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MOLECULAR ASPECTS OF SOYBEAN TOLERANCE AND RESISTANCE TO PHYTOPHTHORA SOJAE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
The Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
by
NamikKaya B.Sc. (Hons), M.Sc. (Hons.)
*****
The Ohio State University 1997
Dissertation Committee: Approved by Dr. T. T. VanToai, Adviser
Dr. J. Streeter, Co-Adviser Adviser Dr. A. F. Schmitthenner
Dr. M. Garraway Adviser
Department of Horticulture and Crop Science UMI Number: 9813281
Copyright 1998 by Kaya, Namik
All rights reserved.
UMI Microform 9813281 Copyright 1998, 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 ABSTRACT
Soybean Glycine max (L.) Merr., is one of the most important crops in the United
States. One of the major disease of soybean is Phytophthora root rot (Prr). Causative agent
of this disease is a fungus called Phytophthora sojae. The fungus causes severe losses on
soybean production each year. One of the most effective way to control the disease is genetic
control which is divided into tolerance and resistance. Tolerance is a quantitative trait and
control by more than one gene. Unlike tolerance, resistance is a qualitative trait controlled
by a single gene. In this study we studied molecular aspects of soybean tolerance and
resistance against Phytophthora sojae.
In the first part of our study, we choose amplified firagment length polymorphism
(AFLP) technique to study disease tolerance. Our specific objective was to identify AFLP
markers and QTL (quantitative trait loci) associated to the disease tolerance. Clark x Harosoy
isoline mapping population was used for molecular marker analysis. Prr tolerance phenotype
was assayed by the inoculum layer tests with three replicates. Total 13 AFLP markers were
added to the previously established Clark x Harosoy soybean map. Of the 13 AFLP markers, only 10 were linked to the existing linkage groups. The map consists of 231 markers on 36
linkage groups and 40 unlinked markers. Six loci were also found significant at the probability level of 0.01 by one way analysis of variance. Two significant QTL (LOD score
ii 3 or above) associated with the P. sojae tolerance were identified on linkage group three (R ^
30.4%) and linkage group eleven (R ^ 62.0%) using MAPMAKER/QTL. This map
including AFLP markers spans 2222.8 cM distance.
In the second part of the study, our specific objective was to determine differential
gene expression patterns and clone differentially expressed genes in response to inoculation
o ff. sojae Race 1 in soyhosn-Phytophthora sojae model system and attempt to clone R psl-k
gene. R psl-k gene provides host resistance to P. sojae Race 1. We used differential display
technique to screen for genes expressed in response to inoculation of P. sojae Race 1
zoospores to the roots of soybean isolines Elgin (susceptible variety without R psl-k gene )
and Elgin 87(resistant variety with R psl-k gene). Total RNA’s of Elgin and Elgin 87 were
isolated at 3 h, 6 h, 13 h, and 17 h post-inoculation with zoospores of Race 1 of
Phytophthora sojae and water as controls. Seven different gene expression patterns have
been identified and 49 differentially expressed bands have been displayed. Three sequences related to the general response of soybean to Phytophthora sojae infection have been cloned and their differential expression was confirmed by northern blot analysis.
Ill Dedicated to pioneers; who enlightened our ways, to my wife, for her patience, help, kindness, and endless support to my parents, for their prayer, support and love to my family, for their cooperation and help
IV ACKNOWLEDGMENTS
I would like to express my gratitude to my advisor, Dr. T. T. VanToai for her guidance and thoughtful suggestions throughout the research. I am also very thankful to the other members of my committee, Dr. J. Streeter, Dr. A. F. Schmitthenner, Dr. M. Garraway for their suggestions and comments. Special thanks to Ginny, Drs. Getachew and Preisner for their help, discussions, suggestions, and also sincere thanks to my fellows Linda and Peng.
I would like to thank to Yuzuncu Yil University in Turkey for their support throughout my studies.
I also acknowledge to College of Food, Agricultural, and Environmental Sciences, OSU for their financial support in Autumn 1997.
I express my sincere appreciation and gratitude to Dr. Alves for his guidance, suggestions, and comments. His frienship and suggestions were unforgetable.
I also would like to express my sincere appreciation and special thanks to Mr. Yetisir for his friendship and suggestions.
My last words for my wife, and my family. Without their support and prayers, this study cannot be accomplished. VITA
August 1, 1966 ...... Bom - Van, Turkiye 1983-1988 ...... B. S., Yuzuncu Yil University (Hons) 1988-1990 ...... M.Sc., Yuzuncu Yil University (Hons) Research Assistant Department of Horticulture Yuzuncu Yil University 1990-1991 ...... Ph.D., Yuzuncu Yil University Research Assistant Department of Horticulture Yuzuncu Yil University 1991-1996 ...... Ph.D., The Ohio State University High Education Council Fellowship 1997-present ...... Research Assistant Agr., Biol., Food Eng. Department The Ohio State University
PUBLICATIONS
Research Publication
1. Kaya, N. 1991. Adaptation studies of several local melon varieties to Van Region.Y.Y.U. Art and Sci. Jour. 3: 10-16
2. Tekintas, E., Kaya, N., Beyhan, O., Karatas, E., Akca, O., 1991. Production of
vi different vegetables under greenhouses and plastic tunnels in Van. EX Annual Productivity Symposium on the Improvements for Eastern Turkey, pp: 100-110
3. Kaya, N., VanToai, T. T., Specht, J., and Schmitthenner, A. F. 1996. Mapping QTL and identification of markers linked to Phytophthora root rot of soybean. International Symposium of Soybean Molecular Genetics. Ill, pp. 135
4. Kaya, N., Alves, J. D., VanToai, T. T., and Schmitthenner, A. F. 1997. Differential display of soybean root genes after inoculation of Phytophthora sojae Race I. ICABERG. pp. 120
5. Alves, J. D., Presiner, J. H., VanTaoi, T. T., and Kaya, N. 1997. Differentially expressed soybean root genes under anoxia. International Brazilian Plant Physiologist Symposium.
FIELDS OF STUDY
Major Field: Horticulture and Crop Science Plant Molecular Biology and Biotechnology Minor Field: Molecular Plant Pathology and Stress Physiology Molecular pathology of Soyhesn.-Fhytophthora sojae interaction and anaeorobic stress physiology, biochemistry, and genetic under the guidance of Dr. Tara T. VanTaoi.
Vll TABLE OF CONTENTS
Eâgs
ABSTRACT...... ii
DEDICATION...... iv
ACKNOWLEDGMENTS...... v
VITA...... vi
LIST OF TABLES...... x
LIST OF FIGURES...... xi
GENERAL INTRODUCTION...... 1
CHAPTER 1
Introduction ...... 16
Materials and Methods ...... 25
Results ...... 31
Discussion ...... 50
Conclusions ...... 55
CHAPTER 2
Introduction ...... 56
Materials and Methods ...... 71
Results ...... 8 6
viii Discussion ...... 97
Conclusions ...... 103
GENERAL CONCLUSIONS...... 104
LIST OF REFERENCES...... 106
IX LIST OF TABLES
Table Page
1.1. Primer sequences used in preselective and selective PCR reactions ...... 35 1.2. Linkage map of markers including 10 AFLP markers (italized-bold) found in this study ...... 38 2.1. Sequences of 5'-primers and 3'-primers used in Differential Display ...... 76 2.2. Differential gene expression patterns observed for Elgin and Elgin 87
in response to 3, 6 , 13, and 17 h post-inoculation with water (control) and zoospores of Phytophthora sojae Race 1...... 89 LIST OF FIGURES
Figure Page
1 . 1 . Soybean seedlings (2 1 days old) inoculated with Phytophthora sojae Race 1...... 32 1.2. Soybean seedlings (21 days old) inoculated with Phytophthora sojae Racel. Illustration of rotted roots of progeny isolines ...... 33 1.3. Bar graph showing distribution of progeny lines with respect to P. sojae tolerance ...... 34 1.4. A sequencing gel autoradiograph of parental and some progeny lines showing polymorphism with the M se\?9 and EcoRIP \primers...... 36 1.5. A sequencing gel autoradiograph of parental and some progeny lines showing polymorphism with the M selP l and EcoRlP% primers...... 37
1.6 . Single Factor Analysis of Variance results showing the most significant six molecular markers associating with Phytophthora sojae tolerance depicted in a main effect plot ...... 42 1.7. Single Factor Analysis of Variance results showing significant molecular markers associations with Phytophthora sojae tolerance depicted in a main effect plot ...... 43 1.8. Linkage group three and eleven showing significant QTL peaks for Phytophthora sojae tolerance ...... 44 1.9. First significant QTL peak for tolerance to Phytophthora sojae Race 1 on linkage group three ...... 45 1.10. Second significant QTL peak for tolerance to Phytophthora sojae
XI Race 1 on linkage group eleven ...... 46 1.11. Main effect plot of Single Factor Analysis of Variance results showing significant loci on chromosome three for tolerance associated with Phytophthora sojae Race...... 1 47 1.12. Main effect plot of Single Factor Analysis of Variance results showing significant loci on chromosome eleven for tolerance associated with race Phytophthora sojae Race...... 1 48 2.1. Agarose gel (2%) of total RNAs isolated from inoculated and control
Elgin and Elgin 87 roots at 3, 6 , 13,17 h ...... 87 2.2. Some of the differential gene expression patterns observed for Elgin and
Elgin 87 in response to 3 , 6 , 13, and 17 h post-inoculation with water (control) and zoospores of Phytophthora sojae Race...... 1 90 2.3. Some of the differential gene expression patterns observed for Elgin and
Elgin 87 in response to 3, 6 , 13, and 17 h post-inoculation with water (control) and zoospores of Phytophthora sojae Race...... 1 91 2.4. Some of the differential gene expression patterns observed for Elgin and
Elgin 87 in response to 3, 6 , 13, and 17 h post-inoculation with water (control) and zoospores oî Phytophthora sojae Race...... 92 1 2.5 Some of the differential gene expression patterns observed for Elgin and Elgin 87 in response to 3, 6,13, and 17 h post-inoculation with water (control) and zoospores of Phytophthora sojae Race...... 1 93 2.6. Probe R1 used in Northern Blot Analysis ...... 95 2.7. Probe R2 used in Northern Blot Analysis ...... 95 2.8. Probe R3 used in Northern Blot Analysis ...... 96
Xll GENERAL INTRODUCTION
Soybean, Glycine max (L.) Merr., is one of the most important economic crops in the United
States. Its importance is due not only to its utilization in animal feeds, human foods, and industrial applications, but also, to its being an ideal system for study of host-pathogen interaction, particularly for plant breeders and pathologists. This is by virtue of having both a potential source for resistance and tolerance against Phytophthora sojae, causative agent of root rot disease in soybean, and having a symbiotic relationship with a number of microorganisms such as genera of Rhizobium and Bradyrhizobium. Recent statistics indicated that about 16 million ha of agricultural land in the U.S. are infested with P. sojae
(Schmitthenner, 1985).
History
The soybean originated in northeastern China (Poehlman, 1987, Delorit et al., 1984) and was domesticated in the 1200's (Shoemaker et al. 1996). At that time it was cultivated as a human food, animal feed and medicinal plant (Smith, 1995). The story of exportation of soybean as a cultigen began in Manchuria, its first place of exportation, where it grew as a local crop. When the Russo-Japanese war started, soybean came into focus and gained world wide attention. In 1908, soybean products were in European markets (Smith, 1995).
1 However, in Europe, as early as 1712, and in the US., around the 1760's (Smith, 1995),
soybean was introduced as a new plant. It was Samuel Bowen, a seaman, who first brought
soybean to the US. Later, Henry Yonge was given credit as the first grower of soybean at
that time (Smith, 1995). In 1853, soybean was introduced in Ohio. By the 1930's it had
become an important crop due to its oil and meal processing capacities (McBlain and
Schmitthenner, 1991). However, soybean, as a recently introduced crop, was not firee firom biological problems. Because, plants are not alone in their ecological niche and evolve with their pathogen coevolutionarily (Staskawickz et al., 1995), an indigenous pathogen,
Phytophthora sojae, appeared on the scene as a prevalent disease of soybean in a biologically short time. P. sojae is a member of Phytophthora genus that belongs to
Peronosporales order of Oomycetes class and causes seed rot, damping off, and root rot on soybean plants (Schmitthenner, 1985).
The fimgus was first recorded in Indiana in 1948 (Schmitthenner, 1985). When the fimgus appeared in Ohio in 1951, its symptoms were initially confused with that of Fusarium and
Diaporthe (Schmitthenner, 1985). In the following year the first report came out; however, it was in 1958 when Kaufinann and Gerdemann first identified and wrote a comprehensive report and named this pathogen P. sojae. At present, there may be more than 45 races of P. sojae infesting the soils in the U.S.A. (Abney, 1997). Properties of the Pathogen
The pathogen has a simple and indistinct sporangiophore (Schmitthenner, 1985) with sporangium ranging in sizes up to 42-65 X 32-53 pm. Oogonium of this homothallic fungus is also large in size and antheridium are either mostly paragynous or rarely amphigynous.
A diploid oospore forms after the occurrence of meiosis in antheridia and oogonia and nuclear fusion in oogonium respectively. Also, the fungus has diploid mycelium and sporangia resulting from germination by oospores. The mature sporangia have a thin, delicate structure that emits zoospores. Zoospores normally have two flagella and are egg shaped. Posteriorly directed flagellum is 4-5 times larger than the anterior one. When the zoospores make contact with the host surface, they transform into a cyst that germinates and forms a germ tube that generally enlarges to develop an appressorium. After a short period, normal growth continues with the penetration into the host (Hardham and Hyde, 1997;
Schmitthenner, 1985).
Disease Symptoms
P. sojae can attack soybean, possibly, in any stage of the development. Susceptible plants are very sensitive and die quickly, especially at the younger developmental stages. Well- known symptoms of the disease are rot, specifically on the roots; but it can occur on the seeds and stems as well. While pre-emergence symptoms can cause damping off, and reduced stands, post-emergence symptoms, rot on the roots and stems, results in wilting and
3 death of seedlings. Wilting may start at the primary leaf stage followed by a water-soaked appearance of the stems. The leaves then turn yellow and the seedling cannot survive any longer. Younger plants are killed more quickly than older plants although vigor in the older plants may be reduced throughout the growing season as well. In older plants, the first symptom is a yellowing of the lower leaves. Soon the upper leaves become chlorotic which leads to a total wilting of the plant. Internal symptoms are in the vascular tissues and cortex.
Discoloration can occur internally and as well as externally. Not only infected tap roots but also some parts of the lower stems and branches become discolored up to th 4th and 5th nodes as well. While lateral and branch roots are completely destroyed, taproots become dark brown.
Biology of the Fungus
The fungus survives in mostly cold, heavy, tightly compacted soils where oospores result from sexual reproduction between antheridia and oogonia. When favorable conditions exist, such as the presence of water, high humidity, and optimum temperature, oospores begin to germinate. This results in sporangia, lemon-shaped and colorless, with sensitive, thin slender, stalks called sporagiophore. Under optimum conditions, the protoplasm in the sporangia begin to divide and result in more or less 30 colorless zoospores having two flagellate.
These zoospores can swim in the vicinity of the soybean roots and are chemotactically attracted to the roots. Encystment follows after the zoospore reaches and attaches itself to the surface of the roots. The encysted zoospores can germinate and penetrate the roots
4 within 2 hours. Mycelium with finger-like or globular haustoria, can penetrate all the
tissues and end up killing the roots and plant. When the host sources are used up by fungus,
oospores are formed as a resting survival spores. This is the final step of the life cycle.
However, many phytophthora species form an asexual spore (chylamydospore) another type
of resting structure. These chylamydospores usually arise from hyphaes and can survive
extended periods in the soil.
Disease Management
Disease management includes several different approaches such as chemical protection, genetic control, and cultinal treatments. The management program has been based on an integrated approach. Among these, genetic control seems the best tactic in defeating the disease. At present, genetic control alone cannot fully control the disease (Schmitthenner, personal communications). Even though resistant soybean varieties are succesflil in preventing the disease, Phytophthora sojae has already become virulent to the deployed resistance genes (Lohnes and Schmitthenner, 1997).
According to Schmitthenner (1985, 1988), in the early 1980's, it was understood that one of general types of the genetic control, tolerance, alone was not sufficient to control the disease. Since resistance, a second general group of genetic control, is defeated by new races in relatively short periods of time, it was thought that it would be essential to combine these genetic control types with some additional strategies, such as chemical and
5 cultural treatments in an integrated management program. In this sense pyroxyfiir and
metalaxyl seed and soil treatments integrated with tolerance would be effective.
