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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6 ” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bdl & Howell Information Company 300 North Zeeb Rood, Ann Arbor MI 48106-1346 USA 313/761-4700 800/321-0600 MOLECULAR CHARACTERIZATION OF THE hrpN
GENE OF ERWIN!A STEWART!I
DISSERTATION
Presented in partial fulfillment of the requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
by
Musharaf Ahmad
B.Sc. (Hons.), M.Sc (Hons.)
The Ohio State University
1996
Dissertation Committee: proved by Dr. D. L. Coplin Dr. T. L. Graham Dr. S. A. Miller Adviser Dr. W. D. Bauer Department of Plant Pathology UMI Number: 9639176
UMI Microform 9639176 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 To my mother, K. J. Afridi
ii ACKNOWLEDGEMENTS
I am very grateful to my advisor. Dr. D. L. Coplin for his thoughtful suggestions, guidance, and encouragement throughout the research. I would also like to express my
gratitude to the other members of my committee, Dr. T. L. Graham, Dr. S. A. Miller, and
Dr. W. D. Bauer for their suggestions and help.
I thank Ms. Doris Majerczak for her technical assistance during the entire course of
this research. I also thank other colleagues in the lab and friends in the department for their
help and friendship.
I greatly appreciate the University Grants Commission, Islamabad, Pakistan for
selecting me for the prestigious Quide-i-Azam Scholarship. 1 also acknowledge The Ohio
State University and Department of Plant Pathology for their financial support for over two
years.
I am especially thankful to Dick Bausman, Director United Christian Centre at the
Ohio State University for selecting me to be one of the resident fellows for a long period of time, and for his personal help and friendship. I also thank other members and friends of the Center for their help and companionship. The association with the Center gave me an experience that I probably would have never gotten otherwise.
Finally, I would like to express my deepest appreciation to my all family members, especially my mom, for their encouragement and prayers, and my wife, Mehnaz, for her patience and cooperation during this work.
iii VITA
April 03, 1964 ...... Bom - Chorlaki, (Kohat, NWFP), Pakistan.
EDUCATION
1983-1987 ...... B.Sc (Hons.) Agric., NWFP Agricultural University, Peshawar, Pakistan.
1987-1989 ...... M.Sc (Hons.) Agric., NWFP Agricultural University, Peshawar, Pakistan.
1989-1991 ...... Research Officer, Department of Agriculture, Government of NWFP, Pakistan.
1991 -199 2 ...... University Grants Commission Fellow, Department of Plant Pathology, Oklahoma State University, Stillwater, OK.
1992-199 3 ...... University Grants Commission Fellow, Department of Plant Pathology, The Ohio State University, Columbus, OH.
1993-199 6 ...... Graduate Research Associate, Department of Plant Pathology, The Ohio State University, Columbus, OH.
PUBLICATIONS
1. Frederick, R. D., Ahmad, M., Majerczak, D. R., Stover, E., and Coplin, D. L. 1994. The wft water-soaking genes of Erwinia stewarni are related to hrp genes, p. 62 in: InL Symp. MoL Plant-Microbe Interact., University of Edinburgh, Scotlannd
2. Ahmad, M., Hassan, S., Ahmad, I. 1989. Pathogenic variation in Macrophomina phaseolina and differential response of some important sunflower varieties to charcoal rot Sarhad Journal of Agriculture (Pakistan), 5: 659-663.
FIELD OF STUDY Major Field: Plant Pathology
Molecular biology of wts genes of Erwinia stewarni under the direction of Dr. David L. Coplin.
iv TABLE OF CONTENTS
PAGE
DEDICATION ...... ii
ACKNOWLEDGMENTS...... iii
VITA ...... iv
LIST OF TABLES...... vi
LIST OF FIG U RES...... vii
CHAPTERS:
I. LITERATURE REVIEW ...... 1
II. CHARACTERIZATION OF HARPIN...... 15 Introduction ...... 15 Materials and Methods ...... 20 R esu lts ...... 29 D iscu ssion ...... 59
III. HARPINes PRODUCTION BY WTS MUTANTS...... 64
Introduction ...... 64 Materials and Methods ...... 6 6 R esu lts ...... 69 D iscu ssion ...... 72
IV. HOST RANGE STUDY AND PRODUCTION OF HARPIN BY PANTOEA STEWARTII. SUBSP. INDOLOGENES...... 73
Introduction ...... 73 Materials and Methods ...... 74 R esu lts ...... 78 D iscu ssio n ...... 80
APPENDIX A. MATERIALS AND METHODS...... 81
LIST OF REFERENCES...... 97
V LIST OF TABLES
TABLE PAGE
1. Bacterial strains and plasmids ...... 21
2. Primers used in the automated sequencing of the hrpN reg io n ...... 24
3. HR assay in Datura and tobacco leaves of cells and cell-free elicitor preparations (CFEPs) of wild-type strain DC283, hrpN mutant DM760, and E. coli DH5d harboring the E. amylovora hrp cluster grown in different media ...... 31
4. HR assay in tobacco leaves of cell-free elicitor preparatios (CFEPs) from hrpN mutants containing Envinia amybvora hrpNEa* clone pCPP1012 ...... 52
5. ED5 0 of wild-type strain DC283 and those of hrpN mutants 54
6 . Disease severity rating of wild-type strain DC283 and hrpN mutants DM760, MAI, MA2, and DM3020Awts on 8 -day-old corn seedlings using three different inoculation m ethods ...... 55
7. Bacterial strains and plasmids ...... 67
8 . HR assay in tobacco leaves of cell-free elicitor preparations (CFEPs) from the wild-type strain DC283(pRF205) and various wts m utants ...... 70
9. Bacterial strains and plasmids ...... 75
10. Host range study of the wild-type strain DC356 and a h rp N mutant MAI ...... 76 LIST OF FIGURES
FIGURE PAGE
1. Genetic map of the Erwinia stewartii wts gene cluster 13
2. Physical and genetic map of pan of the E. stewartii wts region showing the location of the hrpN gene ...... 23
3. Growth of wild-type strain DC283 and hrpN mutant MA1 in tobacco leaves ...... 30
4. Hybridization of pES411 DNA with hrpN^a...... 32
3. Nucleotide sequence of the hrpN region showing open reading frame and putative promoter (hrp box) for the hrpN gene and promoter (hrp box) for the wtsE gene of Erwinia stewartii, and comparison of these hrp boxes with those of other bacteria...... 34
6. Predicted amino acid sequences of HrpN^ and HrpNg, aligned by the clustal V multiple sequence align ment program ...... 39
7. Predicted amino arid sequences of HipNe* and hrpNecb aligned by the clustal V multiple sequence align ment program ...... 40
8. SDS PAGE of electroelution-purified HarpinE,...... 42
9. Western blot of CFEPs from wild-type strain DC283 and several hrpN mutants reacted with antiserum to harpines 43
10. Comparison of biological activities of harping and harpin£* in tobacco leaves ...... 44
11. Western blot analysis of harping, production and secretion by DC283(pRF205) grown at three different temperatures 45
vii 12. Southern blot of genomic DNA from wild-type strain DC283 and hrpN mutants to verify marker-exchange of Tn5 insertions into the chromosome ...... 47
13. HR assay in tobacco leaves at 24 h with purified harpines, wild-type strain DC283(pRF205) and various h rp N m utants ...... 48 14. HR assay in tobacco leaves at 24 h with wild-type strain DC283(pES411) and various hrpN mutants complemented with pES41l and pES411 h rp N/8 9 ::T n 5 ...... 50
15. Immunoblot of cell-free elicitor preparations (CFEPs) from DC283(pES411) and several hrpN mutants complemented withpES411 reacted with anti-harping antibodies ...... 51
16. Immounoblot of cell-free elicitor preparations (CFEPs) from hrpN mutants complemented with £. amylovora hrpN- containing clone pCPP1012 reactedwith harping,, antiserum.... 53
17. Response time of the wild-type strain DC283 and the hrpN mutants on sweet com seedlings ...... 56
18. Growth of wild-type strain DC283 and hrpN mutants in corn seedlings...... 58
19. Immunoblot analysis of harpin production by pRF205- containing wtsF, wtsB, wtsD, and wtsl mutants ...... 71
20. Immunoblot analysis of harpines production by Fantoea stew artii subsp. indologenes...... 79
viii CHAPTER 1
LITERATURE REVIEW
Random transposon mutagenesis of necrogenic and wilt-inducing bacteria has yielded general pathogenicity mutants, called hrp (hypersensitive response and pathogenicity) mutants, that are unable to incite either a hypersensitive response (HR) on resistant hosts or non-hosts, or a pathogenic response on susceptible hosts, hrp mutants are unable to multiply in their host plants, hrp genes were initially identified as major determinants of pathogenicity in Pseudomonas syringae pv. phaseolicola in the mid
1980's (Lindgren and Panopoulos 1986). Since then, they have been found in a wide range of Gram-negative plant pathogenic bacteria including P. syringae pathovars syringae, glycinea, tabaci, tomato, and pisi; many Xanthomonas campestris pathovars;
Pseudomonas soianacearum; Erwinia amylovora; and Erwinia chrysanthemi (Laby and
Beer 1992, Willis e ta l . 1991). These genes are clustered on the chromosome (or the megaplasmid in P. soianacearum), and consist of many complementation groups, which show inter-species DNA sequence homology to each other (Laby and Beer 1992). In some cases, inter-generic sequence homology has also been found (Gough et al. 1992).
The DNA homology between hrp clusters from different bacteria suggests that all hrp genes could have originated either from flagellar genes in a common ancestral plant pathogen or
1 2 as a result of horizontal gene exchange with bacterial pathogens of animals (D. L. Coplin, personal communication).
Among the more interesting hrp clusters are those of P. syringae pv. syringae and E. amylovora. The P. syringae pv. syringae cluster is 36-kb in size and has been cloned on a single cosmid, pHIRll. This clone restores Hrp+ phenotype to all hrp mutants of P. syringae pv. syringae, and it also enables non-pathogens such as P. fluorescens, and Escherichia coli to cause an HR on tobacco (Huang et at. 1988).
Mutagenesis of pHIR 11 with TaphoA revealed eleven complementation groups, two of which appear to encode either transmembrane or exported proteins (Huang et al. 1990).
The hrp region of X. campestris pv. vesicatoria is 25-kb in size and is organized into at least six different complementation groups designated hrpA through hrpF.
Transposon mutations in any one of the six hrp loci abolish symptom formation in susceptible and resistant host plants (Bonas et al. 1991). The hrp cluster appears to be highly conserved among different strains of X. campestris pv. vesicatoria irrespective of their host specificity. Based on genetic and sequence analyses, there could be 21 hrp genes in the hrp cluster of X. campestris pv. vesicatoria. A number of these putative Hrp proteins are likely to be associated with or localized in the bacterial membrane (Bonas 1994).
The hrp cluster of E. amylovora is the largest of all the hrp clusters known so far.
It spans over 39-kb and contains at least nine complementation groups (Bauer and Beer
1991). A full length clone, pCPP430, enables E. coli and other bacteria to produce an
HR in tobacco (Wei et al. 1992a).
Soft rot erwinias also appear to have hrp gene clusters, but they (clusters) seem to have a lesser role in pathogenesis. It was difficult to find hrp genes in E. chrysanthemi because this pathogen secretes several pectate lyases (Pel) that rapidly kill and macerate host tissue. Hie destructive effects of these enzymes mask the ability of E. chrysanthemi 3 to cause Hrp-dependent necrosis. Nevertheless, A p elA B C E , Out- mutants of E. chrysanthemi EC 16 caused a typical HR.
In P. soianacearum, the majority of the hrp genes map in a large cluster, which spans 20-23 kb (Boucher et al. 1987; Arlat etal. 1992). Genetic analysis has shown that this cluster consists of six transcription units. Mutations in transcription units 1-4
(hrpA, B, C, D, E, F, H, I, J, K , N , O, P, Q, T, U, V, W, and X) render the pathogen
HR- on resistant hosts or non-hosts such as tobacco, and non-pathogenic on susceptible hosts. However, mutants affected in transcription units S and 6 have a leaky phenotype with respect to both HR elicitation and pathogenicity (Arlat et al. 1992, Van Gijsegem et al. 199S). A regulatory gene designated hrpB has also been identified within the hrp gene cluster (Genin etal. 1992). This gene positively regulates transcription units 1-4 as well as genes (eg. the popA gene) located in a region immediately adjacent to the left end of the cluster, thereby defining a hrp legulon.
Although substantial progress in terms of molecular analysis of hrp genes was made throughout the last decade, their function remained unknown for a long time. Until recently, the only hrp gene with a known function was hrpS of P. syringae pv. phaseoticola. HrpS shares homology with two component prokaryotic regulatory proteins related to NtiC (Grimm and Panopoulos 1989). In support of a regulatory role, hrpS is required for the transcription of reporter gene fusions within hrpAB, hrpC, and hrpD (Mindrinos et al. 1990), as well as hrpF and hrpE (Rahme et al. 1991).
One of the problems in predicting the biological function of hrp genes has been their hydrophilic nature, i.e. the lack of an N-terminal stretch of 20-25 hydrophobic amino acids and the absence of an N-terminal leader/signal peptide. These characteristics are needed for a protein to be located in the outer membrane or exported via a Sec-dependent
(i.e. proteins employ the Sec machinery to cross the inner membrane) pathway. The products of most of the hrp genes were therefore considered to be cytoplasmic enzymes that had only an indirect role in plant-bacteria interactions. Interestingly, nucleotide sequence comparisons of hrp genes with genes from mammalian pathogenic bacteria
(eg., Yersinia enterocolitica and Y. pestis) that encode proteins involved in the production of pathogenicity determinants revealed a possible common secretion function (Fenselau et al. 1992, Gough et al. 1992). For example, HrpA of P. soianacearum is 34.1% identical to YscC of Yersinia, and HrpI of P. soianacearum is 35.4% identical to YscJ of Yersinia enterocolitica (Gough et al. 1992). Furthermore, YscC, YscJ, HrpA, and HrpI are all predicted to be lipoproteins with a lipoprotein-specific signal sequence, and they have a hydrophobic C-terminal domain followed by positively charged residues (Gough et al.
1992), which is characteristic of integral membrane proteins. Similarly HrpAl, HrpB3, and HrpC2 proteins from X. campestris pv. vesicatoria (Fenselau et al. 1992) showed high identities to YscC, YscJ, and LcrD respectively. Yop {Hers ini a cuter proteins) proteins are secreted via a specific type III (a family of secretion systems involved in the secretion of extracellular pathogenicity proteins in bacterial pathogens of both animals and plants) secretion pathway, and lack a typical signal peptide sequence. The membrane proteins LcrD, YscC and YscJ form part of the Yop secretion apparatus. It was therefore suggested that HrpAl, HrpB3 and HrpC2 could likewise be part of a type III secretion system that is required for the pathogenicity of X. campestris pv. vesicatoria (Michels and Comelis 1991). Such an export system could secrete elicitors that interact with plant cells to induce disease or the HR (Gough et al. 1992).
The above findings prompted S. V. Beer and co-workers (Wei et al. 1992a) to look for an unstable, extracellular elicitor encoded by the hrp cluster of £ amylovora.
PMSF (phenyl methyl sulfonyl fluoride, SIGMA)-treated cell-free elicitor preparations
(CFEP) from £ amylovora strain 321 and £ coli DH5a(pCPP430) revealed a 44-kDa cell-envelope-associated polypeptide, which was named harpin. This protein causes 5 tobacco leaf lamina to collapse and it increases the pH of bathing solutions of suspension-cultured tobacco cells. The N-terminal sequence of harpin was determined and used to design a synthetic oligonucleotide probe. Using this probe, the harpin gene
(hrpN) was mapped to the middle of the hrp cluster of E. amylovora. A mutant carrying a non-polar Tn 5tac mutation in hrpN was non-pathogenic on pear, did not cause an HR in tobacco, and did not produce harpin. The non-pathogenic phenotype of the hrpN mutant suggests that harpin is a primary determinant of pathogenicity in E amylovora . That harpin has an essential role in both susceptible and resistant interactions could be based on differential proteolysis of harpin or differential expression of hrp genes in host and non-host plants (Wei et al. 1992a).
Recently, harpin has been reported to be produced by several other plant pathogenic bacteria, i.e. P. syringae pv. syringae 61 (He et al, 1993), P. syringae pv. tomato, P. syringae pv. glycinae (Preston, et al. 1995), and E. chrysanthemi EC 16
(Bauer et al. 1994). The harpin from P. syringae pv. syringae, designated harping, is a
34.7-kDa extracellular protein, and it is encoded by the hrpZ gene located in the hrp cluster of P. syringae pv. syringae 61. Harpin pss, unlike harping, does not have tyrosine and the nucleotide sequence of hrpZ is different from that of hrpNFa (He etal. 1992). hrp clusters and hrpZ homologs have been found in P. syringae pv. glycinea race 4, and P. syringae pv. tomato DC3000 (Preston et al. 1995). The HrpZpsg (35.3-kDa) and
HrpZp$t (36.5-kDa) proteins, like the HrpZpss protein, lack tyrosine. Harpinech is encoded by the hrpNgch gene of E. chrysanthemi and is 34.3-kDa in size (Bauer et al.
1995). All harpins isolated from different bacteria so far are heat-stable, protease-sensi tive, glycine-rich, acidic, hydrophilic, lack cysteine, cause an HR on tobacco, and lack the N-terminal hydrophobic signal sequence used to target proteins for secretion via the
Sec export pathway (Pugsley 1989). PopA, the HR-eliciting protein of P. soianacearum has not been termed a harpin,
because although it has some characteristics of harpins, it is not the sole elicitor of the HR
and is not required for pathogenicity. popA is co-regulated with hrp genes by hrpB, but
lies outside the hrp cluster. The PopA protein, like harpins, is heat-stable, glycine-rich,
protease-sensitive and elicits HR in tobacco. This protein also lack the N-terminal
hydrophobic secretion signal and its secretion is dependent on hrp genes (Arlat et al. 1994).
Most importantly, the popA mutant has no phenotype. PopA is the most abundant
extracellular elicitor, but it is not the only one, and popA mutants are still Hrp+.
Homology to type III secretion genes in mammalian pathogens suggested that
some of the conserved hrp genes are probably involved in the secretion of one or more extracellular pathogenicity proteins (He et al. 1993). DNA sequence analysis of hrpH
and hrpl from P. syringae pv. syringae revealed that HrpH is similar to outer membrane proteins involved in protein or phage secnetion in many Gram-negative bacteria and that HrpI is a member of superfamily of inner membrane proteins with an apparent function in protein secretion or secretion regulation (Huang et al. 1992, Huang et al. 1993).
Homologs of HrpH and HrpI have also been reported in P. soianacearum (Gough et al.
