor, H.H. 1946 . Genetics of atho enici in Melam sora Iini. J. Agr. Res. 93:335-357. p g ty p Flor, H.H. £1947). Inheritance of reaction to rust in flax. J. Agr. Res. 74:24 -262. Flor, H.H. 1955). Host-parasite interactions in flax rust-its genetics and 0 her implications. Phytopathology 45:680-685. Flor, H.H. 1956 . The com lementa enic stems in flax and flax rust. Adv. enet. 8:29954. ry g sy 20 Fritzemeier, K.-H., Cretin, C., Komb ' E. Rohwer, 17., Taylor, J ., Scheel, D.,.and Hahlbtock, K. (19 7). TranSient mduetion of phen lalanme ammonia-lyase and 4;coun1arate:CoA ligase m As in otato leaves infected With Virulent or aVirulent races of Phytopb ora infestans. Plant PhySiol. 85:34-41. Gabriel, D.W., and Ellingboe, AH. (1982).. High resolution two- . dimensional electrophoreSis of protein from con enic wheat lines giéftering by smgle reSistance genes. Physml. Plan Pathol. 20:349- Gabriel, D.W., Bur es, A. and Lazo, GR. (1986). Gene-for- ene recognition 0 five cloned aVirulence ones from Xan omonas cam pestris pv. malvacearum. bbsgmci c resistance genes in cotton. Proc. Natl. Acad. Sc1., A 83:6415-6419. Gabriel, D.W., Loschke, DC, and Rolfe B.GM(1988)1. Gene-for-gene reco tton: the ion channel model’. In: olecu ar Genetics of Plan -Microbe Interactions R. PalaCios, D.P.S. Verma, eds. APS Press, St. Paul, MN. pp. 3-14. Gabriel, D.W., and Rolfe, B:G. 1990). Working models of specific recognition in lant-micro e interactions. Annu. Rev. Phytopathol. :365-391. Gianinazzi, S. £19842. Genetic and molecular aspects of resistance induced. y in ections or chemicals. : Plant-Microbe . Interactions, Vol. 1, T. Kosuge and .W. Nester, eds. Macmillan, New York. pp. 321-342. Giese, H. (1981). Powde mildew resistance enesjn the Ma and regions on bar ey chromosome 5. creditas 95 :51-62. Habereder, H., Schroder, G:, and Ebel J. (1989). Ra id induction of phenylalanine ammonia-l ase and Chalcone syn ase mRNAs dnrtng fungus infection 0 soybean (lGlycme max L.) roots or elicitor treatment of so bean cell cu tures at the onset of phytoalexm syntheSis. lanta 177:58-65. Hadwiger, LA. and .Wagoner, W. (1983). Electrophoretic patterns .of pea and Fusanum solani proteins syntheSized in Vitro or in mm which characterize the com atible and incompatible interactions. PhySiol. Plant Pathol. 23:15 -162. Hake, S., Vollbrecht, E., and Freeling, M. (1989).. I Cloning Knotted, the dominant mOfiIfiOloglcal mutant in maize usmg D52 as a transposon tag. E O . 8:15-22 Halverson, L.J., and Stacey, G. (1986). Signal exchange in plant- microbe interactions. Microbiol. Rev. 50:193-2 . Heath M.C. (1981). Resistance of plants to rust infection. Phytopathology 71:971-974. 21 Heath, M.C. (1991). The role of gene-for- ene interactions in the oetermination of host speCies spec Clty. Phytopathology 81:127- Hoch, H.C. Staples R.C., Whitehead, B., Comeau, J. and Wolf, ED. 1987). Signalling for growth orientation and cell differentiation y surface topography in Uromyces. Sc1ence 235:1659-1662. Johnson, LE.B. Bushnell, W.R.,. and Ze¥en, R-.J. (1979). Binary . pathwags for analySis of pnmary in ection and host res onse in popula ons of powdery mildew fungi. Can. J. Bot. 57: 7-511. Keen, NT. (1982 . Slpecific recognition in gene-for-gene host-parasite ' systems. A v. lant Patho . 1:35-81. Keen, N.T. Tamaki S., Kobayashi, D. Gerhold, D., Stayton, M., Shen, H., Gold S., ran N. Thordal-Christensen, H., Dahlbeck, D. and Staskawicz, B. I 89). Bacteria expressmg avrrulence gene pt'oduce a s ec1fic e 'citor of the so bean hypersensitive reaction. 01. Plant- crob. Interact. 3:112- 21. Keen, NT. (1990). Gene-for-gene com lementarity in plant-pathogen interactions. Annu. Rev. Genet. 4:447-463. Kerby, K., and Somerville S. (1989). Enhancement of specific . intercellular peroxrd’ases followm inoculation of barle With E i he ammr's f. s . border'. siol. Mol. Plant Pa hol. 35?? -3397 p y Kita, N. Toyoda, H., and Shishiyama, J. (1981). Chronological analysis ofloyttolso $161618 géises in powdery-mildewed barley leaves. . . o . : - . Kobayashi, D.Y., Tamaki, S.J., and Keen, NT (1989). Cloned av1rulence genes from the tomato patho en Psuedomonas firmgae pv. tomato confer cultivar spec1 icity on soybean. Proc. atl. Acad. Sci., USA 86:157-161. Koga, H., Mayama, S., and Shishiyama, J. (1980). Correlation between the deposrtion of fluorescent com ounds in pa illae and resistance in barle a ainst E r' e amr'nrs order'. Can. J. Bot. 58:536-541. y g mp gr Kolster, P., Munk, L. Stolen, 0., and Lohde, J. (1986). Near-isogenic Isaatle 619?)?589‘01‘7th genes for reSistance to powdery mildew. rop ci. : - .. Kolster, P., and Stolen, _O. (1987)..B.arley isolines with genes for reSistance to Erysrplre gamrnrs f. sp. border in the recurrent . parent ’Sin’. Plant Breeding 98:79- 2. Lawton M.A., and Lamb, C.J. (1987). Transcriptional activation of lant defence 1genes by3 fungal ehcitor, wounding, and infection. 01. Cell. Bio . 7:335- 41. . 22 Loegeflng, W.Q. (1966 . The relationship between host and athogen in stem rust of w eat. Proc. 2nd Intl. Wheat Genetics ymp. (Lund, 1963). Hereditas, Suppl. 2:167-177. Mansfield, M.A., and Key, IL. (1987). S thesis of the low molecular weight heat shock proteins in plan 5. Plant PhySlOI. 84:1007-1017. Marineau, C., Matton DP, and Brisson N. 1987). Differential . . accumulation ofcpotato tuber mRNAs mm the h. ersens1tive response induce by arachidonic acrd. Plant 01. 101. 9:335-342. Masri, SS, and Ellingboe, AH. (1966). Germination of conidia and formation of appressoria and secondarg haphae in Erysrphe gramrnrs f. sp. trrtrcr. Phytopathology 5 :3 -308. Mazau, D., and Esquerré-Tugayé, M.T. (1986).Hydroxyproline-rich glycoprotein accumulation in the cell walls of plants infected by various pathogens. PhySlOI. Mol. Plant Pathol. 29:147-157. Moerschbacher, B:M-.i Flott, B.E..,. Noll, U., and Reisener, H.-J. (1989). On the s ec1fic1ty of an elic1tor preparation from stem rus which in uces lignification in wheat leaves. Plant Physiol. Biochem. 27:30 -314. Moseman, J .G. (1966 . Genetics of powdery mildews. Annu. Rev. Phytopathol. 4: 69-290. Moseman, J G (1972 . Isogenic barley lines for reaction to Erysr’phe gramrnrs f. sp. order. Crop Sc1. 12:681-682. Pryor, T. (1987). The origin and structure of fungal disease resistance genes in plants. Trends Genet. 3:157-161. Rama opal, S. (1987). Salini stress induced tissue-specific proteins in arley seedlings. Plant hysrol. 84:324-331. Sachs, M.M., Freeling, M. and Okimoto, R. (1980). The anaerobic proteins of maize. Cell 20:761-767. Schwelzer, P., Hunziker, W., and Mosinger, E. (1989). eDNA cloning, rn vrtro. transcription and partial sequence ana ySlS of mRNAs from Winter wheat Trrtrcurn aestrvum L.) With induced resistance to E 51' e aminr's f. s . trr'trcr'. Plant Mol. Biol. 12:643-654. 'y p gr p Shiriashi, T., Yamaoka, N. and Kunoh, H. (1989). Association between increased phenylalanine ammonia-l ase activ1ty and crnnamic acrd synthe51s and the inductiono temporary inaccessibilit caused b Br 51 he ramrrrrs rima erm tube genetration 6f the barlzy le)af.pPhy%iol. MolPPlantryPgthol. 34:75- 23 SlusarenkOo, A.J., and Lon land A. (1986): Changes in gene activity duringOexpreSSion o the hypersensitive response in Phaseolus vu1garrs cv. Red Mexrcan to an avrrulent race 1 isolate of Pseudomonas s in ac v. haseoIicoIa. Ph siol. Mol. Plant Pathol. 29:79-94? g p p y Sogaard, B., and Jorgensen, J .H. (1982). Supplementary list No. 1 (to master list of barley genes): Genes for reaction to Erysr Ire TEE-21126111115 border (powdery mildew). Barley Genet. News . Staskawicz, B.J., Dahlbeck D., and Keen, NT. (1984). Cloned avrrulence gene of Psuedomonas s Orngae pv.1g}{crnea determines race-specific incom ati mg! on G crne max (L.) Merr. Proc. Natl. Acad. Sci., A 81: 024-60 8. Thordal-Christensen, H., Gre ersen P.L., Andersen, J.B., and O OSmede aard-PetersOen O . (1987). Induction of defense reactions in plan 3. J. Ag. Sc1. Finland 59:231-249. van Loolnl,1L1$6(1985). Pathogenesis-related proteins. Plant Mol. Biol. Whalen M.C., SOtall, RE, and Staskawicz, B.J. (1988). O O Characterization of a gene from a tomato pathogen determining h¥petsensttive reslstance in non-host s ec1es an genetic anal SIS o7‘tl7ns reSistance in bean. Proc. Natl. cad. Sc1., SA 85:674 - Wise, RP, and Ellingboe, AH. (1985). Fine structure and instability at the MI-a locus in barley. Genetics 111:113-130. Yoder, J.I., Palys, I, Alpert, K., and Lassner, MM1988 . Ac s. trans osi ion in transgenic tomato plant 01. en. Genet. 213: 1-296. Yoshikawa, M., YOanOiauchi, K., and Masago,OH. (1978 . Glyceollin: its role in restricting fungal growth in re51stant soy ean hypocotyls infected with Phyto thora megasperma var. sojae. PhySiol. Plant Pathol. 12:73- 2. CHAPTER I Investigation of a genetic instability in barley CHAPTERI Investigation of a genetic instability in barley1 Abstract Progeny of crosses between the near-isogenic lines CI-16151 and CI-16155, carrying resistance alleles M—a6 and M—al3 respectively, were examined in order to identify a reported genetic controlling element. Seeds were analyzed for ADH1 activity and starch content and seedlings were analyzed for disease reaction. More than 1800 seeds were analyzed and 16 were tentatively identified as ADH1- deficient mutants. No starchless mutants or mutants in disease phenotype were identified. Putative ADH1-deficient mutants were subsequently analyzed by a number of methods. Roots of F2 plants were analyzed for ADH1 activity by isozyme gel analysis. DNA was extracted from leaves of these plants and analyzed by Southern blotting for insertion events. Pollen from the putative mutants was also stained for ADH1 activity. ADH1 activity was analyzed by activity stain of the aleurone and isozyme gel analysis of the F3 seed from the putative mutants. All the putative ADH1 mutants displayed wild type ADH1 activity by these analyses. Thus the 16 lines were not true 1Simons, SP. and Somerville, SC. (1988). Investigation of a genetic instability in barley. Barley Genet. Newsletter 18:37-45 and 19:86-87. 24 ADH1-deficient mutants. Further, there was no evidence for the presence of a genetic element, inserted in the ADH1 gene. We conclude that factors other than a controlling genetic element may have been responsible for the observed instability in progeny of the cross between CI-16151 and CI-16155. 