The disease is especially serious under the high moisture and flooding conditions
(Schmitthenner, 1985). Yield loss due to P. sojae can be three times worse in flooded soil than in non-flooded soil (McBlain and Schmitthenner, 1991). Under flooding conditions, soybean cultivars with the prr resistance genes could be subdued by the pathogen. The
possible reasons could be 1 ) the maturation of sporangia and releasing of zoospores are more favorable under the excessive soil water; The possible reason for this is the biological life cycle of the fungus in which oospores, persisting in the soil for long periods of time, can germinate and form sporongia when sufficient moisture is present. When flooding is evident, these sporangia become active and finally release zoospores. This can cause an increased level of inoculum around the roots where zoospores can be chemotactically attracted to the target (Boiler, 1995; Graham, 1994; Schmitthermer, 1985; Schmittheimer and VanDoren, 1985), 2) flooding stress reduces the plant vigor and hence its resistance to the disease; 3) flooding stress and its associated anaerobic conditions may affect the expression of prr resistance {Rps) genes. On the other hand, there are no reports regarding the effects of flooding on reversing the resistance. Therefore, it has become apparent that moisture management has a unique importance in the disease management program.
Furthermore, experiments show that soil treatments (e.g., tillage and drainage) in the program may also play an essential role to limit the disease. Soil drainage, combined with
6 fall plowing and crop rotation might help limit the phytophthora damage for many cultivars
(Schmitthenner and VanDoren, 1985; Schmitthenner, 1985). Schmitthenner (1988)
suggested that, for a long term solution, an integrated approach including cultural (tilling,
crop rotation, drainage) and chemical management programs (e.g., treating seeds with
metalaxyl) and genetic control, specifically utilizing new Rps genes (even this is a short
term solution), pyramiding previously found Rps genes to the varieties with a good yield and
tolerant backgroimd, and combining resistance and tolerance, would resolve the P. sojae
problem (Schmitthenner, 1985, Schmitthenner, 1988; Lohnes and Schmitthenner, 1997).
Genetic Control of the Disease
Genetic control in terms of the mechanism can be divided into two general groups called
tolerance and resistance. Tolerance, as a genetic control mechanism, is different from Rps
resistance or whole plant resistance or single gene resistance (Schmitthenner, 1985). The
difference, in terms of symptoms, is very clear especially in the seedling stage, when the
root rot occurs. Some older plants show a normal growth or even recover when there is rot.
Resistance
Although there may be thousands of plant diseases caused by a wide range of infectious
agents such as fungi, bacteria, viruses, nematodes, and mycoplasms, successful pathogens causing disease represent a comparably small portion of pathogens (Baker et al., 1997). In
7 nature, therefore, it is thought that disease is the exception and resistance is the rule. The mechanism lying behind this is that either somewhat specifically equiped nonhost plant species can recognize these pathogens or pathogens are not able to overcome a general recognition body turning on and off the expression of an array of host resistance or defense systems. This type of resistance is known as nonhost resistance and refers to basic incompatibility at the species level (Callow, 1987). On the other hand, some other species show a susceptible response to certain pathogens which are somehow able to avoid the recognition of the defense system and penetrate. This kind of interaction refer to basic compatibility at the species level (Callow, 1987). However, among these species there are some cultivars which may show resistance to a given race or races of the pathogen or vice versa. This type of interaction is defined as incompatibility and compatibility at the cultivar level, respectively. Improvement of resistance is an essential part of breeding programs. The continuous efforts aim to improve and/or develop risk-firee disease control strategies.
History of Resistance to P. sojae
Phytophthora root rot first appeared in 1948 and interestingly determination of the causal agent, P. sojae, and resistance against the disease, demonstrated as a single gene, were found in the same year, 1954. The gene was named Rps in 1957. Phytoalexin was foimd to be a main mechanism of resistance in 1962. The other Rps genes in soybean, which appear at seven loci with multiple allelic forms at two of these loci, have been reported during the past
30 years. In 1965, resistance was reported as race specific, and, at present, there may be
8 more than 45 races of P. sojae infesting the soils in the U.S. (Abney, 1997). Recently, some
of Rps genes were mapped, some of their inheritance unraveled (Anderson and Buzzell,
1992; Bhattacharyya et al., 1997; Kilen, 1986; Kilen et al., 1974; Kilen and Tyler, 1993;
BCilen, 1986a; Kilen 1986b; Lohnes et al., 1993; Lohnes and Schmitthenner, 1997; Polzin et
al., 1994; Yu et al., 1996).
P. sojae has been known to be very flexible in terms of virulence. Several races of
Phytophthora sojae act in a gene-for-gene manner (Keen, 1982). According to the nature of
the specific interaction between Rps genes and different races, the reaction is defined as
either compatible, referring to one of the races of the pathogen defeating a given Rps gene
or incompatible, referring to a race of the pathogen causing a hypersensitive type of
response.
Genetic Mapping
Genetic mapping is used to provide the relative location of known genes or markers to each other. There are two genetic mapping techniques: 1) physical mapping is the direct localization of genes and markers on DNA or chromosomes either by restriction site mapping, ordered genomic clones of a yeast artificial chromosome (YAC) library, in situ hybridization, or in situ PCR and 2) Linkage mapping established the relative position of genes/markers by the frequency of their co-inheritance (Alberts et al., 1994). The concept of genetic linkage maps was dated back to the work of Gregor Mendel in 1860.
However, the advancement in recombinant DNA technologies in the last decade has significantly renewed the interest in the research and application of genetic linkage mapping
(Tanksley, 1993; Rafalski, 1993). In the recombinant analysis, the percentage of the recombinant is used as a quantitative measure of the linear distance between two genes. The percentage of recombinant, also known as recombinant frequency (RF) is the ratio of recombinant individuals over the sum of all individuals. It has been arbitrary defined that a genetic map unit of 1 centimorgan (cM) is equal to 1 % RF (Griffiths et al., 1993).
Therefore, the closer the two loci are, the smaller their recombinant frequency (Synder and
Hartl, 1985). Knowledge of the RF between genes, hence their map distances, allows the prediction of the frequencies of the genotypes and phenotypes in the F, generation of a given cross (Stoskopf, 1993). From the genetic map of known loci, it is possible to detect the location of unknown loci associated with polygenic traits, a process known as QTL mapping
(Griffiths et al., 1993).
Mapping Population
Geneticists have been using different types of mapping populations including F;, Fjj, backcross, and recombinant inbred lines to construct genetic maps ( Giovannoni et al., 1991 ;
Helentjaris et al., 1986; Kesseli et al., 1992; Michelmore, 1995; Tanksley, 1993).
Determination of linkage between markers and genes have been done mostly in backcross
and F 2 .3 populations. The F 2 population has been preferred for mapping studies due to the
10 high linkage disequilibrium or less opportunity for meiotic recombination (Tanksley, 1993).
Recombinant inbred lines are used only in self-pollinated species that can tolerate high level
of inbreeding (Tanksley, 1993). Michelmore et al. (1991) designed a new method known as
bulked segregant analysis that can determine molecular markers very rapidly and accurately
in a particular region of the genome. Primarily the technique is based on a comparison of
pooled DNAs. Individuals of the segregating population were scored for the trait and divided
into two groups; those with and those without the trait. The DNA isolated from the
individuals within each group are pooled to form two bulked samples. Any polymorphism
detected between the bulked samples is more likely to be linked to the trait. (Michelmore,
etal., 1991).
Molecular Markers
Molecular-marker aided selection offers great promise for the breeding of plants with desirable polygenic traits. Since most of the desirable agronomic traits are polygenic, significant effort has been invested into the construction of genetic maps of many plant species mostly by the restricted fi:agment length polymorphism (RFLP) technique. The list includes maize, arabidopsis, tomato, rice and soybean (Newburry and Ford-Lloyd, 1993;
McCouch and Doerge, 1995; Shoemekar and Specht, 1995; Stoskopf, 1993). The soybean genome map was constructed by Shoemaker and Olson (1993) from a Glycine max x G. sojae cross. Other public soybean maps exist including the map developed from a Minsoy
X Noir cross by Lark et al. at University of Utah (1993) and a Harosoy x Clark map by
11 Shoemaker and Spetch at University of Nebraska (1995). The Harosoy x Clark map
presently has aroimd a hundred RFLP markers. Several microsatellites markers were added
to the map recently by Dr. Cregan’s group, USDA-ARS, Beltsville, Maryland (Akkaya, et
al., 1995).
Restriction Fragment Length Polymorphism (RFLP)
Restriction enzymes cut DNA molecules into smaller fragments at specific sites. Alterations
in DNA sequences can result in either the creation or abolition of restriction sites. The size
differences in DNA Augments of a restriction digest are the basis of the polymorphism
detected by RFLP technique (GrifBth et al., 1993). Since RFLP markers reflect the genotype of a locus, they can be used as reference points in the construction of genetic maps. RFLP markers tightly linked to a gene have been used successfully for gene isolation by chromosome walking (Lewin, 1994). RFLP markers are codominant: the homozygous dominant, homozygous recessive and heterozygous genotypes are easily distinguishable by this technique, since RFLP was first invented to construct the human genetic map (Botstein et al., 1980), it has become a very popular technique and has been used for the genome mapping of many animals and plants including maize, wheat, rice, barley, soybean, tomato, brassica, and arabidopsis (Anderson et al., 1994; Helentjaris, et al., 1985; Newbury and Ford-
Lloyd, 1993; Tanksley et al., 1989; Williams et al., 1991; and Chang et al., 1988). The main disadvantages of this technique are the complex procedure that requires pure, and high molecular weight DNA. Until recently, this technique also requires the use of radioactive
12 probes (Rafalski, 1993).
Randomly Amplified Polymorphic DNA (RAPD)
The RAPD technique is a PCR-based procedure that was designed by Williams et al. (1990)
to detect DNA polymorphism. This technique employs a single primer of arbitrary nucleotide
sequence to amplify genomic DNA in vitro using the PCR reactions. The RAPD products are resulted from the amplification of DNA Augments between the two primer sites that are on opposite DNA strands, but close enough to each other for efficient amplification. The products are separated on agarose gels and visualization by ethium bromide staining
(Beckman, 1988; Rafalski 1993). The RAPD markers are dominant markers that segregate according to the Mendelian rules (Newburry and Ford-Lloyd 1993). This technique has been used successfully to construct the linkage map of pine trees and to find markers closely linked to the Pseudomonas disease resistance gene in tomato for chromosome walking. Also a RAPD marker linked to Rps4 gene was reported (Byrum et al., 1993). Because the RAPD procedure is much faster and easier to do than the RFLP procedure, it has become very popular, especially as a technique for marker-aided selection associated with molecular breeding (Newburry and Ford-Lloyd, 1993; Andersen and Fairbank, 1990; Ragot and
Hoisington, 1993; and Rafalski, 1993). This technique has also been used to locate QTL such as those associated with high oil in soybean (Rafalski, Dupont, per com).
13 Differential Display Analysis (DDA)
This PCR based procedure used to analyze differential gene expression was first proposed
by Liang and Pardee (1992). The chief aim of this method is to detect, isolate, and clone the
differentially expressed genes using the polymerase chain reaction (PCR) (Liang and Pardee,
1992). From 1992 to 1997 differential display became one of the most commonly used gene
isolation technique in the area of molecular biology. The technique is based on amplification
of mRNAs using two primers, one is for poly(A) tails, and the other is a short and arbitrary primer that anneals randomly to the 5 -end of the mRNAs. The products are separated on denaturing sequencing gels. The differentially expressed genes are identified firom the polymorphism of the banding pattern. The DNA band is purified firom the gel, reamplified and cloned (Bauer et al., 1993; Liang and Pardee, 1992; Liang et al.; 1992; and Liang et al., 1993). Many modifications have been made to the original technique such as the use of longer primers to amplify for 3' end of mRNAs, visualization on agarose gels, use of two
Reverse transcriptases instead of one. Even though differential display has been widely used, there have been some problems at different stages of the technique. Most of these problems have been associated with starting material. Especially DNA contamination of RNA is the main source of false positives. Some others are related to cDNA synthesis, use of different visualization methods, RNA blotting and cloning (Liang and Pardee, 1995). The problem of false-positive clones produced by this technique can be corrected by the procedure of Callard et al. (1994).
14 QTLs
Quantitative traits, showing variation perpetually, are identified by segregation of multiple loci. Inheritance for such traits is named polygenic inheritance. Mapping of individual polygenes was first described around 1920's. However, Thoday (1961) established this concept thoroughly (Tanksley, 1993). A linked polygene can be analyzed and its influence can also be estimated by help of single markers dispersed through the genome. This makes likely construction of the genetic maps for polygenes. A population can be divided into the classes owning to the various genotypes at the marker locus; consequently, the analysis and determination of the difference between the trait and various genotypes' individuals as to the same trait will be able to lead us if the polygene is linked to the marker by means of statistical approaches (Tanksley, 1993).
15 CHAPTER I
MAPPING QTL AND IDENTIFICATION OF AFLP MARKERS LINKED TO
PHYTOPHTHORA ROOT ROT OF SOYBEAN
INTRODUCTION
Soybean is one of the dominant economic crops in Ohio since 1987. A major factor diminishing the cash value of soybean production in Ohio since 1955 has been the disease known as Phytophthora Root Rot (prr) caused by Phytophthora sojae (McBIain and
Schmitthenner, 1991). One of the best forms of control of the disease is genetic control. The genetic control of P. sojae can be divided into two general groups; resistance, also called single-gene resistance, referring to whole plant resistance, and tolerance, referring to differential resistance against the disease (Schmitthenner, 1985). Even though the single- gene resistance controls the pathogen efiBciently, it is easier for the pathogen to defeat single gene based resistance (Schmitthenner, 1985). New races of pathogen develop instantaneously, resulting in the conversion of a resistant reaction to a susceptible one. For instance, in the case of the fungal tomato pathogen, Cladosporium Julvurn, just a single base-pair change in a virulence gene of the pathogen can lead to conversion of a incompatible interaction to a compatible one (Joosten et al., 1994). Pathogens now have the ability to avoid the host’s recognition process resulting in an ability to overcome the defense
16 system. Therefore, both pyramiding the resistance genes and tolerance would be an
alternative approach to more durable resistance (Schmitthenner, 1985).
Tolerance
Tolerance, as defined by Caldwell et al. (1958), refers to the relative performance of plants to produce a sufiBcient crop after equal infection by pathogens. Recently, it was understood that this is the narrow sense of the tolerance and was finally referred to as endurance
(Schmitthenner, personal communications). Schafer (1971) defined tolerance as that
“capacity of a cultivar resulting in less yield or quality loss relative to disease severity or pathogen development when compared with other cultivars or crops". However, in later years, Mussell (1980) came up with a broader definition of tolerance stating that “ tolerance is the ability of plants to produce a good crop despite the insults of pathogens" and in more accurate terms the author defines that “tolerance with respect to biotic pathogens, is the ability of a cell, plant, or field to perform acceptably while providing the habitat necessary for the growth and reproduction of pathogens of that cell, plant, or field”. Mussell
(1980) also pointed out the importance of providing the habitat for a pathogen. According to him (1980), this is mostly ignored or an undesired essential characteristic of tolerance.
Furthermore, while using Bateman's (1980) "multiple component hypothesis", Mussell
(1980) characterized some of the aspects of tolerance specifically in terms of environment.
As he stated, the host contributes towards tolerance in terms of having or not having receptor sites for the pathogens, having capacity of deactivation of a pathogen's irritants, and having
17 abilities for compensation for the damage or stress caused by these irritants. He, therefore, proposed that “a host plant's vulnerability or tolerance will be determined primarily by its response or lack of response to these irritants”. In the model, he brought up that vulnerability is the consequence of "stimulus-response" type events happening in an orderly and specific sequence. One of the general features of tolerance to pathogens is the capacity of the host to both yield suitable produce and provide the essential habitat for the pathogen development.
In summary, Mussell (1980) concluded that "two common characteristics of tolerance are that they provide a favorable habitat for the unfavorable effects of the pathogen while minimizing the unfavorable effects of the pathogen on host performance”.