1992) and X. campestris pv. vesicatoria (Fenselau etal. 1992). In P. syringae pv. syringae, two blocks of genes were found to be involved in the secretion of harpinpss: hrpB, hrpC, hrpD, and hrpE in the hrpZ operon, and hrpU, hrpW, hrpO, hrpX, and hrpY in the hrpU operon (Huang et al. 1995). The genes in both operons are colinear with two blocks of ysc genes in Yersinia species (Allaoui etal. 1994). Since the genes in the hrpZ and hrpU operons reveal extensive homologies with proteins associated with the type UI protein secretion pathway of mammalian pathogens, they probably constitute the harpinpls secretion pathway. When hrpC, hrpE, hrpW, hrpX, and hrpY were mutagenized with TnphoA, the mutants were unable to cause an HR on tobacco. To 7
determine the role of these genes in harping secretion, the mutants were grown in Hrp
inducing minimal medium, and the distribution of harpinp^ between extracellular and cell-
bound fractions was determined using Western blots probed with anti-HrpZ serum. HrpZ was secreted by wild-type cells, but accumulated in the cell-bound fraction from' the mutants. These results indicate that these genes are needed for secretion of harpin pss
(Huang et al. 1995).
In E. amylovora, a number of hrp genes, in addition to krpl, were shown to constitute components of the harpinEa secretion apparatus (Bogdanove et al. 1996).
Strains carrying insertion mutations in hrcN, hrpO, hrpP, hrcQ. hrcR , hrcS. hrcT, and hrcU produced harpinEa, but failed to export it to the exterior and were HR- (Wei and Beer
1993; Bogdanove et al. 1996). These genes are arranged colinearly with their homologs in the type III gene clusters of various animal pathogens, especially the Yersinia species.
(The new designation "Arc" indicates that the gene is associated with the hypersensitive response eliciting ability of the bacterium and that it is common to pathogens of both plants and animals.)
X. campestris pv. vesicatoria also has genes encoding proteins for a type III secretion pathway. For example, Wengelnik et al. (1996) used specific polyclonal antibodies and detected HrpAl protein, which is associated with the outer membrane of the bacterium and is a member the PulD family of proteins involved in protein secretion
(Fenselau etal. 1992; Genin and Boucher 1994). HrpAl is most similar to HrpA of P. soianacearum (Gough etal 1992) and to YcsC of Yersinia spp. (Michiels et al. 1991). hrpA has been located near left border of the 25-kb hrp cluster of this bacterium.
Several genes in the hrp gene cluster of P: soianacearum have also been shown to encode proteins that constitute a type III secretion pathway used for the secretion of
PopA and other pathogenicity factors (VanGijsegem et al. 1995). For example, hrpC, hrpE, hrpl, hrpQ, hrpT, hrpU, hrpN, and hrpO all encode proteins that share homology
with components of type III secretion pathways and with proteins involved in flagellum
biogenesis (Gough et al. 1993; VanGijsegem et al. 1995). HrpA and HrpF of P. soianacearum also share homology with type III secretion proteins, but not with flagellar
proteins (Genin and Boucher 1994). HrpA and HrpO proteins were demonstrated to be
essential for the secretion of PopAl (Arlat et al. 1994). Type III secretion pathways differ from the hemolysin secretion system of E. coli
(type I), the pullulanase secretion system of Klebsiella oxytoca (type II), and the Out
system of pectolyic erwinias (type II). Secretion of proteins through a type III pathway is Sec-independent, i.e. proteins do not employ the Sec machinery to cross the inner
membrane, and require a large number of genes, as many as 23 in Yersinia (Forsberg et
al. 1994). In addition, secretion of the Yersinia YopE protein requires SycE, a specific chaperone-like protein, to protect it from denaturation and subsequent proteolytic
degradation before being secreted (Wattiau and Comelis 1993). Sec-mediated
translocation of envelope proteins similarly requires SecB (Pugsley 1993). Hence, it is reasonable to predict that chaperone-like proteins perform the same function in the Hrp
secretion pathway. A few cytoplasmic Hrp proteins with unknown function are potential
candidates as chaperones, for example HrpF, HrpG, HrpK, or HrpA in P. syringae pv,
syringae (Huang et al. 1995).
The proteins exported via type III pathways are diverse. They include flagellar
components of Gram-negative and Gram-positive bacteria, Yops of Yersinia, Ipas of Shigella , InvJ of Salmonella, PopAl and harpins. Once secreted, the proteins either
remain associated with the bacterial cell surface or are released to the culture medium
(Bogdanove et al. 1996). All these proteins lack a classical N-terminal signal peptide
sequence (Salmond and Reeves 1993). So the question remains whether or not the
proteins trafficking type III pathways have any common characteristics involved in 9 secretion targeting. It is likely, however, that each system has its own specificity, because harpin is not exported by the Hrp secretion pathway of E. amylovora, and vice versa (Bauer et al. 1995). A similar problem was encountered with heterologous secretion of pectic lyases and cellulase via the Out pathway in E. chrysanthemi and E. carotovora, two species that are more closely related to each other than E. chrysanthemi is to K amylovora (He et al. 1991; Pye et al. 1991).
In general, hrp genes respond to a complex set of external and internal signals consisting of temperature, pH, osmotic strength, nutrient availability and signal molecule(s) from plants (Goodman and Novaky 1994). Most hrp genes are expressed better in planta than in culture. However, they are expressed in culture media mimicking conditions found inside plant tissue. Different carbon and nitrogen sources, osmolarity, pH, and temperature have varying effects on the level of hrp genes expression, and the optimal conditions for the expression vary from organism to organism. For example, in P. syringae pv. phaseolicola and X. campestris pv. vesicatoria, hrp genes show little or no induction in minimal media containing tricarboxylic acid cycle intermediates such as citrate and succinate (Schulte and Bonas 1992). However, P. syringae pv. syringae 61 hrp genes are induced under these conditions. P. syringae pv. syringae hrp genes are repressed by peptone, but not by glutamine, NH4+ or osmolarity (Xiao et al. 1992), whereas P. syringae pv. phaseolicola is repressed by high osmotic concentrations (Fellay et al. 1991 ) and induced by low pH (pH 5.5 vs 7). The hrp cluster of E. amylovora is repressed by high nitrogen levels, stimulated by low pH, and unaffected by osmolarity
(Wei etal. 1992b).
Most hrp genes are under positive regulation. For example, hrpB of P. soianacearum encodes a positive regulator controlling the expression of all but one of the hrp genes in this bacterium. The transcription of hrpB itself is induced in minimal 10 medium (Genin et al. 1992), so it is in turn regulated in response to environmental signals. Likewise, HrpS is required for the transcription of hrpA-F in P. syringae pv. phaseolicola (Rahme eta l. 1991). hrpS is a 34.5-kDa protein similar to several members of the NtrC family of regulatory proteins found in enteric bacteria, such as
Klebsiella pneumoniae, Escherichia coli, and Salmonella typhimurinum. In P. syringae pv. phaseolicola, HrpS interacts with HrpR and a 54 to activate the hrpL promoter. hrpL encodes an alternate a factor that in turn activates the promoters of each hrp operon
(Grimm et al. 1995). E. amylovora also has a hrpL homolog which is a single transcription unit comprising complementation group VI (Wei etal. 1995). This gene encodes HrpL, a 21.7*kDa, positive regulatory protein. It is a member of the ECF
(extracytoplasmic functions) subfamily of eubacterial RNA polymerase a factors, which also includes HrpL of P. syringae (Xiao et al. 1994). HrpL of E. amylovora controls the expression of other hrp genes including hrpN. It is environmentally regulated and its expression is affected by another regulatory gene, hrpS. However, once HrpL is provided, HrpS is no longer needed for expression of the other hrp genes and elicitation of the HR by the bacteria. In other words, signal transduction goes from hrpS to hrpL, which then functions independently of hrpS in activating expression of other hrp genes
(Wei etal. 1995). However, E amylovora does not have a hrpR gene.
The role of harpin in pathogenesis is not clear yet. He etal. (1993) suggested that harpin may function to release nutrients from plant cells into the apoplast. Nevertheless, harping, in contrast to live bacteria, did not cause necrosis in susceptible apple leaves or immature pear fruit, nor did it elicit an XR (K+ efflux I H+ influx exchange reaction) in apple cell suspension cultures. When added with live cell inoculum, exogenous harping neither restored pathogenicity to hrpN^a mutants nor their ability to grow normally 11
in planta. although it was found to be fairly stable within host tissue (Bogdanove et al.
1994).
hrp genes are also needed for the phenotypic expression of some other genes, such as avr genes, although these two classes of genes have two distinct phenotypes and appear unrelated by sequence homology. For example, AvrBs3 of X. campestris pv. vesicatoria is constitutively synthesized by the avrBs3 gene but the HR in resistant hosts does not develop if the bacteria lack hrp functions. This suggests that an avr product may traffic the hrp export system (Fenselau et al. 1992). There is growing evidence that
Avr proteins are minor pathogenicity factors and can positively affect host range, avr genes that have been shown so far to contribute to virulence on susceptible hosts include avrBs2 of X. campestris pv. vesicatoria (Kearney and Staskawicz 1990), avrb6 of X. campestris pv. malvacearum (Yang et al. 1994), avrRpml of P. syringae pv. maculicola (Ritter and
Dang) 1995), and avrA and avrE of P. syringae pv. tomato(Lorang etal. 1994), Some additional avr genes of X. campestris pv. malvacearum strain HI005 have recently been reported to be required for watersoaking symptoms on a susceptible host. However, most avr/pth genes encoded on pXcmH act synergistically to confer watersoaking on mutant HM2.2S but individually they confer barely detectable watersoaking (Yang et al.
1996).
The genetics of pathogenicity of E. stewartii , a Gram-negative, rod-shaped, non- motile plant pathogenic bacterium that causes a vascular wilt and leaf blight of com, has been studied in detail. In order to identify and clone pathogenicity genes from E. stewartii , McCammon et al. (1985) obtained a number of non-pathogenic mutants of this bacterium. These mutants had two basic phenotypes: EPS - (extracellular polysaccharide) strains, which could not cause wilting but varied in their water-soaking (Wts) ability, and EPS* strains, which could not cause either wilting or Wts and hence were completely non-pathogenic. The latter were tentatively designated as vv/s mutants. Pathogenicity
was restored to many of these mutants by clones from an E. stewartii wild-type library
(Coplin etal. 1986), and three major groups of mutants were identified. The first group
represented a large cluster (19-kb) of cps (capsular polysaccharide synthesis) genes
required for EPS synthesis (Coplin and Majerczak 1990); the second group of mutations
were in res A, a positive regulatory gene of capsule synthesis (Torres-Cabassa et al.
1987); and the third group defined a large cluster of general pathogenicity genes, the wts
genes. These general pathogenicity genes enable the bacterium to cause water-soaked
lesions on young com leaves. The right half of the wts cluster was cloned in a cosmid
designated pES1044 that contains wts A , wtsB. wtsC , and w ts l. The remainder of the
wts cluster was cloned in a cosmid designated pES411. The entire cluster is 28-kb in
size (Fig. 1) and contains seven complementation groups, wtsE, F, Dy A, C, /, and B
(Coplin et al. 1992a; Frederick et al. 1996).
wts A of E. stewartii encodes a HrpS homolog that positively regulates wtsB and
other wts genes. WtsA is a member of the NtiC family of regulatory proteins. NtrC
regulators normally interact with o 54 dependent promoters, and Frederick and Coplin
(1993) demonstrated that wtsB expression in an E. coli background requires o54.
This regulatory gene was cloned in pLAFR3 behind P ^ promotor and designated
pRF205 (Fig. 1) (Frederick et al. 1993). The clone pRF205 was used in different
experiments in this work to enhance the expression of wts genes without which some of
the experiments were very difficult to be conducted.
The vm cluster of E. stewartii is similar to hrp clusters of other bacteria,
especially E. amylovora. Cosmids pES1044 and pES411 share considerable homology
with pCPP430 and pES1044 was able to restore the Hrp+ phenotype to several E. amylovora hrp mutants (Beer et al. 1990). The conservation of function was reflected by 13
i ■ KH 1 r i m n r — * p i p a s k b | Ik H B IHK P M HPH i J.l It II III I ii in i i in nil ii Ml
gfoupi 2Kb IZIH 0E]Q [ H
Fig. 1. Genetic map of the Erwinia stewartii wrs gene cluster. The boxes represent various complementation groups and the arrows underneath them indicate their orientations. H = HindlU, K = Kpnl, B = BamHl, P = Pstl, E = EcoRI, Bg = Bglll. 14
colinear hybridization of portions of pES1044 and pES411 with pCPP430 (Laby and
Beer 1992). This suggested that wts and hrp genes had a common function in
pathogenicity. However, E. stewartii was neither known to cause an HR in tobacco
under normal assay conditions nor was it known to have a hrpN£a homologue.
Moreover, except for wts A, the functions of the remaining wts complementation groups
were not known. We therefore wanted to know if any of the wts genes of E. stewartii encodes proteins having harpin-like functions. This dissertation was an attempt to achieve the following objectives:
1) To find, clone, and sequence a hrpNsa homolog from within the wts
cluster of E. stewartii.
2) To determine conditions under which E. stewartii could cause an HR in tobacco.
3) To determine the role of this homolog as a hypersensitive response (HR) and
pathogenicity determinant in E. stewartii.
4) To examine the potential functions of some of the wts complementation
groups in the synthesis and secretion of harpin. CHAPTER II
CHARACTERIZATION OF HARPIN
Introduction
The invasion of plants by pathogens results in either an incompatible interaction in a resistant host or non-host leading to the elicitation of defense responses, such as the hypersensitive response (HR), or a compatible interaction leading to disease development. The HR due to phytopathogenic bacteria is characterized by a rapid collapse of plant cells, which have come into contact with bacteria (Klement 1982), resulting in inhibition of bacterial multiplication. However, visible necrosis occurs only when >5xl07 cells/ml are infiltrated into resistant leaves; below that inoculum level, single plant cells die in response to individual bacteria (Klement et al. 1964). The HR is an active process (Willis etal. 1991; He et al. 1994) that is correlated with other plant defense responses. The elicitation of the HR requires live, pathogenic bacteria. Any antibiotic that inhibits RNA and protein synthesis blocks the HR if added during the
"induction" period (Sasser 1982). The HR induction period, 30 min in tobacco/
Pseudomonas syringae pv. pisi ( Klement and Goodman 1967) to 4 h in bean/ P. syringae pv. phaseolicola (Roebuck et al. 1978), is the time needed for induction of the genes responsible for the synthesis of the HR elicitor protein (Goodman and Novacky
1994). Pre-inducing bacteria before inoculation eliminates the induction period and the antibiotic effect. Within 24 h after inoculation with plant pathogenic bacteria, plant tissue
15 16 becomes flaccid and turgor is lost. The tissue dries, the bacterial population declines, and any surviving bacteria remain confined to the site of inoculation. As cells undergo the
HR, the plant cell wall thickens, the plasmalemma separates from the cell wall surface and vesiculates in positions opposite to the bacteria, and microfibrils and membrane-bound vesicles aggregate in the space between the plasmalemma and the inner cell wall
(Goodman and Novacky 1994). In addition, lipid peroxidation of the cell membrane results in the formation of activated oxygen species that react with unsaturated membrane fatty acids to generate organic free radicals (Freeman and Crapo 1982). These changes lead to a dramatic decline in the electrical membrane potential accompanied by increased K + efflux and H+ influx (Goodman and Novacky 1994).
The ability of plant pathogenic bacteria to elicit the HR in resistant hosts is correlated with their ability to cause disease in susceptible hosts. Both of these capabilities are controlled by hrp genes (for hypersensitive response and pathogenicity)
(Lindgren e ta l. 1986; Willis et a i 1991). Typical hrp mutants are pleiotropically defective in planta\ they do not elicit the HR on non-hosts and they cannot multiply and cause disease in host plants. In general, hrp gene clusters are large and consist of many complementation groups. Usually, one gene in the cluster encodes a heat-stable, protease-sensitive, and glycine-rich protein called harpin, which has been found in P. syringae pv. syringae, Erwinia amybvora, and E. chrysanthemi and which is required for the elicitation of HR in tobacco and other non-host plants (Wei etal. 1992a). Harpin also contributes to the pathogenicity of P. syringae pv. syringae and E. chrysanthemi, but E. amylovora is the only bacterium in which it is strictly required for pathogenicity. The remaining genes in hrp clusters are needed for secretion of extracellular proteins, hrp gene regulation, and possible "disease specific" extracellular pathogenicity factors.
hrp genes have been found in all genera of Gram-negative plant pathogens except
Agrobacterium. Many of the genes in the hrp clusters are conserved and some are even 17 functionally interchangeable (Willis et al. 1991; Bonas et al. 1994). Most of the conserved hrp genes encode proteins that constitute a Sec-independent secretion apparatus. It is part of a family of secretion systems, termed type QI. that are involved in the secretion of extracellular pathogenicity proteins in bacterial pathogens of both animals and plants. Examples of animal pathogens that have type III secretion genes homologous to hrp genes are Yersinia pestis, Shigella flexneri and Salmonella typhimurinum
(Fenselau et al. 1992; Gough et al. 1992; Rahme et a l 1995).
The first bacterial proteinaceous elicitor of the HR was isolated from E. amylovora (Wei et al. 1992a). A cell free elicitor preparation (CFEP), obtained after boiling and centrifuging a cell sonicate, gave a strong HR when infiltrated into tobacco.
The activity of the CFEP was associated with a 44-kDa protein, termed harpies, This was purified by HPLC and the amino terminus was sequenced. To find the structural gene encoding harpinEa, an oligonucleotide probe corresponding to the ninth to fifteenth amino acid residues was hybridized to the hrp gene cluster. The corresponding gene was located in the middle of the hrp cluster and designated hrpN%a Harping is needed for both the elicitation of the HR on tobacco and other non-hosts and for pathogenicity on apples and pears. Insertion of Tn Stacl into the hrpN gene rendered E. amylovora HR - on tobacco and non-pathogenic on pears (Wei et al. 1992a).
E. chrysanthemi, a wide host range pathogen that rapidly kills and macerates host tissues, also produces a harpin. Although pectic enzyme production in wild-type bacteria masks the HR, A pelABCE Out- mutants of E. chrysanthemi elicit rapid necrosis in tobacco leaves. Tn/O mini-tan mutagenesis of hrpNEch indicated that this gene is required for the HR elicitation in tobacco, and contributes in a minor way to pathogenicity on witloof chicory (Bauer et al. 1994; Bauer et al. 1995). A similar HR-elicitor from P. 18 syringae pv. syringae (He etal. 1993), harpinp8S, encoded by krpZ, is required to cause wilting on peas, but it is not needed for pathogenicity on beans (Loniello et at. 1995).
Another HR-elicitor protein, PopA, is produced by P. solanacearum. ThepopA gene is adjacent to the hrp cluster and is co-regulated by HrpB. However, PopA is not needed for pathogenicity and is not the sole HR-elicitor made by this bacterium (Arlat et al. 1994); popA mutants are fully pathogenic on tomatoes and susceptible petunia lines.
Moreover, the popA mutant still produces a high molecular weight product that elicits an
HR on tobacco and petunia St40 ( Arlat et al. 1994). This suggests that additional proteins, acting as disease determinants, must be secreted via the Hrp pathway.