25 26 Introduction The MI-a disease reaction locus is one of a set of loci which define the response of barley to Erysr'plre gramr'nr's DC. f. sp. border' Em. Marchal, the causal agent of the powdery mildew- disease. In experiments to define the genetic fine structure of this locus, F 3 families with susceptible variants were recovered, from a cross between two resistant lines, at an unusually high frequency (Wise and Ellingboe, 1985). Roughly 0.6% of the F 3 families contained susceptible members; a frequency 10 - 100 times higher than that observed among F3 families of other crosses. Susceptible variants were subsequently identified in an F2 population at a frequency of 1.67%. This apparent high frequency of recombination between alleles at the MI-a locus was only observed in progeny from crosses between barley lines CI-16151 (female) and CI-16155. No susceptible variants were found among progeny derived from the reciprocal cross. Furthermore, susceptible variants often reverted to resistance in subsequent generations. We have repeated the experiments of Wise ' and Ellingboe (1985) in an attempt to confirm their observation and to extend our knowledge of the nature of this genetic instability. A high frequency of apparent recombination and/or mutation is often associated with the action of transp osable elements (N evers et al., 1986), or with paramutation (Brink, 1973). To study the molecular basis of the genetic instability observed in the F2 and F3 progeny, it is convenient to monitor unstable events at a well-characterized gene. Therefore, in addition to the MI-a locus, we screened for variants at the alcohol dehydrogenase isozyme 1 (ADH1) and waxy genes, because rapid phenotypic screens exist and cloned genes are available 27 for use as probes of gene structure. We describe attempts to recover variants at the M—a, waxy or ADH1 genes in F2 (CI-16151 X CI- 16155) progeny. 28 MaterialLQnsLMctthrt Barley lines CI-16151 (M-a6), CI-16155 (MI-313), CI-16138 (ml-a), CI-16137 (MI-a), CI-16141 (M—Ii), and CI-16139 (MI-g) were obtained from R. Wise [see Moseman (1972) for a description of the lines]. The waxy mutant (gamma, field 81) was a gift from Drs. A. Kleinhofs and R. Nilan (Washington State University, Pullman WA), and the ADHI' mutant, CPI-96981-5, was obtained from Dr. Edwards (designated M9 in Harberd and Edwards, 1982). Race CR3 of E. g. border' was obtained from R. Wise. Crosses between CI-16151 and CI- 16155 were made in a growth chamber using standard methods (Starling, 1980). F1 seeds from the various crosses were grown to maturity in a growth chamber, isolated from other genotypes, to produce the F 2 populations utilized for these studies. Seeds from each F1 plant were harvested and analyzed separately. In initial experiments to determine disease reaction phenotypes, seeds were cut in half and the embryo-half was planted directly into jiffypots. However, the number of normal, healthy seedlings recovered was low. Using the method described below roughly 90% of the seeds gave rise to normal seedlings. Seeds were cut in half and the embryo halves were germinated on agar plates containing mineral nutrients (Somerville and Ogren, 1982), in the dark at room temperature for 2 days. The seedlings were transferred to a light rack for one day, and then were transplanted to 3-inch "jiffypots" containing a 1:1:1 mix of vermiculite, perlite and Sphagnum. The seedlings were grown for 1 week in growth chambers with 100-150 uE PAR m'2 s1 (mixed cool white fluorescent and incandescent lights), with a 16 h photoperiod, and day and night temperatures of 22°C and 18°C respectively. 29 Seedlings were then transferred to isolated growth chambers and inoculated with race CR3. The conditions for disease development were 50 uE PAR in'2 5'1 (mixed cool white and incandescent lights), with a 15 h photoperiod, and day and night temperatures of 18°C and 16°C respectively (Masri and Ellingboe, 1966). Disease reaction scores (Moseman, 1972) were determined 2 weeks following inoculation. Lines CI-16138, CI-16137, CI-16141, CI-16151 and CI- 16139, each of which carries a different resistance allele, were tested at the same time to verify the identity of race CR3. Endosp erm quarters from each seed were tested for the waxy phenotype by staining with iodine (Ho et al., 1980). Starch lacking amylose, the waxy' phenotype, stains a red-brown color, while the waxy+ phenotype gives a black color in this assay. Endosp erm quarters were incubated in a 96-well microtiter plate in 200 pl of KI-Iz solution (8.67 mM KI, 0.56 mM 12 in 0.04 N HCl) for 60 min with shaking (100 rpm) at room temperature. The seed pieces were then scored visually for black color. The waxy mutant was included as a negative control and the two parental lines served as positive controls. ADH1 phenotype was determined by incubating endosperm quarters in 24-well plates in 400 pl of freshly prepared ADH activity stain (25 mM Tris-Cl pH 8, 2.5 mM NAD"', 2.5 mM Nitro Blue Tetrazolium, 25% ethanol) with shaking for five minutes. The reaction was stopped by the addition of 1 ml 0.01 N HCl, and the endosperm piece was observed at 25X magnification using a stereomicroscop e. In the aleurone layer, ADH1 is the only ADH isozyme expressed constitutively at significant levels (Hanson et al., 1984). ‘A dark purple ring of cells around the endosperm is indicative of wild-type levels of 30 ADH1 activity (Harberd, 1982). The ADHI' mutant, CPI-96981-5, was included as a negative control and the parental lines were included as positive controls. _ Barley roots were harvested from 3-week-old' Seedlings grown hydrop onically to determine their ADH isozyme profiles. Isozymes of ADH were induced by bubbling N2 through the hydroponic medium for 3 days prior to harvest. Roots were ground in 0.15 M Tris-Cl pH 8, 10 mM dithiothreitol (2 ml/g roots) at 4°C; the extract was clarified by centrifugation; and 45 ul of supernatant was analyzed on native polyacrylamide gels. Extracts from endosp erm quarters from dry seeds were prepared and analyzed as described for root tissue. Electrophoresis and staining for ADH activity was carried out as described by Hanson et a1. (1984). Lines CI-16151 and CI-16155 have distinct herdein banding patterns which are useful for checking the genotypes of these lines (Wise and Ellingboe, 1985). Hordeins were isolated from half a seed and separated by polyacrylamide gel electrop horesis under denaturing conditions according to the protocol of Doll and Andersen (1981). DNA was isolated from 2-4 week old seedlings by a miniprep method (Dellaporta et al., 1983). Southern blots were prepared using standard procedures (Maniatis et al., 1982). In brief, DNA was digested with various restriction endonucleases, separated by electrophoresis in 0.9% agarose gels and transferred to nylon membranes (Zetaprobe, Biorad) by the alkaline transfer method (Reed and Mann, 1985). Filters were probed with the 6-kbp EcoRI insert of pM9-15.1, which contains most of the coding sequence and 31 about 4-kbp of 5 ’ noncoding region of the barley ADH1 gene (Trick et al., 1988). The 6-kbp insert was purified from 1% low melting agarose gels on a NACS column (Prepacs, BRL), and labelled by the random primer method (Feinberg and Vogelstein, 1983) to a specific activity greater than 108 cpm/pg (Oligolabeling kit, Pharmacia). Hybridization was carried out overnight at 42°C in 50% formamide, 5X SSC, 5X Denhart ’5 solution, 1% sodium dodecyl sulfate, 500 u g salmon sperm DNA/ml and 10% Dextran Sulfate. The filters were then washed at increasing stringencies culminating with a wash in 0.1X SSC, 0.1% sodium dodecyl sulfate at 68°C. Filters were dried and exposed for 4-6 days to Kodak XAR-S X-ray film at -80°C using intensifying screens. 32 Results F2 seeds from crosses between CI-16151 and CI-16155 were screened for ADHl' and waxy‘ variants as well as for variants susceptible to E. g. border; race CR3. As shown in Table 2, no waxy or susceptible variants were identified. Two suscep tible variants were recovered from field grown stocks of CI-16151, however these were shown to be contaminants based on their hordein phenotypes. All subsequent tests were done using growth chamber-grown stocks. Twenty ADH1' seeds were identified. The recovery of ADH“ variants was only slightly less frequent than the recovery of susceptible variants, as described in previous work (Wise and Ellingboe, 1985). Eight of the 16 ADH1 variants from the F2(CI-16151 x CI-16155) progeny survived to produce seed. About 16 F3 seeds from each of the 8 putative mutants were tested for ADH1 activity in the aleurone. All progeny exhibited wild-type levels of ADH1 activity. Seeds from the ADHl' control plants, grown under the same conditions, displayed a higher than normal degree of staining. Therefore, we investigated whether ADH2 or ADH3 activity had been induced during development of these seeds, thereby creating the false impression that the variants had ADH1 activity. The ADH isozymes were separated by native polyacrylamide gel electrophoresis and identified using the ADH activity stain. Progeny from the F3 lines displayed significant levels of ADH1 activity in both anaerobically- and aerobically-grown roots, and in dry, aerobic seeds (Figure 2), confirming the results of the aleurone stain. Structural alterations at the ADH1 gene, such as insertions and deletions characteristic of transposon-induced mutations, should be .eoEmomfi $852034 33 .8308 “autonomous v mo Mono Sod 0:028 803 >5on owe Soo< w .mEofiw 5020: a muomEEwEOU on 8 8588qu n d8 9 worse—omen: 23:... see 2e seen? .Eo< $22-5 x £8-65 m use Ease -Eo< 3320 2i e UoEEBoU co: m .m seek AB : eeeweam eee 8; :5: as. N . A: see: e32 N ow 2: a 5 MS as. 982 «.3 05 um emboooao 5 Eats, 80? €23 moms—UEEE mo owfiooeoo be: 8 282 ocosvocm H seed o am e em... 222-6 x 58.6? e some e E e e22 922.5 x 52-6mm .m etc o e e on. 32-6 .4 same a a e we. 53-6 .m see c oz oz e5 NomsEo x 32-6va .N see; NN oz eoz ammo Nfiflezo x $338 No A Hwooosvem 03530me 93% b8? coon—WSW” moo: Eon—m ho Hon—8:2 400— Mi: mugs Hug «Au/fix: 2.5 an UOHO>OOOH muflmmuflxl .N Wing? 34 Figure 2. ADHlO isozymes fromOseeds of putative ADH1 mutants, separated by native polrgacrélamide gel electroohoreSis. Lanes: 1-4, outative mutants from I-16151 x CI-1615 3; 5, CPI-96981-5 negative control); 6, CI-thngS (positive control . 