In respect to the P. jq/ae-soybean model system, resistance and tolerance have different meanings. The term resistance implies total or whole plant resistance, that means “immunity” associated with a single gene causing a hypersensitive response. This also refers to race specific resistance. On the other hand tolerance was defined as “the relative ability of plants to survive root infection, either natural or artificial, without showing severe symptom development such as death, stunting, or yield loss and called as race non-specific resistance”
(Walker and Schmitthenner, 1984). Tolerance has been named field tolerance (Buzzel and
Anderson, 1982), field resistance (Irwin and Langdon, 1982), rate-reducing resistance
(Tooley and Grau, 1982), and age-related resistance (Lazarovits et al., 1981). Tolerance has
been divided into several groups; 1 ) race-specific root resistance, 2 ) race-nonspecific slow rotting, and 3) endurance. However, it is recognized that some race-specific mechanisms are also involved in the general definition (McBlain and Schmitthenner, 1991). There have been
18 several techniques to evaluate tolerance. While the slant board test (Olah and Schmitthenner,
1985) has been used in growth chamber evaluations, in inoculum layer test (Hobe, 1981) was
used to evaluate greenhouse experiments. Basically, both techniques test the seedling
response in respect to root infections. Rating vigor of seedlings at the growth stage in the
field is another form of evaluation by which mature plants responses are surveyed (Walker
and Schmitthenner, 1984).
Race specific root resistance is thought to be race-specific and conferring low infection, a
lesion, in inoculum layer test (McBlain and Schmitthenner, 1991). This response occupies
a place in between whole immunity and extensive rot. This root resistance can be
differentiated firom the resistance by hypocotyl inoculation test in greenhouse or root
inoculation tests. Ripley, a root resistant variety, responded differently to the hypocotyl
inoculation tests and slant-board tests. Hypocotyl inoculations of Ripley gave different
results with compatile races ( 1 0 0 % killed) and incompatible races (partially killed).In the slant-board test, Ripley gave less than 10 mm of root rot (McBlain and Schmitthenner,
1991).
Slow rotting is also called rate reducing resistance (McBlain and Schmitthenner, 1991) which is an epidemiological term that describes a reduction of sporulation and symptoms such as lesion size. In addition, slow rotting is referred to as field tolerance (Buzzell and
Anderson, 1982) due to evaluation in plant loss or rate reducing resistance (Tooley and Grau,
1982). However, in respect to P. sojae, it implies a reduction in the amount of rot in a
19 specific period of time but does not indicate a lower colonization of the pathogen. Slow
rotting mechanism usually does not distinguish slow rotting plants firom fast rotting plants
since total oospore production may be the same in both varieties. This mainly implies an equal amount of oospore production in both tolerant and susceptible plants. Conrad, a slow rotting variety, showed a score of 4-5 in inoculum layer tests. In the slant-board tests, rot on the roots was less than 30 mm. This type of tolerance seems to constrain the development of some strains of P. sojae. Recently slow rotting has attracted intensive attention to improve genetic control programs with respect to race nonspecificity and inheritance of tolerance as a quantitative trait (McBlain et al., 1991a; McBlain and Schmitthenner, 1991;
Walker and Schmitthenner, 1984). After several attempts to increase slow rotting, rot ratings
3 to 10 (see Material and Method for ratings) have been achieved through the efforts of the
Schmitthenner group (Walker and Schmitthenner (1984); McBlain and Schmitthenner,
1991). However this rating was not quite as good as the one that can be achieved by race- specific resistance.
The last component of tolerance is endurance. In a narrow sense, endurance refers to equal disease with differential yield loss. Theoretically, it implies no overlapping with resistance or other components of tolerance, but practically it is very difficult to distinguish endurance firom some forms of slow rotting in the field. Cultivars having endurance may produce better yield than expected from their seedling response in the field under disease conditions. This type of tolerance still remains as an ideal system to control P. sojae (McBlain and
Schmitthenner, 1991)
2 0 Molecular M arker Studies
The development of molecular marker technology, which is based on polymorphisms found in proteins, DNA or RNA, has greatly facilitated mapping studies. Lately, attention has increasingly focused on DNA and RNA as a source of informative polymorphism. Because the DNA sequence of each individual is unique, a variety of techniques to visualize polymorphism between or within different species, populations, and even close individuals have been developed in the last decade. These techniques have also been used for the identification and mapping of quantitative trait loci (QTL).
A genetic marker can be identified as a gene whose phenotypic expression is easily discriminated and this expression can be used to identify an individual or a cell that carries it as a probe to mark a nucleus, chromosome or locus. There are two criteria for a genetic marker to be useful; 1) The marker must distinguish between the parents, and 2) the marker must be precisely reproduced in the progeny of the parents (Paterson et al., 1996). Discovery of the genetic linkage by Morgan (1911), that Mendelian genetic factors called genes lying close together on an inherited material can be cotransmitted from generation to generation or parents to progeny gave an idea that a marker could function as a proxy for the genes in close proximity to the trait of interest (Griffiths et al., 1993). The first report of quantitatively inherited traits was reported around 1920 by Sax. Sax (1923) described the association of seed coat pattern and pigmentation with seed size differences in Phaseolus vulgaris. From his findings, the author concluded that there is a linkage between the single
21 gene controlling seed color and polygenes controlling seed size. Polygenes in this context are termed as genes controlling quantitative traits, meaning that the traits’ genotypic variation is continuous, and are believed to have effects that are small relative to the other sources of variation. Also, polygenes can be used interchangeably with QTL (Tanksley,
1993). Mutations related to continues range of phenotype (that is quantitative traits) can be placed on a genetic maps as it is the case for mutations imparting qualitative traits. Mapping populations including 200 or more individuals can be helpful to map QTLs. Using “complete genetic maps, such QTLs can be assigned to likelihood intervals spanning 10-30 cM
(Paterson et al., 1988; Lander and Botstein, 1989). The basic principle of determination the approximate chromosomal locations of QTLs is to test correlation between marker genotypes and quantitative phenotypes.
Molecular Marker Techniques
Discovery of allozymes or isozymes (allelic forms of proteins) opened a new era of molecular marker studies in the life sciences. The generation of molecular markers based on protein polymorphism can be achieved by the separation and specific staining of the proteins on electrophoretic gels (Andersen and Fairbanks, 1990).
One of the most commonly used molecular markers is restriction fragment length polymorphism (RFLPs). RFLPs detect differences in the length of specific DNA fragments.
Detection of RFLPs relies on two basic steps; digestion of DNA samples with specific
2 2 restriction endonucleases and Southern blot analysis of the restricted DNA by a specific
probe (Botstein et al., 1980). RFLPs are known to be extremely useful, reliable and
informative due to enabling the detection of both dominant and codominant patterns
(Rafalski and Tingey, 1993). However, the technique itself is very laborious, expensive and
time consuming (Andersen and Fairbanks, 1990). Another commonly used technique is
random amplified polymorphic DNA (RAPD). RAPD is based on DNA amplifications with
primers of arbitrary sequence but requires no prior knowledge of the target DNA sequence.
The technique is simpler, less expensive and five times quicker than RFLP (Andersen and
Fairbanks, 1990). It also does not require the use of radioactive isotopes for visualization
of amplified DNA products.
One of the best techniques is called Amplified fragment length polymorphism (AFLP) discovered several years ago (Zebeau, 1993; Vos, 1995) and used succesfully for soybeans
(Keim et al., 1997; Maughan, et al., 1996; VanToai et al., 1996). The main advantages of
AFLP over other PCR-based fingerprinting methods are using stringent reaction conditions and the detection of a large number of polymorphism in a single PCR reaction. We have used the original technique with some modifications to identify and map QTL associated with P. sojae tolerance (Lin and Kuo, 1995; VanToai et al., 1996).
A number of dominant gene or alleles conferring resistance controlled by single loci have been introduced into cultivars. This type of resistance was soon overcome by new races of the fungus having genes compatible with the resistance alleles of cultivars. Therefore,
23 current breeding strategies concentrate on development of more durable, race-nonspecific resistance which is polygenically inherited and for which the defeat is difficult. Since tolerance is a quantitative trait (McBlain et al., 1987; McBlain et al, 1991b; McBlain and
Schmitthenner, 1991) and its defeat by the disease is more difficult compare to the race- specific disease resistance, it is very essential to identify Quantitative trait loci associated with the tolerance by the help of molecular marker technology such as AFLP or RFLP.
24 MATERIALS AND METHODS
Plant Material
The Clark x Harosoy mapping population was provided by Dr. Jim Spetch of University of
Nebraska. Initially the population of near isogenic lines (NILs) of Clark and Harosoy was
established for the purpose of integrating many segregating classical and molecular markers
into the public RFLP map (Shoemaker and Specht, 1995). Parental mating of these cultivars
were performed in the greenhouse during the 1990-1991 winter season. 10 randomly
harvested F, seeds from each of six F, plants grown in the field during the summer of 1991
were germinated, grown to maturity in a greenhouse during the winter of 1991-1992, and harvested. Fourty-five Fj seeds were randomly selected from the progeny of each of the 60
Fj plants and planted in 60 separate rows of a field nursery on the East Campus, University
of Nebraska at Lincoln, NE. The same procedure was again repeated to provide F 2.3 seeds which were placed in a cold storage for further research use.
DNA Extraction
For DNA extraction, dry leaves of 58 Fz^ lines were provided by Dr. Lohnes (The Ohio State
University, OARDC, Wooster, Columbus, Ohio). DNA extraction was performed by the
CTAB method (Saghai-Maroof et al., 1984). Two grams of lyophilized ground leaf tissue
25 was used per 9 ml CTAB (Hexadecyltri-methylammonium bromide) extraction buffer (100
mM Tris pH 7.5,0.7 M NaCl, 1% CTAB, 10 mM EDTA and 1 % P-mercaptoethanol). The
mixture was incubated at 65 °C for 30-90 minutes with occasional gentle shaking to denature
proteins. After removal from the waterbath and cooling for 5-10 min, 4.5 ml of
chloroformrisoamyl alcohol (24:1) was added to the solution and mixed gently for 5 min by
inversion. The emulsion that formed was centrifuged at 2000 rpm for 10 minutes. The
supernatant was poured through miracloth into a 15 ml tube containing 6 ml cold
isopropanol and mixed gently by tube inversion to precipitate DNA. The precipitated DNA
was removed with a sterile glass hook and transferred to 1.5 ml eppendorf tubes containing
1 ml of washing solution (76% ethanol and 0.2 M NaOAc). After washing up and down
occasionally for 20 minutes, the hooked DNA was dipped in a rinse solution (76 % ethanol
and 10 mM NH^OAc) and simultaneously washed up and down 5 times. DNA of each
sample was transferred to a sterile 1.5 eppendorf microflige tube containing 400 pi TE (10
mM Tris, pH 8 , and 1 mM EDTA) and agitated up and down to release the DNA. The tubes
were incubated overnight at 4 °C with gentle shaking at 70 rpm. DNA concentration was
than determined by spectrophotometer, and samples stored at - 20°C.
DNA Purification
Each DNA sample (20 pg) was diluted to 200 pi and precipitated with 2 volumes of phenohchloroformrisoamyl alcohol, and centrifuged at 4°C for 2 min. One himdred and seventy pi of the aqueous phase of each sample was transferred to clean tubes. This
26 extraction procedure was repeated twice. One tenth of 1 volume of 3 M sodium acetate (pH
7.00) was added to the aqueous phase of the samples and precipitated with 2.5 volume of
100% ethanol. After 30 min incubation at -80°C, samples were centrifuged at 4“C for 10 min.
The supernatant was carefully discarded and the pellet washed with 80% ethanol and dried
under vacuum. The pellet was resuspended in 50 pi TE.
Restriction and Ligation of Purified DNA Samples
DNA samples were restricted and ligated to adapter by adding 2.5 pg of genomic DNA to the mixture containing restriction and ligation buffer (10 mM Tris-HAc pH 7.5, lOmM
MgAc, 50 mM BCAc, 5 mM DTT), 17 pmol EcoRL adapter, 170 pmol Msel adapter, 20 units
EcoRl, 16 units Afeel, 1 unit T 4 DNA ligase and 0.1 pM ATP and then incubated at 37°C for
4 hours.
Labeling Primers
Msel primers having 3 selective bases and used in selective PCR reactions were end-labeled by using ^-P or ^^P-yATP radioactive isotope according to Zabeau (1993). The mixture
containing or ^^P-yATP (10 pCi), Msel (30 ng), 10 units T 4 kinase and buffer (125 mM
Tris-HCl, 50 mM MgCl^, 25mM DTT pH 7.5) was incubated at 37 °C for 30 minutes.
27 DNA Amplification
Two successive rounds of PCR were used in the pre-seiective and selective amplification, to amplify the EcoRL-Msel DNA fragments. The first PCR amplification, known as
preselective PCR, contained 25 |il total reaction mixture including 6 pi of 10-foId diluted restriction and ligation products, 40 ng of Msel and £coRI primers having one selective base,
2 mM MgCl;, 0.2 mM dNTPs, 2 units Taq polymerase, and PCR buffer (10 mM Tris-HCl, pH 8.3,50 mM KCl). Amplification reactions were as follows; 94 °C for 1 min, followed by
20 cycles of 94 °C for 30 sec, 55 °C for 30 sec and 72°C for 1 min. The second PCR or
selective amplification, was carried out by using 6 pi of 1 / 1 0 0 diluted products of the first
PCR reaction. In these reactions, EcoRL and Myel primers having three selective bases were used. The PCR program was very similar to the first profile except 40 cycles were used and the annealing step was at 62 °C. PCR products were kept at -20 °C for further use.
Gel Electrophoresis
A 5% denaturating polyacrylamide gel containing 8 M urea in Ix TBE was used to analyze the PCR products. An equal volume of loading buffer (98% formamide, 10 mM EDTA,
0.01% (w/v) xylene cyanol) was mixed with PCR products and then incubated at 100 °C for
3 minutes and quenched on iced for 2 min. 5-6 pi of DNA samples were loaded onto each lane. The gel was electrophoresed at 80 watts for 90 min. It was washed in a solution containing 10% acetic acid and 10% methanol for 30 min and dried overnight at room
28 temperature. Autoradiograms were developed from the gels after 1 -2 days of exposure.
Inoculum Layer Test
The test with three replicates was performed on 2 parental lines and 58 progeny lines as
described by Schmitthenner and Bhat (1994). For the determination of Phytophthora
tolerance, the seeds provided from Dr. Specht were surface sterilized according to Hwang
and VanToai (1991). The mycelia of Race 1 of P. sojae was grown on dilute lima bean
plates for 4-7 days until the colony nearly covered the surface. They were then placed on the
surface of previously prepared 32-oz polystyrene containers having three holes 5 mm in
diameter in the bottom and containing 11 cm of wet coarse agricultural vermiculite. After
adding 2-3 cm more of coarse vermiculite, 5 seeds of each cultivar were placed on top of the
vermiculite and covered with 2 cm of the coarse agricultural vermiculite and watered. After
three weeks, plants were removed from the containers and scored according to Schmitthenner
and Bhat (1994).
Symptom Rating
Symptom rating or rot rating system established by Schmitthenner and Bhat (1994) was based on the rotted areas on the root surface and plant survival in the polystyrene containers.
The adopted rating system was based on a 1-10 scale as follows: 1 = no root rot, 2 = trace of root rot, 3 = bottom third of root mass rotted, 4 = bottom 2/3 of root mass rotted, 5 = all
29 roots rotted, 10% seedling kill, slight stunting of tops of plants, 6 = 50% seedling kill and
moderate stunting of tops, 7= 75% seedling kill and severe stunting of tops, 8 = 90% seedlings killed, 9 = all seedlings dead, 10= all seedlings killed before emergence.
Mapmaker
The computer software Mapmaker/EXP 3.0 (Lincoln et al., 1992a) and Mapmaker/QTL
(Version 2.0.) (Lincoln et al., 1992b) were used to construct the linkage maps. The map was based on previously available data contributed by R. C. Shoemaker, P. B. Cregan, D. Lohnes,
J. E. Specht and P. B. Cregan.
Statistical Analysis
The statistical analysis was done with Minitab version 10 for Windows. One-way analysis of variance was then performed for each marker.
30 RESULTS
The Clark x Harosoy mapping population was provided by Dr. Jim Spetch of University of
Nebraska, USA. Initially the population of near isogenic lines of Clark and Harosoy was established for the purpose of integrating many segregating classical and molecular markers into the public RFLP map (Shoemaker and Specht, 1995). All 57 isolines and their parent
(Clark X Harosoy) used in this study were tested for disease reaction to race 1 of
Phytophthora sojae. Inoculation of the soybean plants with race 1 of Phytophthora sojae was performed as described by Schmitthenner and Bhat (1994).
The trait chosen was tolerance, a quantitatively inherited type of resistance (McBlain and
Schmitthenner, 1991) to Phytophthora sojae. Symptom rating or rot rating established by
Schmitthenner and Bhat (1994) was based on the visual scoring of rotted areas on the root surface and of plant survival in the polystyrene containers. Differential response of the progeny have been observed (see Figure 1.1-1.2). Recovery of the tolerant isolines at later stages have been observed after the rating at 21th day of inoculation (data not shown). The test with three replicates indicated the existence of normal distribution with a mean score of
6.45 (see Figure 1.3.).