Harpins, despite differences in their amino acid sequences, have several physical and chemical properties in common; they are protease-sensitive, have open structures
(i.e., contain no cysteine), and are glycine-rich, heat-stable and acidic in nature (He et al.
1993; Arlat eta l. 1994). However, they seem to be quite different from each other regarding the length of the peptide needed for HR elicitation and the trans location recognition signal of the protein. The N-teiminal 128 amino acids of harpinea are needed for elicitor activity, and the C-terminus is responsible for secretion (Laby et al. 1994). In contrast, the elicitor activity of harpinEch and harpin pss is not confined to any one region of the protein (Alfano et al. 1994). In addition, harpinpss has two directly repeated sequences in its C-terminus, which are lacking in harpin(He etal. 1993). Secretion targeting signals also appear to differ. hrpN mutants of E. amylovora complemented with hrpNEck, and vice versa, could synthesize the heterologous harpin but were unable to secrete it to the exterior (Bauer et al. 1995).
E. stewartii also has a hrpAike gene cluster, termed wts (jyaier soaking), which is required for lesion formation and wilting on com (Coplin et al. 1986). This cluster is
28-kb in size and consists of seven complementation groups. The wts genes of E. 19
stewartii hybridize with hrp genes of E. amylovora and P. syringae pv. phaseolicola,
and some wts subclones functionally complement certain hrp mutants of E.
amylovora (Frederick etal. 1996; Laby and Beer 1992). This suggests that many of the
wts genes are structurally and functionally similar to hrp genes of E. amylovora.
However, wild type E. stewartii does not cause an HR in tobacco under normal assay
conditions, so we did not originally call them hrp genes. Nevertheless, we have recently
been able to demonstrate that E. stewartii can indeed incite an HR on tobacco and Datum
when we engineer increased expression of the wts regulon by putting wtsA under the control of a constitutive promoter (Frederick et al. 1996). Under a recent proposal to revise
hrp nomenclature, the wts genes will be renamed as hrp (Bogdanove etal. 1996). Since hrpN gene of E. amylovora which encodes harpin, a pathogenicity determinant, is located
in the part of the hrp cluster that colinearly hybridizes with thewts cluster of E. stewartii , we hoped to find a hrpN£a homolog in the wts cluster and wanted to investigate if this homolog would encode proteins with harpin-like functions.
In this study, we report the molecular cloning, characterization, and mutagenesis of the E. stewartii hrpN gene, characterization of harpin^, and an assessment of the role of harping in pathogenicity on com and multiplication in related grasses. 20
Materials and Methods
Bacterial strains, plasmids, and media
Bacteria and plasmids used in this study are listed in Table 1. All E. stewartii strains were derived from DC283 or DC356, which are spontaneous nalidixic-acid-resistant
(Nalr) and rifampicin-resistant (Rifr) mutants of wild-type strain SS104 (Coplin etal.
1981), respectively. Culture media, growth of bacteria, and mating conditions for E. stewartii have been described previously (Coplin 1978; Coplin et al. 1986). The following antibiotics were used in selective media in the amounts indicated (pg/ml); tetracycline, 20; kanamycin, 20; ampicillin, 100; nalidixic acid, 20; and rifampicin, 50.
Inducing medium (IM), which is optimal for expression of w/s genes, consisted of 100 mM 2-[N-Morpholino]ethanesulfonic acid (MES; Sigma Chemical, St. Louis), 2 mM
(N H ^SfX t,0.1% casamino acids (Difco, Detroit, MI), 1 mM potassium phosphate (pH
7.2), 1% sucrose, and 1 mM MgSO^ The pH of the medium was adjusted to 5.5 with
NaOH and then it was autoclaved for 20 min. Plasmids were mobilized from £. coli
HB101 into E. stewartii by pRK2013::Tn7 (Coplin et al. 1986) or introduced by electroporation using a BIO-RAD Gene P ulsarT M (Model 1652076) according to the manufacturer's protocol. Anti-harpinEl antibodies were a kind gift from Dr. S. V. Beer
(Cornell University).
General DNA manipulations.
Plasmid DNA isolation, agarose gel electrophoresis, restriction analysis, transformation, ligation, Southern hybridization, and random primer labelling were performed according to standard protocols (Sambrook et al. 1989). Non-radioactive
Southern blots were done with the Photogene Nucleic Acid Detection System (version 2.0) as described by the manufacturer (GIBCO BRL) except that Western Blue™ Stabilized 21
TABLE 1. Bacterial strains and plasmids
Bacterial strains or plasmid Relevant characteristics1 Reference or source
Bacterial strains Erwinia stewartii DC283 SS104 NaP Coplin et of. 1981 DC356 SS104 RiP Coplin et al. 1992 b DM760 DC283 hrpN189r.TnJ Frederick et al. 1996 D M 3020 DC283 Atvis Coplin et al. 1992b MAI DC283 krpN123: T nJ This study MA2 DC283 krpN254::Tn5 This study Escherichia coli HB101 thr leu Ihi recA hsdR hsdM pro Boyer etal. 1969 SM 17-1 Xpir Xpir lysogen of S I7-1thipro recA hsdR- hsdM+ S. V. Beer41 RP4-2-Tc::Mu SmrTpr BL21(DE3) hsdS gal (Xclts 857 indl 5am7 nin5 Studier and Moffatt lacUVS-T7 geneI) 1986 Plasmid pM A l 1.8-kb Hindm fragment cloned in pBS (SK), Apt This study pMA2 1.8-kb Nifldm fragment cloned in pT7-7, Apr This study pMA3 pMA2 with vectorrbs and ATG deleted. Apt This study pM A6 1,3-kb HituSSU fragment (containing This study hrpNEa) cloned in pBS (SK). Apt pRK2013::Tn7 ColEl mob* Smr Spf Tpt Jktw::Tn7 Dennis Deanb pDM2513 3-kb HindHUBamHl hrpN* fragment of pES411 cloned in pBluescript pKS+, Apt Doris Majerczak* pD M 2530 Same as above but cloned in pGP704, Apt Doris Majerczakc pD M 2510 1.1-kb HittdSUlBamW. fragment of pMAl cloned in Doris Majerczak* pBluescript SK '. Apt pDM2501 0.7-kb Hindlll/firunHl fragment of pMAl cloned in Doris Majerczak' pBluescript SK~, Apt pCPP430 Spt, hrp gene cluster o fErwinia amylovora Ea321 Beer et al. 1991 pCPP1012 4.2-kb Bom HI fragment (containingh r p N ^ from pCPP430 cloned in pBluescript KS', Apt S. V. Beer* pE S411 wts* clone (wisA. C, /, D, F, N, and E) in pVKlOO from E. stewartii chromosome Coplin et al. 1992a pRF205 1.8-kb WindUl fragment (containingwtsA) from pES1044 in pVKlOO Coplin et al. 1992b pT7-7 Cloning vector. Apt Stan labor0 pBluescript KS+ and SK' Apt Stratagene pLAFR3 Tcr cos IncP Knauf and Nester 1982 pGP704 OtiR6K tttob gp4, Apr. suicide vector Miller and Mckalanos 1988
* Nalr. Rif', Tcr, Smr, Tp4, Apr, Spr: resistant to naladixic acid, rifampicin. tetracycline, streptomycin, trimethoprim,ampicillii), and gpectinomycin, respectively. b Virginia Polytechnic Institute and State University, Blacksburg. * Department o f Biological Chemistry, Harvard Medical School. Boston. d Department o f Plant Pathology, Cornell University, Ithaca. e Department of Plant Pathology, The Ohio State University, Columbus. 22
Substrate for Alkaline Phosphatase (Promega Corporation, Madison, WI) was used for the detection of the hybridizing bands.
To obtain the nucleotide sequence of the 1.8-kb //indIII fragment containing hrpN. eight custom primers were designed from the previously sequenced DNA (Fig. 2). To sequence the hrpN promoter region, a primer was designed from the DNA sequence just upstream of the start of the ORF. The single-stranded DNA sequence obtained was compared to hrp boxes of other bacteria (Wei et al. 1995). All primers were synthesized by DNAgency, Malvern, PA (Table 2). Double-stranded DNA sequence templates were prepared as described by Applied Biosystems, Inc. (ABI, Foster City, CA). The PCR- based dye-terminator labelling reactions and the preparation of the labelled samples for sequencing were done as recommended by ABI. Automated sequencing was done on an
ABI Model 373A sequencer by the OSU Biological Instrumentation Center. Comparison of HrpNgg, HrpNg, and HrpNeCh was done with the Clustal V multiple sequence alignment program (Higgins et al. 1992).
Transposon mutagenesis and marker-exchange.
E. coli S I7-1 A,/?/r(pDM2530) was mutagenized with XTn5 as described by
Dolph et al. (1988). Two plasmids were identified that had insertions in the 1.8-kb ///ndlll fragment. Both of these plasmids were mobilized into DC283 and selected for Kim The pDM2530i.:Tn5 plasmid could not replicate in DC283 and integrated into the chromsome by a single cross-over. To allow for the excision of the integrated plasmid to occur, the
Apr Kmr transconjugants were grown in LB-Km broth for two days.
Harping* purification and Western blotting.
E. coli BL21(DE3/pMA2) cultures were grown overnight to stationary phase in
100 ml Terrific Broth containing 200 Hg/ml ampicillin (Sambrook etal. 1989) at 37°C. 23
MA2 A i 1 MAI DM760 r. — ? _ J Z r B J pDM2510 T i IT T3 pDM2501
D T7
Fig. 2. Physical and genetic map of part of the the £. stewartii wts region showing the location of the hrpN gene. The DNA sequencing strategy for hrpN is shown by the arrows below the expanded map of the 1.8-kb ffmdlll fragment. The lettered arrows represent sequence obtained from the primers listed in Table 2. The shaded arrow above the expanded map indicates the hrpN ORF and the direction of transcription. The shaded box indicates the 3-kb pDM2513 clone used for Tn5 mutagenesis. Filled circles show the location of Tn5 insertions hrpN123, hrpN189 and hrpN254 in mutants MAI, DM760 and MA2, respectively. B = BamWV, K = Kpnl\ H = //indlll. TABLE 2. Primers used in the automated sequencing of the hrpN region.
Primer Sequence %GC
A 5' CTCGTCATCAGGTCTTCG 3 ' 55
B 5 ' CCACTTCCCTGGCCAGCG 3 ' 72
C 5 ' GTGCCGAGCCTGAACCAC 3 ' 66
D 5 ' GTGGTTCAGGCTCGGCAC 3 ' 66
E 5 ' CGCTGGCCAGGGAAGTGG 3 ' 72
F 5 ' CGAAGACCTGATGACGAG 3 ' 55
G 5 ' CTGCCTGCCTTATGGTGC 3 ' 61
H 5 ' GCACCATAAGGCAGGCAG 3 ' 61
P 5 ' GATGTGCCCAGCGGACTC 3 ' 66 25
Cells were disrupted by sonication (sonicater model MS2T, Jewell Electrical Instruments,
Inc.) using 40% duty cycle and output control of 4, the sonicate was heated at 1(X)°C for
10 min, and then it was centrifuged to remove the denatured proteins (Wei et al. 1992a).
The resulting cell-free elicitor preparation (CFEP) contained partially purified harpin Es.
To further purify harping, the CFEP was separated on 8% preparative SDS-PAGE gels
(Laemmli 1970). The 43-kDa harpin e5 band was visualized with ice-cold 0.25 M KC1
(Hager and Burges 1980). The protein was electroeluted from gel slices for 4 h at 200 V
in Tris/glycine/SDS (25 mM Tris, pH 8.3; 192 mM glycine; 0.1% SDS) buffer using an
Elutrap apparatus (Schleicher and Schull). SDS was removed by precipitating the protein
with 80% acetone at -20°C (Hager and Burgess 1980) and then resuspending it in 50 mM
potassium phosphate, pH 6.5. Protein concentration was determined according to
Bradford (1976). However, harpinEt and harpinea used for biological activities
comparison in tobacco were quantitated by scanning the clearly visible bands of these
proteins on their Coomassie blue-stained PAGE gels with a densitometer (Hoefer Model
GS300) and comparing peak heights using known amounts of purified harping, as a standard.
To compare the biological activities of harping» and harping,, known amounts of the two proteins were infiltrated into panels of tobacco leaves and the proportion of the infiltrated area showing necrosis (%HR) after 24 h was recorded. Two independent experiments were carried out using at least three leaves for each dose of harping, and harping,. These data (%HR) were transformed to probit form (a method of data transformation that makes an S-shaped curve linear) and then a non-parametric t test was used to analyze these transformed data. Within each experiment, the amount of harping, needed to cause 50% HR was compared, for statistical significance, to the amount of harping, needed to incite 50% necrosis in tobacco leaves. 26
To extract harping, from E. stewartii strains containing pES411, the cells were
grown in 100 ml LB (with appropriate antibiotics) at 28°C to an A $4 0 of 0.8. Cells were pelleted and washed in 10 ml of IM, resuspended in 100 ml of the same medium, and incubated with shaking overnight For extraction from strain DC283(pRF205), the bacteria were grown overnight in LB at 28°C. CFEPs were prepared as above. To obtain extracellular harping, culture supernatants were boiled for 1 0 min immediately after centrifuging at 3,840 x g for 10 min, cooled to room temperature, and mixed with an equal volume of 10% TCA. The mixture was held on ice for 15 min and then centrifuged for 10 min at 20,400 x g. The precipitate was dissolved in 50 mM potassium phosphate buffer (pH 6.5).
For Western blotting, CFEPs were electrophoresed and electroblotted on an
ImmunoSelect (GIBCO BRL) nitrocellulose membrane (Sambrook et al. 1989). The filter was probed with anti-harping, serum and immunodetection of the bands was performed with rabbit alkaline phosphatase-conjugated secondary antibody (ProtoblotR II
AP system kit) according to the manufacturer's (Promega) protocol.
Production of anti-harping, antibodies.
Harping, antibodies were produced in two adult white New Zealand rabbits (3-4 kg body weight) in response to injections with harping, according to Ausubel et al. (1992) protocol. Electroelution-purified, acetone-precipitated harping, (1.5 pg/pl) suspended in
50 mM potassium phosphate buffer, pH 6.5 was used for this purpose. For primary immunization, 400 pg of the antigen was emulsified in 2 ml mixture (1:1) of Freund’s complete adjuvant and PBS (137 mM NaCl, 2.7 mM KC1, 4.3 mM Na 2HP0 4 -7 H20 . and 1.4 mM KH 2PO 4 , pH 7.4) and two 0.5 ml aliquots of the emulsion were injected subcutaneously into the hips of each rabbit. Four weeks later, two booster injections
(200 andlOO fig antigen respectively/rabbit emulsified in 2 ml 1:1 mixture of incomplete 27
Freund’s adjuvant and PBS), at 2 week intervals were delivered intramuscularly in the hips of each rabbit. Eight days after the second booster injection, the rabbits were bled from the marginal vein of the ear and small samples of blood were collected. IgG titer was measured by direct ELISA (Ausubel eta l. 1992), and the antiserum was harvested after 8 weeks by heart puncture. To partially purify the IgG, 18 ml antiserum was precipitated with concentrated ammonium sulfate (Ausubel et al. 1992), resuspended in 2 ml of PBS buffer and dialyzed against PBS containing 0.2 mM phenyl methyl sulfonyl fluoride (PMSF) to remove excess salt. The reactivity of the antiserum was confirmed by reaction with harping, in Western blots.
Inoculation of corn seedlings and corn*related grasses.
Sweet com seedlings (Earliking) were grown at 28'C and 16 h daylight in
20x10x2.5 flats containing soil:vermiculite:peatmoss (1:1:1). In order to assay the virulence of E. stewartii strains, 8 -day-old seedlings were inoculated with DC283 and hrpN mutants, using three different inoculation procedures as described in Appendix A.
The data obtained were used for calculating ED 50, response time, and doubling times etc. for the mutants and the wild-type. A paired t test was used for the analysis of ED 50 data and a non-parametric t test was used to analyze the response time data.
A diversity of corn-related grasses were tested for their ability to support the multiplication of wild-type strain DC356 and those that supported bacterial growth to >
105 CFU/gm or higher were selected (see Chapter IV). All grasses were grown in flats as described for corn. Twenty plants/strain/host were inoculated with DC356 and MAI hrpN123::Tn5, and bacterial populations were determined after various time intervals
(see Chapter IV). The relative numbers of the wild-type and mutant bacteria at different times were compared using paired t test with ni+n 2-3 d egrees of freedom. 28
HR assay in tobacco plants
Tobacco plants (Nicotiana tabacum L. var. Wisconsin) were grown in a greenhouse and then transferred to a controlled environment chamber several days before use. The chamber was maintained at 28 “C, 90% relative humidity, 16 h light and 8 h dark cycle. Bacteria were prepared by pelleting and resuspending overnight cultures in 10 mM phosphate buffer at a concentration of 5xl0 8 cells/ml (A54 0 = 0.52). Tobacco leaves were inoculated by pricking them with a dissecting needle and then forcing inoculum into the wound using the open end of a 3-ml disposable plastic transfer pipet pressed against the lower leaf surface (Bauer et al. 1994). The margins of the water-soaked infiltrated areas were marked and the plants were rated for HR development at 24 h. The percent of
HR for different strains was calculated as the proportion of the infiltrated area showing necrosis. 29
Results
E. stewartii can elicit an HR in tobacco
E. stewartii has not been reported to cause the HR in tobacco under normal assay conditions, although bacterial populations remain above SxlO7 CFU/g for at least 12 h, which is sufficient cell concentration and time to elicit an HR (Fig. 3). We hypothesized that this might be due to poor expression of the wts genes in tobacco or instability of a possible elicitor protein. Two approaches were used to increase elicitor production. In the first, plasmids pES411 and pRF205 were introduced into the wild-type strain DC283 to increase the copy number of certain wtt genes. Both plasmids contain wts A, a positive regulatory gene. pES411 contains the left half of the wts cluster (wtsE-F) and pRF205 contains wtsA+ transcribed from the pLAFR3 lac promotor, which is expressed fairly well in E. stewartii. The second approach involved growing cells in an inducing medium
(IM), which was developed to maximize wtsBv.lacZ expression (Coplin and Majerczak, unpublished). When DC283 and DC283(pES411) were grown in IM and infiltrated into tobacco and Datura, typical HR symptoms were observed within 24 h. DC283(pES411) gave a strong HR, whereas DC283 alone gave a weak and patchy HR on both plant hosts (Table 3). When DC283(pRF205) cells were grown in LB and used to inoculate tobacco leaves, a strong HR was observed within 24 h.
Molecular cloning and sequencing of theE. stewartii hrpN gene.