35 observed as changes between the parents and progeny in the banding pattern of genomic DNA following digestion with restriction endonucleases and hybridization with the cloned ADH1 gene. No restriction fragment length polymorphisms were observed among the variants and the parental lines (Figure 3). The blots in Figure 3 were also overexposed, so that minor bands and bands in underloaded lanes could be observed, and no polymorphisms were found. Thus, it appears that notrue mutants were recovered in the original screen, and that the ADH1 screen is less stringent than the waxy or disease reaction screens. 2.3’ 2.0’ Pi re 3. Southern blot of genomic DNA cut with BamHI and robed Wi pM9-15.1 which contains a genomic clone of barle ADH6. Lanes: 1, CI-ll61I_I’511, parental line; 2, CI-161515, parental l3, utative ants fromF (1C1-.-16151 x CI- -;16155) 67, ADH1+ iii ividual from 1312(c1u 16151 x or 1% E';_ U 37 Discussion We report that we were unable to repeat the observation made by Wise and Ellingboe (1985) of a genetic instability in progeny derived from the cross CI-16151 X CI-16155. We tested the stability of three different loci and recovered no true mutants. It is difficult to reconcile these contradictory reports other than to suggest some environmental or genetic factor, absent in our experiment, is required to activate the destabilizing process in these lines. Environmental stresses have been implicated in the activation of quiescent transposable elements (N evers et al., 1986). Certain chemical treatments have been reported to activate excision of P- elements (Osgood and Seward, 1989) and hybrid dysgenesis induced by P-elements is also sensitive to temperature (Engels, 1983). Unlike the materials we used, the barley seeds used by Wise and Ellingboe were harvested prematurely and dried in order to reduce the generation time. It is possible that either the premature harvest or the heat treatment may have activated the genetic instability they observed. . A second possible explanation for the discrepancy between the two results is that a genetic factor required to activate the instability at the M-a locus was missing in the parental lines utilized in the experiment described in this report. Although CI-16151 and CI-16155 were derived from the same seed stocks utilized by Wise and Ellingboe, the lines used in this study were subjected to a generation of single seed descent to ensure their genetic purity. A genetic factor, necessary for the expression of the instability, may have been present 38 in a subset of either the CI-16151 or CI-16155 stocks, and may have been lost in this process. In conclusion, we were unable to find evidence for an active transposable element system in these barley lines. 39 I . C l Brink, RA. (1973). Paramutation. Annu. Rev. Genet. 7:129-152. Dellaporta, S.L., Wood, J., and Hicks, J.B. (1983). A plant DNA minipreparation: versron II. Plant Mol. Biol. Rep. 1:19-21. Doll, H., and Andersen B. (1981). Preparation of barle storage protein, Hordein, for analytical sodium dodOe l su ate- polyacrylamide gel electrophoreSis. Anal. Bioc em. 115:61-66. Engels, W.R. 1983). The P family of trans osable elements in Drosop 3. Annu. Rev. Genet. 17:31 -344. Feinber , A.P.O and Vogelstein, OB. (1983). A technique for O ra iolabefing DNA restriction endonuclease fra ents to Ohigh specific activitg. Anal. Biochem. 132:6-13 [see a dendum in Anal. iochem. 137: 66-267 (1984)]. Harberd, NP, and Edwards, K.J.R. (19O82 . A mutational anal sis of the alcohol dehydrogenase system in arley. Heredity 48: 87-195. Hanson, AD., Jacobson, J .V., and Zwar, J.A. (1984). Re lated expreSSion of three alcohol dehydrogenase genes in arley aleurone layers. Plant Physrol. 5:5 -581. Ho, D.T.H., ShOih, SC, and Kleinhofs, A.O(O198O0). Scteening for barley mutants With altered hormone sensrtiv1ty in their aleurone layers. Plant Physrol. 66:153-157. Maniatis T. Fritsch E.F., and Sambrook, J. (1982). Southern transfer. in: Molecular Cloning: A Laborato Manual. Cold Spring arbor Press, Cold pring Harbor, .Y. pp. 382-390. Masri, SH, and Ellingboe, AH. (1966 . Primary infection of Wheat and barley by Erysrphe yamrnrs. hytopathology 56:389-395. Moseman, LG. (1972). Isogenic barle lines for reaction to Erysr'pbe gammrs f.sp. border. rop Sc1. 1 :681-682. Nevers, P., She herd NS. and Saedler H. (1986). Plant transposable elements. dv. Bot. Res. 12:104-203. Osgood, C., and Seward, S. (1989). The use of molecularly tagged P elements to momtor spontaneous and induced frequenCies of trans oson excision and transposition. Prog. Nucl. Acid. Res. Mol. Biol. 6:59-67. Reed, KC, and Mann DA. (1985). Ra id transfer of DNA from agarose gels to nylon membranes. ucl. Acrd Res. 13:7207-7221. 40 Somerville, CR, and Ogren, W.L. (1982). Isolation of O hotorespiration mutants in Arabrdo srs tbalrana. : Methods in oroplast Biologty. M. Edelman R. . Hallick an .H. Chua, eds. ElseVier, Ams erdam. pp. 129-138. Starlin T.M. fi980)HBarley. an: Hybridization of Crop Plants. W.R. Fe and .H. adle e . American Socrety of Agronomy Publishers, Madison, . pp. 189-202. Tric M., Dennis, E.SO., Edwards, K.J.R., and Peacock, W.J. (1988). olOecular analysrs of the alcohol dehydrogenase (ADH) gene family of barley. Plant Mol. Biol. 11:147-1 . Wise, RP, and Ellinghoe, A.H. $985), Fine structure and instability at the M-a locus in barley. enetics 111:113-130. CHAPTERII Identifying polypeptides associated with disease resrstance usmg near-isogemc lines CHAPTERII Identifying olypeptides associated with disease resrs ance usmg near-isogenic lines Abstract Near-isogenic lines of barley were examined by two-dimensional gel electrophoresis for polypeptide differences associated with race- specific resistance mediated by the MI-a disease reaction locus. Two novel sets of polymorphic polypeptides were observed in coleoptiles of the isolines AlgR and AlgS carrying alleles M-aI and mI-a respectively. One set of three low molecular weight polypeptides of pI 6.2 was found in AlgS but not in AlgR and, thus, is unlikely to be involved in the disease response. A second polymorphism of pI 6.7 and high molecular weight was also detected. Two additional isolines carrying the MI-aI allele were used to determine whether the pI 6.7 polymorphism was associated with the disease reaction locus. We demonstrated that this polymorphism was not associated with the MI-a locus. The use of different sets of near-isogenic lines carrying the identical resistance allele provided an easy method for assessing the association of a polymorphism with the MI-a disease reaction locus. 41 42 Introduction One working model of the gene-for-gene hypothesis is that resistance results from the interaction between a receptor protein encoded by a host resistance allele and a complementary ligand specified by a pathogen avirulence allele (Albersheim and Anderson- Prouty, 1975). This recognition event triggers a cascade of host defensive responses, which may include the deposition of papillae and hypersensitive necrosis at the site of attempted penetration (Ellingboe, 1972, Johnson et al., 1979). The putative receptor encoded by the resistance allele is often presented as being constitutively expressed. The identification and isolation of the protein product of a resistance allele would permit a critical evaluation of this model. The M—a locus of barley (Hordeum vulgare 1...), which conditions resistance to the powdery mildew pathogen Erysr'phe gramr'nr's DC. f. sp. border’ Em. Marchal, is highly polymorphic, with more than 30 resistance alleles identified (Giese, 1981). The various alleles at MI-a have been incorporated into a series of paired near-isogenic lines (NILs) in which the resistant and susceptible members of each pair share a homogenous genetic background (Moseman, 1972). Comparison of NILs is advantageous for identifying differences associated with a specific resistance allele (Young et al., 1988; Gabriel and Ellingboe, 1982). Since the polypeptide gene product of the M—a locus is expected to be polymorphic among the various genotypes, a high degree of polymorphism can also be used to identify polypeptides that may be gene products of M-a. Two additional sets of NILs have been established in barley in which various powdery mildew resistance alleles have been incorporated into either Siri or Pallas (Kolster et al., 43 1986; Kolster and Stolen, 1987). This base of genetic materials provides a unique opportunity to screen for biochemical or molecular markers associated with specific powdery mildew resistance genes. With three sets of NILs in which the same resistance allele has been incorporated into different genetic backgrounds, it is possible to quickly focus on a limited number of polymorphic polypeptides that are associated with MI-a-mediated resistance to E. g. horder'. Enrichment for polypeptides related to the disease response can be partially attained by examining epidermal cell extracts. Powdery mildew infection is confined to the epidermis of aerial parts of the plant, yet epidermal protein accounts for only 6% of total leaf protein (Gershenzon et al., 1987). Coleoptiles are composed primarily of epidermal tissue and respond to infection by E. g. horder' in a manner similar to leaves (Bushnell et al., 1967). By enriching for epidermal tissue, we reasoned that we would enrich for the MI-a gene product. The objective of this study was to identify a polypeptide polymorphism that was associated with the MI-aI‘resistance allele by comparing the coleoptile profiles of the three sets of NILs with the M1-a1 allele. Our strategy was to first compare the polypeptide profiles of AlgR (CI-16137) and AlgS (CI-16138), two isolines from the Moseman collection, because the paired lines of this set are expected to be more homologous than NILs of the Siri or Pallas collections. AlgR, with the MI-al resistance allele, and AlgS, with the mI-a allele, are related by four generations of backcrossing to Manchuria followed by 14 generations of selfing, selecting the heterozygote at each selfing generation. The isolines are derived from the resistant and susceptible homozygous progeny from a single 44 heterozygous individual following the final selfing generation and are estimated to be >99% homologous. Any polypeptides that are polymorphic between AlgR and AlgS were then evaluated in the P- 01/Pallas and S-01/Siri NILs. P-01 contains the M-a] allele in the Pallas