31 TOLERANT
Figure 1.1. Soybean seedlings (21 days old) inoculated with Phytophthora sojae Race 1. A: Progeny line #13 rated as one of the most susceptible one among all 58 progeny lines. B: Progeny line #9 rated as one of the most tolerant one among all progeny lines.
32 Figure 1.2. Soybean seedlings (21 days old) inoculated with Phytophthora sojae Race 1. Illustration of rotted roots of progeny isolines. A: Progeny line #13. B: Progeny line #9.
33 PHYTOPHTHORA ROOT ROT TOLERANCE
i u> -li. È I Oh
PRR Scores
Figure 1,3. Bar graph showing distribution of progeny lines with respect to P. sojae Race I. No Primer Name Primer Sequence
1 EcoRl PI 5'-ACTGCGTACCAATTC caa
2 EcoRl P2 5'-ACTGCGTACCAATTC c* 3 EcoRl P3 5'-ACTGCGTACCAATTC ca 4 EcoRl P4 5'-ACTGCGTACCAATTC cag 5 EcoRl P5 5'-ACTGCGTACCAATTC cgt
6 EcoRl P6 5'-ACTGCGTACCAATTC etc 7 EcoRl P7 5'-ACTGCGTACCAATTC ccg
8 EcoRlP8 5’-ACTGCGTACCAATTC egg 9 EcoRlP9 5'-ACTGCGTACCAATTC ctg
1 0 MselPl 5'-GATGAGTCCTGAGTAA gaa
1 1 MselP2 5’-GATGAGTCCTGAGTAA g*
1 2 MselP3 5'-GATGAGTCCTGAGTAA ga 13 MselP4 5'-GATGAGTCCTGAGTAA get 14 MselP5 5'-GATGAGTCCTGAGTAA ggc
15 MselP6 5'-GATGAGTCCTGAGTAA gtc 16 MselP7 5'-GATGAGTCCTGAGTAA gac 17 MselPS 5'-GATGAGTCCTGAGTAA gag 18 MselP9 5’-GATGAGTCCTGAGTAA gtg * primers for preselective amplification
Table 1.1. Primer sequences used in preselective and selective PCR reactions
35 16 1514 13 121110 9 8 7 6 5 4 3 2 1 HC ^ -
m # w
Figure 1.4. A sequencing gel autoradiograph of parental and some progeny lines showing polymorphism with the MseIP9 and EcoRlPl primers. C: Clark; H: Harosoy; 1-16: Progeny lines; Arrows: Polymorphic bands.
36 _16 151413 1211 JO 9 8 7 6 5 4 3 2 1 H C
Figure 1.5. A sequencing gel autoradiograph of parental and some progeny lines showing polymorphism with the MselP7 and £co/?IP 8 primers. C: Clark; H: Harosoy; 1-16: Progeny lines; Arrows: Polymorphic bands.
37 Linkage Groups Loci
A702T_2, A702T_1, 0, Dl, Pdl, B219V_2
MSE4mr3, PI, E014H_2, M7E8mr3, Sat020, A668H_1
Y9, A661V_2, A069D_2, A646D_1, A454D_2, A656H_1, A702I_4, A 702IJ, K274D_2, R013I_1, Satt045, pcr21101, A598T_2, OPG06, A597D_1, A136V_2
K644V_3, K644H_6, Ln, Enp
W 5 A505I l,Lfl,A690D 1.A117H 1 00 —
A748V_1, Sct028, P029T_1, A538I_1, T, KOI 1T_3, R092V_3, Sat076, L148V_1, A635T_1, A031I 2
E2, LBC, Sat038, Pgml, Sail 09
A461T_1, Dtl, Satt006, UBC015
B046I_1, Im, LI, A071V_1, Sattl43, UBC090
10 A374HJ, A036H_2, BLT027I_2, A329I_2, A487I_2, L050I_4, A381I_2, A847H_1, Aco4, A588I_1
(continued) Table 1.2. Linkage map of markers including 10 AFLP markers (italized-bold) found in this study. Table 1.2, (continued).
1 1 A681H_1, Mpi, L183H_1, A378H_1, A586T_2, A235H_1, T005V. 2, KOI 1T_4
1 2 A199H_2, A233D_1, A724T_1, OPFIO, OPEOl
13 A095T_1, A401T_2
14 A816V_1, A121H_2, Slel_l, Satt012, Sattl38, UBC423, All2D_l
15 M9Elntr3, A519T 1, A593H 1, A516H 1, Satt063, Satt020, M 9Elmrl, Satt070, OPF04, Satt009, A584V_2
16 Satt036, Satt077, UBC204, A691T_1, B214T_1, S45035 w A352I_1,B142D_1 VO 17 18 A109HJ, A655H_1
19 A664H_2,B039H_1, LPSI2
2 0 K647I_2, Satt042, A083H_2, A065V_2
2 1 K644H_4, K644V_5, HSP176, K002H_1, K002D_2
2 2 Rps7, Satt009, A280D_1, Sat084, Sat033, B162T_1, ABAB
23 1, GMEN0D2B, UBC767, A020H 2, M8E6mr3, Sattl45, GMRUBP, Satt030, OPA04, M8E6mrl, A018V_2, A183V_2, A203V_2, Al 18V_2 Table 1.2. (continued).
24 A676I_2, A036I_3, A3291_3, A401I_3
25 A605V_1, Satt005, UBC300, A199H_1, Sat043, OPA20, Sat044, SatOOl, Sat046, RPRPl, Satt055
26 MSE4mr2. M7E8mrJ, M7E8mr2, Idhl, Sat069
27 Satt002, Mdh, SattOM
28 0PE14, Sat039
29 A060T_1, Sct046
30 A586T_3, Satt095 o 31 Bng064D2, Sat042
32 M9Elmr2, K258H_2, Satt082, SatOOl
33 pcr21901, Sct065
34 Sat022, Satt031
35 B148T_l,Sattl42
36 T092T_2, Satll2 AFLP procedure originally invented by Zabeau (1993) and modified by Lin and Kuo, (1995)
was performed in the preselective and selective amplification steps as described in Materials and Methods. Five different primer combinations were used in the selective amplification step whereas only one primer combination (MreIP2 and EcoR\?l) was used in the preselective amplification (see Table 1.1. for primers).
Even though the use of silver staining to visualize the AFLP bands on a sequencing gel has been developed (Bassam et al., 1991) and optimized (VanToai et al., 1996), radioactive labelling for primers was used to visualize the AFLP banding pattern in this study. Only clear, unambiguous polymorphic bands were counted as markers and used in the Mapmaker and statistical analysis. On the average, three polymorphic markers were detected firom each
AFLP reaction (see Figure 1.4-1.5).
A total of 13 AFLP markers were provided from AFLP reactions and ten of these markers were added into the existent 36 linkage groups of the previously established public soybean map (see Table 1.2) (Shoemaker and Specht, 1993). Three AFLP markers whose map positions could not be located to any linkage group were not mapped. The 231 markers covered 2222.8 cM area in the public map. Unassigned markers were included in the phenotype-genotype association analysis using single factor analysis of variance but not the
Mapmaker QTL analysis.
41 Tolerance to Phytophthora Root Rot
7.4
i 6.8 i
H 5.6 \J:‘
5.0 —T :------r— r—'— r- r "I------1------:------1— '— r “ t — i — '— t------1------1— — i------r —i------r - 0125 0125 0 1 3 024 0 1 3 0125 A454D-2 A635T-1 A668H-1 KOllT-3 Slel-l SatOOl
Figure 1.6. Single Factor Analysis of Variance results showing the most significant six molecular markers associating with Phytophthora sojae tolerance depicted in a main effect plot. Y axis: Rot rating scores. X axis: Molecular markers and numerical values of polymorphic banding pattern codings. If Harosoy has the polymorphic band and Clark does not, then Clark was coded as A (1) and Harosoy was coded as C (3), all progeny lines were entered either C, A, or - (missing data: 0: Absence of the band in both parents). If Harosoy does not have the band and Clark has the band, then Harosoy was coded as B (2) and Clark was coded as D (4) and all progeny lines were entered as either B, D, or -.
42 Tolerance to Phytophthora Root Rot bl u z 8.8 ■ i 7.6 w 6.4 ■ 1 - 1 x \ . u o 5.2 -1 H 4.0 - i I : ^ i I 12 5 1 2 5 2 4 0 2 4 0125 0125 0125 0 2 4 01 3 01 3 LI Mpi OPE14 OPFIO A063I-2 A280D-IA454D-2 A487I-2 A538I-I A593H-1 w u 8.8 ! i : z 7.6 A I \ . .T : . 6.4 V 1 ■ ■ ...... \ 1 t . w V 11 5.2 1 ’ u I 1 ' o 4.0 - i ! : i 1 ! H 1 , 11 0125 01 3 0 2 4 0125 0125 0125 0Z 4 0125 01 3 0 1 3 A635T-1 A668H-1 B219V-SLT0I3TBngl40T2E014H-2 KOI lT-3 K644V-3 L183H-1 SleI-1 u i ! u 8.8 ! z 7.6 I *\ \ ;. w 6.4 . ■ ■ . - ■ 'V'»-» ■ ■ ■ / • • I - • u 5.2 : i X ^ i : 4.0 / I g ...... ! : i ii 0 125 0125 0125 0125 01 25 01 25 0125 01 25 0 2 4 0 2 4 SatOOl Sat003 Sat038 Sat076 Satt006 Satt012 San030 Satt070 M9EImrlM7E8inr3
Figure 1.7. Single Factor Analysis of Variance results showing significant molecular markers associations with Phytophthora sojae tolerance depicted in a main effect plot. Y axis: Rot rating scores. X axis: Molecular markers and numerical values of polymorphic banding pattern codings. If Harosoy has the polymorphic band and Clark does not, then Clark was coded as A (1) and Harosoy was coded as C (3), all progeny lines were entered either C, A, or - (missing data: 0: Absence of the band in both parents). If Harosoy does not have the band and Clark has the band, then Harosoy was coded as B (2) and Clark was coded as D (4) and all progeny lines were entered as either B, D, or -.
43 Linkage Group Three
21.2 ■ Y9 5.8 ■ A66IV_2
37.9- A069 D_2 3.9 ■ A646D_I A454D_2 38.6 A656H_1 25.2 \. A702L4
\ A702I3 Linkage Group Eleven
A681H_1 345.4- 4 .7 ------3.2 ____ L183H_1 •A378H_I 12.5____ A586T_2
■A235H_I
16.6
K274D_2 T005V_2 ^ R 0 1 3 I_ 1 8.5 att045 KOI IT_4 A598T_2 OPG06 A597D_1 AI36V_2
Figure 1.8. Linkage group three and linkage group eleven showing significant QTL peaks for Phytophthora sojae tolerance
44 LOD score
4^ (VI
20
i i i
Figure 1,9. First significant QTL peak for tolerance to Phytophthora sojae Race on 1 linkage group three LOD score
40 --
4i. 3 0 0 \
30
I I 6 g
Figure 1.10. Second significant QTL peak for tolerance to Phytophthora sojae Race on 1 linkage group eleven Tolerance to Phytophthora Root Rot
u 7.0 - . : U A • A . , \ \ ■ “ 6.4 ■ \ - i f • ■ \ ' * ' : \ • : #\ S 6.1 • : • • 5.8 1 1 2 5 01 25 01 25 01 25 0125 01 25 01 25 0 2 4 0 2 4 01 25 Y9 A661V-2 A069D-2A646D-1 A454D-2A656H-1 K274D-2 R013I-1 pcr21101 A598T-2
fcj 7.0 - Z 6.7 -
2 6.4 6.1
0 1 3 0125 0125 OPG06 A597D-I AI36V-2
Figure 1.11. Main effect plot of Single Factor Analysis of Variance results showing significant loci on chromosome three for tolerance associated with Phytophthora sojae Race I. Y axis: Rot rating scores. X axis: Molecular markers and numerical values of polymorphic banding pattern codings. If Harosoy has the polymorphic band and Clark does not, then Clark was coded as A (1) and Harosoy was coded as C (3), all progeny lines were entered either C, A, or - (missing data: 0: Absence of the band in both parents). If Harosoy does not have the band and Clark has the band, then Harosoy was coded as B (2) and Clark was coded as D (4) and all progeny lines were entered as either B, D, or
47 Tolerance to Phytophthora Root Rot
7.0 - I
u 6.7 - i z < 6.4 -1 o 6.1 -
5.8 - ;
A681H-I Mpi L183H-I A378H-I A586T-2 A235H-1 T005V-2 KOI IT-4
Figure 1.12. Main effect plot of Single Factor Analysis of Variance results showing significant loci on chromosome eleven for tolerance associated with race Phytophthora sojae Race I. Y axis: Rot rating scores. X axis: Molecular markers and numerical values of polymorphic banding pattern codings. If Harosoy has the polymorphic band and Clark does not, then Clark was coded as A (1) and Harosoy was coded as C (3), all progeny lines were entered either C, A, or - (missing data: 0: Absence of the band in both parents). If Harosoy does not have the band and Clark has the band, then Harosoy was coded as B (2) and Clark was coded as D (4) and all progeny lines were entered as either B, D, or -.
48 The probability level was determined as 0.05 to declare a significant association between
marker loci and tolerance. Each markers interaction with tolerance was analyzed with single
factor analysis of variance by using MINITAB software version 10 for Windows (MINITAB,
Inc.). Single factor analysis of variance has revealed significant association of 6 molecular markers (A454D_2, A635T_1, A668H_1, KOI 1T_3, Slel l, SatOOl) with tolerance at the
0.01 probability level. Two AFLP markers (M9Elmrl, M7E8mr3) and other 28 markers has been found to be significant at the 0.1 probability level (See Figure 1.6-1.7).
Linkage groups were established with MAPMAKER/EXP 3.0 (Lincoln et al., 1992a). Two significant QTL were detected by MAPMAKER/QTL (Version 2.0.) (Lincoln et al.,
1992b). One region covering two markers (A454D 2 and A646D 1) on the linkage group three found to be significant for tolerance in MAPMAKER/QTL analysis with a LOD score of above 3. Another region covering KOI 1T_4 and T005V2 markers on the linkage group eleven was detected as a significant QTL in the same analysis (See Figure 1.8-1.11 ).
49 DISCUSSION
Many different tests are available to analyze the pathogenicity of fungi. Among these, several
root inoculation methods have been used such as inoculum layer test and slant-board test.
The inoculum layer test developed by Walker and Schmitthenner (1984), modified by
Schmitthenner and Bhat (1994) was used to differentiate between susceptible and tolerant plants under the disease condition (McBlain and Schmitthenner, 1991). The inoculum layer test also helps to distinguish slow rotting plants from root resistant plants. In this study inoculum layer test was used to screen the Clark x Harosoy population response to
Phytophthora sojae race 1 infection. Differential response among the lines observed and the population showed a distribution with a mean score of 6.45. Similar result was reported
McBlain et al., (1991a). In their study, progeny from the cross of Ripley (a high tolerant cultivar) x Harper (a low tolerant variety) was tested against Phytophthora sojae. The frequency distribution was continuous and skewed.
This study is original in the determination of the tolerance response of the Clark x Harosoy population. However, it could be more significant if the results of inoculum layer test can be repeated with another test such as slant-board test and/or under different environmental conditions such as greenhouse. This is because there are some reports indicating inconsistency of QTL across different environments (Goldman et al., 1993, 1994; Berke and
Rocheford, 1995). In this study, interval analysis based on MAPMAKER/QTL (2.0.)
(Lincoln etal., 1992) (see Materials and Methods) and single factor analysis of variance were
50 used to identify QTL. Some other software such as MAPMANAGER and QGENE recently
has become available. These programs use the same principals of MAPMAKER/QTL to map
the markers and identify QTL. However these are only available for Macintosh computer.
Shoemaker and Olson (1993) and Shoemaker and Specht (1995) created a public RFLP map
that combined a previously available data from the Glycine max x G. max mapping
population, data from the G. max x G. sojae mapping population and data from the newly
established Clark x Harosoy isoline mapping population. The map includes 231 molecular
and classical markers. The Rps 7 gene was located in linkage group 22 in Clark x Harosoy
map and linkage group N in the public map. Another important aspect of this study is that
it is the first report of the identification of QTL in the same map associating with tolerance
of soybean to Phytophthora sojae.