A 1.3-kb HindUl fragment from cosmid pCPP430, containing hrpN, was used to probe Southern blots of cosmid pES411 DNA restricted with HindUl, BamHl, and Kpnl
(Fig. 4A). The smallest pES411 fragment strongly hybridizing with the probe was a
1.8-kb H indIU fragment (Fig. 4B) located in the region of the wtt cluster that was expected to be colinear with hrpN ^ This fragment was subctoned into pBluescript SK 30
pcaaane -a-DcattM M — mai/ib -m — mai/im
9- -
6
0 5 10 15 20 25 30 40 50 HOURS
Fig. 3. Growth of wild-type strain DC283 and hrpN mutant MAI in tobacco leaves. Both strains were grown overnight in either LB or IM (pH 5.5), adjusted to 5xI08 cells/ ml (As4o= 0.52) and then infiltrated into tobacco leaves as described in the text. CFU/g (fresh weight) were determined at 0, 3, 6, 12, 24, and 48 h after infiltration by cutting out 1 cm diameter discs (two discs/sample and three samples/strain/interval) from the infiltrated area, grinding them in 0.85% saline, and dilution plating. The experiment was repeated once with similar results. Error bars indicate standard deviations from the mean. Only DC283/IM caused a visible HR at 24 h. 31
TABLE 3. HR assay in Datura and tobacco leaves of cells and cell-free elicitor preparations (CFEPs) of wild-type strain DC283, hrpN mutant DM760, and Escherichia coli DHSa harboring the Erwirna amylovora hrp cluster grown in different media.
Strain Growth HR on Datura^ HR on Tobacco^ medium cells® cells CFEPb
DC283 IM ++ + +/-
DC283 LB -- -
DC283(pES411) IM +++ +++ ++
DC283(pES411) LB -- nt*
DC283(pRF205) IM nt +++f ++
DC283(pRF205) LB nt +++ ++
DM760 IM - --
DM760(pRF205) IM ---
DH5a(pCPP430) LB +++ +++ ++
Phosphate buffer — ---
a Cells were grown overnight at 28°C in IM or LB, centrifuged, and nesuspended either in IM (pH 5.5) or 10 mM phosphate buffer (pH 7.2) respectively. Cell concentration was adjusted to 5x108 cells/ml. t> CFEPs were extracted from the respective strains (Wei etal. 1992) and then infiltrated into tobacco leaves. c Two-month-old pre-flowering Datura plants were used. d Two-feet tall tobacco plants (Nicotiana tabacum L. var. Wisconsin) were transferred from a greenhouse to a growth chamber (28°C) at least 1 week before infiltration. e nt = not tested; - = no HR; -/+ = weak HR (< 30% of infiltrated area necrotic); + - moderate HR (30-49%); ++ = strong HR (50-69%); +++ = very strong HR (70-100%). r Each value is the mean of three replicates and the experiment was repeated once with similar results. 32
K H 8BH K H B BH *
* 4 - b
B
Fig. 4. Hybridization of pES411 DNA with hrp N £a A = Restriction enzyme digestion of pES411 DNA. B. Southern blot of the gel in A probed with 32P-labelled hrpN&i DNA, Bands hybridizing with the probe (a - 2.5 kb, b = 1.9 kb, c = 1.8 kb, and d = 0.7 kb) are indicated by arrows. The two other hybridizing bands (1 kb in size and indicated by the asterisk) are of unknown origin. Gel B has been cropped just below the well. K = K p n, lH = //indlll, B = BamHl, and BH =BamW\l H indm double digest. 33 and the resulting clone was designated pMAl. However, the insert in pMAl is oriented opposite to the vector P tac promotor and all attempts to reclone this in the other orientation failed. Therefore, this clone could not be used for the complementation of hrpN mutants.
The nucleotide sequence of the entire 1.8-kb //indlll fragment of pMAl was determined and is shown in Fig. 5a. A 1.146-bp open reading frame was identified by homology with the E. amylovora and E. chrysanthemi hrpN genes and designated hrpN. A typical ribosome-binding site, consisting of GAGGAA was located 6 bases upstream of an ATG translational initiation codon. A promotor sequence, hrp box, (Fig.
5b and 5c) was found 74-bp upstream of the start of the hrpN ORF. The hrpN open reading frame encodes a 382 amino acid polypeptide that has a predicted molecular mass of 43-kDa and a pi of 4.28 (Chou and Fasman 1978). The polypeptide is rich in glycine (20%), has only three tyrosines and lacks cysteine. Unlike harpinp„, harpine* does not have C-terminal direct repeats, and as expected, has no apparent N-terminal signal sequence for Sec-dependent secretion. The protein is highly hydrophilic and does not show any transmembrane domains. Comparison of the amino acid sequences of the predicted harping and harpinga proteins revealed significant homology throughout their entire lengths except for a stretch of 10 amino acids in the N-terminus, and two regions of 9 and 11 amino acids in the C-terminus (Fig. 6). The overall identity and similarity of harping to harpinea is 58% and 78% respectively. HarpinEs is also homologous to harpinEch- The two proteins are 41% identical and 66% similar to each other with higher homology occuring in their C-terminal halves (Fig. 7).
Expression of h rp N in E. coli.
To confirm the production of harpin e& hy its predicted open reading frame and determine its molecular size, the 1.8-kb //tndlll fragment containing hrpN was cloned into vector pT7-7 behind the T7 promotor. This clone, designated pMA2, was used to 34
Fig. 5. Nucleotide sequence of the hrpN region showing open reading frame and putative promoter {hrp box) for the hrpN gene and promoter ( hrp box) for the wtsE gene of Erwinia stewartii, and comparison of these hrp boxes with those of other bactera. A. Nucleotide sequence of the hrpN region and deduced amino acid sequence of HrpNt*. A putative ribosome binding site (rbs), the translational start and stop codons and a potential terminator (for hrpN), and the translational start codon forvm£ are underlined. The locations of the Tn5 insertions in MAI, MA2, and DM760 are indicated by asterisks. B. Nucleotide sequence of the promotor region ( hrp box) of the hrpN gene o f E. stewartii. The promotor sequence 'is underlined. Only one strand was sequenced. C. Comparison of putative E. stewartii hrp promoter sequences ( hrp boxes) with those of other bacteria. The start of the E. stewartii hrpN and wtsE promoter sequences arc located 74-bp and 68-bp upstream of their respective translation start codons. N = any base, lowercase letter = £75% conserved, Es = Erwinia stewartii, Ea = E. amylovora (Wei and Beer 1993, Bogdanove et al. 1996), Ech = E. chrysanthemi (B aueretal. 1995). 35
1 AAG CTT TCC CCG GAC ACT CAC AT* GOG CAC CGT ATC CAA ACT AAT CAT CAC TTA TTC AAA
61 7AG GAA GAG GAC ATC AGT ATG AAT ACG ACT CCG CTG GGC ACA TCT GCG CTA CAA GTT ACT MSMNTSPLGTSALQVT 16
121 TTC GGT GGC AAT AAT GGT TTG ATG GGA ACG GAT TTA COT ACC GAC GGA TTG OGA TTA CTT LGGNNGLMCTDLRTDGLCLL 36
i s i TCC CAG CCA GOG CTC GGC GAG GOG AAA GGT CAC AAT GAA AGC ATC GAT CTG CTT GCA GOT SQPGLGEGKGHNES I DLLAA 56
241 GCC CTC ACT GGC ATG ATG ATC ATG ATC AGC ATG ATG GGC GGC GGC GGC CTT AGC AGC CTT A'LTGM M HHM SM HGGGGLSSl. 76
301 TTA GGT TCA GGC ACT GGG ATG GGG AAC TCA CCT TTC GGT GGT TCA GGC TCC OCA CCC GGG LGSGTGMGNSPFCCSGSAPG 96
361 AAC ACG CTG AGT GGA ACA TCG GOT GGT TCA CCT GGA GGT ACT ADC GGG GCT GGT TCG TCG N TLSG TSG G SPG G TTG A G SS 116 * MAI 42 1 CTA GGA CTT GAT CCG ACC CAG ACG GGT GAT GAT TCT CTC TCA GOC GCT GGC CAA ACA TCC LGLDPTQTGDDSLSGAGQTS 136
481 GGT ATG AGT CCG ATG GAA CAG TTG ATG AAA ATA TTC GCC GAT ATT ACG CAA AGC CTG TIT GM SPM EQLM KIFADITQSLF156
541 GGC GAT CAG GAT GGC GCA TCG GGA GGC AAC GCA GGG CGT CAA CCT TCT CAG GAT GAG CAA GDQDGASGGNACRQPSQDEQ176 * DM760 601 AAT GCA TAT AAA AAA GGT GTT ACG GAT GCC CTG ACT GOC T IT ATG GGA GGA GGC CTG AGC N A Y K KG VTDA LTA FMGGG LS 196
661 CAG GTT GCT GGA AAC GGA TCC GAA GGT GGA CTG GAT GGA GGC ATC GGG CTT GGT GGC GGT Q V A G NGS E G G L D G G M G LG G G 216
721 AAT GGA CTC GGC GGA AAA GGG TTA CAG GAT CTC AGT GGT CCT GCT GAC TIT CAG CAG TTC NGLGG KGLQDLSG PADFQCL 236 * MA2 78 1 GGT AAT GCG ATC GGT ACC GGT GTC GGT ATC AAA GCG GGC ATT GAG GCC CTC AAC AAT ATT GNAIGTGVGM KAGIEALNN I 256
8 4 1 GGT ACA CAC AGC GAT AGC AGT ACT CGC TCT TIT ATT AAC AAA GAG GAT CGC GCG CTG GCC GTHSDSSTRSFINKEDRALA 276
9 0 1 AQG GAA GTC OGA CAG TIT ATC GAT CAA TAT CCC GAA ACT TTT GGC AAA CCC CAG TAC CAA REVGQFM DQYPETFGKPQYQ 296
961 AAA AAC GCA GAT TCG GCA CTA AAG ACC GAT ACG AAA TCC TCG GCT GAA GCA CTC AGT CAA KNADSAVKTDTKSWAEALSQ316
1021 CCA GAC GAT GAC GGT ATC ACG CCT GOC AGC ATC GAG CAA TTT AAT AAA GCC AAA GGC ATC PDDOGMTPASMEQFNKAKG I 336 36
1081 ATC AAA AGC OCT ATG GCT GGC GAT AAT GGC AAC ATC AAC CTT CAG GCA CGT GGC GCA GGC T K S AM AGDMG N r N L Q AR GAG
1141 GGA TCA TCA ATG GGT ATT GAT GCC ACC CTC ACG GGA GAT GCC ATA AAC AAT ATG GCA CTG GSS MG ID AT LT GDA I NN MA L
1201 ACC AGA CTC AGT GCG GCA TAA AGC ACC ATA Afifi s c a s s g ACT .ZEC. 2BC. ,£££. T R LS A A #
1261 TCA CGG ACC TCC AAT AAA TOC TCG CPC CGC GAA AGT CTT TCC GGG TAA ATA AAT CAT GTT
1321 ACT TCA CCA IT T GCC CGG TOC OCC TTC CCA GCA ATG ATT CCC CCC TCG GAC CGC TAA TCA
1381 TCA TTC TCA CGC ATT CCT TAT T IC ATC GTT ATA AGA ACA CGC OCC ATA AAT ATC ATT TCA
1441 CTA TAG GGA AAA AGT TTT GGG TAC ACG TTC TCA AAC ACT GTT CAT CCT TCG C IT GAG GCT
1501 TTA TTC AAC AAA GTC GAA CTA TCC ACG CAA ACT CAC CAC ACA AAG ATA ATT AAT GTT.LTT
1561 TTC GGA AGA CCT GAT GGC GAG GGG GGA TTA TCA GAT GAA T IT GCA CGC AAC AGA AAA GAA
16 2 1 AAC AAC CGT TCA GAA TCT CGA AAA TCC TAA TAA TTC TAC AAT CCC GCC ACT GCA ACA GGG
1681 AAG CAG CAG CAG CGC CCC TCA GGC GTC AGG CGG AAC GCT AGC CAG CGA GGG TAA AAA CAT
1741 TOC CAG TAT OCC CGC TAT TCG TCA GCA IT T ATC CGC TCA TCG TCA AAA TCG TCC TCC CCG
18 0 1 OCA AAA AAA TAA AAG CTT
A. Nucleotide sequence of the hrpN region and deduced amino acid sequence of HrpN^s. A putative ribosome binding site ( rbs ), the translational start and stop codons and a potential terminator (for hrpN), and the translational start codon for wtsE are underlined. The locations of the Tn5 insertions in MAI, MA2, and DM760 are indicated by asterisks. 37
1 CTG T TATC TC TGTTTGG AC AAGTGAG AACAAACTTT CTTGCTGGTATCAGT TAAGGGAAG
61 AGGGCGCTGATGACAAGCATCAGATTGCACAACTTTTTCAGCTAGCCGGCATCGAAACTG
121 AAGTCTGGATCTGAGTAAGTTTCATCTTTTATGATCTTCCACCTACCTCTGCTAACCCAC
181 TACGCTTTGTATTTACAGCTTTTTTCCACATCGCTGTGCCGCTTTTATTTGCAAATTTCG
2 4 1 T T CTTC T GACCCAAAC GAAAG TCACGC CC T GT AAACGTCAAAT TCCCCCATCACG TGTGA
3 0 1 TTATTTTC AT T TCAGACAGGAACCAGCACCGCGATAGC GGCACTTAATAAAAGC TTTGCC
361 CGGACACTCACATAGCGCACCGTATGCCAAGTAATCATCACTTATTCAAAGAGGAAGAGG
421 ACATGAGTATGAATACG
B. Nucleotide sequence of the promotor region (hrp box) of the hrpN gene of E. stewartii. The putative promotor sequence and the start codon of the hrpN ORF are underlined. Only one strand was sequenced. 38
Es hrpN TTTCAGACAGGAACCAGCACCGCGATA-GCGGCACTTAATAAAAG Es WtsE C AAC AAAGTGGAACTATCC AC GC - AAAC TC AC C AC AC AAAG ATAA Ea hrp Consensus CNNNNcNNNGGAACNNNNNNNNNNNNNNNCNcCACNNAatNNNNg Ea hrpN CAC TTCGCCGGAACCAGAGCGG - AATAACCAGCACTCAATATAAG Ea hrcC CTTCACCACGGAACTCCGCCACGCCCGAACCCCACTCAAAGACAG Ea hrcJ CGCCGCGTGGGAACCGATCGAA-ACTGCCCGCCACTTAATTAACG Ea ArcV CATGGCCAGGGAACCGATGGCT - C AATCGCACC AC AC AATGACAA Ech Ar/W TAACGGTGAGGAACCGTTTCAC-CGTCGGCGTCACTCAGTAACAA
C. Comparison of putative E. stewartii hrp promoter sequences (hrp boxes) with those of other bacteria. The start of theE. stewartii hrpN and wtsE promoter sequences are located 74-bp and 68-bp upstream of their respective translation start codons. N = any base, lowercase letter = £75% conserved, Es = Erwinia stewartii, Ea = E. amylovora (Wei and Beer 1993, Bogdanove et al. 1996), Ech = £. chrysanthemi (Bauer et al. 1995). 39
H fJ?!68 MSMNTSPLGTSALQVTLGG NNgLMGTOT.RTrvrr.rrr.TgQ pC T ^g^g^ MSLKTSGLGRSXMQISIGGAGSHNGUJGTSRQMat **.*** **.*..*...** ****** * *** * • *** * * * ^I^E e MESXOIJAAftLTQ——iStMGSGgL-RgT^^CCTg^BMSPFffffP^P^Pg “PNeb NPTVWDlLAGIJ.TGMWiStMGGGGLMGGGT.ms^ y .^ i ^ ^ ; yfaTr.CTCT,c * **- **************** * * * * * ** * * • HrpNes NTLS------GTSGGSPGGTTGAGS------SLGLDPTQTGDDSLSGAG I lr PNEa NALNDMLGGSLNTLGSKGGHNTTSTTNSPLDQALGINSTSQNDDSTSGTD *. ** * •* « • * *** ** HrpNes QTSGMS-PMEOLMKIFADITQSLFGD-ODGASGGNAG-ROPSODEQNAYK H rpN es STSDSSDPMQQtLKMFSEIMOSLFGDGODGTOGSSSGGKOPTEGEQNAYK ** ^ * w*^**.*.*^* ****** *»* _ * * _ ** ___ ****** HrpNes KGVTDALTAFM3GGLSQVAGNGSEGGLDGGMGLGG— GNGLGGKGLQDLS HrpNe* KSVTDALSGLMSNGLSQLLGNGGLGGGQGGNftGTGLOGSSLGGKGLRGLS ******* . . . ** **** ^ ***_ ** _ «•* * **««-*** ** * * HrpN£6 GPADFQOLGNAIGTGVGMKAGIEALNNIGTHSDSSTRSriNKEDRAIARE HrpNea gpvdyqqlghavgtgigmkagioalndigthrhsstrsfvnkgdramake HrpNEs VGQFMDQYPETFGKPQYQKNADSAVKrDTKSWAEALSQPDDDGMTPASME H rpN es IGQFMDQYPEVFGKPQYQKGPGQEVKTDDKSWAKALSKPDDDGMTPASME • ******** • ***** ***# *** ************ i H rpN es OrNKAKGIIKSAMAGDNGNINLQARGAGGSSMGIDATLTGDAINNMALTR H rpN es QFNKAKGMIKRPMAGDTGNGNLOH------AVPWL------R ********* _**** ** *** * * HrpNes LSAAX H rpN es WVlHP Fig.6. Predicted amino acid sequences of HrpN^ and HrpN^, aligned by the Clustal V multiple sequence alignment program. Stars indicate identity and dots show similarity of corresponding amino acids. 40 H rpN ga MSMNTSPLGTSALQVTLGGNNGLMGTDLRTDGLGLLS— QPGLGEGKGH- HrpNgch MQI------TIKAHIGGDLGVSGLGLGAQGLKGLNSAAS SLGSSVDKL * * * * HrpNes NESIDLLAAALTGMMMMKSMMGG-GGI.SSLLGSGTGMGNSFFGGSGSAFG HrpNgch S STIDKLTSALTSMMFGGALAQGLGAS SKGLGMSNQLGQS -FGNGAQGAS * * * • * *• HrpNes NTLSGTSGGSPGGTTGAGSSLGLDPTQTGDDSLSGAGQTSGMSPMEQLMK HrpNech NLLSVPKSG ------GDALSKMFDKALDDLL------GHDTVTKLTN HrpNEa IFAD ITQSLFGDQDG ASGGNAGRQP SQDEQNAYKKGVTDALTATMGGGLS HrpNgch QSNQLAMSMLN------ASQMTQGNMNAFGSGVNNALSSXLGNGLG H rpNss QVAGNGSEGGLDGGMGLGGGNGLGGKGLQDLSGPADFQQLGNAIGTGVGM H rpN sch QSMSGFSQPSLGAG ------GLQGLSGAGAFNQLGNAIGMGVGQ * * * HrpNe* KAGIEALNNIGTHSD S S TRSFINKEDRALAREVGGFKDQYFETFGKPQYQ HrpNrch NAALSALSNVSTHVDGNNRHFVDKEDRGMAK£IGQFMDQYPEIFGKPEYQ . * . * * * * * * * •* * *t** « - * • *« HEPNes KNADSAVKTDTKSWAEALSQPDDDGMTPASMEQFNXAKGIIKSAMAGDNG HrpNecii Iffl GWS5 PKTDDKSWAKALS KPDDDGMTGASMDKFRQAMGMIKSAVAGDTG * . . *. *** *»*• **W *««*»** * V« * * • • «*• * HrpNes HIHLQARGAGGSSMGIDATLTGDAIHNMAI.TIU.5AAX HrpNsch NTNLNLRGAGGAS LG ID AAWGDKIANMS LGKL-ANA Fig.7. Predicted amino acid sequences of HrpNgs and HrpNEcb aligned by the Clustal V multiple sequence alignment program. Stars indicate identity and dots show similarity of corresponding amino acids. 41 express harpin es in E. coli BL21(DE3) (Studicr and Moffatt 1986). The estimated molecular mass of this protein, as determined by SDS-PAGE, was 44-kDa, which agrees with sequence data (Fig. 8 ). Purification of harping, and Its biological activity The electroelution-purified, acetone-precipitated harpin es produced in E. coli was electrophoiessed several times on SDS-PAGE (12%) gels to check its purity. The protein was found to be almost 85-90% homogeneous (Fig. 8 ) and was used to raise antibodies in rabbits. The resulting serum reacted with the 43-kDa harping band in Westerns, but not with the pre-immune serum. Likewise, the antiserum did not react with the CFEP from a Awrs mutant (Fig. 9). In addition, antibodies to harping cross reacted with harpinEs and vice versa. The CFEPs prepared from E. coli BL21(DE3/pMA2) and E. coli BL21 (DE3/pMAl) caused a typical HR in tobacco leaves within 18 h, whereas those identically prepared from E.coli BL21(DE3/pT7-7), and E. coli BL21(DE3/pBluescript SK) did not produce any necrosis. In comparison of harpinES and harpinEa on the same tobacco plants under similar conditions, 22 jig/ml of harping were needed to cause a 50% HR but only 12 |ig/ml of harpinF.a produced the same result. In another independent experiment, 49 jig/ml of harpinEs were needed to cause 50% HR in tobacco leaf panels whereas only 14 pg/mi of harpin Ea incited same amount of necrosis. Therefore, the HR-elicitor from E. amylovora was considered to be 2 to 2.5 fold more active than that from E. stewartii (Fig. 10). This difference in activities within each experiment was statistically significant. When the concentrated culture supernatants of DC283(pRF205) cells grown overnight in LB at 25 “C, 28*C, and 32*C were used for Western blots, the anti-harpin serum detected harpin in all of the samples (Fig. 11). 