background and S-01 carries the MI-a1 allele in the Siri background. If a given polymorphic polypeptide is an MI-a1 gene product, then the same isoform of the polypeptide will occur in the three genotypes, AlgR, P-01 and S-01. Using these methods, we identified two sets of polymorphic polypeptides, which differ constitutively between AlgR and AlgS, and assessed their relationship to M—a. 45 MW AlgR (CI-16137) and AlgS (CI-16138) are a congenic pair (Table 3), derived by backcrossing the MI-aI resistance allele from Algerian (CI-1179) into the recurrent susceptible parent Manchuria (CI-2330) (Moseman, 1972). The two lines are estimated to be greater than 99% homologous. P-01 and 801 are derived by backcrossing the Ma] resistance allele into the recurrent parents Pallas and Siri, respectively (Kolster et al., 1986; Kolster and Stolen, 1987). P-01 is estimated to be 99% homologous with Pallas and S01 is estimated to be 96% homologous with Siri. The barley lines used in this study were kindly provided by Drs. Moseman and Kolster. Surface disinfested barley seeds (Klecan et al., 1990) were imbibed under axenic conditions for 24 h on 50 ul water containing a mixture of 35S-labelled methionine and cysteine (50 uCi/seed, Tran3sS label, >1000 Ci mmol'1 from ICN), and then transferred to moistened filter paper on the inner surface of the lid of a sterile GA7 box (Magenta Corp., Chicago, IL). The boxes were used to cover the seedlings, which were then placed in a dark cabinet at room temperature for 3 days. Two coleoptiles were harvested for each sample. The primary leaf was discarded and the coleoptile was ground in 300 [.11 of cold 10% Trichloroacetic acid, 0.07% 2-mercaptoethanol (2MB) in acetone in a microcentrifuge tube (Damerval et al., 1986). Following incubation for 45 min at -20°C, the sample was pelleted at 13,000 x g for 10 min. The pellet was gently washed in 0.07% 2MB in acetone on a rocker for 1 h at 4°C and, then, resuspended in solubilization buffer (9.5M Urea, 1.25% (w/v) SDS, 2% (w/v) 2MB, 2% (w/v) Bio-Lyte 3/10 [Biorad, 46 Richmond, CA], 6% (w/v) Triton X-100). Solubilized protein samples were stored at -80°C. Prefocussed tube gels (4% T, 0.5% C acrylamide-bisacrylamide, 4% (WV) Bio-Lyte 5/7, 1% (w/v) Bio-Lyte 3/10, 9.5M urea, 2% (w/v) Triton X-100) were loaded with 4.5X105 dpm of sample and run for 10,800 V-hr (O’Farrell, 1975). Alternatively, samples were run by non- equilibrium pH gel electrophoresis (NEPHGE), which allows observation of more basic proteins (O’Farrell et al., 1977). Briefly, 5% (w/v) Bio-Lyte 3/ 10 is used in place of the Bio-Lyte 5/7 + Bio-Lyte 3/ 10 combination. The NEPHGE gels are run for 2500 V-hr towards the basic end on non-prefocussed gels. Tube gels were equilibrated in 125mM Tris-HCl pH6.8, 0.1% (W/v) SDS, 2% (w/v) 2MB and run in the second dimension on 10% SDS- PAGE slab gels or 10-20% linear gradient slab gels. Proteins were transferred electrophoretically from the gels to nitrocellulose (T owbin et al., 1979) and visualized by a three day exposure on X-ray film. Autoradiograms of polypeptides transferred to nitrocellulose are of higher quality than exposures of polypeptides in fixed gels. The autoradiograms were analyzed with a Visage 110 image analyzer (Bioimage, Ann Arbor, MI). At least three gels of each type were analyzed. Hordein seed storage proteins were isolated from endosperm seed halves by the method of Doll and Anderson (1981). Endosperms were crushed in extraction buffer containing 41 mM Tris pH 8.6, 40 mM borate, 5 mM dithiothreitol, and 50% (v/v) isopropanol. After a one hour incubation at room temperature, the debris was pelleted for 5 minutes at 12,000 X g in a minifuge and the supernatant taken. 47 Iodoacetamide was added to a final concentration of 10 mM, the solution was heated at 50°C for 30 minutes and the protein was precipitated by the addition of one ml H20 followed by incubation at 4°C overnight. The precipitate was resuspended in sample buffer (41 mM Tris pH 8.6, 41 mM borate, 10% sucrose, 1% SDS, 50 mM dithiothreitol, bromophenol blue) and boiled. Proteins were separated on a 12% acrylamide, 0.1% SDS slab gel (Doll and Anderson, 1981). 48 Resulfi Coleoptile polypeptide patterns were analyzed by two dimensional gel electrophoresis in an effort to identify polypeptides which differed between AlgR and AlgS. Different gel systems were used such as NEPHGE in the first dimension and 10-20% gradient gels in the second dimension. These gel systems allow observation of basic polypeptides and polypeptides of low molecular weight. Apprordmately 300 polypeptides were observed per gel ranging in intensity from more than 3 OD. units to 0.004 O.D.' Two previously undescribed sets of differences between the isolines were noticed. A set of three polypeptides of 37, 38, and 40 kD and pI 6.2, designated p6.2, were present in coleoptiles of the susceptible AlgS line but not the resistant AlgR line (Figures 4 and 5, box). A polymorphic, high molecular weight, polypeptide at pI 6.7, designated p6.7, also differed constitutively between the two lines (Figures 4 and 5, arrow). In the susceptible line, a polypeptide of 103 kD was expressed while the resistant line had a polypeptide of 101 kD. A doublet was identified in the expected position on gels of F1 (AlgR X AlgS) progeny. The p6.7 polymorphic polypeptide was observed in five other paired NILs possessing different alleles of the MI-a locus (Table 3). One additional isoform was identified in the Franger pair. This polypeptide was 101 kD with an altered pI of 6.8 (Table 3). Both members of the Durani and Long Glumes pairs exhibited the AlgR form of p6.7, while the AlgS form was expressed in both members of the Multan and Rupee pairs. The triplet of polypeptides with an apparent pl of 6.2 followed. the same pattern (Table 3). NILs showing the AlgS form of p6.7 also expressed p6.2. pH—> 49 7 6 5 V V V Figure 4. Autoradiogra: protein profile from co separated in the first d] n demonstrating constitutive differences in leoptiles of AlgI 5(CI-16138). Pol eptides were Omensron on p -7 tube gels an in the second dimension on 1 % D. (arrow) and p6.2 (box) i-PAGEO. The polymorphic polypeptides p6.7 are indicated. 50 ’3'... -. . . Figure 5. Autoradiogram demonstrating constitutive differences in protein profile from coleootiles of Alg (CI-16137). Polypeptides were so aratted aszdescribed in igure 4. 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V) ‘O h + + * - v “082:: Em + - + - o :«_:0w:< ~23: Si 380 m 038. 53 The p6.2 triplet was absent in the NILs with the AlgR form of p6.7 as well as the Franger pair. Two cultivars carrying MI-aI in different genetic backgrounds were examined. The susceptible parent Siri possesses an isoform of p6.7, similar to that of Franger (Figure 6a). In line S-Ol, containing the MI- a1 allele, the AlgR isoform of p6.7 replaced the Siri form (Figure 6b). In the susceptible line Pallas, the same isoform is found as in Franger and Siri (Figure 6c). However, P-Ol did not contain the. AlgR form of p6.7, but rather, retained the form of the parent Pallas (Figure 6d). Progeny of the F1 (AlgR X P-Ol) exhibited both the AlgR and the Pallas forms of p6.7, identified as a doublet with the Pallas form to the left of the AlgR polypeptide (Figure 6e). Hordein patterns of each of the NILs were also examined (Table 3) to help identify the introgressed region containing the genes for p6.2 and p6.7 (Appendix A). The resistant lines of the Rupee and Multan pairs possess the AlgS forms of the two sets of polymorphic polypeptides and the Manchuria form of Had. The susceptible members of the Long Glumes and Durani pairs are also informative. These lines express the AlgR forms of p6.2 and p6.7 along with HorZ patterns that differ from Manchuria and presumably represent the HorZ of the resistant parental lines. These data are in agreement with a placement of the proximal end of the segment of chromosome 5 containing the genes encoding the polymorphic polypeptides between M-a and HorZ (Figure 7 and Appendix A). The distal end of the chromosomal segment carrying the p6.2 gene cannot be determined from the data in Table 3. However, the S-Ol line is informative. The AlgR form of p6.7 is expressed in this line which also carries MI-a] 54 figure 6. Autoradio ams of l—labeggd coletolpte f 0 es in e re on o p héjpfie arrow identle hepolgmorphisc polypgtefitlide EtBaltlSasfn d)XaI;-(S)i1n’atr)1 F‘gR1(Al XP d1):l See re 4 gfor a description of ge methods. 55 How 7.9.5 (3.6) H07]? 7.9.4 (3.6) HOTC' 7.9.2 (3.6) HOT-5 77.7 (3.5) H072 76.8 (3.5) Ml—CL 69.5 (3.5) H077 64.2 (3.5) Y74 63.4 (3.7) H074 63.1 (3.5) Ml—k 67.7 (3.5) Figure 7. Linka e map of the short arm of barley chromosome 5 in the region of 73. Ada ted from Jensen, 1989. Genetic distances are given in ma umts dista to the centromere shown as a circle at the ottom of e map. Standard errors are given in parentheses. 56 and the Algerian form of H011. The Hor2 pattern is that of Siri, indicating that the distal limit of the chromosomal segment containing the p6.7 gene is also localized proximal to H012. Therefore, the gene encoding the polymorphic polypeptides p6.7 is located between MI-a and H012, and provides an additional marker on barley chromosome 5. 