Molecular marker techniques vary from PCR-based approach to the protein analysis
approach and hybridization-based approaches. Some of these techniques are more efficient than others. Lately PCR based-methods such as RAPD, SSR, DAF, and AFLP became much more widely used for detection of DNA polymorphisms. There have been lately many reports that AFLP is a very reliable and powerful DNA fingerprinting technique for bacteria as well as plants, nematodes, viruses and fimgi (Colwyn et al., 1995; Folkertsma et al., 1996;
Keim et al., 1997; Lin and Kuo, 1995; VanToai et al., 1996; Vos et al., 1995; Zebeau, 1993).
In this study the technique was also very successfully performed. The technique efficiency in terms of reliability was very high when the conditions were set properly as mentioned by
51 VanToai et al. (1996). The same results was achieved with different PCR reactions from the
same beginning material or two separate reactions from the same master mixture (which are
the basic tests for the reliability). The problems that VanToai et al. (1996) defined and
unraveled have had happened in this study and solved with the same approaches as well
(VanToai et al., 1996). In my personal experience, I prefer AFLP over RAPD due to the fact
that AFLP works very well imder high stringency conditions which may be very difficult
to achieve with RAPD because of the use of arbitrary short decamers and being very
susceptible to the template DNA concentration (Ellsworth et al., 1993). This may lower
RAPD’s efficiency in PCR reproducibility.
Another advantage of AFLP over other PCR-based techniques was its high efficiency
(Colwyn et al.., 1995; Keim et al., 1997; VanToai et al., 1996; Vos et al., 1995). A large
number around 50-100 restriction fragments were detected per amplification reaction which
provides a higher chance to identify polymorphisms between different samples (Zebeau,
1993 and Vos et al., 1995). However in this study even though I detected similar numbers of restriction fragments (60-80) per reaction, the average polymorphisms for each primer combination were not as high as it was reported previously (VanToai et al., 1996). The reason may be due to the primer combinations that was used in this study or the subset of the selected fragments may not have enough polymorphism. Therefore, most of the possible polymorphic bands may not be selected at the first PCR.
52 All loci identified by the MAPMAKER/EXP 3.0 (Lincoln et al., 1992a) span a distance of
529.5 cM on chromosome three and 45.6 cM on chromosome eleven. T005V 2 and
KOI 1T_4 were found to have the biggest contribution on the QTL found on chromosome eleven whereas A646D1 and A454D_2 found on chromosome three was significant in the
QTL analysis of MAPMAKER/QTL (Version 2.0.) (Lincoln et al., 1992b). These QTL do not contain any known Rps genes of soybean that have been mapped in différent linkage groups at this map or other known soybean maps. This may indicate that the tolerance response to Phytophthora sojae differs from single gene controlled resistance. One of the reason is that the single gene resistance confering complete resistance is different from root roting. Moreover, P. Sojae-soyhean interaction in terms of root roting seems a compatible interaction not an incompatible one. Therefore it is most probable that the known Rps genes on the molecular maps may not associate with tolerance response of soybean. This may also be relevant to the idea that many different strategies of hosts employed against pathogens are controlled by several different loci as a cumulative response.
Other loci revealed significant by single factor analysis of variance was found (see Figures
1.6-1.7). However, only one locus A454D_2 in chromosome three was identified as significant by both single factor analysis of variance, and analysis of MAPMAKER/EXP
(Version 3.0) (Lincoln et al., 1992a) and MAPMAKER/QTL (Version 2.0.) (Lincoln et al.,
1992b) (See Figures 1.6-1.11).
53 These findings can be supportive data towards the understanding of P. ^q/cre-soybean interaction, particularly in terms of tolerance response. However, further studies may be needed to investigate similar aspects of P. jq/ae-soybean interaction with different approaches in order to confirm our findings. Overall, to the best of our knowledge, this study is first original study to unravel the tolerant aspect of P. sq/ae-soybean interaction at the molecular level and hopefully will illuminate or help those who would seek clarify this interaction in future research.
54 CONCLUSIONS
In this study, Clark x Harosoy isoline mapping population was used for molecular marker
analysis. Prr tolerance phenotype was assayed by the inoculum layer tests with three replicates. Total 13 AFLP markers were added to the previously established Clark x Harosoy soybean map. Of the 13 AFLP markers, only 10 {M7E8mr3, M5E4mr3, M9Elmr3,
M9Elmrl, M8E6mr3, MSEômrl, M5E4mr2, M7E8mrl, M7E8mr2) were linked to the existing linkage groups. The map consists of 231 markers on 36 linkage groups and 40 unlinked markers. Six loci (A454D_2, AD635T_1, A668H_1, KOI 1T_3, Slel_l, and SatOOl) were also found significant at the probability level of 0.01 by one way analysis of variance.
Two significant QTL (LOD score 3 or above) associated with the P. sojae tolerance were identified on linkage group three (R ^ 30.4%) and linkage group eleven (R ^ 62.0%) using
MAPMAKER/QTL. This map including AFLP markers spans 2222.8 cM distance.
55 CHAPTER H
DIFFERENTIALLY EXPRESSED SOYBEAN ROOT GENES IN RESPONSE TO
PHYTOPHTHORA SOJAE INFECTION
INTRODUCTION
Soybean root rot disease caused by Phytophthora sojae is a major soybean disease in the
U.S. It has the ability to infect all vegetative organs of the plants and can lead to total
destruction of production (Sinclair, 1982). Resistance determined by major dominant Rps
genes seems to be the best effective method to control the disease. However, the mechanism
of resistance and the starting point of the hypersensitive response (HR) still remains obscure
(Graham, personal communication). HR leads to cell and tissue death at the site of infection
(Baker et al., 1997). In the HR, cell walls at the point of attack and/or surrotmding area can
be reinforced by accumulating phenolic compoimds (Graham and Graham, 1991) resulting in lignification and cross-linking of hydroxyproline rich proteins. The cells also synthesize phytoalexins, and induced pathogenesis-related proteins (Lamb et al., 1989).
The Rps genes of soybean are mediated by seven different loci with multiple alleles at two loci (Athow et al., 1987; Buzzell et al., 1987; Ploper et al., 1985). The genes interact with
56 more than 45 races of P. sojae (Abney, 1997) in a gene-for-gene manner (Keen, 1975; Keen,
1982). This interaction has been a model system for biochemical and genetic studies on host- pathogen interactions (Graham and Graham, 1990). It has been shown that soybean plants are equipped to respond in a constitutive or inducible manner to a variety of elicitors of biotic and abiotic origins (Graham et al., 1990; Graham, 1991a; Morris et al., 1991; Keen and
Dawson, 1992). UV light, some specific synthetic detergents, and heavy metal salts are examples of abiotic elicitors whose mechanisms and effects have not been well characterized. Biotic elicitors are very broad and can be subdivided into general and specific elicitors (Keen and Dawson, 1992; Beckman and Roberts, 1995).
The elicitors referred to as general elicitors (Keen and Dawson, 1992), non-host elicitors
(Atkinson, 1993) or non-race specific-elicitors (Lamb, 1994) can originate from plants or pathogens. One of these elicitors is cell wall glucans. The smallest molecule isolated and characterized from the cell wall of P. sojae was a heptaglucan which was very active in terms of activating HR and phytoalexin production in several soybean cultivars (Keen and
Dawson, 1992; Graham and Graham, 1991). This heptaglucan consists of a five membered
(3-1,6 linked glucose chain with p-1,3 linked glucose molecules on the second and fourth residues (Keen and Dawson, 1992). Keen and Yoshikawa (1983) reported that as plant originated glucan elicitors, endoglucanase in soybean tissue is expressed constitutively at very high levels. The P-1,3-endoglucanases are able to attack the pathogen’s cell walls and, as a result, releasing glucan and glucomannan elicitors.
57 Another example of general elicitors are chitin and chitosan present in the cell walls of
Fusarium solani and elicit callose deposition, plant lignification, phytoalexin synthesis, and
the induction of proteinase inhibitors (Hahn et al., 1993). These two elicitors, especially
oligomers of chitin with four to six N-acetyl glucosamine residues, are very active and
directly inhibit the growth of certain pathogenic fimgi and invoke lignification in wounded
leaf tissues of wheat plants. Even though they elicited defense responses in pea as well as wheat and rice, they were not determined as active agents in some plants such as soybean
(Keen and Dawson, 1992).
Major elicitors from Phytophthora infestans, arachidonic and eicosapentaenoic acids, were detected in invaded potato tuber tissues. These belong to a very distinct class of elicitors because of their fimction as 20 and 22 carbon fatty acids with unsaturations at specific positions in the carbon chain. Specifically, arachidonic acid invoked a general increase in protein synthesis in potato tubers while repressing different sets of host active defense genes
(Keen and Dawson, 1992).
Proteins and oligogalacturonides are another group of biotic elicitors. Treatment of plant tissues or cells with specific enzymes such as exo-polygalacturonase, pectate lyase, and xylanase stimulates an array of defense responses, including the synthesis of pathogenesis related proteins, ethylene, proteinase inhibitors, lignin, activated oxygen and phytoalexins, the inhibition of protein synthesis, the stimulation of membrane K^'/H"^ exchange, and necrosis (Baron and Zambryski, 1995; Cutt and Klessig, 1992; De Wit, 1992a; De Wit, 1995;
58 Keen, 1990; Keen, 1992). A variety of proteins have been documented as elicitors. Some of these are 10 kDa proteins elicitors of HR from culture fluids of Phytophthora capsici, P. cryptogeae, and certain isolates of P. parasitica and elicitor of HR in leaves of some wheat cultivars, as well as 67 kDa glycoprotein from germ tubes of Puccinia graminis f. sp. tritici.
Linear, alpha linked oligomers of D-galacturonic acid in oligogalacturonides, which are plant originated elicitors and also called endogenous elicitors, are of special interest. Generally, oligogalacturonide elicitors elicit the accumulation of phytoalexins and proteinase inhibitors, and the stimulation of pathogenesis related (PR) proteins, e.g., P-glucanase and chitinase, and lignification (Hahn, 1996). Residue range of these elicitors is of special interest, since
10-12 galacturonic acid residues showed optimum elicitor activity. Upon infection, the pathogen releases a hydrolase which digest the host pectic firactions of cell wall to 10-12 residues of oligomers which are active elicitors of the host defense system (Keen and
Dawson, 1992).
Specific elicitors are named as race-specific elicitors. Because they trigger the defense mechanisms of the host plants with corresponding resistance genes which are specific to the gene elicitor. There are a number of reports of pathogenic fungi and bacterial avirulence iavr) genes that have been shown to provide good evidences of efficiently stimulating defense responses only in the plant cultivars having the complementary resistance genes (De
Wit, 1992b; Kombrink and Somssich, 1995). Accumulation of phytoalexins in a bean cultivar resistant to a race was stimulated by a partially purified galactose/mannose-rich glycoprotein from the same race of Colletotrichum lindemuthianum (Tepper and Anderson,
59 1986). Another example of specific elicitors is a necrosis inducing polypeptide isolated fi-om
intercellular spaces of tomato leaves infected with Cladosporium fulvum. This polypeptide
exhibits suitable race-cultivar specificity on differential tomato cultivars containing the
resistance gene complementary to the C. fulvum ovrA9 gene ( De Wit et al., 1988). There
has been no evidence of race-specific elicitor associated with P. sojae ( Schmitthenner,
1988). On the other hand, the evidences of non race-specific P. sojae elicitors have been
identified. A 90 kDa parsley plasma membrane protein has been found as a receptor for the
42 kDa protein elicitor of pathogen. A 70 kDa protein has been identified as a receptor for
the P. sojae elicitor in the soybean root membrane (Hahlbrock et al, 1995; Numberger et al,
1994; Numberger et al, 1995; Cosio et al, 1990; Cosio et al, 1992).
The first step in the elicitor-induced transduction pathway is the perception of the signal
molecule by a specific receptor molecule (ligand). For example, cell wall hydrolysis are not
only important for the pathogen to break down the plant cell wall resulting in gaining access
to vital nutrients, but also essential components of the recognition events by which the plant
recognizes the presence of a pathogen. Therefore, this perception is a prerequisite for the
activation of the host’s defense system. In most cases, it seems that plants do not perceive the extracellular cell wall hydrolysis directly, with the exception of xylanase (Sharon et al,
1993). There has been evidence of the presence of membrane-localized glucan binding sites on the plasma membrane of soybean (Ebel et al., 1995). Recently, a candidate membrane protein (70kDa) has been identified. Since binding sites for glucans are inactivated by heat and protease activity, it is thought that they are proteinaceous in nature (Casio et al., 1992).
60 The combined results of ligand-binding studies provide evidence that for the successful
binding of the glucan elicitor to its target molecule depends on the precise structural requirements for elicitor activity (Cheong et al., 1993).
Most of the inducible defense responses are due to transcriptional activation of specific genes. The interaction of elicitor molecules with membrane receptors involves a complex response in which a number of events are stimulated , leading to an increase in gene expression (Dixon et al, 1994). General aspects of these responses can be summarized and grouped as follows; 1) immediate, initial defense responses which are thought to involve recognition and initial signaling events, 2) locally activated defense responses which are considered to have direct detrimental effects on the attacking pathogens, and 3) systematically induced post defense responses.
The examples associated with immediate early plant defense responses are the heptaglucoside (P-glucan) elicitor from P. sojae, the 42 kDa glucoprotein from the same pathogen and oligopeptide of 13 amino acids derived from them. Ligands of these elicitors have been identified on the plasma membrane of the soybean, tomato, parsley and rice cells
(Ebel and Cosio, 1994; Numberger et al, 1994). The very immediate responses of the parsley cells to the elicitor of P. sojae were changes in the H^, Cl", and Ca-^fiuxes across the
plasma membrane and the formation of H 2 O2 (oxidative burst), occurring within 2-5 minutes
(Numberger et al., 1994). Within the next 5-30 minutes, Ca^"^-dependent phosphorylation of proteins in vivo determined with quick biosynthesis of ethylene and transcriptional
61 activation of the most rapidly induced defense mechanism genes (Dietrich et al., 1990). In
parsley and bean cells, the early transcripts of phenylpropanoid biosynthetic genes associated
with hypersensitive response accumulates maximally in 3-4 hours (Lawton and Lamb,
1987). In addition to these responses, there are some evidences of cytological changes, such as cytoskeleton disorganization (Kobayashi et al., 1995) as well as the production of reactive
oxygen intermediates, e.g. superoxide anion (O; ), and hydrogen peroxide (H 2 O2 ) (Apostol et al., 1989). Generation of and other active oxygen species, such as superoxide, and hydroperoxyl, may not only act as components of the induced signaling pathways but also behave directly to antimicrobial cytotoxicity of the nature of these molecules. These active oxygen species also contribute to the generation of bioactive fatty acid-derivatives and being involved in the cross linking of cell wall-bound proline-rich proteins (Dixon et al, 1994). Due to their very quick production and accumulation, it has been suggested that active oxygen species may be the first line of defense against the pathogen invasion (Hammond-Kosack and
Jones, 1996).
After very early steps of the recognition of the pathogen, the host employs an array of defense responses resulting in transcriptional activation of specific genes, some of which are associated directly with HR (Atkinson, 1993) and others activated by elicitors that do not cause an HR or are expressed to similar levels in compatible and incompatible interactions
(Schroder et al., 1992; Atkinson, 1993; Jakobek and Lindgren, 1993). Locally activated gene products in most cases, directly respond adversely on pathogens and include essential enzymes of general phenylpropanoid metabolism, such as phenylalanine ammonia-lyase
62 (PAL) and 4-coumarate; CoA ligase (4CL), many proteins involved in the biosynthesis of phytoalexins and secondary metabolites, hydroxyproline rich proteins (HRGP) and glycine- rich proteins (GRP), intra and extracellular PR proteins, including chitinases and l,3-(3 glucanases, peroxidases, lipoxygenases, proteinase and polygalactiuronase and antimicrobial proteins ( Kuc, 1995; Lawton and Lamb, 1987; Pontier et al., 1994). Most of these components are shown to be transcriptionally activated in parsley cells by P. megasperma and in parsley leaves upon inoculation with spores of the same fungus ( Kawalleck et al.,
1992,1993,1995; Somssich et al., 1986; Somssich et al., 1988; Schmelzer et al., 1989). In this category, phenylpropanoid metabolisms or pathways have significant importance.
Because it has been shown that it was activated in virtually every plant-pathogen model systems analyzed to date (Nicholsan and Hammerschmidt, 1992). This pathway and its different branches, a large variety of compounds with diverse function are derived, including pigments, antibiotics (phytoalexins), UV protectants, signal molecules and structural compounds, e.g. lignin suberin and other cell wall compounds.