42 Fig. 8 . SDS PAGE of electroelution-purified Harping,,. Lane M, molecular weight markers with sizes in kDa shown to the left; Lane H, Harping. Fig. 9. Western blot of CFEPs from wild-type strain DC283 and several ftrpN mutants reacted with antiserum to harpin e«- All strains contained pRF205 to increase harpin production. Harpin is indicated by the arrow. The low molecular weight cross-reacting bands in lane A are due to harpin degradation. H » harping, A =DC283(pRF205), B = MAl(pRF205). C = MA2(pRF205), D = DM760(pRF205), E = DM3020(pRF205), F = harping The gel has been cropped below the well. 44 100 75- g 50- 25- 0 10 20 30 40 50 60 70 Ug/ml Fig. 10 Comparison of biological activities of harpinEs and harping in tobacco leaves. Harping was extracted (Wei et al. 1992) from E. coli BL21(DE3/pMAl) and harpinEa from E.coli BL21(DE3/pMA6). Both harpins were quantitated by scanning their Coomassie blue-stained bands on SDS PAGE with a densitometer using known amounts of purified harping as a standard. The figure shows that 44 jig/ml of harpinEs were needed to cause 50% HR in tobacco leaves whereas only 11 pg/ml of harping produced the same result indicating that harping* is four-fold more active than harpinE*. A second independent experiment indicated that harpinEl was two-fold more active than harpinEs. A = % HR caused by harpin ej (n = 3 i.e. three tobacco leaves/dose/data point were used, Standard error ranged from 16 to 56%). B = % HR incited by harpinEa (n = 3, Standard error ranged from 3 to 22%). % HR = the visually estimated % of necrotic tissue in the infiltrated area. 45 Fig. 11. Western blot analysis of harpinEs production and secretion by DC283 (pRF205) grown at three different temperatures. Harpin is indicated by the arrow. Cells were pelleted and discarded. Equal volumes of culture supernatants (CS) were immediately boiled, then cooled down and concentrated as described in the text. The concentrated samples were used for SDS PAGE and Westerns, h = purified harpin; a = CS from cells grown at 25'C; b = CS from cells grown at 28°C; c - CS from cells grown at 32°C. The get was cropped at well. 46 Mutagenesis of h rpN Our lab previously isolated a Tn5 insertion mutation near the center of the putative hrpN ORF and marker-exchanged it into wild-type strain DC283 to produce strain DM760 (Frederick etal. 1996). This mutant was HR- on tobacco but fully pathogenic on com (E. Stover, unpublished). Although this result suggested that hrpN might not be required for pathogenesis, additional hrpN :: Tn5 mutants were needed to rule out the possibility that the pathogenicity of DM760 was due to the production of a truncated harpinEs molecule that was still biologically active. Two new Tn5 insertions into hrpN were isolated and marker-exchanged, were marker-exchanged into the genome of wild-type strain DC283 to produce strains MAI and MA2. Marker-exchange of the transposon mutations and excision of pDM2530 were confirmed by Southern blotting (Fig. 12). Direct sequencing placed the respective mutations in DM760, MAI and MA2 at amino acids 189, 123 and 254 from the N-terminus of the HrpNe& peptide. To confirm that the mutants did not produce truncated harpin£s proteins, the CFEPs from the mutants and wild type strains containing pRF205 were assayed for harpin in Western blots using harpin e* antibodies. The CFEPs from the mutants did not contain any cross-reacting proteins, whereas a protein ladder, typical of harpin, was observed in the parent strain. In tobacco leaves, the mutants failed to elicit any necrosis at 2.5 x 10s cell s/ml, even when wrs gene expression was increased by the introduction of pRF205. In contrast, DC283(pRF205) caused a strong HR within 18 h (Fig. 13). To complement the hrpN mutants, the wild-type cosmid pES411 and a derivative containing hrpN\.Tn5 were introduced into them by triparental matings. The HR phenotype of the hrpN ::Tn5 mutants, with and without pES411, was tested in tobacco. Panels of leaves were infiltrated with 2.5x108 cells/ml of 47 H in d III Bam H I 1234 56709 Fig. 12. Southern blot of genomic DNA from wild-type strain DC283 and hrpN mutants to verify marker-exchange of Tn5 insertions into the chromosome. Genomic DNA was digested with //jndHl or BamHI, electrophoresed through an 0.8% agarose gel, stained with ethidium bromide, photographed and then used for Southern transfer. The blot was probed with the 1.8-kb //mdlll insert from pDM2507 containing the hrpN region. 1.8-kb ///ndlll fragment (lower bands in lane 1 and 9) and 2.4-kb BamHI fragment (lower band in lane 5) have been replaced by two Tn5 junction fragments of the predicted sizes in the hrpN mutants. Lane 1 and S = DC283 DNA; lane 2 and 6 = MAI DNA; lane 3 and 7 = MA2 DNA; lane 4 and 8 = DM760 DNA; and lane 9 = //mdlll-d igested pDM2507 DNA bands (upper = 3-kb vector and lower = 1.8-kb insert DNA fragment). Gel has been cropped just below the well. 48 Fig. 13. HR assay in tobacco leaves at 24 h with purified harpinEs, wild-type strain DC283(pRF205) and various hrpN mutants. HarpinEs (90 pg / ml) was suspended in 50 mM KPO4 , pH 6.5 and bacteria were suspended (2.5x10s cells/ml) in inducing medium pH 5.5. Tobacco leaves were infiltrated with bacteria or harpin as described in the text. The experiment (three replicates/strain) was repeated twice with similar results. 1 = HarpinEs, 2 = DC283(pRF205), 3 = DM3020(pRF205), 4 = MAl(pRF205), 5 = MA2 (pRF205), 6 = DM760(pRF205). 49 DC283(pES411) and those of the mutants, MAl(pES411), MAl(pES411 hrpN 7S9::Tn5), MA2(pES4U), MA2(pES41 IhrpNJ 89::Tn5), DM760(pES411) and DM760(pES41 \hrpN189::Tn5). Only DC283(pES411), MAl(pES411), MA2(pES411), and DM760(pES411) produced a necrotic response (Fig. 14). Likewise, when the CFEPs from the above strains were tested, only the CFEP from DC283(pES411) and those from the mutants with pES411 gave an HR or reacted with anti-harpin£g serum in Western blots (Fig. 15). These results indicate that the hrpN gene is required for harpin production and the elicitation of the HR by E. stewartii . The phenotype ofkrpN£t mutants could not be complemented by HrpNta. Electroporation of the plasmid pCPP1012 into MAI, MA2, and DM760 did not enable any of these mutants to cause an HR on tobacco at 5x108 cells'ml, nor did the concentrated culture supernatant from any of the three transformants produce necrosis in tobacco tissue (data not shown). However, when cells of the interspecifically complemented mutants were disrupted by sonication and the CFEPs were infiltrated into tobacco leaves, all three extracts caused a strong HR in tobacco leaves within 18 h (Table 4). Furthermore, the CFEPs revealed a 44-kDa protein band in SDS-PAGE gels, which reacted with both anti-harpin (Fig. 16) and anti-harping sera (data not shown) in Western blots. HarplnEs'deficient mutants of E. stewartii are fully pathogenic on corn. The pathogenicity of mutants MAI hrpN J23::Tn5, MA2 hrpN 254::Tn5 . and DM760 hrpN 189-TnS on com was compared to that of wild-type strain DC283 with respect to ED 5 0 (Table 5), disease severity (Table 6 ) , and response time (Fig. 17). ED 5 0 ranged from 17 to 26 cells/plant and were not significantly different (P = 0.05). The differences between the response times for mutants and the wild-type were not statistically 50 Fig. 14. HR assay in tobacco leaves at 24 h with wild-type strain DC283(pES411) and various hrpN mutants complemented with pES4II and pES411 hrpN 189::Tn5. Bacteria were suspended (2.5x10* cells/ml) in inducing medium pH 5.5. pES41 I hrpN 189::Tn5 has a transposon insertion in the hrpN ORF at amino acid position 189. Tobacco leaves were infiltrated with bacteria as described in text. The experiment (three replicates/strain) was repeated once with similar results. 1 = DC283(pES411), 2 = DM3020(pES411 hrpNI89::TnS). 3 = MAl(pES411), 4 = MAl(pES411 hrpN 189:^5), 5 = MA2 (pES411), 6 = MA2(pES411 hrpNJ89::Tn5)t 7 = DM760(pES411), 8= DM760 (pES411 hrpN189::Tn5). 51 Fig. 15. Imraunoblot of cell-free elicitor preparations from DC283(pES411) and several hrpN mutants complemented with pES411 reacted with anli-harpinEs serum. pES41 \hrpNI89v.Jn5 has a transposon insertion in the ORF of hrpN gene at amino acid position 189, Harping* is indicated by the arrow. The low molecular weight cross-reacting bands are due to degradation products of harpin. W = DC283(pES411), A = MAI (pES411), B = MAl(pES411 hrpN189::Tn5 ), C = MA2(pES411), D = MA2(pES41 \hrpN189:\Tn5)y E = DM760(pES411), F = DM760(pES411 hrpN 189::Tn5), H = HarpinEs The gel has been cropped below the well. 52 TABLE 4. Tobacco HR assay of cell-free elicitor preparations (CFEPs) from E.stewartii hrpN mutants containing Erwinia amylovora hrpNga+ clone pCPP1012. % HRb by CFEPs* Strain Expt. 1 Expt. 2 Expt. 3 Mean±S.D MAl(pCPP1012) 100c 1 0 0 1 0 0 1 0 0 MAl(pRF205) 0 0 0 0 MA2(pCPP1012) 85 90 70 81 ± 1 0 MA2(pRF205) 0 0 0 0 DM760(pCPP1012) 90 8 8 95 91 ± 3 DM760(pRF205) 0 0 0 0 harpin 1 0 0 70 95 8 8 ± 16 * CFEPs were extracted from bacteria grown overnight at 28"C in IM(pH 5.5) according to Wei et al. (1992) and then infiltrated into tobacco leaves. b % HR was determined by estimating the percentage of the infiltrated area that was necrotic at24h. c Each value is the mean of three replicates± Standard deviation from the mean. 53 Fig. 16. Immunoblot of cell-free elicitor preparations from E. stewartii hrpN mutants complemented with E. amylovora hrpN-containing clone pCPP1012 reacted with harpinEa antiserum. The low molecular weight cross-reacting bands are due to harpin degradation. The experiment was repeated once with similar results. H = harpinEa* A = DM760(pCPP1012), B = DM760, C = MAl(pCPP1012), D s MAI, E - MA2 (pCPPlOl 2), F = MA2. The gel was cropped just below the well. 54 TABLE 5. ED50S of wild-type strain DC283 and hrpN mutants on com seedlings. ED50* Strain Experiment lb Experiment 2b Average DC283 17 2 0 18±2 DM760 26 27 26±1 MAI 26 40 33±10 MA2 2 0 27 24±5 •Number of bacteria/plant needed to cause disease symptoms in 50% of the inoculated plants. *>Plants were inoculated (pseudo-stem inoculation method) with different concentraions of the appropriate strains as described in the text Thirty plants/strain/concentration were used. ED50 values were determined graphically by plotting dose (bacterial cell numbers) vs the probit of the proportion of the inoculated plants showing disease symptoms eleven days after inoculation. TABLE 6 . Disease severity rating of wild-type strain DC283 and hrpN mutants DM760, MAI, MA2, and DM3020Awrr on 8 -day-old com seedlings using three different inoculation methods. Strain Pseudo-stem* Tooth-pickb WhorK DC283 4.3d ±0.8 A* 4.98 ± 0 . 2 A 2.77 ± 0.3 A DM760 4.6 ±1.1 A 4.97 ± 0 .2 A 2.64 ±0.7 A MAI 4.2 ±1.2 A 4.88 ±0.3 A 2.67 ±0.6 A MA2 4.4 ±1.1 A 4.98 ±0.3 A 2.53 ±0.7 A DM3020 1.1 ±0.4 B 0 . 0 0 B 0 . 0 0 B * Five |il of inoculum (1,250 cells/plant) were pipeted onto the cut ends of decapitated 8 -day-old seedlings (grown as described in the text) and plants were rated 1 0 days after inoculation using a 1-5 scale: 1= no symptoms, 2 = scattered lesions, 3 = slight wilting, 4 = severe wilt, and 5 = dead. bThe seedlings were inoculated with sterile toothpicks dipped into fresh bacterial cultures and inserted into the plants 1 cm above the soil line; the plants were rated as above 1 0 days after inoculation. cTwo hundred M-l of inoculum (10 7 cells/ml) in 0.01 M potassium phosphate buffer (pH 7.0) containing 0.2% Tween 40 were placed in the whorls of the seedlings: the plants were rated 1 0 days after inoculation using the following 0-3 scale: 0 = no symptoms, 1 = a few lesions but no ooze. 2 = many lesions and some ooze, and 3 = coalescing lesions and ooze. d Each value is the average of 25 plants rated ± standard deviation from the mean; the experiment was repeated once with similar results, e Means followed by the same letter are not significantly different at P = 0.05 level according to the paired t test 56 1 D C 283 MA2 Days Fig. 17. Response lime of wild-type strain DC283 and hrpN mutants on sweet com seedlings. Plants were inoculated (pseudo-stem method) with 1,250 cells/plant as described in the text. Thirty plants per strain were used. At intervals of 1, 3, 5, 7, 9, and 11 days after inoculation, any plants showing disease symptoms were counted. The experiment was repeated twice with similar results. At any of the above intervals, the proportion of DC283-inoculated plants showing disease symptoms was not significantly different from the proportion of DM760-, MA1-, or MA2-inoculated plants showing disease symptoms using a non-parametric t test with n i+n 2 - 2 degrees of freedom and P = 0.05. 57 significant at P = 0.05. The results show that mutations in hrpN did not affect the pathogenicity of E. stewartir, the mutants were neither quantitatively nor qualitatively different from the wild-type controls. To measure the ability of the hrpN mutants to grow and persist in planta„ 7-day-old com seedlings were inoculated with DC283, MAI and MA2 grown in IM (pH 5.5). Bacterial populations were determined from 0 to 192 h after inoculation. There was not a significant difference between the wild-type and mutants in their ability to grow in planta (Fig. 18). Populations increased up to 48 h with doubling times of 7, 7, and 6 h for DC283, MAI and MA2, respectively, and then leveled off. Similar results were obtained by E. Stover (unpublished) for DM760. 58 —• —MAI —* —MA2 0C283 10 9 . 5 8.5 - 7.5 0 20 00 80 100 120 180 180 2 0 0 HOURS Fig. 18. Growth of wild-type strain DC283 and hrpN mutants in com seedlings. Plants were inoculated (pseudo-stem inoculation method) with DC283 hrpN+, MAI hrpN J23::Tn5 , and MA2 hrpN254::Tn5 as described in the text. At intervals entire plants were sampled (two plants/sample and three samples/strain/interval) as described for grasses in Chapter IV. The experiment was repeated once with similar results. Bacterial populations are given as the log CFU/g (fresh weight). Error bars indicate standard deviations. 59 D iscussion We demonstrated that E. stewartii produces a harpin which is homologous to harping throughout its entire length and to the C-terminal half of harping- Comparison of these three proteins suggests that the E, stewartii and E. amybvora harpins are more closely related to each other than they are to harpin from E. chrysanthemi. Nevertheless, all three proteins share many physical and chemical properties with harpins from other plant pathogenic bacteria. w tsB , w tsD , w tsF , and wtsE have been shown to be regulated by wtsA (Frederick et al. 1996; Frederick and Coplin, unpublished). The observation that the harpin content of CFEPs is greatly increased by the presence of a wtsA + plasmid suggests that hrpN is part of the same regulon as the other wts genes. hrpN appears to constitute a separate transcription unit. This is supported by the following observations: i) mutations in the upstream complementation group wtsF do not have a polar effect on hrpN, ii) mutations in hrpN do not have a polar effect on the downstream operon w tsE , iii) Tn5 insertions mapping immediately upstream and downstream of the hrpN are still HR + and pathogenic (Frederick et al. 1996), and (iv) the hrpN ORF is preceded by a hrp consensus promoter (hrp box) and is followed by a fairly strong terminator, and another hrp box, 5' to wtsE. The detection of harpin Eg in culture supernatants from DC283(pRF205) cells grown at three different temperatures suggests that harpinEs secretion is not restricted to any of the temperatures tested. This is in contrast to the finding of Wei et al. (1993) that E. coli DH5a(pCPP430) produces harpin£a at both 37 °C and 25°C but exports it to the medium only if cells are grown at 25*C. In £. amylovora, hrp gene expression is 60 favored by low temperature (Wei et al. 1992b). Nevertheless, our results are only qualitative, so it is possible that we missed a more subtle effect of temperature on harpine$ secretion. Although harpins have extensive sequence homology, it appears that the secretion signal is species-specific. E. stewartii synthesized harping but could not secrete the heterologous protein, suggesting that the property of harpinEs which targets it for secretion is different from that of harping. In similar experiments, Bauer etal. (1995) reported that E. chrysanthemi and E. amylovora could not secrete each other's harpin. A similar problem has been noticed with heterologous secretion of pectic lyases and cellulases via the Out pathway in E. chrysanthemi and E. carotovora. (He et al. 1991; Pye e ta l . 1991). Although E. stewartii produces harpin, it does not cause HR in tobacco unless it is either pre-induced by growing the cells in IM or genetically induced by introduction of plasmids that increase the level of WtsA. This could be due to the following reasons: i) the bacterial cells quickly die following infiltration into tobacco tissue; ii) harpinEs is unstable in tobacco tissue; iii) the wts genes are poorly expressed in tobacco; or iv) harpinEs is less active on tobacco than other harpins. Failure of the bacteria to grow is probably not a factor because populations remained above 5xl0 7 CFU/g for at least 12 h, which is sufficient cell concentration and time to elicit an HR. Since pre-induced cells cause an HR, this rules out the possibility that harping, is especially unstable in tobacco. Therefore, the third and fourth possibilities i.e. the poor expression of hrpN and less biological activity of HrpNg, in tobacco appear to be appropriate. This is also supported by the observation that constitutive expression of WtsA allows E. stewartii to cause an HR in tobacco and, we have observed that harping, has at least 3-fold less activity in tobacco than harping,. The questions why E. stewartii hrpN is poorly expressed and 61 why harping* is less active than harping, in tobacco as well as why harpinEs causes less membrane depolarization in tobacco suspension-cultured cells than harping, at equivalent protein concentrations (Novacky, A. personal communication) remain to be answered. Mutagenesis of the E. amylovora hrpNga gene has clearly indicated that it is required for both elicitation of HR and pathogenicity (Wei eta l. 1992a). However, its role in pathogenesis is not yet clear. He eta l. (1993) suggested that harpin likely functions in pathogenesis to release nutrients from plant cells into the apoplast. Nevertheless, purified harping,, in contrast to live bacteria, does not cause necrosis in susceptible apple leaves or immature pear fruit. Nor does it elicit a K+ efflux / H+ influx reaction in apple cell suspension cultures. In an extracellular complementation test, harping, added to inoculum could neither restore pathogenicity to hrpNEa mutants nor their ability to grow in host plants, although it was found to be relatively stable inside host tissue (Bogdanove et al. 1994). This suggests that harpin probably requires additional co-acting proteins in order to affect host cells, or it must be introduced directly into apple cells by the Hrp secretion system. Currently, there is no direct evidence for the transfer via a type III secretion system of elicitor proteins from a plant pathogen into a plant cell. However, the extensive similarity between the hrp gene cluster of E. amylovora and virulence loci of Yersinia spp. strongly suggests this possibility (Bogdanove et al. 1996). Type 111 protein secretion systems have been found in a number of enteric bacteria pathogenic to mammals (Hutcheson et al. 1996; VanGijsegem et al. 1995) and have been associated with production of filamentous structures for the apparent direct injection of secreted proteins into mammalian cells (Parsot et al. 1995). Direct transfer of elicitor proteins of plant pathogenic bacteria to