57 Disc—ussim We identified two sets of previously undescribed, constitutive, polymorphic polypeptides in the AlgR/AlgS pair. Previous efforts to identify polypeptide differences between barley NILs (Manners et al., 1985) and between wheat NILs (Gabriel and Ellingboe, 1982) using leaf tissue were unsuccessful. The analysis of coleoptile polypeptides rather than total leaf proteins is the likely explanation for our ability to observe differences in the polypeptide profile of these barley lines that had not been previously described. The set of differences at pH 6.2 are probably unrelated to disease resistance for the following reason. The p6.2 polypeptides were absent in the resistant line, AlgR and present in the susceptible isoline, AlgS. The F1 progeny (AlgR X AlgS) are resistant, however, p6.2 is present in these individuals. Thus, the p6.2 polypeptides do not correlate with susceptibility or resistance. The original identification of the polymorphic polypeptide of pI 6.7 encouraged us to believe that it could represent the gene product of the Ml-a locus. The polymorphic nature of the polypeptide is similar to what would be expected from the gene product of this highly polymorphic locus. Replacement of the Siri isoform of this polypeptide with the AlgR form in S-01 also supported this supposition. However, our analysis of the third set of NILs, P-Ol/Pallas, demonstrated that p6.7 is not the MI-a] gene product. It is significant that we were able to arrive at this conclusion without having to resort to detailed mapping experiments. Although p6.2 and p6.7 are not gene products of the Ml-a locus, they must be linked to this locus, and thus provide additional genetic 58 markers for this region of the barley genome. An examination of the Hor1, Hor2, and M—a loci from the Moseman series of NILs reveals that p6.2 and p6.7 must lie distal to MI-a on chromosome 5 (Table 3). Moseman has estimated that the introgressed region of AlgS and AlgR is about 7 cM (Moseman, 1972). Since recent mapping has placed Hor2 about 7.2 i 3.5 cM away from M—a (Doll and Jensen, 1986), the p6.2 and p6.7 genes likely lie within the MI-a to H012 interval or a short distance distal to H012. There is no good estimate of the number of polypeptides expressed in epidermal tissue of barley leaves or in coleoptiles. Shoot tissues of Tradescantia has been estimated to contain 30,000 different transcripts (Willing and Mascarcnhas, 1984). Potentially then, there are as many as 300 polypeptide polymorphisms between two NILs differing by 1%. We observed four polypeptide differences (p6.7 and three at p6.2) out of 300-400 polypeptides. This is in accord with the expected 1% difference. Recent reports suggest that the number of polypeptides resolved in this experiment is within the expected values for plants. Szerszen and Pettit (1990) resolved 257 polypeptides from infected peanut cotyledons and Ramagopal (1988) was able to resolve approximately 400 polypeptides from salt-stressed barley seedlings. Our results concur with a report of Meyer et al. (1988) that resolution of plant proteins by the O’Farrell method is less than proteins from other organisms due to low proteinzpolysaccharide ratios and due to the fact that plant proteases are active in the NP40-urea solubilization buffer. Therefore, it is likely that the inability to identify a polypeptide product of the MI-a locus is due to a lack of sensitivity with this method. Although other explanations may apply, such as the 59 polypeptide product may not be labelled by 35S-methionine/cysteine or the allelic variation between MI-a alleles cannot be detected by these methods, it is most likely that the M—a polypeptide is not abundant enough to be observed by two dimensional electrophoresis. 60 I’t C ! Albersheim, P., and Anderson-Prouty, AJ. (1975). Carbohydrates, proteins cell-surfaces, and the biochemistry of pathogenesrs. Annu. ev. Plant Phy51ol. 26:31—52. ' Bushnell, W.R., Dueck, 1,, and Rowell J.B.(1967).1.ivin haustoria and hi hae of E ipbe graminis f.s(p. border With in ct and cum vulgare. Can. J. Bot. artial dissecte host cells of Hor 5:171 -1732. Damerval, .C., deVienne, D., Zi , M. . and Thiellement, H. (1986). Technical 1m rovements in o-dimenSional electro horesm . increase the evel of genetic variation detected in w eat seedling proteins. Electrophoresrs 7:52-54. Doll, H., and Andersen, B. (1981). Pre aration of barle storage protein, hordein, for analytical s . rum dodecyl sul ate- polyacrylamide gel electrophoresrs. Anal. Biochem. 115:61-66. Doll, H,, and Jensen HP. (1986). Localization of owdery .mildew ggsrstance gene M-ra on barley chromosome . Hereditas 105:61- Ellin boe, AH. 1972 . Genetics and h iolo of rim infection gy Emiphe(gram)ihils. Phytopatho ogyS 62:501-406. ary Gabriel, D.W., and Ellingboe, AH. (1982). High resolution two- . dimensional electrop .Ol‘eSlS of protem from con enic wheat lines ggfgenng by Single reSistance genes. PhySiol. Plan Pathol. 20:349- Mechanical techniques for the selective extraction 0 e from plant epidermal glands. Anal. Biochem. 163:159-1 Giese, H. (1981). Powdery mildew resistance genes in the Ma and regions on barley chromosome 5. Hereditas 95:51-62. Gershenzon,.J., Duffy, M.A. Karp, F. and Croteau, R. 1987). es Jensen, J. (1989). Coordinator’s report: chromosome 5. Barley Genet. Newsl. 19:74-77. Johnson, LE.B., Bushnell, W.R., and .Zeyen, RJ. 1979). Binary . pathwa. for analysrs of primary infection an host res onse in popula ons of powdery mildew fungi. Can. J. Bot. 57: 97-511. Kolster, P.L., Munk, .L., Stolen, 0., and Lohde, J. 1986). Near- . isogemc barle?! hnes With genes for reSistance o powdery mildew. Crop Sci. 26: 03-907. Kolster, P.L., and Stolen O. (1987). Barle isolines with genes for reSistance to E1361 e grammis f.s . order in the recurrent . parent "Sin". Plan Breeding 98:7 -82. 61 Klecan, AL, Hippe, S.,.and Somerville, SC. 1990 .. Reduced growth of Elysrphe £3111” f. sp. border induce by ilIetiopsw pallescens. ytopathology 80:325-331. Manners, J .M., Davidson, AD, and. Scott, K.J. (1985). Patterns of ost-infectional protein syntheSis in barley cat-filing different lgenes an . or resistance to the owde mildew fun . Mol. Bio 4:275-283. p ry gus Meyer, Y., Grosset, J ., Chartier, Y, and Cleyet-Marel, J -C 1988). Pregaration b two-dimenSionoal electro horesis of ro ems for . anti ody (pig) uction: Antibodies agains rotems w ose syntheSis Y is reduce auxm in tobacco mesophy protoplasts. ElectrophoreSis 9:704-712. Moseman, J. G. (1972). Isogcenic barle lines for reaction to Emipbe grammis f. sp. border. rop Sci. 2:681—682. O’Farrell, PH. (1973). “(5h resolution 2D electrophoresis of proteins. J. Biol. Chem. 0:4 7-4021. O’Farrell P.Z., Goodman, _H.M., and O’Farrell, PH. (1977). High resolution two dimenswnal electrophoreSis of [33810 as well as acidic proteins. Cell 12:1133-1142. Ramagopal, S. (11987). Salini stress induced tissue-specific proteins in barley seed gs. Plant P ys1ol. 84:324-331. Szerszen, J .B., and Pettit, RE. 1990).. Detection and partial characterization of new po ypep ides in Peanut co ledons associated with earl stages of infection by Asper us spp. Phytopathology 80: 432- 438. Towbin, H., Stachelin, J. and Gordon J. (1979). Electrophoretic transfer of proteins from polyaciE/lamide els to mtrocellulose sheets: rocedure and some app cations. roc. Natl. Acad. Sci., USA. 6:4350-4354. Willing, RP, and Mascarcnhas, J .P. (1}984). Analysis of the complexity and diversity of mRNAs from po en and shoots of Tradescantia. Plant PhySiol. 75:865-868. Young,.N.D., .Zamir, D., Ganal, M.W., and Tanksleg, sp. 8988). Use 0 isogenic lines .and snnultaneous probing to l entify NA rlnzaglggiés 5tghtly linked to the Tin-23 gene in tomato. Genetics CHAPTERIII Examination of polypeptide patterns from coleoptiles of resistant and susceptible barley lines following inoculation with Elysipbe gaminis f. sp.borde1’ CHAPTERIII Examination of pol eptide patterns from coleoptiles. of reSistant and suscep 1e barley lines followmg inoculation With ErySiphe grammis f.sp. border Abflmfl The 35S-labelled polypeptide profile from coleoptiles of near- isogenic lines of barley differing in the MI-a disease reaction locus were examined by two-dimensional gel electrophoresis. Gels of polypeptides from resistant and susceptible plants were examined at two hour intervals over a 12 hour time course in an effort to identify changes in protein expression during the early stages of the interaction with the powdery mildew pathogen, Erysipbe gramim's f. sp. hordei. Technical difficulties were encountered during these experiments that prevent a definitive statement as to the utility of analyzing two- dimensional gels to identify polypeptides involved in the early interaction of barley and E. g. bordei. The results of these experiments are discussed in relation to the problems encountered. 62 63 Man The development of a series of paired near-isogenic lines of barley differing in their dominant powdery mildew resistance genes (Moseman, 1972) has greatly facilitated study of the disease reaction in barley. Consequently, the cytological aspects of this system have been studied extensively (Ellingboe, 1972; Johnson et al., 1979; Kita et al., 1981; Koga et al., 1980) and, recently the system has been investigated at the molecular level (Davidson et al., 1987; Davidson et al., 1988; Manners et al., 1985). Nevertheless, the early signalling events involved in recognition and race-specific resistance remain unknown. The paired near-isogenic lines carrying alleles of the M-3 resistance locus are useful for examining the temporal changes that occur following inoculation with the barley powdery mildew organism. This locus conditions one of the earliest and most effective resistance responses by barley to this pathogen. Extensive cytological evidence has been accumulated concerning the sequence of events leading to resistance (Ellingboe, 1972; Johnson et al., 1979; Kita et. a1, 1981; Masri and Ellingboe, 1966). At 10-12 hours post inoculation (hpi), approximately 95% of the conidia are excluded at penetration of the epidermis in the resistant line. Termination of penetration pegs occurs in cell wall appositions termed papillae, which occur in both resistant and susceptible lines yet are more abundant and halt penetration more completely in the resistant line (Johnson, et al., 1979). However, other research disputes the involvement of this response in MI-a- mediated resistance (Koga et. a1, 1990). Incompatibility is further manifested in the resistant line by formation of small and distorted 64 haustoria in the fungus, accompanied by hypersensitive cell death in the host at 21124 hpi. ‘ ' Two-dimensional gel electrophoresis has been used to examine many physiological responses to stress, including heat shock, salinity, anaerobiosis, and fungal infection (Hadwiger and Wagoner, 1983; Mansfield and Key, 1987; Ramagopal, 1987; Sachs et al., 1980). This technique was used by Scott and coworkers (Davidson et al., 1988; Manners et al., 1985) to study changes in protein and mRNA expression that occur during infection by E. g. hordei' of resistant and susceptible lines of barley differing at the MI-a locus. Infection-related protein differences occurred in both the resistant and susceptible lines at 24 hpi. Infection-related mRNA differences were identified at 12 hpi. Resistance-related changes in mRNA, those which differ between the resistant and susceptible lines, are not observed until 24 hpi. Resistance-related