One of the most rapid responses of the defense system is the incorporation of phenolic compoimds into the cell wall. These rearrangements and other cell wall modifications, such as lignification or incorporation of structiual proteins, such as hydroxyproline rich proteins are presumably catalyzed by peroxidases. The purpose of all these defense responses is mainly to render the cell wall less permeable to pathogens (Nicholson and Hammerschmidt,
1992; Kuc, 1995; Lamb et al., 1989). Among the major biochemical compounds that are induced locally, phytoalexins, a diverse group of chemical compounds with broad spectrum
63 antibiotic activities, have been extensively studied. Phytoalexins are characterized as low
molecular weight antimicrobial compounds and are not detected in significant amounts in
healthy tissues. Each plant species produces a characteristic set of phytoalexins which are
derived firom a secondary metabolism and most often constitute phenylpropanoide,
tertenoide, or polyacetylene derivatives . Their key enzymes are usually induced
sequentially, such as PAL and 4CL, and are involved in the formation of parsley phytoalexm
(fiiranocoumarin). One rapid response is the synthesis of callose which is not regulated at the
transcriptional level but through allosteric activation of the biosynthetic enzyme, 1,3-P-
glucan synthase. Callose deposition on cell walls in the form of papillae is a highly localized
defense system occurring aroimd the pathogen’s penetration site (Gross et al., 1993) Because
HR mediated resistance launches from a clearly defined position within the host tissue,
temporal and spatial expression patterns of the host defense system becomes very essential.
This localized mobilization of the defense mechanisms following initial recognition of the
pathogen is essential to limit invaders to a relatively small area.
Local infection leads to the stimulation of a general line of defense at the distal cells in
uninfected parts of the plants. This phenomenon has been called systemic acquired
resistance. Typically, this type of resistance is effective against a wide spectrum of
pathogens, including viruses, bacteria, and fimgi. Recently, it has become apparent that this type of resistance associates with localized responses through the signal transduction pathway initiated locally at the infection front (Draper, 1997; Kessman et al., 1994; Ryals et al., 1996; Staskawicz,1995). Lately a number of difiusible molecules have been identified
64 and their involvement in the signal transduction pathway leading to general plant disease resistance was well documented (Lamb, 1994). Salicylic acid (SA) and jasmonic acid (JA) are some of the most-studied endogenous signal molecules (Delaney et al., 1994; Creelman and Mullet, 1997).
Surprisingly, SA is also a product of the phenylpropanoid pathway (Raskin, 1992). The strongest evidence supporting the involvement of SA in plant-induced resistance has been provided by Gaffiiey et al., (1993). The nahG gene from Pseudomonas putida that encodes for salicylate hydroxylase converts salicylic acid to catechol, a phenol that is unable to induce resistance, was constitutively expressed on transgenic tobacco plants (Gaffiiey et al.,
1993). The authors concluded that the transformed plants have a decreased capacity to form resistance mechanisms as compared to the control plants that are not transformed with this gene. Even though the involvement of SA in SAR has been well established (Delaney et al.,
1994; Ryals et al., 1996; Staskawickz et al., 1995; Uknes et al., 1996), it is now known that
SA is not the translocated signal (Vemooji et al., 1994; Ryals et al., 1994; Ryals et al., 1996;
Uknes et al., 1996). Therefore, it is hypothesized that local infection leads to the induction of a mobile component whose function may depend upon SA in distal tissues for the expression of SAR (Klessig et al., 1994 ).
In the case of soybean, Graham and co-workers ( 1989, 1990, 1991a, 1991b, 1994, 1996) defined at least three distinct zones that appear to be involved in the complete reaction complex. The first zone is the HR cell lesion which is likely to be only one cell thick. This
65 type of cell is in immediate contact with the launching hyphae of the fungus. The response
mechanism of this first zone can be mimicked by woimding experiments. Another zone is
the proximal cell zone which is presumably the 2-4 cell thickness just before the outside of
the HR region. The host in this zone mobilizes phenolic polymer deposition (Graham and
Graham, 1991), hydrolyzes isoflavone conjugates into genistein, a directly adverse
isoflavonoid molecule to P. sojae (Rivera-Vargas et al., 1993), and daidzein, a precursor of
glyceollin (soybean phytoalexin, pterocarpan antibiotic), and accumulates glyceollin (Ebel,
1986; Ebel and Grisebach, 1988). The last zone in healthy cells is distal to the HR zone and
responds to elicitor treatments with an increased accumulation of conjugates of daidzein and
genistein (Graham and Graham, 1991). This massive build up of isoflavone conjugates
stimulates the defense potential of these distal cells. In the HR zone, dying cells release some
wound-associated signals which are necessary for some of the proximal cell responses
(Graham, 1994; Graham and Graham, 1994). These wound-associated signals are soybean
in origin and may not associate distal cell responses (Graham, 1994; Graham and Graham,
1994).
HR is an immediate early response of incompatible reaction and is characterized by the death of the cells in contact with the pathogen. In the surrounding cells of this immediate contact zone, phenolic polymer deposition and very rapid and massive accumulation of phytoalexins restrict the invading pathogen. There is strong evidence that phytoalexins, such as coumestrol and glyceollin, accumulate in infected soybean plant tissues. It has also been shown that the cessation of growth of the pathogen in resistant plants is associated with the
66 accumulation of soybean phytoalexin, glyceollin, and correlates well with race-specific resistance (Graham, 1989, Graham et al., 1990, 1991; Hahn et al., 1985; Keen and
Yoshikawa, 1983; Ward, 1989) but not with race non-specific resistance (Schmitthermer,
1988). It was proposed that the mechanism for the toxicity of glyceollin was associated with the injury of the plasmalemma and tonoplast ATPase activity. In addition to glyceollin and coumestrol, genistein and daidzein (isoflavonoids) that are present constitutively in large quantities are produced in infected soybean tissues (Graham, 1989; Graham et al., 1990;
Graham et al., 1991). It was proposed that daidzein may play an important role in the overall accumulation of glyceollin since it is the first immediate precursor of glyceollin (Graham et al., 1990). Besides the daidzein, genistein may be directly adverse to P. sojae (Graham,
1989). Conjugates of daidzein are rapidly hydrolyzed to firee daidzein during the incompatible interactions of soybean with P. sojae. Conjugates of both daidzein and genistein also accumulate in response to the pathogen attack after 8 hours when compared to the glyceollin that begins to accumulate after 12 hours. Unlike incompatible reactions, the release of precursors is delayed in compatible reactions and low levels of glyceollin accumulate after the infection (Graham, 1989; Graham et al., 1990; Graham et al., 1991).
Moreover, accumulation of phenolic compounds induced by elicitors of P. sojae (cell wall glucans) have been observed in cells immediately ahead of the infection fi-ont (Graham and
Graham, 1994). This rapid and massive induction was observed in soybean cotyledon cell walls proximal to the point of elicitation. Within 4 hours of treatment, the accumulation of these phenolic compounds in the wounded controls was around 10 times less than in the stimulated tissues and continued to increase by 24 hour in the elicited tissues. This
67 deposition was the highest among those including glyceollin and isoflavonoid compounds
and correlate with the induction of a specific group of peroxidases that are considered to be
involved in the final step of phenolic polymer deposition in the cell wall at the expense of
H2 O2 (Graham and Graham, 1994).
Presence of competency factors (wound-associated factors) were also demonstrated in response to the P. sojae wall glucan elicitors induced in soybean tissues. ( Graham and
Graham, 1994). The authors suggested that these competency factors may play a role in cell to cell signaling and are released fi-om dying or dead cells during HR in resistant plants.
Interestingly, there is no hydrolysis of pre-formed isoflavone conjugates, but rather an accumulation of these competency factors are apparent in distal cells. It is possible that they may act as a long distance stimulator and build up a defense response in distal cells. All these responses in the proximal and distal cells of resistant plants are induced sequentially and spatially with temporal fashion by P. sojae cell wall glucan elicitor ( Graham and Graham,
1994).
Since, HR mediated response initiates from a clearly defined cellular position within the soybean plant tissues as well as in other plants, such as parsley and bean, both temporal and spatial expression of defense mechanisms are vital. Hence, due to their rapid and localized activation at the site of infection followed by the first point of perception, these localized responses can be suggested as a second line of defense.
68 Recently, many genes for resistance to viruses, bacteria, nematodes and fungi have been
cloned (Bent, 1996; Hammond-Kosack et al., 1996; Hammond-Kosack and Jones, 1996;
Hammond-Kosack and Jones, 1997; Kunkel, 1996; Kunkel et al., 1993; Staskawicz et al.,
1995) including the M locus in flax conferring resistance to Melampsora Uni, the rust disease
on which Flor (1971) based his famous gene-for gene hypothesis (Anderson et al., 1997).
Interestingly, all cloned R gene products share high level structural similarities, such as
leucine-rich repeat (LRR) motif or serine-threonine kinase domain, regardless of whether
they confer resistance to viral, fungal, or bacterial diseases or nematodes. Hence recognition
of a virus by a resistant host does not look to be essentially different to recognition of a
bacterial, fungal pathogen or a nematode. Such structural similarities suggest a functional
role for the R gene products as membrane receptors and/or components of signal transduction
pathways (Jones and Jones, 1997; Gebhardt, 1997). Also, some R gene analogs in soybean
and potato were identified by help of such structural similarities (Kanazin et al., 1996;
Leister et al., 1996).
R genes can be classified according to their common features as follows: The first class includes RPMl and RPS2 having a nucleotide binding site (NBS) and LRR domain and encode cytoplasmic receptor-like proteins (Bent et al., 1994; Mindrios et al., 1994; Grant et al., 1995). The second class of R genes, (e.g., Ptd), encodes serine-threonine kinase.
Interestingly, Pto needs the action of Prf encoding an LRR and NBS containing protein to function properly (Martin et al., 1993; Salmeron et al., 1994). The third class of R genes encodes transmembrane proteins with LRR domains. This group includes Cf-2, Cf-9, and
69 (Dixon et al., 1996, Jones et al., 1994; Daguang et al., 1997). The fourth class,
represented by Xa21, encodes a presumed transmembrane receptor having an intracellular
serine-threonine kinase domain and extracellular LRR domain (Song, et al., 1995). The
fourth class includes N, L6, and RPP5 genes products. These are intracellular proteins with
amino terminal domain (homology with Drosophila Toll protein), nucleotide binding site,
and leucine-rich repeats domains (Whitham, et al., 1994; Lawrence, et al., 1995). The last
class is represented by HMl (Johal and Briggs, 1992). HMl encodes a reduced form of
nicotine amide adenine dinucleotide phosphate (NADPH)-dependent reductase that
inactivates the toxin produced by Race 1 of Cochliobolus carbomim (Hammond-Kosack and
Jones, 1997; Baker et al., 1997; Jones and Jones, 1997). Recently, interaction of R and Avr gene products, which is the basic implication of revised gene-for-gene theory has been proven by using the yeast two hybrid system (Scofield et al., 1996; Tang et al., 1996).
However, it is not yet clear whether this relationship holds true for each HR specific relationship or R-Avr interaction.
In this study, our goal is to determine differential gene expression patterns and clone differentially expressed genes in response to inoculation of P. sojae Race 1 in soybean-
Phytophthora sojae model system and attempt to clone Rpsl-k gene.
70 MATERIALS AND METHODS
Two isolines with/without Rpsl-k gene, Elgin 87 and Elgin respectively, have been used for
inoculation, screening, and differential display analysis in this study. These isolines were
provided by Schnaitthenner (OSU, OARDC, Wooster, Ohio).
Growing Phytophthora sojae zoospores for Inoculation
Medium for growth (dilute lima bean agar) was prepared according to Schmitthermer and
Bhat (1994). Pieces of the agar (1 mm) from the stock tube were placed on fresh lima bean
agar plates and left on the lab bench at room temperature for 4 days (until it grows the size
of a quarter). Twenty pieces of agar (1 mm) from the outside edge of the colony were cut and
placed in 25 ml of fresh lima bean broth. After growing for 4 days at room temperature,
the solution in the Bellco flask was poured off and replaced with 25 ml of Chen Zentmayer
salt solution (Schmitthermer and Bhat, 1994). After 15 min, the solution was changed again.
This procedure was repeated for a total of 4 changes. The last solution was replaced with 25 ml distilled water. The flasks were placed overnight on the bench. Zoospores were counted the following morning by viewing under a microscope. The zoospores were then used for inoculation (Bhat and Schmitthermer, 1993a; Bhat and Schmitthermer 1993b).
71 Liquid Culture of Race 1 of Phytophthora sojae (Lima Bean Broth)
25 g of lima beans (Fordhook lima beans) were placed in a container containing 250 ml water. After autoclaving for 20 min, the beans were mashed with a spatula and passed through a sieve into a beaker (1000 ml). The solution was then filtered through 2 cm of celite and 2 Whatman papers (#2). It was filled to 500 ml with water and aliquoted into 250 ml bellco flasks of 25 ml each which were then autoclaved for 20 min.
Inoculation of Roots with Zoospores
All equipment including seed containers, plastic germination bags, pipet tips, drinking straws, and glass beakers were sterilized by autoclaving. Drinking straws were cut in 2 cm length and filled with 100 pi of zoospore solution ( containing approximately 1000 zoospores) or sterile water for controls. Roots of 48 hour germinated Elgin and Elgin 87 seed
( with 2-4 cm root length) were inserted into the straws containing 100 pi of zoospore solution. After 3,6,13,17 hour intervals of inoculation with zoospores or water, roots were taken out and cut from the areas that were inoculated. Root sections were immediately frozen in liquid nitrogen and placed in a - 80 °C freezer.
RNA Isolation
The glassware and pestle and mortar were baked at 180 “C overnight prior to use. All
72 solutions were treated with diethylpyrocarbonate (DEPC). Plasticware used was either from sterile unopened packs or DEPC treated packs. Isolations were performed for four different
times (3 h, 6 h, 13 h, 17 hours) after Phytophthora sojae inoculation.Root tissues (1-3 g) from healthy and Race 1 inoculated plants were used for RNA extraction. Tissues were ground to a fine powder with a pestle in a mortar filled with liquid nitrogen. Frozen
powdered tissue was transferred to 2 0 -ml disposable polypropylene tube (resistant to 1 0 0 0 0 rpm) containing 10 ml of RNA extraction buffer (100 mM Tris-HCl (pH 9.0), 10 mM EDTA
(pH 9.0), 2% SDS, 100 mM LiCl, 1 % SDS). After vigorous shaking for 10 sec, 3-9 ml of the supernatant (depending upon the initial amount of the material 1:3 ratio) was extracted once with phenol:chloroform:isoamylalcohol (24:24:1). After 5-10 min of vortexing at high speed, the samples were centrifuged at 10 000 rpm for 20 min at 4 “C. The aqueous phase was collected in a sterile polypropylene tube. RNA was selectively precipitated by addition of LiCl to a final concentration of 2M and stored at 4 °C overnight. The next day, samples were centrifuged at 10000 rpm for 30 min at 4 °C, the DNA left in the supernatant was discarded. To remove any remaining DNA, the pellet was mixed with 1:10 volume of 2 M sodium acetate (pH 5.6) and then washed once with 2 volumes of ethanol (100%). The samples were stored at -20 °C for 2-3 hr. Precipitated RNA was collected by centrifugation at 10000 rpm at 4 “C for 20 min and washed once with 70% ethanol. After the final centrifugation at 10000 rpm at 4 °C for 5 min, the ethanol was discarded and the RNA pellet was left to dry for 10-20 min. The pellet was then redissolved in 100 pi sterile DEPC-treated water and quantitated by a spectrophotometer (Beckman, DU-50). Approximately, 300-800 pg RNA was obtained with this method. RNA (3 pg) was tested on a denaturing formamide-
73 agarose gel (1.5%) to check the integrity of the RNA. RNA from the fungus and plant
materials has been isolated according to the method mentioned above.
Removal of DNA from RNA
RNA (50 (ig) in a mixture of 10 units of human placental RNase inhibitor, 10 units of
RNase-free DNase 1,0.1 M Tris-HCl, pH 8.3,0.5 M KCl, 15 mM MgCl; was incubated at
37 °C for 30 minutes. Phenolrchloroform (3:1) was added to the mixture, vortexed and
centrifuged 2 min at maximum speed in order to deactivate DNase I before cDNA synthesis.