the plant cytosol may explain why it has been difficult to isolate avr gene products from extracellular media and why the predicted products of 62 many of the plant resistance genes cloned to date appear to be cytoplasmic (Staskawicz et al. 1995) and Avr proteins have no extracellular activity on plant cells. We have been unable to show even minor effects of a hrpN mutation on the ED 5 0 , response time or symptom severity of E. stewartii in com. This finding suggests that additional pathogenicity proteins, possibly acting as cell leakage factors, must be produced and secreted by the ms/hrp system. This is supported by our observation that w tsE mutants are HR+ but non-pathogenic on corn (Frederick and Coplin, unpublished). These mutants still produce and secrete harpinEs but cannot cause water-soak ing or wilting on com seedlings. Therefore, genes in the wtsE complementation group may encode extracellular disease determinants that are secreted in the same way as harpinEs. An important question is why does E. stewartii retain the ability to make harpin, if it has no obvious role in pathogenicity? One possibility is that E. stewartii has not co evolved with com, and we are not testing it on its original host. The bacterium reportedly can cause latent infections in a number of hosts (Poos 1940) suggesting that it is really an endophyte or minor pathogen of North American grasses, and it only later became problem on modem cultivars of sweet com and maize after they were introduced. For this reason, it is possible that hrpN is needed to colonize a host other than com. We therefore, selected a number of grasses (Chapter IV) that could support endophytic population of wild-type E. stewartii at levels > 10s cells/g, and tested if a hrpN mutant could colonize them as well as the wild-type. hrpN was apparently not required for the ability of E. stewartii to colonize these grasses. Similar observations were made by L. Try (unpublished) in our lab, who tested DC283 and DM760 on several of the same grasses. Nevertheless, this study examined a limited number of species, so it is possible that none of these are a natural host for E. stewartii for which harpin is required. The notion that harpin is, in some way, advantageous to the bacterium is also supported by the fact that it is produced by related Erwinia sp. and Pantoea stewartii subsp. indologenes strains isolated from grasses. CFEPs (Chapter IV) and the culture supernatants (data not shown ) from these strains reacted with harpinEs antibodies. A second possibility would be that hrpN gene of E. stewartii may have only weak selective value that is difficult to assay individually. CHAPTER III HARPINes PRODUCTION BY WTS M U T A N T S Introduction The hrp clusters of many plant pathogenic bacteria are known to have genes encoding proteins for regulatory and export functions. For example, nine Hrp proteins that have export functions have been found in E. amylovora. These proteins constitute the Sec-independent type III secretion pathway that is used for the secretion of harpins and possibly other extracellular proteins. The first regulatory protein, HrpS, was found in P. syringae pv. phaseolicola (Grimm and Panopoulos 1989). HrpS is a 34.5-kDa protein similar to several members of the NttC-family of regulatory proteins, which is present in enteric bacteria such as K. pneumoniae, E. coli, and S. typhimurinum. HrpS interacts with HrpR and a 5 4 to activate the hrpL promoter. hrpL encodes an alternate sigma factor, which in turn activates the remaining hrp operons of P. syringae pv. phaseolicola (Grimm etal. 1995). Since the E. stewartii wts cluster has a gene for harpin production and hybridizes to the hrp clusters of E. amylovora and P. syringae pv. phaseolicola, we expected it to also provide the Hrp secretion and regulatory functions. The wts cluster of E. stewartii has a hrpS homolog, wtsA, which positively regulates other wts genes (Frederick et al. 1993). However, the function of the remaining complementation groups was unknown. In 64 65 this work, we use harpin production and secretion to determine which of the remaining genes are involved in extracellular protein production, secretion, or regulation. 66 Materials and Methods Bacterial strains and media Bacterial strains used in this study are listed in Table 7. All E. stewartii strains were derived from DC283 or DC356, which are spontaneous nalidixic acid-resistant (Nalr) and rifampicin-resistant (Rifr) mutants of wild-type strain SSI04 (Coplin e ta l. 1981), respectively. pRF205 was mobilized from E coli HB101 into E. stewartii strains containing mutations in wtsF, wtsB, wtsD, and wtsl complementation groups in triparental matings using the helper plasmid pRK2013::Tn7 as described previously (Coplin et al. 1986). Culture media, growth of bacteria, and mating conditions for E. stewartii have been described previously (Coplin 1978; Coplin et al. 1986). The following antibiotics were used in selective media in the amounts indicated (pg/ml); tetracycline, 20; ampicillin, 100; and nalidixic acid, 20. The inducing medium (IM) developed by Majerczak and Coplin (unpublished) consisted of 100 mM 2-N-morpholino ethanesulfonic acid (MES; Sigma Chemical, St. Louis, MO); 2 mM (NH 4 )2 S 0 4; 0 . 1 % casamino acids (Difco, Detroit, MI); 1 mM potassium phosphate (pH 7.2); 1 % sucrose; and 1 mM MgS04. The medium was adjusted to pH 5.5 with NaOH. Sucrose and MgS0 4 were added after autoclaving. Protein manipulations and Western blotting Harpinps was extracted from DC283(pRF205), and E. stewartii strains carrying mutations in complementation groups wtsF, wtsB, wtsD, and w tsl (each containing pRF205). For this purpose, the cells were first grown in 100 ml LB (plus antibiotics) at 28 °C to A 54 0 - 0.8. Next, the cells were pelleted and washed in 10 mi of IM, resuspended in 100 ml of the same medium, and incubated with shaking for 24 h. Then, the cells were pelleted and disrupted by sonication (Wei et al. 1992a). The sonicate TABLE 7. Bacterial strains and plasmids Bacterial strain or plasmid Relevant characteristics Reference or source Bacterial strain Erwinia stewartii DC283 SS104 Nat' Coplin et al. 1981 DM760 DC283 hrpNl89::Tn5 Frederick et al. 1996 MEX101 DC283 wtsFl 0/ ::TnJHoHoI This lab MU 141 DC2&iwtsBI41::Mu kan pf7701 McCammon et al. 1985 MEXI05 DC283 wtr/;05::TnJHoHoI This lab DM4031 DC283 wtsD403I‘.:Tn5 This lab Escherichia coli HB101 thr leu thi recA hsdR hsdM pro Boyeretal. 1969 Plasmids pRK2013::Tn7 ColEl mob* Stnr Spf Tp* Jtaa::Tn7 Dennis Deanb pRF205 1.8-kb HiruJIH fragment containing Pjac'.-.wtjA inpVKlOO Coplin et al. 1992b *Nalr, Smr, Tpr. Sp1: resistant to naladixic acid, streptomycin, trimethoprim, and spectinomycin. respectively. b Virginia Polytechnic Institute and State University, Blacksburg. 68 was boiled for 10 min and centrifuged to remove the denatured proteins. The resulting cell free elicitor preparation (CFEP) contained partially purified harpin E s • f o concentrate harpin, the CFEP was mixed with equal volume of 10% TCA, iced for 15 min and centrifuged at 20,400 x g. The pellet was dissolved in 200 pi sample denaturation buffer. For Western blotting, the CFEPs of different strains were subjected to PAGE (Sambrook et al. 1989) and electroblotted on ImmunoSelect (GIBCO BRL, Gaithersburg, MD) nitrocellulose membranes. Filters were probed with an anti-harpinEs serum, and immunodetection of bands was performed with a rabbit alkaline phosphatase-conjugated secondary antibody { ProtoblotR II AP system kit) as described by the manufacturer's (Promega , Madison, Wl) instructions. HR assay in tobacco plants Tobacco plants (Nicotiana tabacum L. var. Wisconsin) were grown under greenhouse conditions and then transferred to a controlled environment chamber maintained at 28°C, 90% relative humidity, 16 h light and 8-h dark cycle. Bacteria were prepared by pelleting overnight cultures grown in IM or LB and resuspending the cells in 10 mM phosphate buffer at 5x10* cells/ml (A54o = 0.52). Tobacco leaves were infiltrated with bacteria or CFEPs by pricking them with a dissecting needle and then pressing the open end of a 3 ml disposable plastic transfer pipet (Denville Scientific, Denville, NJ) against the lower leaf surface and forcing the inoculum into the wound (Bauer et al. 1994). The margins of the water-soaked infiltrated areas were marked and the plants were rated for HR development at 24 h. The percent of HR for different strains was calculated as the proportion of the infiltrated area showing necrosis. 69 R esults Because E. stewartii neither produces detectible quantities of harpin nor does it give HR under normal assay conditions (see Chapter II), we first introduced plasmid pRF205 into the wtsF, B, D, and 1 mutant strains. pRF205 contains wtsA* cloned behind the vector P ^ promotor, so it enhances harpin Eg production and the expression of other Wts functions, thereby enabling wild-type strains to give an HR. When live cells of the wild type strain DC283(pRF205) and the wts mutants containing pRF205 were grown in either IM or LB, and infiltrated into tobacco and Datura, only DC283(pRF205) produced typical HR symptoms within 24 h, whereas the wtr mutants containing pRF205 did not give any HR . However, when the cells were disrupted by sonication, a strong HR was observed with CFEP from DC283(pRF205) as well as with those from wtsF, wtsB, and wtsD mutants, but not from the hrpN and wtsl mutants (Table 8). Moreover, the HR was correlated with the presence of intracellular harpin in the CFEPs as shown by Western blots (Fig. 19). 70 TABLE 8. HR assay in tobacco leaves of cell-free elicitor preparations (CFEPs) from the wild-type strain DC283(pRF205) and various wts mutants. % HRb by CFEPs* Strain Compl. group Expt. lc Expt. 2« Expt. 3« Mean ± S.D DC283(pRF205) 80 95 nt 87 ± 10 DM760(pRF205) hrpN 0 0 0 0 MEX101(pRF205) wtsF 62 54 92 69 ±20 MU 141(pRF205) wtsB 58 85 45 62 ± 20 DM403l(pRF205) wtsD 97 82 nt 89 ± 10 MEX 105(pRF205) wtsl 3 0 0 1 ± 2 a CFEPs were extracted from the respective strains grown overnight at 28*C in EM (pH 5.5) according to Wei et al. (1992) and infiltrated into the panels of tobacco leaves. b % HR was determined by estimating the percentage of the infiltrated area that was necrotic at 24 h. c Each value is the mean of at least three replicates and nt = not tested. 71 Fig. 19. Immunoblot analysis of harpin production by pRF205-containing wtsF, w tsB,w tsD , and w tsl mutants. HarpinEs is indicated by the arrow. The low molecular weight cross-reacting bands are due to harping degradation products. 1 = harpinEs, 2 * DC283(pRF205)wrs+, 3 = DM760(pRF205)w/sAf, 4 = MEX101 (pRF205)vmF, 5 = MU141(pRF205)w/rfl , 6 = DM403 l(pRF205)w/sD , 7 = MEX105(pRF205)wfs/. The gel has been cropped at the well. 72 D iscussion The model for hrp gene regulation in P. syringae pv. phaseolicola suggested that theE. stewartii wts cluster might include several positive regulatory genes in addition to wrsA. In this study, we found that a wtsl mutant was HR-and non-pathogenic and could not synthesize harpin intracellularly. This pleiotropic phenotype indicates that it is defective in the secretion and synthesis of harpin and other pathogenicity proteins and wtsl may therefore encode a regulatory protein. Preliminary sequence analysis indicates that Wtsl is related to HrpL (Coplin and Majerczak, unpublished) and we are postulating that it acts between wtsA and the individual operons. wtsC mutants have a similar phenotype and may also be regulatory (Coplin and Stover, unpublished). The observation that the cells of E. stewartii strains containing mutations in wtsF, wtsB, and wtsD did not cause an HR in tobacco and accumulated harpin intracellularly suggests that the genes in these complementation groups encode proteins needed for the export of harping. A similar phenotype has been reported for harpin secretion mutants of other bacteria. For example, the export of harpinpgs of P. syringae pv. syringae is dependent on hrpH (He etal. 1993), hrpWt and hrpY (Huang et al. 1995) and possibly other genes. However, it is not known whether WtsF, WtsB, and WtsD function directly in export or regulate the export process. It may be that some genes in these complementation groups encode proteins that are the constituents of export system while others synthesize proteins having export-related regulatory roles. Since harpin* mutants of E. stewartii are still pathogenic, the genes in the wtsF, wtsB, and w tsD complementation groups are probably used not only for the secretion of harpin ns but for other pathogenicity proteins as well, because mutants in these groups are completely non- pathogenic. CHAPTER IV HOST RANGE STUDY AND PRODUCTION OF HARPIN BY PANTOEA STEWARTII SUBSP. INDOLOGENES Introduction Because of the colinear hybridization between the hrp cluster of E. amyolvora and the wts of E stewartii, we used the £. amylovora hrpN (1.3-kb //indlll fragment) as a radioactive probe and located E. stewartii hrpN. However, unlike E. amylovora, E. stewartii hrpN mutants were not different from the wild-type in terms of ED 5 0 , response time, or symptom severity. This raises the question why E. stewartii retains this gene if it is not needed for pathogenicity on com. E. stewartii is able to colonize a number of North American grass hosts (Poos 1940). This suggests that it is an endophyte or a minor pathogen of these grasses and has not co-evolved with com. Therefore, it is quite possible that hrpN is needed to colonize some other plant. Moreover, if this gene is useful in some way to the bacterium, we can expect other strains of £. stewartii to have the gene. In this work, we tested a number of selected grasses to determine if a hrpN mutant could colonize them. In addition, we tested three strains of P. stewartii subsp. indobgenes (Mergaert etal. 1993) which is closely related to E. stewartii, for harpin production and secretion. 73 74 Materials and Methods Bacterial Strains and Media Bacterial strains used in this study are listed in Table 9. pRF205 was mobilized from E. coli HB101 into E. stewartii, P. stewartii subsp. indologenes, and E. herbicola in tripaiental matings using the helper plasmid pRK2013::Tn7. Culture media, growth of bacteria, and mating conditions for E. stewartii have been described elsewhere (Coplin et al. 1986). The antibiotics used in selective media were tetracycline (20 jig/ml), nalidixic acid (20pg/ml), rifampicin (50 pg/ml), and kanamycin (20 pg/ml). In order to avoid fungal contamination, benomyl and PCNB fungicides (20 pg/ml) were added to LB Nal agar when reisolating bacteria from inoculated plants. Protein manipulations and dot blots Harping, was extracted from the wts + strain DC283(pRF205) and all other test strains (each containing pRF205) as described in Chapter III, except that after precipitating the CFEP with 10% TCA, the pellets were dissolved in 300 pi 50 mM potassium phosphate buffer (pH 6.5). To perform dot blots, 8 pi of each sample was pipeted onto an ImmunoSelect nitrocellulose membrane (Life Technologies BRL, Gaithersburg, MD) and air-dried. The samples were pipeted onto the membrane as two 4 pi aliquots, to avoid spreading. Then, the filters were probed with an anti-harpin serum, and immunodetection was carried out according to the instructions given in Promega's manual for their Protoblot*1 II AP System kiL Inoculation of grasses The various grasses listed in Table 10 were grown in flats as described in Appendix A. The bacterial strains used for inoculation, wild-type DC356 and MAI hrpN123::Tn5, were grown overnight in 5 ml L-broth at 28°C, pelleted and 75 TABLE 9. Bacterial strains and plasmids strain or plasmid Relevant characteristics Reference or source Bacterial strain Erwinia stewartii DC283 SS104 Naif Coplin et at. 1981 DC356 SS104 RiP Coplin et at. 1992b DM760 DC283 hrpN189::Tn5 Frederick et al. 1996 Pantoea stewartii subsp. indologenes NCPPB 1845c Wikl-type Mergaertet al. 1993 NCPPB 2275c Wild-type Mergaertet al. 1993 NCPPB 2280c Wild-type Mergaertet al. 1993 Escherichia coli HB101 thr leu thi recA hsdR hsdM pro Boyeretal. 1969 Plasmids pRK2013::Tn7 ColEl mob* tra* Smr SpfTpf fajn::Tn7 Dennis Deanb pRF205 1.8-kb HindlU fragment containing inpVKlOO Coplin et al. 1992b •NaP, Rift, Smr, Tpf, Sp1: resistant to naladixic acid, rifampicin, streptomycin, trimethoprim, and spectinomycin, respectively. b Virginia Polytechnic Institute and State University, Blacksburg. c Original names for these strains wereErwinia ananas (NCPPB 1845), E. herbicola (NCPPB 2275), and E. herbicola (NCPPB 2280) from the National Collection of Plant Pathogenic Bacteria, Harpendon, England. 76 TABLE 10. Host range study of the wild-type strain DC356 andhrpN a mutant MAI. Strain* Hostb CFU/gc Common Name Scientific Name 0 days* 3 days 9 days 15 days DC356 Johnson Sorghum 5.0± 1.4x105 0.410.1x107 1.110.5x10s 1.310.4x10s MAI grass halepense (L.) 4.414.2x10s 1.510.5x107 3.812.3x10s 2.510.5x10s DC 3 56 Rio S. vulgare 2.5±0.2x 106 0.31.05x10s 0.910.6x10s 0. 