polypeptide differences between the two lines are observed even later, at 30 hpi. While differences that appear after the hypersensitive response has occurred are potentially interesting, resistance-related differences that occur prior to this cytological response are more likely to be components of the series of biochemical responses leading to the expression of resistance. Several induced changes have been reported at early times following inoculation in other host-pathogen ' interactions. Hadwiger and coworkers (Riggleman et al., 1985) analyzed seven cDNA clones which were induced in pea 8 hours after inoculation with the bean pathogen Fusarium (Fusarium soIani (Mart.) Sacc. f. sp. phaseoli (Burkholder) W.C. Snyder & H.N. Hans). In another non-host situation, nine infection-related changes in products 65 of in vitro translated mRNA were observed by two-dimensional electrophoresis (Gregersen et al., 1990). These mRNA products increased as early as three hours following inoculation of barley with the wheat powdery mildew, E. g. tn'tici. The authors contend that some of these mRNAs also increase in the race-specific interaction between wheat and E. g. tn'lici. Two in vitro translation products were observed to increase transiently in the race-specific response of Phaseon vulgan's cv. Red Mexican to race 1 of Pseudomonas syringae pv. phaseolicola (Slusarenko and Longland, 1986). The products are visible on SDS-PAGE at 6 hpi but not at 4 or 8 hpi with the avirulent race 1. Races 2 and 3 of the pathogen do not induce the two changes. These results suggest that the identification of polypeptides involved in the race-specific response of barley to E. g. hordei can be reasonably expected by examining the polypeptide profiles of inoculated plants. The powdery mildew disease is confined to the epidermis, presenting the possibility that the inability of Scott’s group to observe protein differences at time points preceding hypersensitive cell death may be due to the use of whole leaf tissue. Coleoptiles are likely to be a more useful organ for the observation of protein changes occurring at early time points as they are composed almost entirely of epidermal tissue (Bushnell et al., 1967). . We have used coleoptiles of near-isogenic lines of barley, differing for single alleles of the MI-a locus, to study changes occurring at the protein level during the response of barley to infection by the barley powdery mildew organism in an effort to identify resistance associated factors and to characterize the molecular events of infection in a temporal fashion. 66 MatenalsmMethgds The resistant CI-16137 (AlgR) and susceptible CI-16138 (AlgS), barley lines used in this study are a congenic pair, estimated to be 99% homologous (Moseman, 1972). They were. derived by crossing Algerian type resistance (M—aI) into the recurrent susceptible parent Manchuria. . Erysipbe gmminis f. sp. bordei, race CR3 (R. Wise, Michigan State University) was maintained as a gnotobiotic culture on leaf segments in Petri dishes containing 1.2% agar, 1mM Ca(NO3)2, and 1mM benzimidazole (Carver and Phillips, 1982). Cultures were transferred weekly. Surface disinfested barley seeds (Klecan et al., 1990) were imbibed under axenic conditions for 24 h on 50 pl water containing a mixture of 35S-labelled amino acids (5 uCi/secd, Tran35S label, >1000 Ci mmol'1 from ICN), and then transferred to moistened filter paper on the inner surface of the lid of a sterile GA7 box (Magenta corp., Chicago, IL). The genotypes were arranged in a random block experimental design with at least 2 replicates. The boxes were used to cover the seedlings and were then placed in a dark cabinet at room temperature for 2 days. The coleoptiles were inoculated with conidia of race CR3, using a settling tower, to a density of 10-20 conidia mm'z. Conditions for germinating conidia were modified from the procedure developed by Masri & Ellingboe (1966). Inoculated coleoptiles were placed in a dark cabinet for 1 h, then placed in an incubator set at 19°C, 60 1113 m'2 s'1 for 5 h, followed by the standard dark cycle of 14 h. Uninoculated controls were treated identically, including a mock 67 inoculation without conidia. All manipulations were performed under axenic conditions. Results from experiments utilizing 50 uCi/seed and/or 3-day old coleoptiles were also analyzed. Samples were prepared as described in chapter II except that the coleoptiles were wiped with a Kimwipe to remove the fungus prior to extraction. Gels were run as per chapter 11 and the autoradiograms were analyzed with a Visage 110 image analyzer (Bioimage, Ann Arbor, MI). At least three gels were analyzed at each time point. Development of conidia was followed on labelled coleoptiles, inoculated as described above. Samples were taken at 4, 8, 12, and 24 hpi. Three replicates were analyzed at each time point, except at 24 hpi. The coleoptiles were dipped in 5% (w/v) collodion in 75% ether, 25% alcohol and allowed to dry for 5 minutes. The collodion was then peeled off and stained on a microscope slide with Aniline Blue- Lactophenol (0.33g phenol ml‘l, 33% (v/v) glycerine, 28% (w/v) lactic acid, 0.7mg aniline blue ml‘l) (Shipton and Brown, 1962). Slides were examined by DICI‘ optics at a final magnification of 400X. Conidia were evaluated for the following stages: 1° germ tube: single short germ tube; appressorial germ tube: 1° germ tube and second, long (greater than length of conidia) germ tube; appressoria: appressorial germ tube possesses a distinct swollen, hooked structure at distal end of tube. 68 Results. Initial experiments were performed on coleoptiles that had been imbibed on 50 iiCi of an 35S-methionine and cysteine mixture and grown for 3 days prior to inoculation. A number of changes were observed in basic proteins observed by NEPHGE as early as 4 hpi. One protein of pI 7.3 decreased over the first 12 hpi, while two others at pI 7.3 and pI 8.2 increased by 12 hpi. These changes were not different from the mock inoculated control plants exposed to the same night/day regimen and, therefore, are not infection-related changes. Coleoptiles treated with 50 pCi of radioisotope were stunted, the primary leaf often emerged by the second day, and the plants did not grow beyond 5 days. Primary leaves of non-labelled seedlings, by comparison, emerge at 5-6 days in the dark, and few seedlings are stunted. Consequently, the experiments were repeated with 5 uCi of 35S-methionine per seed. The coleoptiles grown under these conditions were nearly normal in growth, although the primary leaf emerged from the coleoptile by the third day in many cases. To avoid potential developmental changes related to primary leaf emergence, 2-day-old coleoptiles were used in all subsequent experiments. Polypeptide changes observed in experiments based on 50 pCi of radiolabel were not found in experiments using the modified conditions. Thus, changes in the polypeptides of pI 7.3 and 8.2, described above, were most likely due to radiation stress. The development of E. g. hordei' on labelled coleoptiles was also analyzed. The rates of appressorial germ tube appearance and appressoria formation (T able 4) were similar to those previously described using non-radioactively labelled leaves 69 8:: vm ::00x0 480: 08:: :0«0 :« 00w«:0>« 0:03 :0_:::00:00 v 80:: 33:0: 08:. :80: 08:: :0«0 :« 3:00: 0: :0 00« 08: 0:023 :0«0 :0 :08«> .008: :«::08:0_0>00 :0«0 :« 80800 00:«88:0w :0 800 :0 0:: :« :80: 080 :0«0 :« 00::0: x0 0:« :00M:« m .0«: : bm0:000: 0880:20 : m: “00:: :0 %:0 _«:::0 :« 0:325: 000—00: .:0m03: 88:80 :0::0::0: 00: 8:0: :«::0::0:::« ”8:080: « .003 8:0 30800 :0 50:0: :« : 880:0: :0: 0:000: 0:« 0 3 8:0 0: ”09:: 8:00 :«::0::0:::« ”00:: 8:0» 80:: 0:08: “00:: 8:0 0: ”30:0: :« 008.:00 0:« :0 S: 05:. N 8008000880: 8002 : 0 0 0.00 m.m0 0.m: mam 0.0 S. rum 0 0 :00 0:: Wm: NS 0.0:: N.:m m: 0 0 20:: m.:m m.:m N0m Gum m.~.m w 0 0 0 0 0.: ms :0: Wm: v 0 0 0 0 0 0 0 :0 0 8% M03 :33 Maud :03: 3 :03 M91: mmlm 8:0::0::.:< 000:. 8:00 _«::0::0: < 003:. 8:000: N009: _«::08:0_0>0n: .WWMDIOfl . w 0:« :02 :0 85:00:00 080 :00«_:008 :0::« 0:0 00«: .008: .m .m :0 0:8: :«::08:0_0>0Q .v mam?“ 70 (Kerby and Somerville, 1989). Germination of the fungal spores was slightly slower on labelled coleoptiles than on unlabelled leaves. Appressorial initials appeared by 8 hpi and were more abundant on the susceptible than the resistant line. Nearly two-thirds of the germinated conidia formed appressoria on AlgS by 12 hpi. It is therefore reasonable to assume penetration occurred during the 10—12 hpi time frame as expected. The conidia were unable to advance beyond the initial penetration stage on either host. Even after extended periods of time of 42 hours, no haustoria or elongating secondary hyphae were observed on AlgS. Development of the fungus was similarly halted on non-radioactive coleoptiles. The fungus developed normally on leaves, suggesting that the conditions inside the closed boxes, necessary for maintaining gnotobiotic conditions, were detrimental to normal pathogen development. The polypeptide profiles of the susceptible isoline and the resistant isoline were analyzed by both NEPHGE and pH 5-7 IEF gels at 2 hour intervals following inoculation with race CR3 of the fungus. Approximately 300 polypeptides could be resolved on either IEF gels or by NEPHGE. Polypeptide patterns of inoculated and umnoculated plants of each genotype were compared at each time point. Inoculated plants of both genotypes were also compared. Although many changes in polypeptide intensities were observed, no polypeptide was observed to change consistently during the first 12 hpi. Polypeptide patterns of both isolines were also analyzed at 24 hpi. Fifteen polypeptides changed in a consistent manner at this time point (Figure 8 and Table 5). Six polypeptides decreased in the susceptible 71 line but not the resistant line indicating changes in protein expression in the compatible interaction. Three polypeptides were observed to decrease in both lines and four polypeptides increased in both lines suggesting a possible general response to infection. These polypeptides can be classified as infection-related. Two polypeptides appeared to be resistance-related in that they increased in inoculated AlgR coleoptiles and not in inoculated AlgS coleoptiles. 72 NH 2 s e 194» - - '* ‘ g ' ' [k0 - - .- d' - .