The upper phase was transferred to a sterile tube containing 3 M sodium acetate and 100%
ethanol. It was incubated for 30 min at -70 ®C to precipitate the RNA and then centrifuged at high speed for 10 minutes. Subsequently, the supernatant was removed and the remaining pellet was washed once with 70% ethanol. The RNA pellet was then dissolved in 20 pi
DEPC-treated water, quantitated by measurement with the spectrophotometer, and 3 pg of
RNA was electrophoresed on denaturating formamide-agarose gel (1.5%) to check the integrity of the RNA to be used in differential display analysis.
cDNA Synthesis
Purified RNA samples were reverse transcribed using First Strand cDNA Synthesis KIT
(Pharmacia Biotech). 3 pg of purified total RNA was diluted in water (0.15 pg/pl) and incubated 10 minutes at 65 °C to denature the secondary structures on mRNAs. The samples
74 were chilled on ice for 5-10 minutes and reverse transcribed at 37 “C for 1 h in a total
volume of 33 |il reaction mixture containing 3 mM DDT(Pharmacia), 0.006 pg first strand
primer (Pharmacia), and 11 pi of buffer for the first strand reaction mixture (Pharmacia).
PCR Amplification of cDNAs
DNA amplification was carried out in a 20 pi reaction mixture containing PCR buffer (50
mM KCl, 10 mM Tris-HCl, pH 8 .8 , 1.5 mM MgCl;, 3 mM DTT) and 200-400 ng of cDNA,
10 pCi of ^-P (dCTP), 0.2 pM arbitrary decamer, 1 pM degenerate anchored oligo(dT)
primer (T,g or TnMN), 2 pM 4dNTP and 0.5 units Taq polymerase (Perkin Elmer). The
PCR program was 40 cycles of dénaturation, annealing, and extension steps with the
following temperatures respectively: 30 sec at 94 °C, 60 sec at 40 °C, 60 sec at 72 °C, and one
final cycle of 5 min at 72 °C. See Table 2.1. for primers used in PCR amplifications.
Gel Electrophoresis
5 pi of the PCR product was mixed with an equal amount of formamide loading buffer (98% formamide, 10 mM EDTA, 0.01% w/v bromophenol blue, 0.01% xylene cyanol FE) and incubated for 3 min at 95 °C and quenched on ice for 2 min, centrifuged briefly, and loaded
on the gel (5% polyacrylamide with 8 M urea) and electrophoresed for 1.5 hour at 80 W.
75 No Oligonucleotide Name Oligonucleotide Sequence (lObp) Anchored Oligo d(T) Primers (13bp) 5' Arbitrary Primer Set 3'-T-Tailed Anchor Primer Set TTTTTTTTTTT (M)(N) I 425 5'-CGT CGG GCC T TTTTTTTTTTT A A
2 427 5'-GTA ATC GAG G TTTTTTTTTTT G A 3 437 5'-AGT CGG GTG G TTTTTTTTTTT G A
4 445 5'-TAG GAG GIT G TTTTTTTTTTT A G
5 448 5'-GTT GTG GGT G TTTTTTTTTTT G G
6 463 5'-AGG GGG AAG G TTTTTTTTTTT G G o\ 7 465 5'-GGT GAG GGG T TTTTTTTTTTT A G
8 471 5'-GGG AGG GGA A TTTTTTTTTTT G G 9 475 5'-GGA GGG TAT T TTTTTTTTTTT G G
1 0 478 5’-CGA GGT GGT G TTTTTTTTTTT A T
1 1 485 5'-AGA ATA GGG G TTTTTTTTTTT G T
1 2 499 5’-GGG GGA TGA T TTTTTTTTTTT G T
Table 2.1: Sequences of 5'-primers and 3'-primers used in Differential Display. Silver Staining of Polyacrylamide Gels
Silver staining originally described by Bassam et al. (1991) was performed with minor
changes. The developer solution (30 g/L NazCO], 0.055 % formaldehyde, 400 pg/L sodium
thiosulfate prepared as lOmg/ml) was prepared 1 hour before use and chilled at -20 °C.
Depending on the time available, either the fast or slow fixation technique was used. In the
fast technique, the gels were fixed with 1 0 % acetic acid with gentle agitation for 20-30 min
or until the dye on the gels disappeared. In the slow fixation technique, the gels were
incubated in 10% acetic acid overnight without agitation. After fixation, the gels were
washed 3 times with 2 L of double distilled water for 2 min each and then transferred to a
silver impregnation solution (containing silver nitrate 0.73 g/L and 0.055 % formaldehyde)
for 30 min. The gels were then rinsed with double distilled water for 6-7 sec and immediately
transferred to the cold developer solution. When the bands appeared, the gels were fixed in
10% acetic acid and rinsed with double distilled water for 1 min. As a final step, the gels
were left to dry at the room temperature before they were exposed to Kodak AFC paper for
20-60 second (depending on the intensity of bands) using fluorescent ceiling light. The photographs were developed with occasional shaking for 5 min in 1 liter of developer solution (Kodak), 30 sec in 1 liter of stop solution (Kodak), 30 sec in 1 liter of fixer solution
(Kodak) and a final rinse in water for 5-10 min. The gels were hung to dry.
77 Removal of Differentially Expressed Bands
One or two drops of elution buffer (containing 50 mM KCl, 10 mM Tris-HCl, pH 9.0, and
0.1% Triton X-100) were placed on the band of interest. After 15-20 sec, the band was
excised with a sterile blade and transferred to 0 . 6 ml microtubes containing 1 0 0 pi of the elution buffer. The microtubes were incubated at 95 ®C for 20 min and then centrifuged for
5 min at 15 000 rpm. Supernatants were transferred to new microtubes and stored at - 20 °C.
2-6 pi of the eluted bands were reamplified for one or two times as described above. The reamplified PCR products were electrophoresed on 2% agarose gels and at 70-90 V for 2-3 h to confirm the product size. The bands with positive identity were used in labelling reactions for Northern hybridization.
RNA Samples Preparation
RNA samples (10 or 20 pg) were diluted in 6 pi DEPC-treated water and mixed with 12.5 pi of formamide, 2.5 pi of lOx MOPS, 4.0 pi of formaldehyde (37.7%) and 2 pi of ethidium bromide (0.5 mg/ml). The solution was incubated at 65 °C for 5 minutes and quenched on
ice. 5 pi of DEPC-treated 6 x loading buffer (0.025% bromophenol blue and 40% sucrose) was added to each sample. Samples were loaded onto the agarose/formaldehyde gel as described elsewhere in this paper.
78 Agarose/Formaldehyde Gel Electrophoresis
Seakem agarose (1.8 g) (FMC BioProducts) was dissolved in 109.5 ml water in a 200 ml
glass flask with 0.1% DEPC added to the solution. The flask was shaken at a moderate
speed overnight. The following day, 15 ml of 1 Ox MOPS (concentration) was added to the
agarose solution and heated in the microwave for 3 minutes. After cooling to 60 “C, 25.5 ml
of formadehyde (37.7%) was added. The gel was poured into a DEPC treated electrophoresis
tray and after gelling was placed in an electrophoresis tank washed several times with DEPC
treated water. A sufRcient amount of 1 X MOPS was added to the tank as a running buffer.
RNA samples were first mixed gently by vortexing or hand shaking, and then centriftiged
briefly to collect the liquid. The samples were loaded onto the gel and electrophoresed at 90-
100 V for 2.5-3.0 hours until the bromophenol blue dye migrated one-half to two-thirds the
length of the gel. The gel was removed from the tray and trimmed of excess gel to the size of membranes. RNA integrity was checked under UV light at 315 nm. The gels were oriented by cutting a small portion of the upper left side of the gel for recognition during
RNA transfer to the membranes and then directly used for transfer.
Transfer of RNA from Gel to Membranes
Two layers of buffer (2x SSC) wetted Whatman 3 MM paper were laid over a plexiglas plate(8.2xl5.5 cm) with the ends extending into a plastic tray (11.5x20.0 cm) filled with 2x
SSC buffer. The cut gel was placed on the two layers of Whatman 3 MM paper. Air bubbles
79 were squeezed out by rolling a glass pipet (washed with DEPC-treated water) over the surface. A Hydrobond™-N+ (Amersham) membrane the exact size of the gel, including the
marked edges position, was laid over the gel and covered with a sufficient amount of 2 x
SSC. Bubbles under the membrane were removed as mentioned above. Two layers of
Whatman 3 MM paper wetted with 2x SSC buffer were placed on top of the membrane surface which was wet with 2x SSC. Plastic wrap covering the whole apparatus was placed over the Whatman 3 MM papers to prevent the evaporation of buffer and a window with the exact size of the gel was cut. Paper towels were cut to the same size as the membrane and stacked (4-5 cm thick) on top of the Whatman 3 MM paper. A glass plate was laid over the paper towels and supported with a weight (500 gram). The transfer was performed overnight.
Radioisotope Labelling of Reamplified Bands
Labelling of the probes was performed with the Megaprime Labelling Kit ( Amersham).
The labelling reactions with a-^^P [dCTP] radioactive nucleotide (10 pCi/pl) in a total volume of 50 pi were carried out as recommended by the manufacturer.
Preparation of Membranes for Hybridization
Paper towels were removed and Hydrobond™-N+ (Amersham) membrane was rinsed with
2x SSC and placed on a sheet of Whatman 3 MM paper to dry. RNA on the dried membrane was placed RNA-side-down on a UV transilluminator and immobilized with irradiation for
80 30 second as recommended by the manufacture. The membrane was covered with the plastic wrap and stored at -20 °C for further use.
Hybridization of Hydrobond™-N+ Membranes with Labelled Probes
Hydrobond™-N+ (Amersham) membranes with total RNA from inoculated and control plants were placed RNA-side-up in a hybridization tube (either a glass tube manufactured by Robbins Scientific or plastic 50 ml certrifuge tube) and 20 ml of hybridization solution
(5x SSC, containing 175 gr/L NaCl, 88.2 g Na3-citrate, 20 ml/L 0.5 M EDTA, 10 mM Tris-
HCl, pH 7.5,50% v/v formamide, 10% w/v wheat germ tRNA, 0.25% milk nonfat powder,
0.1% sodium dodecyl sulfate, 1 mM EDTA) was added. The tube then was placed in a hybridization incubator (Robbins Scientific, Model 310) and incubated with rotation for 1-3 hour at 42 “C. The hybridization solution was discarded and replaced with 20 ml of hybridization solution containing 50 pi of the labelled probe. Hybridization was performed overnight or for 6-12 hours at 42 “C . This solution was replaced with 20 ml of washing solution 1 (2x SSC, 0.5% SDS) and washed with rotation at 42 “C for 20 minutes. Washing solution I was poured off and a second washing was repeated exactly as described above.
The next washing contained 20 ml of washing solution II (O.lx SSC, 0.5% SDS) and was carried out in a 62 °C water bath for 20 minutes (Forma Scientific, Model 2568). The washing was repeated one more time with 20 ml of fresh washing solution II exactly as described above (if necessary, the washing was repeated at 62-65 “C several more times).
Washing solution II was discarded and the membrane allowed to dry on a clean paper towel.
81 The dried membrane was covered with plastic wrap and placed in a intensifying screen with
the RNA-side-facing up position. An X-ray film was then placed on the membrane and
developed at -80 °C overnight
Cloning of Differentially Displayed Sequences
The sequences that showed positive results on the northern blot analysis were PCR amplified
to be used in the ligation reaction. The probe was ligated to the pCR™ 2.1 vector (Original
TA Cloning Kit, Invitrogen) in a ligation reaction (Ix ligation buffer, 50 ng pCR™ 2.1
vector, 4 Weiss units of T4 DNA Ligase, 50-150 ng of fresh PCR product) which was mixed gently and incubated overnight at 14 “C. Competent cells (One Shot™ cells, Invitrogen) were removed from -80 °C and immediately placed on ice. When the cells defrosed, 50 pi were
aliquoted to 0 . 6 ml microtubes. 2 pi of ligation reaction was added to each microtube and incubated on ice for 10 minutes. Heat shock was applied for exactly 30 second at 42 °C in
PTC-100™ Programmable Thermal Controller (MJ Research, Inc.). The microtubes were placed back on ice and 400 pi of autoclaved LB medium (0.5% w/v yeast extract, 1% w/v tripton, 1% w/v NaCl pH 12-1 A) was added to each microtube. The tubes were then incubated 0.25-2.0 hour at 37 “C in a waterbath (Forma Scientific, Model 2568) or in the thermocycler (MJ Research, Inc., PTC-100™ Programmable Thermal Controller).
Simultaneously, 30 pi X-Gal and 30 pi IPTG were spread onto previously prepared and labelled LB plates with a sterile glass hook and dried for 10 minutes. An aliquot of each transformation (50-200 pi) was spread on the plates with a sterile glass hook. After the
82 liquid was absorbed (5-15 mins), the plates were placed in a 37 °C incubator overnight for
colony development. The plates were removed to a refrigerator (4 °C) for 2-4 hours to
enhance the color of nontransformed colonies. Clearly distinguishable white colonies were
picked with sterile toothpicks and spread on previously prepared X-Gal and IPTG LB plates.
For each white colony transfers, a blue colony was also transferred to the same plates as the
positive control. The plates were placed in the incubator at 37 °C overnight as described above.
Small Scale Preparation of Plasmid DNA
Sterile liquid LB medium (3-5 ml) containing carbenicillin (50 mg/L) was poured into sterile glass tubes (30 ml). A single bacterial colony was transferred to the glass tube and incubated overnight at 37 °C with vigorous shaking (3000-4000 rpm) on an orbital shaker. Inoculated liquid cultures were removed from the shaker and placed on ice. 1.5 ml of the liquid culture was removed to a microtube and centrifuged at 5000 rpm for 2 min at 4 °C to pellet the bacteria. The supernatant was discarded and the bacteria resuspended in 100 pi of TGE (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0). The tube was vortexed and 200 pi of NSE (0.2 N NaOH, 1% SDS) were added followed by a brief vortexing. The DNA was precipitated by adding 150 pi of cold 3 M NaOAc. The tube was incubated for 5 min at 4 °C and centrifuged at 5 °C at 15000 rpm. The supernatant was removed and transferred to a new
1.5 ml micro tube. Equal amounts of phenol were added to the tube to extract the DNA. The tube was then centrifuged for 5 min at 4 “C at 10000 rpm. Chloroform extraction was
83 performed in the same manner. The upper phase of the solution was transferred to a new 1.5 ml microtube and 2.5 volumes of 100% ethanol was added. After incubating at -20 °C at least for 1 hour, the tube was centrifuged for 10 min at maximum speed (15000 rpm). The supernatant was discarded and the pellet washed with 100 pi of 70% ethanol. The tube was centrifuged for 2 minutes at maximum speed. The ethanol was discarded and the pellet air dried, resuspended in 60 pi of sterile water, and mixed with 0.5 pi of DNase-free RNase
(lU/pl). The sample was briefly vortexed and stored at -20 °C for further use.
Removing of Inserts
DNA (3 pi) from the small scale preparation (miniprep) was mixed with Ix CA buffer (1 M
Tris-HCl, pH 7.5, 1 M MgCl2 , 2 M KCl, 1% bovine serum albumin, 0.07% 2- mercapthaethanol added prior to use) and 2 units of EcoRl. The solution was incubated for
1-3 hours at 37 °C. The size of insert was checked on an agarose gel (2%) or stored -20 °C for further use.
PCR Analysis of the Cloned Inserts
The plasmid samples were diluted 100 times. For PCR, 2 pi of lOOx diluted sample was mixed with the appropriate primers (M l3 reverse primer, T7 promoter primer). In a total volume of 25 pi, the reaction mixture consisted of 1 pM M l3 reverse primer, 1 pM T7 promoter primer, 0.25 pM each of the four dNTPs, 150 pM MgClj, Ix PCR buffer, and 1
84 unit of Taq DNA polymerase. After an initial dénaturation step for 5 min at 95 °C, amplification was performed for 29 cycles at 92 °C for 30 sec, 58 “C for 30 sec, 72 °C for 30 sec and an extension period at 72 “C for 5 min. An aliquot of the resulting PCR product (10 pi) was electrophoresed on 2% agarose gel stained with ethidium bromide. DNA firagments were visualized under UV light at 315 nm. The remaining PCR product was sent directly to the DNA Sequencing Facility, Molecular Biology Department, Iowa State University,
Ames, lA where sequencing was performed using fluorescent labelled primers and dideoxy termination protocols developed by Applied BioSystem, Inc. (Foster City, CA), for ABI automated sequencing equipment. Sequencing results were analyzed with Intelligenetics
Sequence-Management Software (Genetics Computer Group, Inc., Madison, WI).
Sequence Analysis in Database
Sequence analysis were compared with known sequences in the current database (GenBank) using BLAST through Internet (http://www.ncbi.nlm.nih.gov/Web/Search/irx.html) in
National Center for Biotechnology Information Homepage.
85 RESULTS
Inoculation experiments, as explained in Materials and Methods section of this chapter, were performed for two times. The symptom development was observed on roots of inoculated samples and controls of Elgin and Elgin 87. Five to ten samples from each treatment were evaluated for symptom development.