410.4x10s MAI Sorghum cv. 1.9±0.2x10s 3.012.0x10s 0.510.1x10s .051.01x10s DC356 Pearl pennisetum NT> 0.41.06x10s 1.810.1X104 NT MAI millet gtaucum (L.) NT 4.31.09x10s 1.41.01X104 NT DC356 Atlas S. vulgare 1.5±0.2x 10s 0.310.1x10s 0.110.1x10s 2.010.6x10s MAI sorghum cv. 1.810.2x10s 3.511.5x10s 0.110.0x10s 0.51.05x10s DC356 Large crab Digiiaria 1.4±1.2x10s 8.711.8x10s 8.711.7x10s 1.110.7x107 MAI grass sp. 0.810.4x10s 5.810.4x10s 5.215.7x10s 0.210.2x107 DC356 Green fox Alopecunts 0.610.3x10s 1.3i0.6xl07 1.210.6x10s 0.210.1x10s MAI tail pratensis (L.) 1.110.3x10s 1.111.2x107 2.911.4x10s 2.012,Ox 10s DC356 Switch Pamcum 1.310.1x10s 0.610.3x10s 1.210.9x10s 1.910.1x10s MAI grass virgatum (L.) 1.911.1x10s 0.610.2x10s 3.013.0x10s 1.81.01x10s DC356 Witch Pamcum 1.910.3x10s 1.2l.05xl07 0.410.3x107 7.010.7x10s MAI grass capillare (L.) 2.410.6x10s 1.010.2x107 1.310.1x107 3.012.0x10s DC356 Champ Triticum 5.112.2x10s 1.31.08x107 1.411.4x10s 0.21.03x10s MAI spelt speita (L.) 2.711.6x10s 0.910.8x107 0.410.1x10s 0.31.01 xlCH DC356 Garland Avena 9.214.7x10s 4.113.5x10s 0.510.5x107 3.211.0x10s MAI oats sativa (L.) 8.316.4x10s 5.710.7x10s 3.113.0x107 3.413.0x10s DC356 Broom com Pamcum 5.811.2x10s 2.911.4x107 1.311.2x107 3.611.4x10s MAI millet miliaceum (L.) 9.815.0x10s 1.21.05x107 4.111.2x107 8.011.1x10s ■ Bacterial cells were grown overnight in 5 ml L-brotfa at 28‘C, centrifuged and resuspended in 10 mM potassium phosphate buffer (pH 7.2). Cell concentation was adjusted to 10° cells and then used for inoculation. bTest hosts were grown in 20x10x2.5 inches Hats filled with a mixture of soil: venniculite : peat moss (1:1:1) and inoculated with the eye end of a #4 sewing needle dipped in bacterial suspension. e Were determined by homogenizing two entire plants / sample (2 samples / host) in 5-20 ml 0.85% saline using the formula described in the text. 4 NT ■ not tested e values of a single experiment; the experiment was repeated once with similar results. 1 standard deviations from mean. resuspended in 0.01 M potassium phosphate buffer (pH 7.2) and adjusted to A 540 = 0.57. Twenty plants were inoculated for each strain/host combination. Stems were punctured with the eye end of a #4 sewing needle dipped in the bacterial suspension. The eyelet held about 1 p.1 of inoculum. At 0, 3, 9, and 15 days after inoculation, populations were determined for the mutant and the wild-type by homogenizing two entire plants per sample in 5-20 ml of 0.85% saline using a Tissue-mizer (TekmarR, Jewell Electrical Instruments Inc.). The homogenate was serially diluted in saline and 100 ill of appropriate dilutions were spread on selective media plates. Two samples per host per interval were taken. The following formula was used to calculate CFU/gm of plant tissue: CFU/gm = (average count) (drop factor) (homogenizing volume) (dilution factor)/(weight in gms); where average count is the average number of colonies on three plates; homogenizing volume is the volume of saline used for tissue grinding; drop factor is a ml divided by the amount added to each plate; and the dilution factor is the reciprocal of the dilution. The population dynamics of the wild-type and the hrpN mutant at different time intervals for each experiment were compared using a paired t test with ni+n 2-3 degrees of freedom and P = 0.05. 78 Results Growth of a wild-type strain and an isogenic hrpN mutant was examined in a number of selected grasses. Although many grasses supported the multiplication of both the wild-type strain and the mutant to high levels (>105 CFU/gm), no symptoms were observed on any inoculated plant. The growth of the wild-type strain DC356 did not differ (P = 0.05) from that of the hrpN mutant MAI in any of the hosts except in Atlas sorgham. In this host, the mutant population was significantly (P = 0.05) higher than that of the wild-type 3 days after inoculation. However, this result was not consistent and could not be reproduced. Therefore, it is concluded that harpin does not appear to affect the ability of this bacterium to multiply endophytically in other potential hosts. In order to determine if bacteria closely related to E. stewartii have also retained the hrpN gene, CFEPs were extracted from three strains of P. stewartii subsp. indologenes and used to perform dot blots, using harpin es antiserum (Fig. 20). The CFEPs from two of these strains reacted with the harpin e* antibodies, indicating that these strains have a functional hrpN. 79 Fig. 20. Immunoblot analysis of harping, production by Pantoea stewartii subsp. indologenes (containing pRF205 to increase harpin production). The experiment was repeatd once with similar results. A. Western blot of cell-free elicitors preparations (CFEP) from wild-type strain DC283 and P. stewartii subsp. indologenes. 1 = NCPPB 2280(pRF205); 2 = NCPPB 2275(pRF205); 3 = NCPPB 1845(pRF205); 4 = DM760 (pRF205); 5 = DC283; and 6 = DC283(pRF205). B. Dot blot of CFEPs from wild-type strain DC283 and P. stewartii subsp. indologenes. a = DC283(pRF205); b = DM760 (pRF205); c = NCPPB 1845(pRF205); d = NCPPB 2275(pRF205); and e = NCPPB 2280(pRF205). 80 D iscu ssion Our results that harpine* is neither required for the pathogenicity of E. stewartii on com nor its multiplication in related grasses are quite surprising. Similar observations were made by Lisa Try in our lab who also compared the multiplication ability of wild-type strain DC283 and a different hrpN mutant, DM760, in some of the grasses. In this study however, we used a limited number of host species, so it is quite possible that none of them are a natural host for E. stewartii. Moreover, in our studies, we homogenized entire plants to determine CFU/g (fresh weight) of the mutant and the wild-type in order to see if the wild-type grew better than the mutant in different grasses tested. It is quite possible that the wild-type multiplies better than the mutant in certain tissues of the test-hosts which we might have missed. Our finding that the CFEPs from P. stewartii subsp. indologenes reacted with harpings antibodies in dot blots and Western blots suggests that harpin is produced and exported by these strains. This also shows thathrpN might have some value for the pathogen. The two positively reacting strains, NCPPB 2275 and NCPPB 2280 isolated from Pennisetum americanum and Setaria italica , respectively, were originally E. kerbicola and the negative one, isolated from pineapple was £. ananas (Mergaert et a l 1993). APPENDIX A Materials and Methods Taq DyeDeoxyTM Terminator Cycle Sequencing The following procedure was used to obtain a dsDNA template for automated sequencing. The procedure is a modification of ABI's (Applied Biosystems Inc.) protocol that removes almost all trace of contaminating proteins and, therefore, gives best sequencing results. Cells containing the plasmid to be sequenced were grown overnight in 1 1. Terrific Broth (Sambrook et al. 1989) plus antibiotics. The cells were centrifuged for 15 min al 1,880 x g, resuspended in 30 ml solution 1 (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0), and incubated at room temperature for 20 min. The cell suspension was mixed with 60 ml of solution II (0.2 N NaOH, I % SDS), and kept on ice for 10 min. Then 60 ml of solution III (60 ml 5 M potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml water) were added to the cell suspension, and the mixture was iced for 20 min. To remove the debris, the mixture was spun at 1,880 x g for 15 min, and the supernatant was passed through two layers of cheesecloth. To precipitate the DNA, 90 ml of isopropanol were added to the supernatant, and the mixture was incubated at room temperature for 20 min. To pellet the precipitated DNA, the mixture was spun at 9,820 x g for 15 min. The pellet was resuspended in 5 ml double distilled water, and 2.5 ml of 7.5 M ammonium acetate (to precipitate proteins) were added to the DNA solution, and the mixture was iced for 10 min. To pellet the precipitated proteins, the mixture 81 82 was spun for 10 min at 3,840 x g, the pellet was discarded, and three volumes of absolute ethanol were added to the supernatant. After thorough mixing and cooling at -70°C for 15 min, the mixture was spun at 12,400 x g for 15 min, and the pellet was resuspended in 1 ml TE. To remove RNA, 10 |il of 10 mg/ml RNase A solution were added to the DNA solution, which was then incubated at 50 °C for 30 min. To precipitate the plasmid DNA, 300 pi of 5 M NaCl, and 325 pi of 30% polyethylene glycol 8000 (w/v), 1.5 M NaCl were added to the DNA solution, the components of the mixture were gently mixed, and then the mixture was iced for 30 min. The DNA solution was spun at 4°C for 15 min, and the pellet was resuspended in 200 pi TE. To dissolve the plasmid DNA in TE, the pellet was vortexed for 5 min, and 10 pi of 20 mg/ml proteinase K were added. The solution was incubated at 37 "C for 30 min, and then the volume was increased to 500 pi with TE. Extraction with an equal volume of 1:1 phenol- chloroform was carried out until the interphase was clean. To remove traces of phenol, the solution was extracted twice with an equal volume of chloroform. One tenth volume of 3 M ammonium acetate, and two volumes of absolute ethanol were added to the plasmid DNA solution, and the mixture was then iced for 20 min. The solution was spun for 15 min in a microfuge, the resulting pellet was washed three times with 1 ml 70% ethanol, and then it was resuspended in 0.5 ml water. The DNA concentration was determined by A 2 6 o as well as by comparison with a DNA mass ladder (Life Technologies, Inc., Gathersburg, MD) on agarose gels. A total of 8 primers (Chapter II), synthesized by DNAgency, (Malvern. PA) were used to sequence the entire l. 8 -kb //indlll fragment that contains hrpN. Partial sequence previously determined by D. Majerczak, was used as a starting point to design the primers. In addition to gene specific primers, universal T7 and T3 primers were also used (Chapter II). 83 The following procedure (Applied Biosystems, Inc. Foster City, CA) was used to label and prepare the samples for automated sequencing in the ABI sequencer (Model 373A) at the OSU Biological Instrumentation Center. The reagents of the kit (Taq DyeDeoxyTM Terminator Cycle Sequencing Kit) were thawed, briefly vortexed and centrifuged. For convenience, 4X premixes were prepared in a 0.6 ml Denville microcentrifuge tube, using the following reagents: 16 pi 5X TACS Buffer, 4 pi dNTP Mix, 4 pi DyeDeoxyTM A Terminator, 4 pi DyeDeoxyTM T Terminator, 4 pi DyeDeoxyTM g Terminator, 4 pi DyeDeoxy™ C Terminator, and 2 pi AmpliTaq DNA Polymerase. To prepare the samples for PCR-labeling, the following reagents, in a total volume of 20 pi, were mixed in a 0.6 ml Denville PCR tube: 9.5 pi reaction premix, 7 pi (0.19 pg /pi) ds DNA template, 1 pi (40-80 ng /pi) primer, and water to bring the volume to 20 pi. pGEM (1 pg) with M13 (18 ng) primer (provided in the kit) was used as positive control. However, for > 80 kb plasmids, 3-fold more template was used. The reagents were mixed together and centrifuged, and then the tube was placed in a thermocycler, (model PTC 100, MJ Research Inc., Watertown, MA) for 25 cycles of labeling. The thermocycler was programmed to perform the following steps during each cycle: denaturation at90*C for 30 s., annealing at 50°C for 15 s., and extension at 60°C for 4 min. In order to remove the unincorporated dyedeoxy terminators, phenol/chloroform extraction was performed as described below. At the end of thermal cycling, 80 pi of water was added to the reaction mixture to bring the volume to 100 pi. Then, 100 pi of phenol: waterxhloroform (68:18:14) was added and the mixture was briefly vortexed and centrifuged. The lower organic phase was discarded and the upper inorganic phase (120 pi) was transferred to a clean tube. The inorganic phase (120 pi) from the above step was extracted again with 1 0 0 pi of phenol: waterxhloroform mixture, and the upper inorganic 84 phase (140 pi) was saved. To precipitate the extension products, 15 pi of 2 M sodium acetate and 300 pi of 100% ethanol at room temperature were added to the inorganic phase (140 pi) saved from the previous step. The mixture was then centrifuged for 15 min at room temperature, and the resulting pellet was washed twice with 70% ethanol. The pellet was dried for two hours at 37‘C. For automated sequencing, the samples were prepared as described below just before loading on the gel. A mixture of 5 pi deionized formamide and 1 pi 50 mM EDTA, pH 8 was prepared, 4 pi of this mixture was added to each sample, and the tube was vortexed vigorously to dissolve the dry residue. The samples were briefly centrifuged to bring the contents to the bottom. Just prior to loading, each sample was heated at 90°C to denature it and then it was immediately transferred to ice. Protein manipulations and Western blots. All the extraction procedures, except the one for making cell free elicitor preparation (CFEP), have been described in Chapter II. In order to make CFEPs from E. coli strains the following steps were performed according to Wei et al (1992a). Bacterial cells were grown overnight in 100 ml LB-broth plus antibiotics, pelleted at 3,840 x g for 10 min and resuspended in 10 ml of 50 mM potassium phosphate buffer (pH 6.5). Next, the cells were transferred to sterile metal tubes, and 100 pi of 0.1 M PMSF (Phenyl methyl sulfonyl fluoride, a serine protease inhibitor) were added. The cells were immediately sonicated for 10 min (Jewell Electrical Instruments, Inc., model W-225R) at a power output of 4 and 40% duty cycle. The tubes were kept in ice-water during sonication to avoid excessive heating. Following sonication, the cell lysates were boiled in the same metal tubes for 10 min to denature proteins. Then, the samples were cooled on ice, transferred to 30 ml glass Corex tubes, and 10 pi of 0.1 M PMSF were added to each sample. The samples were centrifuged at 20,400 x g for 30 min, the pellet was discarded and the supernatant was passed through 0.2 pm Acrodisc syringe filters 85 (Gelman Sciences). PMSF (35 jil of 0.1 M solution) was again added to each sample, and they were stored at -70°C for later use. CFEPs from E. stewartii strains were extracted as described above, except that the cells were grown overnight (A 5 4 0 = 0 .8 ) in 100 ml LB-broth, transferred to 100 ml IM, and then incubated with shaking for another 20-24 h in order to induce the wts genes. Due to very low harping yield, it was necessary to concentrate the CFEPs from E. stewartii strains by precipitation with an equal volume of 10% TCA on ice for 20 min. The precipitate was collected by centrifugation at 20,400 x g for 15 min, and the pellets were dissolved in IX sample buffer [5 ml stacking buffer (0.5 M Tris-HCI, pH 6 .8 , 0.4% SDS), 0.7 ml glycerol, 0.5 ml fi-mercaptoethanol, 3.8 ml 10% SDS, 10 ml water]. CFEPs were subjected to SDS-PAGE in a Mini-ProteanR II Dual Slab Cell (Bio- Rad, Richmond, CA). The 12% separating gel and 5% stacking gel were prepared according to Laemmli (1970) from a stock solution of 30% (w/w) acrylamide and 0.8% (w/w) N, N'- bis-methylene acrylamide. The separating gel, which contained 0.375 M Tris-HCI, pH 8 .8 , was polymerized by the addition of 0.025% (v/v) of tetramethylethylenediamine (TEMED) and 50 ^tl of 10% ammonium persulfate. The stacking gel contained 0.125 M Tris-HCI, pH 6 . 8 and was prepared as above. Electrophoresis was performed in running buffer (0.025 M Tris base, 0.192 M Glycine, pH 8.3, 0. 1% SDS) for 45 min at 200 volts. The gel was then stained with Coomassie brilliant blue R-250 [2.5 g/liter of destaining solution (40% methanol, 9% acetic acid)] for 1 h and destained in destaining solution for 2 h to visualize the bands. Duplicate gels intended for Western blots were not stained. In order to transfer proteins from the polyacrylamide gel to ImmunoSelectTM nitrocellulose membranes (Gibco BRL, Gaithersburg, MD), a Mini Trans-BlotR electrophoretic transfer cell (Bio-Rad, Richmond, CA) was used. For this purpose, the 86 membrane, Mini Trans-Blot Filter Papers (Bio-Rad), sponges, and the unstained gel were prewet in the transfer buffer (0.025 M Tris base, 0.192 M glycine, 20% methanol , and 0.1% SDS) for 15 min, and then the transfer chamber was assembled according to the manufacturer's instructions. The assembled chamber was then placed in the buffer tank containing 400 ml of refrigerated transfer buffer, which was kept cool by a Bio-Ice Cooling unit, and electroblotting was continued at 100 volts for 1 h. After electroblotting, the margins of the membrane were marked, and it was air-dried, wrapped in Saran wrap, covered with aluminum foil, and stored at 4°C. Western blotting was performed according to the manufacturer of the Pro to B lotR II AP System (Promega, Madison, WI). The dried membrane containing the antigens was floated on TBST (20 mM Tris-HCI pH 7.5, 150 mM NaCl , and 0.05% Tween 20) until evenly wet, submerged and rinsed briefly in the same buffer. To saturate the non specific protein binding sites, the membrane was incubated in TBST containing 1% BSA for 30 min. The blot was then incubated in 50 ml primary antibody solution (a 1:25,000 dilution of anti-harpinES serum was made in TBST) in a small plastic container for 60 min with gentle agitation. To remove the unbound antibody, the membrane was washed for 5-10 min three times in TBST. The blot was then transferred to the alkaline phosphatase conjugated secondary antibody (Sigma, St. Louis, MO) solution (a 1:5,000 dilution was made in TBST) and incubated at room temperature for 30 min with gentle agitation. A total of 40 ml secondary antibody solution was used in a small plastic container. To remove the unbound secondary antibody, the membrane was washed three times ( 5 - 1 0 min each) in TBST. The blot was then briefly rinsed twice in TBS to remove Tween 20 from its surface. Finally, the blot was incubated in enough Western Blue Stabilized Substrate for Alkaline Phosphatase to cover the membrane until the bands of interest reached the desired intensity. 