- l£111» 00: ,_.i 010- :--- 00. _.- O f"- i - ‘ -1773“, i , .‘ 131.0... ‘ .:- 2 ‘ ' 8" 3" 58’; _ f. _o . -. . ': t- ‘9 ‘0 . - . ~” ;’--' a ".0 - "$-53”? ’ ' - (>533! ‘ . dd 0 .. «93 ~‘ 00 . .0" ' [Q , Q .- _ ~0 .» .0 ‘ ‘ CO ‘ . P -- - - c , 9 - - --~_ - - .' O - f . - - J. .. O- _.. A s‘-- 99 - gas-v: - ..<->-<'- ~ -4. 9‘ "- r?--- _ *- - - ".59, ,:-. 2%?» .. -._ 9 'f 0 Z. O ' z‘ ’3? ‘0 ° 0 v ‘ . " . w "3’ 0' -- ‘ - 0 _ 9— ' . - g -Q - 0 (pg .40 o . <9 - ._O_O.- ’ , O O o. . ”'7 ‘ ’ — Figure 8. Electrojphoretic patterns of coleoptile pol eptides from unmoculated an 1noculated barley lmes 2 hpl Wlt race CR3 of Egg. bordez. A umnoculated AlgR, B) AlgR 24 hpi, C) unmoculated Al , and D) gS 24 hpi. Polypeptldes were se arated b pH 5-7 IEF tu e els 1n the first dunensmn and-10% SDS- AGE 1n he second . imension. Clrcles, numbered m A, Identifylpol eptldes that d1ffer between .24 h '1 and umnoculated controls. um ers correspond. to _ polypeptides .sted m Table 5. The arrow and box 1dent1fy constltutlve polypeptlde d1fferences between AlgR and AlgS (see chapter II). 73 Table 5. Summa of pol eptides from A158 and AlgS that chanfigd in intensity at 24 ours a ter 1nocu1at10n M E. g. hordez, race C . Eolypeptide1 Barley genotype Al__g_R2 A185 1 -3 32 0.8 2 - 33 4.7 3 - 19 7.7 4 - 0.0 5 - 18 5.7 6 - 28 7.8 7 50 5 7 30 3.7 8 54 2.0 67 5.3 9 0.0 50 12 10 214 76 734 6 1% 313 30 290 175 13 199 14 183 $312) 1% 204 18 1Q 1 P01 peptides labelled as per Fi ure 8. 2 Va ues, quantltated usmg the 1sage 110 gel analyzer, are the average of two expenments and are expressed as er cent of . values w1th standard dev1at10n 1n parentheses. 0 changes w. ch were con.51stent 1n both ex erlments are re orted see Appendix B). 3 Polypeptldes that d1d not 1ffer m a con31s ent manner from mock 1nocu1ated controls. . . #ThlS pol peptlde wasnot present 1n mock moculated control coleopt1 es of elther hne and appeared 1n both .at 24 hpi. * Thls pol pe t1de was present 1n AAgR at 24 h 1..It was not resent 1n mocu ate or mock moculated gS or moc moculated gR. 74 D . . Changes in protein expression in barley during infection by the barley powdery mildew organism were observed at 24 hpi or later (Manners et al., 1985). These changes correspond to late events in the interaction between host and pathogen, which occur after the hypersensitive response is expressed in the resistant line (Ellingboe, 197 2). Infection-related changes in mRNA expression in both resistant and susceptible lines occurred by 12 hpi, but resistance-related differences between the two lines were not detected until after 24 hours (Davidson et al., 1987; Davidson et al., 1988). Cytologically, differences between the responses of susceptible and resistant barley lines have been reported to occur as early as 10-12 hpi (Ellingboe, 1972; Bushnell, 1981). One would expect to find differences in protein and mRNA expression prior to the appearance of cytological changes. One reason changes at these earlier times may not have been observed is that previous investigators examined changes in inoculated leaf tissue. While such tissue is easy to work with, it is composed primarily of chloroplasts and other mesophyll components. Coleoptile tissue, with its predominance of epidermal cells, can eliminate problems with the detection of epidermal polypeptides due to the interference of mesophyll components. The identification of constitutive polypeptide differences (Chapter II) demonstrates the efficacy of using coleoptiles to view the molecular changes occurring in the disease reaction. Our initial hope was that we would be able to observe the early signalling events which occur between the fungus and host plant. Despite the elimination of a great deal of interfering proteins, no 75 differences in polypeptide expression were detected in the early time course of the barley-E. g. bordei interaction. Normal development of the fungus has been observed on barley coleoptiles (Bushnell et al., 1967), therefore, one would expect to observe changes in the early " time course of infection. Consequently, we analyzed our system to verify that development of the fungus occurred within the expected time frame. Unfortunately, E. g. bordei did not develop paSt the appressorial germ tube stage even after extended periods of time. It is likely that the conditions within the closed boxes, in which the coleoptiles are axenically grown, are unfavorable to the establishment of a successful infection. However, the development of the fungus did appear normal during the first 12 hpi suggesting that the inability to observe differences within this time frame was not due to the experimental conditions. Our desire to minimize manipulations for the sake of maintaining an axenic environment led us to use non-excised coleoptiles. This differs from the method described by Bushnell et a1. (1967). In his system, coleoptiles were removed from 7-day-old plants which were grown in the light. The excised coleoptiles were floated on media and inoculated on the inside surface. With non-excised, etiolated coleoptiles, it is more difficult to consistently attain a uniform, heavy inoculum density. It is therefore likely that no consistent changes in protein expression were observed because differences in inoculum density between plants caused different protein profiles. One cannot rule out the possibility that the early signalling events are of too transitory a nature to be detected by 2 hour time points or the early polypeptide changes occur at levels too low to be detected by our methods. 76 We examined protein profiles in the two isolines at 24 hpi to show that differences arising as a result of infection could be detected in the coleoptile system. By this time point, cells of the resistant isoline should be responding with hypersensitive cell death while compatibility and successful infection by the fungus should be established in the ‘ susceptible isoline. Therefore, we expected to see changes in the polypeptide profiles at this time. Indeed, we observed at least 15 changes in polypeptide expression at 24 hpi (Table 5). These experiments indicate that we may have made the first identification of resistance-related polypeptides in the barley-barley powdery mildew interaction, in contrast to the results of Manners et a1. (1985), who were only able to identify infection-related changes. Unfortunately, the inability of the fungus to develop to the proper stage at 24 hpi reduces the importance that may be assigned to the polypeptide changes we have reported. It is possible that these changes were initiated in the resistance response during the first 12 hpi while development of the fungus was normal. Alternatively, the polypeptide changes observed at 24 hpi could be stress-related responses resulting from the conditions under which the coleoptiles were grown. Many plant defense proteins are triggered by other stresses such as wounding, uv irradiation, chemical poisoning, and ethylene (Dixon and Harrison, 1990). However, the fact that the polypeptides we observed changed in a differential fashion between the two isolines only after challenge by the fungus suggests that they may play some role as infection-related and resistance-related proteins. 77 I. C ! Bushnell, W.R., Dueck, J ., and Rowell, J .B. (1967).. Living haustoria and h hae of Erympbe grammzs f.s . horde; W1th mtact and gr?“ 1d7i§s2ected host cells of Hor eum vulgare. Can. J. Bot. Bushnell W.R. 81981). Incompatibihgfl conditioned by the M—a gene in owdery mil ew of barle : the h in cytoplasmlc streammg. hytopathology 71:1062- 066. Carver, T.W., and Philli s, M. (1982). Effect of Ehoto eriod and level of irradiance on pr uctlon of haustona by 13151 e gramzms f. sp. bordez. Trans. Br. Mycol. Soc. 79:207-211. Davidson, AD” Manners, J.M_., Simpson, RS, and Scott, K.J. (1987). cDNA clonm of mRNAs mduced 1n resrstant barley during isnsfection by rymphe gaminis f.sp. bordel. Plant Mol. B101. 8:77 - Davidson, A.D., Manners, J.M., Simpson, R.S.,'.and Scott, K.J. (1988 . Altered host gene expresswn 1n near;1sogen1c barle condltloned y different cues for re51stance durin mfectlon b r i be minis f. sp. bor8e1'. Physiol. Mol. Plant athol. 32:127XI39.” p gra Dixon, RA, and Harrison, M.J. 1990). Activation, structure and organization of enes involve in microbial defence in plants. Adv. Genet. 28:165- 4. Ellin boe, AH. 1972 . Genetics and h iolo of rim infection b? Erysipbe 1éram12115. Phyt0patholggyys62: 406? ary Greggrsen, P.L., Collinge, DB. and Smedegaard-Petersen, V. (1990). r1 mductlon of new m As accompames the resistance reac ion of park to the wheat atho en, Erysiphe gaminis f. sp. tntzcx. Phys101. 01. Plant Patho . 36: 1-481. Hadwiger, LA., and Wa oner, W., (1983). Electrophoretic patterns of pea and Fusanum so am proteins synthesnzed 1n wtro or m VIVO which characterize the com atible and incompatible interactions. Phys101. Plant Pathol. 23:15 -162. Johnson, LE.B., Bushnell, W.R., and Zeyen, R.J. 1979). Binary. pathwa. for analys15 of pnmary mfectlon and ost res onse 1n popula 10118 of powdery mildew fungl. Can. J. Bot. 57: 7-511. Kerby, K., and Somerville, S. 1989). Enhancement of s ecific mtercellular perox1dases fo owm mpculatlon of bar e with 3531;61pr gramzms f. sp. border. ysnol. Mol. Plant Pa hol. 35:323- Kita, N., Toyoda, H., and Shishiyama, J. (1981). Chronological analysis Bf ctzytsg Ci galllyggonses 1n powdery mildewed barley leaves. Can. J. o . : - . 78 Klecan A.L., hHippe, S. and Somerville, S. C. (1999). Reduced of Erys ysiph e gramimsf. ngl, hordei induced 111etiops1s p escens. Phytopathology 80: 325 Ko a, H. ,Busl1nell, W. R., and Zeyen, RJ. (1990). Specifici of cell e and timing of events associated W1 h papilla] forma on and the hypersensitive reaction in leaves of Hordeumvu vu21§are2352 attacked by rysipbe graminis f. sp. bordei. Can. J. Bot. 68: Kogla], H. M,ayama, S. and Shishiyama, J. 1980). Correlation between e de 051tio11 of fluorescent comp oun in apill ac and resistance in bare ey against Erysipbe gamims bordei. J. Bot. 58: 536-541. Manners, J. M. Davidson, AD, and Scott, K.J. (1985). Patterns of post-mfectional protein synthesis 1n barl ey yeah-3mg different lgenes or 71'5esist3ance to the powdery mildew fungus Mol. Bio Mansfield, M .,A. and Key, J. L. (1987) .S thesis of the low molecular weight heat shock proteins in plants. lant Physiol. 