Symptom development (small brown specks) occurred on Elgin 87 but not on Elgin roots.after 13-17 h of zoospore treatments indicating the presence of HR. In Elgin, symptom development started after 17 hour of treatment, and by 48 hours, it peaked.
Total RNA were isolated from inoculated and control Elgin and Elgin 87 roots, at 3, 6 , 13, and 17 h of the treatments. In Figure 2.1., total RNAs isolated from some of the samples are shown. The integrity and purity of RNA was checked by agarose gel (2%) electrophoresis
(Figure 2.1.). High quality RNA is essential for cDNA synthesis and northern analysis.
Each differential display reaction produced on the average 60 bands (representing partial mRNAs) ranging in size from 100 to 1200 bp. The highest average numbers of the bands displayed per gel were detected by G and C subset of anchored oligo d(T) primers respectively.
86 Figure 2.1 : Agarose gel (2%) of total RNAs isolated from inoculated and control Elgin and
Elgin 87 roots at 3,6, 13, 17 h. L: 0.24-9.5 Kb Ladder. The highest molecular weight is 9.5
Kb.
87 Of the around 11000 bands produced, 49 were differentially expressed and were classified
into one of seven pattern. Pattern 1 : expressed in inoculated roots of both Elgin and Elgin
87, not expressed in control roots; Pattern 2: expressed only in control and inoculated Elgin
87 roots, not expressed in Elgin roots; Pattern 3: expressed only in control Elgin 87 roots;
Pattern 4: expressed only in inoculated Elgin 87 roots; Pattern 5: expressed only in control
Elgin and Elgin 87 roots. Patterns 6 and 7: expressed at low level in control Elgin and Elgin
87 roots, level of expression increased in inoculated roots of both Elgin and Elgin 87.
Most of the bands were equally abundant in control and inoculated samples of Elgin and
Elgin 87 at 3 and 6 hours of post-inoculation. Two polymorphic bands have been detected unique to the inoculated samples at these hours. These were constitutively expressed.
The polymorphic bands specifically induced after the infection (gene pattern 1, see table 2.2) were the most dominant polymorphic bands ( 69.4%). Most of these bands appeared at 13 and 17 h post-inoculations in both Elgin and Elgin 87. These genes are specific for inoculations with the zoospores. 91.2% of these appeared at 13 h and continued to expressed at 17h. 5.9% of these just appeared at 13 hour treatments. cDNAs that appeared to be differential in both inoculations and controls at the different intervals have been utilized as probes for northern hybridizations. Three of these probes (Rl, R2, and R3) were confirmed by northern analysis (see Figures 2.5,2.6, and 2.7). These sequences expressed at 13 hours inoculated in both Elgin and Elgin 87, but were not expressed in control roots.
88 Gene Elgin Elgin Elgin 87 Elgin 87 Total Percentage of Action Control Inoculated Control Inoculated Polymorphic Polymorphic Pattern Bands Bands
1 - ■l'++ - +-H- 34 69.4
2 - - -H- ++ 2 4.1
3 - - + - 1 2 . 0 4 -- - -H-+ 7 14.3
5 + - "h - 2 4.1
6 + ++ + 4*+ 2 4.1 oo VO 7 + +++ 4**H- 1 2 . 0
+ weak band strong band very strong band band not present
Table 2.2. Differential gene expression patterns observed for Elgin and Elgin 87 in response to 3, 6 , 13, and 17 h post inoculation with water (control) and zoospores of Phytophthora sojae Race 1. 8
— — uw ma
Figure 2.2. Some of the differential gene expression patterns obser\'ed for Elgin and Elgin
87 in response to 3. 6 . 13. and 17 h post-inoculation with water (control) and zoospores of
Phytophthora sojae Race L: I. 100 bp DNA ladder; l:Elgin, 13 hour control; 2: Elgin, 13 hour post-inoculation; 3:Elgin 87, 13 hour control; 4:Elgin 87. 13 hour post-inoculation;
5:Elgin, 17 hour control; 6 : Elgin, 13 hour post-inoculation; 7:Elgin 87, 13 hour control;
8 :Elgin 87. 13 hour post-inoculation.
90 tÉÜ-f
Figure 2.3. Some of the differential gene expression patterns observed for Elgin and Elgin
87 in response to 3, 6 , 13. and 17 h post-inoculation with water (control) and zoospores of
Phytophthora sojae Race L: 1. lOObp DNA Ladder; IrElgin. 13 hour control; 2: Elgin, 13 hour post-inoculation; 3:Elgin 87, 13 hour control; 4:Elgin 87. 13 hour post-inoculation.
91 I
ir i 65 ia sd f kj M ^ flui ILj iiri
Figure 2.4. Some of the differential gene expression patterns observed for Elgin and Elgin
87 in response to 3 , 6 , 13, and 17 h post-inoculation with water (control) and zoospores of
Phytophthora sojae Race /. L: 100 bp DNA ladder; 1: Elgin, 6 hour post-inoculation; 2:
Elgin 87,6 hour post-inoculation; 3: Elgin, 13 hour post-inoculation; 4: Elgin 87, 13 hour
post-inoculation; 5: Elgin 17 hour post-inoculation; 6 : Elgin 87, 17 hour post-inoculation.
92 m l
a lî
(continued)
Figure 2.5. Some of the differential gene expression patterns observed for Elgin and Elgin
87 in response to 3 . 6 . 13. and 17 h post-inoculation with water (control) and zoospores of
Phytophthora sojae Race L: I. 100 bp DNA ladder; 1: Elgin. 6 hour post-inoculation; 2:
Elgin 87, 6 hour post-inoculation; 3: Elgin, 13 hour post-inoculation; 4: Elgin 87, 13 hour
post-inoculation; 5: Elgin 17 hour post-inoculation; 6 : Elgin 87. 17 hour post-inoculation.;
7: Elgin. 17 hour post-inoculation; 8 : Elgin 87, 17 hour post-inoculation.
93 Figure 2.5. (continued)
&
H
fT '
94 Figure 2.6. Probe Rl used in Northern Blot Analysis. 1: Elgin, 13h inoculated; 2: Elgin 13 control; 3: Elgin 87. 13h inoculated; 4: Elgin 87, 13h control.
Figure 2.7. Probe R2 used in Northern Blot Analysis. 1: Elgin. 13h inoculated; 2: Elgin 13. control; 3: Elgin 87. 13h inoculated; 4: Elgin 87, 13h control
95 12 3 4
Figure 2.8. Probe R3 used in Northern Blot Analysis. 1 : Elgin, 13h inoculated; 2: Elgin, 13h control; 3; Elgin 87, 13h inoculated; 4; Elgin 87, I3h control.
96 DISCUSSION
Plants defend themselves to pathogens by mobilizing their mechanisms of resistance that
rely both on preformed protective defense mechanisms or inducible defenses (Dixon and
Lamb, 1990; Atkinson, 1993; Greenberg, 1997). Attempted infection by avirulent pathogen
activates many responses which include hypersensitive response, production of phytoalexins,
proteinase inhibitors, hydrolytic enzymes, and deposition of hydroxyproline-rich
glycoproteins (Jakobek and Lindgren, 1993). As mentioned in the introduction, after very
early steps of the recognition of the pathogen, the host activates an array of defense
responses resulting in transcriptional activation of specific genes some of which are
associated directly with HR (Atkinson, 1993) and others stimulated by elicitors that do not
lead an HR or are expressed to similar levels in compatible and incompatible interactions
(Schroder et al., 1992; Atkinson, 1993; Jakobek and Lindgren, 1993). Now, with the recently cloned resistance genes we have more idea about the HR type of response in
incompatible reactions. It has been well established that HR is the one of earliest response of a race-specific resistant plant to a corresponding pathogen (Baker et al., 1996; Dangl,
1995; Greenberg, 1997; Heath and Skalamera, 1997; Hammond-Kosack and Jones, 1997;
Jones and Jones, 1997; Lamb and Dixon, 1997, Staskawicz et al., 1995). For example, the very immediate responses of the parsley cells to the elicitor of P. sojae were changes in the
H*, K'’, CL, and Ca^* fluxes across the plasma membrane and the formation of HjOj
(oxidative burst), occurring within 2 to 5 minutes (Numberger et al., 1994). Within the next
97 5 to 30 minutes, Ca^Mependent phosphorylation of proteins in vivo determined with quick biosynthesis of ethylene and transcriptional activation of the most rapidly induced defense mechanism genes (Dietrich et al., 1990). However, at the later stages of infection, in some cases, the response to the certain pathogens, such as, bacteria (Jakobek and Lindgren, 1993) or fungi (Schroder et al., 1992), can be indistinguishable in compatible and incompatible interactions.
The main objective of this study was to investigate the pattern of the gene or genes
associated with hypersensitive reaction and/or general defense responses against race 1 of
Phytophthora sojae. For this reason, two different isolines having (Elgin 87) or not having
(Elgin) Rpsl-k gene were used in the inoculation experiments with the zoospores of the pathogen and water as controls. The approach was based on the technique called differential display, a novel technique enabling to screen many tissue samples on the same sequencing gel which gives an advantage of identifying and isolating the genes differentially expressed in various cell types or under altered conditions (Liang and Pardee, 1995). The gene expression patterns at post-inoculation intervals were detected and analyzed by using differential display.
One of the main ideas of our approach was to be able to detect the first responses which may be linked to HR. Several gene expression pattem have been identified. However, these gene patterns other that number 1 (see Table 2.1.) cannot be confirmed by northern analysis. It was likely that the genes associated with HR were very low abundant messages; therefore,
98 they are very low in copy number. Since the mRNA in northern analysis was isolated from
2-cm root sections, they were diluted. Future research using more sensitive methods such as
RT-PCR or Ribonuclease protection assay is needed to confirm the expression of these sequences. The gene pattem 1 was directly associated with the inoculation irrespective for incompatible and compatible interactions and was confirmed with the northern blot analysis of three probes detected by differential display.
Our findings, in general terms, are in accordance with the observations of Schroder et al.,
(1992) and Jakobek and Lindgren (1993) in terms of timing and co-ordination of the gene expression patterns of some components of general defense mechanisms in both compatible and incompatible interactions. It is also possible to detect the same response from both resistant and susceptible varieties of soybean at certain post-inoculation intervals
(Schmitthenner, personal communications).
The possibility regarding the detection of the gene expression pattem 1 but not other gene expression patterns on the northem blot analysis may lie on northern blot analysis. It is possible that the northem blot analysis was not performed properly. However, this possibility seems not reasonable. Since three probes have been detected on the same analysis with the same methodology. The analysis repeated several times to confirm the results. Also, northem analysis is not as sensitive as ribonuclease protection assay by which very low abundant mRNAs can be detected.
99 Another possibility is that the bands referring to gene expression patterns other than number
one were false or fake bands as it has been recorded in several reports (Callard et al., 1994;
Liang et al., 1993; Liang and Pardee, 1995; Shoham et al., 1996). However we repeated each
step in differential display (cDNA synthesis, PCR and Northem) several times to reduce the possibility of having fake bands. Related to this possibility, the followings can be assumed;
First one is that hypersensitive response related gene expression may take place at the very early stage of the interaction before three hours post-inoculation. This may be possible since
Phytophthora sojae zoospores are able to penetrate through the cell wall of the roots of soybean plants within 20 minutes (Schmitthenner and Graham, personal communications).
This implies that as soon as the pathogen begins to penetrate to the roots, hypersensitive response related components may become activated. This may take very few minutes following penetration (Numberger et al., 1994). Therefore the time of post-inoculation after first twenty minutes or so may be the most important period to detect hypersensitive response related gene expression pattem at the mRNA level.
It is probable that even 3 h post-inoculation may be late to detect these messages at mRNA level. This may be related to the fact that mRNA stability is very variable and in some cases it is very unstable in some organisms (Alberts et al., 1994). In fact eukaryotic mRNAs are known as very stable molecules. However, some proteins such as lymphokines in higher eukaryotes specifically mammals are needed for short period of time. Because they are required for the coordination of cell to cell interactions between the cells that are involved in the immune response of mammals. These proteins are synthesized in bursts. Therefore,
100 the mRNAs encoding these proteins have very short half-lives (Lodish et al., 1995). It may
be possible that hypersensitive response of plants may have similar potential mechanism of
programmed cell death in animals (Jones and Dangl, 1996; Dangl et al., 1996). This similarity may include the mRNA stability of immune responses in animals for which the half-live of these molecules may be very short.
It is also very important to note that the interval between 6 h and 13h post-inoculation periods is very long compare to the other intervals. This is a potential drawback. Since, in the light of above explanations regarding mRNA stability, it is most likely that this period is very long to detect initial signals induced after HR. Because most of the clear bands (the polymorphic
bands arise at 3h and 6 h were not as clear as that of 13h and 17h.) started to appear at 13h.
Maybe a very initial mRNA signal of HR was induced and disappeared at this interval.
Moreover, the signal concentration may not be high enough to be detectable with differential display technique. This may lead us to state following hypothesis; First, primer combinations may not be enough to determine polymorphisms. Since these combinations were covered only 20-30% of the whole mRNA population in the isolated samples.
Therefore, the target was basically missed. Second, the number of zoospore infection was not as high as it is necessary for the detection of the target by differential display. Since target mRNA was eluted by RNA of uninfected regions under the straws. Therefore, increased number of inoculum should be used in further investigations if differential display technique would be the choice.
101 Overall, this study gives very important clues for further studies which aims to clone R psl-k
gene. As a next step, the analysis can be performed with very high inoculum and with shorter post-inoculations specifically after 1-6 hours of infection. Also the sequences that are specific to the gene expression pattem 2 and 4 should be analyzed with more sensitive methods such as RT-PCR or ribonuclease protection assay.
102 CONCLUSIONS
A novel method of cloning differentially expressed genes was used to screen for genes involved in the soybean host defense system against Phytophthora sojae and classifying the gene expression pattem in soyhean-Phytophthora sojae model system. The screening is based on selection of the mRNAs expressed in response to inoculation of P. sojae zoospores to the roots of susceptible and resistant soybean isolines Elgin and Elgin 87, respectively.
The gene expression pattem is identified by comparing the banding patterns of the controls and inoculated samples of Elgin and Elgin 87. Total RNA’s of Elgin and Elgin 87 were isolated at 3 h, 6 h, 13 h, and 17 h post-inoculation with zoospores of Race 1 of
Phytophthora sojae and water as controls. Seven different gene expression pattem have been identified and 49 differentially expressed bands have been displayed. Three sequences related to the general response of soybean to Phytophthora sojae infection have been cloned and their differential expression was confirmed by northem blot analysis. The sequences that expressed specifically in Elgin 87 roots after 13 h of inoculation were more likely related to the hypersensitive response to Phytophthora sojae. Future research using RT-PCR or ribonuclease protection assay will be needed to confirmed the expression of these sequences.
103 GENERAL CONCLUSIONS
In the first part of this study, Clark x Harosoy isoline mapping population has been used for
molecular marker analysis. Phytophthora tolerance phenotype was assayed by the inoculum
layer tests, with. 13 AFLP markers were added to the previously established Clark x Harosoy
soybean map. Of the 13 AFLP markers, only 10 (M7E8mr3, M5E4mr3, M9Elmr3,
M9Elmrl, M8E6mr3, M8E6mrl, M5E4mr2, M7E8mrl, M7E8mr2) were linked to the
existing linkage groups. The six loci (A454D_2, AD635T1, A668H_1, KOI 1T_3, Slel l,
and SatOO 1 ) were also found significant at the probability level of 0.01 by one way analysis
of variance. Two significant QTL on linkage group three (R*: 30.4%) and linkage group
eleven (R-: 62.0%) were found. This map including AFLP markers spans 2222.8 cM
distance.
In the second part of the study, differential display technique was used to screen for genes involved in the soybean host defense system against Phytophthora sojae Race 1 and classifying the gene expression pattem in soybosorPhytophthora sojae model system. The screening is based on selection of the mRNAs expressed in response to inoculation of P. sojae zoospores and water (controls) to the roots of susceptible and resistant soybean iso lines
Elgin and Elgin 87, respectively. The gene expression pattem at durations of 3 h, 6 h, 13 h, and 17 h of inoculations was identified by comparing the banding pattems on sequencing gels. Total seven different gene expression pattem have been identified and 49 differentially expressed bands have been displayed. Three sequences related to the general response of
104 soybean to Phytophthora sojae infection have been cloned and their differential expression
was confirmed by northem blot analysis. The sequences that expressed specifically in Elgin
87 roots after 13 h of inoculation were more likely related to the hypersensitive response to
Phytophthora sojae. Future research using RT-PCR or Ribonuclease protection assy will be needed to confirmed the expression of these sequences.
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