87 Transposon mutagenesis of the hrpN gene. In order to mutagenize the hrpN gene with Tn5, the 3-kb insert of pDM25I3 (Chapter II) was removed as an XbaliSall fragment and ligated into the suicide vector pGP704. The resulting plasmid, pDM2530, was introduced into E. coli S 17-1 Xpit and then the transconjugant was mutagenized with XTn5 according to Dolph et at (1988). The target strain was grown overnight at32“C in 5 ml LB-broth containing 10 mM MgSO+t 0.2% maltose and 200 pg/ml of ampicillin. The cells were pelleted and resuspended in 5 ml of the same L-broth as above. Next, 250 jil of XTn5 (titer = 6.2 x 109 PFU/ml) were added to 1.5 ml of the target strain and incubated at 30°C for 30 min. Then, 0.5 ml of the mixture was plated on LB Ap Km plates, and incubated overnight at 37*C. The Apr Kmr colonies were pooled in 5 ml L-broth, and plasmid DNA was isolated as described elsewhere in this Chapter. Four |xl of the mutagenized plasmid DNA was used to transform E.coli S17-1 Xpir competent cells and transformants were selected on LB Ap Km plates. E. coli S17-1 Xpir (pDM2530) was used as control. The plasmid DNA was isolated from all the individual transformant colonies (10- 15/plate). The DNA samples were restricted with /ftndlll and screened for the loss of the 1.8-kb //indlll fragment. A total of 300 colonies were screened and a few colonies were found in which pDM2530::TnJ was missing the 1.8-kb Hindlll fragment. Only two of these colonies had insertions within hrpN ORF. These were introduced into DC283 by electroporation. Since DC283 does not supply the Pir protein and pDM2530 cannot replicate without it, selection for only KmR resulted in either integration of the plasmid into the chromosome or exchange of the insert DNA by a double cross-over. To avoid problems with secondary transpositions of Tn5, we first selected for integration of 88 pDM2530 (Apr Kmr) transconjugants and then tested for the stability of the Apr marker in the absence of selection. Stable Apr Kmr colonies were grown in LB-broth-Km for two days to allow for excision of the integrated plasmid to occur, and then Kmr colonies from this culture were screened for Ap*. Marker-exchange of the transposon mutations and loss of pDM2530 were confirmed by Southern blotting. Inoculation of corn seedlings Sweet com seedlings (Earliking or hybrid sweet com variety Seneca Horizon) were grown in 20x10x2.5 inches flats filled with a mixture of soil:vermiculile:peal moss (1:1:1). The seeds were germinated in growth chambers at 28°C, and 16 h daylight. In order to assay the virulence of E. stewartii strains, 7-day-old seedlings were inoculated, using the following three procedures, i) Pseudo-stem inoculation: Sterile toothpicks were dipped into fresh bacterial cultures grown on L-agar plates, and inserted into the young com seedlings 1 cm above the soil line. Symptoms were recorded at 3 and 10-days after inoculation, using a 1 to 5 scale (1= no symptoms, 2 = scattered lesions, 3 = slight wilting, 4 = severe wilt, and 5 = dead), ii) Whorl inoculation: Eight day-old sweet com seedlings, grown as above, were used. Appropriate E. stewartii strains were grown overnight in 5 ml of LB-broth at 28“C in an incubator-shaker. The cultures (1x10® cells/ml) were diluted 100-fold in 0.01 M potassium phosphate buffer (pH 7.0) containing 0.2% Tween 40. About 200 |il of inoculum was placed into the whorls of the young seedlings without wounding them. Plants were rated for lesions at 3 and 10 days after inoculation, using a 0 to 3 scale (0 = no symptoms, 1= a few lesions but no ooze, 2 = many lesions and some ooze, 3 = a lot of lesions and ooze), iii) Quanta! response lest: To determine the infectivity of different E. stewartii strains at different cell concentrations, 8 -day-old seedlings, grown as above, were used. The seedlings, which were not watered the day before the inoculation, were decapitated with a razor blade 89 approximately 2 cm above the soil line. Five pi of inoculum were immediately applied to the cut ends, and the seedlings were not disturbed until the droplet had been drawn into the stem. Inoculum concentrations were 1250, 250, 50, and 10 cells/plant. At 3, 5,7, 9, and 11 days after inoculation, the plants were rated for symptoms. Any plant showing a symptom was scored as positive and the percent infection was calculated. Ten days after inoculation, the plants inoculated with the highest concentration of cells (1,250 cell s/plant) were rated for disease severity, using the above 1 to 5 scale. ED 5 0 S were determined graphically by plotting log dose against the probit of the proportion of the plants responding. The response time data were analyzed using a non-parametric t test statistics given below: t = Pw - Plunder root of the (SEw ) 2 + (SEm) 2 with Nw+Nm* 2 degrees of freedom, where Pw and Pm are the proportions of wild-type- and proportions of the mutant-inoculated plants respectively showing disease symptoms. SEw and SEm are the standard errors for proportion of the wild-type- and proportions of the mutant-inoculated plants respectively showing symptoms. These standard errors are calculated as follows: SEw = under root of the Nwx Pw x (1-Pw) and SEm = under root of the Nm x Pm x (1-Pm ) where Nw and N m are the numbers of plants inoculated with wild-type and mutant respectively. Radioactive hybridization procedure For large scale isolation of plasmid DNA from E. coli strains, cells were grown overnight in 500 ml LB-broth (plus antibiotics) at 37*C, centrifuged for 10 min to pellet the cells, and resuspended in 18 ml water. Cells were lysed by the addition of 35 ml of solution II (0.2 N NaOH, 1% SDS) to the cell suspension, mixing the components of the mixture by inverting, and then incubating the mixture for 5 min at room temperature. To remove chromosomal DNA and SDS, 26.5 ml of solution III (60 ml 5 M potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml H 2 O) were added to the lysed cells, the 90 mixture was inverted many times, and then incubated for 5 min at room temperature. Debris was removed by centrifugation at 7,520 x g for 15 min, and the supernatant was poured through two layers of cheesecloth. To precipitate the DNA, 50 ml of isopropanol were added to the DNA solution. The mixture was mixed, incubated at -20°C for 20 min, and then centrifuged at 12,400 x g for 15 min. The supernatant was discarded and the pellet resuspended in 2 ml of TE. Next, 3.3 gm CsCl, and 100 pi ethidium bromide (10 mg/ml) were added. The DNA solution was transferred to 2 ml Beckman centrifuge tubes. The tubes were sealed and centrifuged at 95,000 rpm in a Beckman TLl(K) ultracentrifuge with a TLA 100.3 rotor for 24 h. The lower plasmid DNA band was removed with an 18 gauge syringe needle and extracted with sec-butanol until the pink color was no longer visible. The DNA solution was then dialyzed three times against one liter TE. The DNA concentration was determined from A 2 6o- The restriction enzymes tf//tdIII, BamHl, and Kpnl used in this study were purchased according to the manufacturer's recommendations using 1 0 - 2 0 units of enzyme per pg of DNA at 37“C. The reactions were terminated after 2-3 h by heating the mixture at 65®C for 10 min. The digested DNA preparations were electrophoresed in 0.8% agarose gels in TA (40 mM Tris-HCI, 20 mM acetic acid, 2 mM EDTA, pH 8.0) buffer at 1.5 V/cm for 10 h. To visualize the DNA bands, the gels were stained in 0.5 pg/ml ethidium bromide solution for 30 min and photographed on Polaroid Type 55 film with ultraviolet light using a red filter. \ DNA digested with EcoRI and Hindlll was used as molecular weight standards. DNA fragments were transferred from agarose gels to filters by the procedure of Southern (1975) as modified by Wahl etal (1979). To hydrolyze the DNA. the gel was soaked in 0.25 M HC1 for 15 min. Next, the gel was placed in a solution of 1.5 M NaCl, 91 0.5 M NaOH for 30 min to denature the DNA fragments. The denaturation step was repeated once using fresh solution. To neutralize the DNA* the gel was submerged in 1 M Tris-HCl (pH 8.0), 1.5 M NaCl for 30 min . The neutralization step was repeated once. All of the above steps were performed at room temperature with gentle shaking. The nitrocellulose membrane (Schleicher & Schuell) and three Whatman filter papers (cut slighdy larger than the gel) were pre-wet in water for 10 min and then in 10X SSC (IX = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) for 20 min. Then, the gel was placed on three pre-wet filter papers, and the pre-wet membrane (with its top right comer cut to show the orientation) was placed on the top of the gel. Air bubbles between the membrane and the gel were removed. A dry filter paper and a 5-cm-thick stack of paper towels was then placed on the top of the membrane. A glass plate with a 500-g weight was placed on the top of the paper towels. After 24 h, the filter was removed and the completeness of the DNA transfer was checked by staining the gel in ethidium bromide and observing it under UV light. The membrane was washed for 5 min in 2X SSC, placed between two filter papers, and dried for 1 h in a vacuum oven at 80°C. To label the probe with 3 2 P, 500 ng of purified probe DNA was boiled for 2 min and then placed on ice for 5 min. Next, 4 pi of each dNTP solution (GTP, CTP, TTP), 3 pi of 32P dATP, 5 pi of primer, 5 pi of Klenow buffer, 2 pi of Klenow fragment, and a few pi of water were added to bring the total volume up to 50 pi. The labeling reaction tube was placed in a shielded container at room temperature for 3 h. Then 400 pi of TE was added to the labeled probe, and 225 pi of the probe was boiled for 10 min. The denatured probe was placed on ice for 5 min and then added to 50 ml of hybridization solution. Hybridization solutions and conditions have been described by Sambrook et a i (1989). The baked nitrocellulose membrane was placed in a very small closed plastic box 92 containing 50 ml of warm (65*C) prehybridization solution ( 6 X SSC, 0.5% SDS, 5X Denhardt's solution, 10 mM dithiothreitol, 100 pg/ml denatured salmon sperm DNA). Denhardt's solution consisted of 0 . 1 % Ficoll, 0.1% polyvinylpyrrolidone, and 0 . 1% BSA. The plastic container was incubated in a waterbath at 65°C for 8 h. The membrane was carefully removed with forceps and placed in 50 ml hybridization solution ( 6 X SSC, 0.01 M EDTA, 225 pi of 32P labeled denatured probe DNA, 5X Denhardt's solution, 0.5% SDS, 10 mM dithiothreitol, and 100 pg/ml denatured salmon sperm DNA). The reaction was incubated at 65'C for 16 h. Then, the membrane was washed in 2X SSC and 0.5% SDS for 5 min at room temperature with slow shaking. The membrane was again washed in 2X SSC, 0 . 1 % SDS for 15 min at room temperature, and finally in 0 .1 X SSC, 0.5% SDS at 65°C for 2 h. After the last wash, the membrane was placed on a piece of Whatman filter paper and air dried. The dried membrane, on a filter paper, was wrapped in a plastic wrap, and taped in the center of an X-ray film cassette. Then, under a red safety light, a piece of X-ray film ( X-OMAT™ AR, Kodak) was placed on the top of the membrane, the cassette was closed and locked, and placed at -70°C for 34 h. Non-radioactive hybridization procedure A Photogene Nucleic Acid Detection System version 2.0 (Life Technologies Inc., BRL, Gaithersburg, MD) was used to verify marker exchange of Tn5 mutations. For this purpose, genomic DNA was isolated as follows. Bacteria were grown overnight in 10 ml LB-broth at 288C, and then the cells were collected by centrifugation at 3,840 x g for 5 min. The cell pellet was resuspended in 1.8 ml TES (10 mM Tris-Cl, pH 7.5, 100 mM EDTA, 150 mM NaCl), and 100 pi of proteinase K (20 mg/ml in 0.1 M Tris, pH 7.4) and 100 pi of 20% SDS were added. The mixture was incubated at 55°C for 5 h. To remove proteins, 0.5 ml of phenol was added to the mixture, the mixture was briefly 93 vortexed, and then it was centrifuged for 2 min. The supernatant was transferred to a clean microfuge tube and extracted once with 0.5 ml 1:1 phenolxhloroform. The supernatant was again saved and extracted once with 0.5 ml chloroform. Then, the supernatant was mixed with one tenth volume of 3 M sodium acetate, pH 5.2 and two volumes of 95% ethanol, and incubated at -20’C for several hours. To pellet the DNA, the mixture was spun at 31,000 x g for 15 min, and the pellet was dried at 37 “C. The pellet was resuspended in 400 |il of TE and incubated overnight at 55°C. The DNA concentration was determined by A 2 6 0 /2 8 0 * and its purity assessed by restriction endonuclease digestions. To label the probe with biotin, the PCR Nonradioactive Labeling System (Life Technologies Inc., BRL, Gaithersburg, MD) was used, following the manufacturer's instructions. Since this procedure uses lacZ forward and reverse primers, it can only be used for the amplification of inserts cloned in /acZ-containing vectors. To PCR-amplify and label the 1.8-kb HindUl (in pMAl), the following reagents from the labeling kit, were mixed in a microfuge tube: 23 pi DEPC-treated water, 5 pi 10X PCR buffer, 2.5 pi 50 mM MgCl, 1 pi dATP solution, 2.5 pi dCTP solution, 7.5 pi biotin- 14-dCTP, 1 pi dGTP solution, 1 pi dTTP solution, 2.5 pi lacZ forward primer, 2.5 pi lacZ reverse primer, and 1 pi (1 ng/pl) supercoiled plasmid DNA. The total volume was 49.5 pi. Next, the mixture was heated at 100°C for 10 min to denature the supercoiled template. After denaturation, the mixture was placed on ice for 5 min and then 0.5 pi of Taq DNA polymerase was added to it. The contents of the tube were mixed, briefly centrifuged, and the tube was then placed in a thermocycler (model PTC-100, MJ Research, Inc., Watertown, MA). TTiirty cycles of PCR amplification were performed, using the following temperatures and times for different steps: denaturation at 94°C for 30 s, annealing at 60°C for 1 min 15 s. and extension at 72 °C for 2 min. Finally, an additional incubation at 94 72°C for 10 min allowed completion. Unincorporated biotinylated nucleotides were removed by Sephadex G-50 chromatography. Sephadex G-50 was swollen overnight in column buffer (IX SSC, 0.1% SDS). The column was prepared by blocking the pointed end of a Pasteur pipet with siliconized glass wool, attaching a plastic tube to the lower end of the pipet, pinching the tube with a clip to control the flow rate, and slowly filling the pipet with the swollen resin. A space for about 200 pi sample was left at the top of the pipet. The column was equilibrated with column buffer for 10 min. Next, blue dextran was added to the biotinylated PCR amplified probe, and the sample was added at the top of the column. The column was then eluted with the same buffer and all the blue fractions ( 6 drops each) were collected in microfuge tubes. Biotinylated DNA elutes in the void volume with the blue dextran. The purified labeled probe was quantitated by running a small portion of it on an agarose gel. Four pi of DNA Mass Ladder (Life Technologies Inc., BRL, Gaithersburg, MD) were also loaded on the gel. The intensity of the probe band was compared with those of the Mass Ladder after staining the gel in ethidium bromide. To perform the DNA hybridization, the prehybridization solution (50% form amide, 6 X SSPE, 5X Denhardt's solution, 1% SDS), at the rate of 0.25 ml/cm 2 of membrane, was poured into a very small plastic container and warmed to 42°C. IX SSPE consists of 8.76 g NaCl, 1.38 g NaH 2P0 4 , 0.37 g EDTA, pH 7.4. Next, sheared salmon sperm DNA (10 pg/pl) at the rate of 20 pi per ml of prehybridization solution was transferred to a microfuge tube, and the tube was placed in boiling water for 1 0 min to denature the DNA. The warm, denatured salmon sperm DNA was then added to the prewarmed prehybridization solution. The baked membrane was then carefully placed into the prehybrization solution, the lid of the plastic container was tightly closed and the solution incubated for 4 h at 42*C with gentle agitation. The membrane was removed 95 from the prehybridization solution after 4 h, and placed into the hybridization solution (10% dextran sulfate, 50% formamide, 6 X SSPE, 5X Denhardt's solution, and 1% SDS) at the rate of 0.1 ml/cm2 of the membrane, and prewarmed to 42°C. Then, denatured salmon sperm DNA, as described above, was added to the hybridization solution and mixed well. Next, the labeled probe at the rate of 50 ng/ml of hybridization solution, was put into a microfuge tube, and the tube was placed in boiling water bath for 1 0 min to denature the probe. The probe was then cooled on ice and added to the prewarmed hybridization solution. The membrane was incubated overnight at 42°C in the hybridization solution with gentle agitation. The membrane was removed from the hybridization solution and washed twice, 5 min each, with 2 ml/cm 2 of prewarmed (65°C) 5X SSC, 0.5% SDS. Then, it was washed with 2 ml/cm2 of prewarmed (50°C) 0.1X SSC, 1% SDS for 30 min. Finally, the membrane was washed with 2 ml/cm2 2X SSC for 5 min at room temperature. In order to block the unoccupied sites on the membrane, the still wet membrane was placed in small plastic container with enough prewarmed (65“C) blocking solution (3 g BSA/100 ml of TBST) to cover the membrane. The blocking reaction was incubated at 65°C for 1 h with gentle agitation. The next step was binding of streptavidine-alkaline phosphatase (SAAP) conjugate to the biotinylated probe that was fixed to the membrane. For this purpose, the SAAP conjugate was centrifuged at 12,000 x g for 30 min at 4°C, the supernatant (at the rate of 7 pi / 100 cm2 of membrane) was removed, and diluted to 1:1,000 in TBST (100 mM Tris base, 150 mM NaCl, and 0.05% Tween 20, pH 7.5). The membrane was carefully placed in the diluted SAAP and incubated at room temperature for 1 0 min with gentle agitation. Next, the membrane was washed with TBST at the rate of 1 ml / cm2 for 15 min at room temperature with gentle agitation. Finally, the membrane was washed with final wash buffer (100 mM Tris base, 100 mM 96 NaCl, and 50 mM MgCh 6 H 2O, pH 9.5) at the rate of 1 ml / cm 2 for 60 min at room temperature with slow shaking. The hybridizing bands were detected by submerging the membrane in Western Blue™ Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, WI) for 2 min. 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