84:1007-1017. Masri, S. S., and Ellingboe, AH. (1966). Germination of conidia and formation of a pressoria and second hyphae 1n Erysiphe graminis f. sp. tici. Phytopathology 5 :304-308. Moseman, J. G. (1)972 150 enic barle lines for reaction to Erysiphe gramim'sf. sp. 1101')dei. rop Sci. 1 68'1.-682 Ramagopal, S. (19871}. Salinity stress induced tissue-specific proteins in bar ey seedlmgs lant Physiol. 84: 324-331. Ri leman, R. C., Fristensky, B. and Hadwige1',L.dA.1985).Thed isease resistance res onse in pea is associated wi mcreased levels of specific m As. Plant Mol. Biol. 4:81-86. Sachs, M. M., Freeling,M and Okimoto, R. (1980). The anaerobic proteins of ma1ze. Celf 20' 761-767. Shipton, W. A., and Brown, J. F. (1962). A whole leaf clearing and staining techni ue to demonstrate hostipathogen relationships of wheat stem rus. Phytopathology 52' 13 Slusarenko, A.J., and Longland, A. (1986) Changes 1n ene activity during expression of the hypersensitive response in haseolus vulgaris cv. Red Mexican to an avirulent race 1 isolate of 1299911713d8‘11nonas syringae pv. phaseolicola. Physiol. Mol. Plant Pathol. SUMMARY AND FUTURE DIRECTION Three attempts to isolate a plant disease resistance gene or gene product have been discussed. The results demonstrated that the methods used were not sufficiently developed or sensitive enough for these experiments to be successful. Further development of the transposon tagging method and different approaches to identifying differences between near-isogenic cultivars may yield useful results in the future. Transposon tagging may be a useful method in the future for cloning disease resistance genes. In order for these experiments to work, a clone of a low copy number transposable element from the host plant of a genetically well described plant/pathogen interaction must be secured. Alternatively, it may be possible to transform plant species that are amenable to transformation and regeneration with one of the elements currently available. For this technique to be successful, it is necessary that the transposon be mobile when introduced into the new plant species. The Ac element of maize, for instance has been shown to be active when introduced into tomato (Yoder, 1988). In all cases, the target gene must be of low natural mutability to give a reasonable probability that mutants in the disease reaction are caused by the introduced element. It may, therefore, be practical to target a disease reaction gene which is not multi-allelic (Pryor, 1987). 79 80 Future efforts at identifying molecular factors associated with the disease reaction by screening subtracted cDNA and genomic DNA libraries may be successful. Clones for low abundance transcripts have been isolated from subtracted cDNA libraries (Travis and Sutcliffe, 1988) and this approach has been enhanced through the use of PCR (Akowitz and Manuelidis, 1989). Increasing the concentration of driver cDNA by PCR amplification, forces the renaturation reaction toward completion more quickly. Additionally, the sensitivity of clone recovery is enhanced by amplifying the product of the subtraction prior to library construction. This technique has also been applied to construction of subtracted genomic libraries. Straus and Ausubel (1990) were able to isolate genomic clones from yeast corresponding to a 5 kbp deletion at the 1y52 locus. By using an excess of DNA from the deletion mutant as driver, and amplifying the final product using PCR, wild-type sequences corresponding to the deleted segment were preferentially recovered after three rounds of subtraction. Amplification by PCR to increase the concentration of driver DNA may make this technique applicable to organisms with large genomes. The transposon tagging and subtraction methods described above can be applied to the barley-barley powdery mildew interaction. Transposon tagging will require the identification of a suitable transposable element in barley or development of methods for transformation and regeneration of barley. Although genomic subtraction has not been successfully demonstrated in organisms with large genomes, the barley isolines are unique since the resistant and susceptible cultivars of each pair differ genetically by only 1% (Moseman, 1972). This still represents about 55000 kbp as compared to the 5 kbp deletion target in yeast, however, 81 putative deletion mutants of barley have been recovered that affect the MI-a-mediated disease reaction phenotype (S. Somerville, personal communication). These mutants likely provide a much smaller target for the subtractive hybridization technique. A subtracted cDNA library of infected barley tissue has a good likelihood of success. Travis and Sutcliffe (1988) recovered cDNA clones from monkey brain cortex that represent 0.001% of total mRNA in that tissue. The least abundant polypeptide observed in our gels was approximately 200 fold more abundant than these cDNAs. Similar results can be expected from barley, increasing the sensitivity of the system for identifying differences between the near-isogenic lines accordingly. The technique can be enhanced by using mRNA from barley coleoptile tissue if conditions for suitable development of the powdery mildew fungus can be developed. As discussed in Chapter III, synchronous development of E. g. bordei on barley provides the opportunity to correlate recovered clones with specific stages of disease development. The primary difficulty in studying race-specific resistance at the molecular level is the inability to use standard cloning techniques, which require antibody to the gene product or a heterologous probe, to isolate a resistance gene. The subtractive cloning methods described above are designed to identify a gene product. In the absence of an identified gene product, techniques that rely only on a genetic knowledge of the gene must be used. Transposon tagging is one method which relies only on a knowledge of the resistance phenotype and the genetics of the element being employed. A cloning method that relies on knowledge of genetics alone is chromosome walking. This method has been used to clone the Cystic fibrosis (Ct) gene from the human genome (Rommens et al., 82 1989). The major drawback to chromosome walking in barley is a lack of markers adjacent to the M—a locus from which to conduct and pr0perly direct a chromosome walk. Recently, a method for quickly generating large numbers of markers (RAPD) has been developed (Williams, et al., 1990). Random oligonucleotide primers are used to amplify genomic DNA, generating polymorphic fragments which can be mapped by traditional segregation analysis. This technology has been applied to bacterial, fungal, human, and plant genomes (Williams, et al., 1990; Caetano-Anollés et al., 1991). Recent results in this laboratory indicate the RAPD method will work in barley (J. Weller, personal observation). Bunce et a1. (1986) have estimated physical distances in barley of 1500- 3000 kbp/cM. Isolation of Cl" required walking and jumping a distance of 550 kbp. Therefore, markers need to be identified within 0.1 cM of the M—a locus in barley in order for chromosome walking to be a successful method for cloning M-a. The barley-barley powdery mildew system has many unique advantages that make it the logical plant-pathogen interaction with which to pursue further efforts at identifying the molecular basis of race-specific disease resistance. The base of genetic and cytological knowledge in this interaction will be indispensable in the analysis of the molecular and physiological basis of race-specific disease resistance. Additionally, advances in molecular techniques indicate that it is only a matter of time before a race-specific resistance gene is isolated. Molecular characterization of an isolated resistance gene will lead to a determination of the function of resistance genes, and identification of the associated signalling pathways and principal biochemical changes which result in race-specific resistance. 83 'ter 'ted Akowitz A., and Manuelidis, L. (198925. A novel cDN CR strate for ggftiscient cloning of small amoun of undefined A. Gene 8 :295- Bunce, N.A.C., Forde, B.G., Kreis, M., and Shewry, RR. (1986). .DNA restriction fragnent polymorph1sm at Horde1n loc1: A phcatlon to identi ‘ an fin e r1ntin barle cultivars. Seed c1. Technol. 14:419-fy4r2§. g rp g y Caetano-Anollés, G., Bassam, B,J., and Gresshoff, RM. (1991). DNA amplification fingerpnnnn usmg ve short arbltrary oligonucleotide pr1mers. B10 echnol. :553-5 7. Moseman, J .G. (1972). Isogenic banle lines for reaction to Etyszpbe grammis f. sp. borde1. Crop SCI. 2:681-682. Pryor, T. 1987). The origin and structure of fungal resistance genes in plan . Trends Genet. 3:157-161. Rommens J.M., Iannuzzi, M.C. Kerem, B.-S. Drumm, M.L. . Melmer, G., Dean, M., Rozmahel, R, Cole, J .L., Kennedy, D., I-Itdaka 1N., IZTSSlg?’19\8I9’ Buchwald, M., R10rdan, J.R. Tsu1, L.-C., and Coflms, ). Identification of the Cyst1c fibrosis gene: chromosome walkmg and jumpmg. Sc1ence 245:1059-1065. Straus D. and Ausube1, RM. 1990). Genomic subtraction for cloning DNA corres ondmg to elet10n mutants. Proc. Natl. Acad. Sc1., USA 87:188 -1893. Travis, G.H., and Sutcliffe, J .G. 1988). Phenol emulsion-enhanced DNA- driven subtractive cDN clonin : isolation of low abundance n16grék1ey080rtex-spec1fic mRNAs. roc. Natl. Acad. Sci., USA 85: Williams, J .G.K Kubelik, AR, Livak, K.J., Rafalski, J .A., and Tingey, SN. 1990 . DNA 01 o h15ms am lified b arbitra nmers “1.1.11. 1.1.1: $1.23.. 10.11:... mamasglizsas. Yoder, J .I., Palys,.J., Alpert, K., and Lassner, M. (19881. Ac trans osition 1n transgemc tomato plants. Mol. Gen. Genet. 13:291—2 6. APPENDIX 84 APPENDIX A Model of barle chromosome 5 sho ' g the genetic in erpretation of Tablewglil barley line Ho'ri Ml-a Hor2 Algerian R Algerian S Multan R I Multan S Durani R Durani S Franger R Franger S Rupee R J I Rupee S 1 _] L__ Long Glumes R Lon Glumegs s 1 I 1 85 barley line Hori Ml-a Hor2 p6.7 s-01 : - Siri + + + + + - + P-Ol : + Pallas + + + + Horizontal bars represent the short arm of barley chromosome 5 between the oenenc markers Her] .and Hor2. - and + represent, respect1velv, tlie res1stant and suscept1b1e arental henotvpes at each of the markers, des1gnated by the vert1ca hnes. e p6.7 henotyge of each 11ne 15 shown in the rlght hand column. 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