INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. U n i v e r s i t y M icro film s In tern a tio n a l A Bell & H o w e ll Inform ation C o m p a n y 3 0 0 N o rth Z e e b R o a d . A n n Arbor, Ml 4 8 1 0 6 - 1 3 4 6 U S A 3 1 3 /761-4700 8 0 0 /521-0600 O rder N u m b er 9314665 M olecu lar ch a ra cteriza tio n o f d sR N A -a sso cia ted h yp oviru len ce in M ich igan iso la tes o f Cryphonectria parasitica D urbahn, Christine Mary, Ph.D. Michigan State University, 1992 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 MOLECULAR CHARACTERIZATION OF dsRNA-ASSOCIATED HYPOVIRULENCE IN MICHIGAN ISOLATES OF CRYPHONECTRIA PARASITICA By Christine M. Durbahn A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1992 ABSTRACT MOLECULAR CHARACTERIZATION OF dsRNA-ASSQCIATED HYPOVIRULENCE IN MICHIGAN ISOLATES OF CRYPHONECTRIA PARASITICA By Christine M Durbahn Cryphonectria parasitica, the causal organism of chestnut blight, was responsible for the demise of the American chestnut in eastern North America. Throughout Europe and in several locations in North America, including Michigan, chestnut trees are surviving even though they are infected with C. parasitica. This biological control of chestnut blight is thought to be due to the appearance of hypovirulent strains of the fungus which contain double-stranded RN A molecules that vary in size, concentration, and homology. The objective of this project was to gain an understanding of the biology and molecular structure of the dsRNA genomes involved in dsRNA-associated hypovirulence in Michigan strains of C. parasitica. This study focused on the biological function, genomic structure, and genomic organization of the dsRNA found in strain GH2. A strain containing two different dsRNA genomes was constructed, which was less virulent than the parental strains and which yielded asexual progeny that contained one, both, or neither of the parental genome types. Overlapping cDNA clones spanning nearly the entire length of the largest segment of dsRNA purified from the Michigan hypovirulent isolate GH2 were generated, and the nucleotide sequence of cloned inserts was determined. The combined nucleotide sequence of these clones, totaling 9,608 nucleotides, includes 8,625 base pairs, which encode one open reading frame (ORF). The deduced amino acid sequence of this O R F (2,874 amino acids) includes putative RN A helicase, RNA-dependent RNA polymerase, and protease domains with a genomic organization similar to that observed for O R F B of hypovirulence-associated dsRNA molecules isolated from two other C. parasitica strains including one from Europe and one from New Jersey. Absent from the Michigan dsRNA molecule is a sequence similar to O R F A, which is apparently responsible for the down-regulation of the fungal enzyme laccase in European hypovirulent isolates. Correspondingly, isolate GH2 and other Michigan hypovirulent isolates accumulate the enzyme laccase at levels similar to those of virulent strains. Further study of the protein products encoded by the dsRNA may lead to a better understanding of the mechanism responsible for dsRNA-associated hypovirulence. ACKNOWLEDGEMENTS To all my friends in the D epartm ent of Botany and Plant Pathology and in the Plant Research Lab, I thank you for your knowledge, time, patience and for use of your equipment. I thank my co-workers in Dennis Fulbright’s lab for putting up with me over the last five years. 1 am especially grateful to A1 Ravenscroft for teaching me how to work with fungi and for making the lab a fantastic place to work. I thank Dr. Ray Hammerschmidt for the use of his sequencing apparatus, for his knowledge of biochemistry, for his friendship, and for his Pilsner Urquell. Barb Sears and her co-workers were extremely helpful with advice on molecular techniques and on life in general. I am grateful to Dr. Donald Nuss and his co-workers at the Roche Institute of Molecular Biology for their help with cloning and sequencing, and to Dr. Bradley Hillman and his co-workers at Rutgers University for protocols and the nucleotide sequence of the NB58 dsRNA. I thank my guidance committee, Drs. Rebecca G rum et, Patrick Hart, Barbara Sears, and Jonathan Walton for their support, encouragem ent, and interest in my project. I would not be a plant pathologist if it were not for my friend and major advisor Dr. Dennis Fulbright, whom I thank for giving me a fascinating project to research and for his support and continued enthusiasm for my project. Finally, I must thank my family and my husband Larry for all of their support and encouragement. TABLE OF CONTENTS Page LIST OF TABLES......................................................................................... viii LIST OF FIGURES............................................................................................. U ST OF ABBREVIATIONS...................................................................... ix xi Chapter I. Introduction........................................................................................ History of chestnut blight Review of fungal viruses Ty elements in yeast Killer viruses in yeast Viruses of plant pathogenic fungi The killer system in Ustilago maydis Disease-factors in Ophiostoma ulmi dsRNA-associated virulence in Rhizoctonia solani The Reoviridae O ther dsRNA viruses found in plants dsRNA in Cryphonectria parasitica Hypovirulence in Michigan Objectives of this study Literature cited Chapter II. Characterization of a Cryphonectria parasitica strain infected with multiple dsRNA genom es............................... Introduction M aterials and methods Cultures and growth conditions Strain construction Single-conidial isolation and virulence assays Double-stranded RNA isolation and cDNA cloning N orthern blot analysis Results Multiple infection Identification of dsRNA molecules present in single-conidial isolates v 1 32 Discussion Acknowle dge me nts Literature cited Chapter III. Molecular characterization of a dsRNA molecule from the Michigan Cryphonectria parasitica isolate GH2 . Introduction M aterials and methods Cultures and growth conditions Double-stranded RNA isolation and cDNA cloning Sequence analysis of cDNA clones Results Cloning and sequence analysis of dsRNA from isolate GH2 Com puter alignments of deduced amino acid sequences Discussion Acknowledgements Literature cited Chapter IV. Presence of laccase in hypovirulent strains of Cryphonectria parasitica recovered from Michigan Introduction M aterials and methods Cultures and growth conditions Bavendamm assay for phenol oxidase activity Assay for laccase activity Native polyacrylamide gel electrophoresis Isolation of dsRNA Results and discussion Conclusions Literature Cited Chapter V. Discussion........................................................................ Literature cited Appendix A Virulence of Cryphonectria parasitica strains with various combinations of dsRNA molecules.......................... Introduction M aterials and methods Cultures and growth conditions dsRNA analysis and culture conversions Stem inoculations Results and discussion Characterization of culture morphology and dsRNA content Effect of dsRNA genomes on virulence Literature cited Appendix B. Possible nuclear influence on dsRNA-associated hypovirulence.................................................................... Analysis of single-conidial isolates Analysis of ascospore progeny Conclusions Literature cited LIST OF TABLES Table Page 2.1 Isolates of C. parasitica used in this study 35 2.2 Characteristics of C. parasitica strain CL1-16 after infection with dsRNA genomes 40 dsRNA content of single-conidial isolates of strain CL1-16(GH2/RC1) 46 4.1 Strains used in this study 80 4.2 Laccase activity assays 85 2.3 viii LIST OF FIGURES Figure Page Culture morphology of C. parasitica strains used in this study 36 Banding patterns of dsRNA from converted strains of C. parasitica 41 Diagrammatic representation of dsRNA banding patterns in ethidium bromide stained polyacrylamide gels 42 2.4 N orthern blot analysis of dsRNA 44 2.5 N orthern blot analysis of dsRNA probed with the cDNA clone pGH234 45 3.1 M ap of cDNA clones of dsRNA from C parasitica strain GH2 59 3.2 Nucleotide and deduced amino acid sequences of GH2 dsRNA cDNA clones 60 O pen reading frames in the six reading frames of the cDNA sequence 64 3.4 Alignment of putative protease domains 66 3.5 Alignment of putative RNA-dependent RNA polymerase domains 67 3.6 Alignment of putative RNA helicase domains 68 3.7 Genomic organization of protein coding regions of dsRNA from EP713, NB58 and GH2 70 Virulent and hypovirulent isolates of C. parasitica grown on Bavendamm medium 83 Laccase activity in a native protein polyacrylamide gel 86 2.1 2.2 2.3 3.3 4.1 4.2 ix Figure 4.3 Page Banding patterns of dsRNA from Michigan and European C. parasitica isolates 89 Al Culture morphology of isolates used in this study 107 A2 Ethidium bromide-stained dsRNA isolated from each strain inoculated into chestnut stems 108 M ean canker area produced by each strain at the Frankfort plot 110 M ean canker area produced by each strain at the Russ Forest plot 111 Summary of banding patterns of single conidial isolates 117 A3 A4 B1 x LIST OF ABBREVIATIONS ABTS 2,2’-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) BaYMV barley yellow mosaic virus bp base pairs cDNA complementary DNA DM OP 2,6-dimethoxy-phenol dsRNA double-stranded RNA kb kilobases kd kilodaltons ORF open reading frame PA G E polyacrylamide gel electrophoresis PDA potato dextrose agar sci single-conidial isolate VLP virus-like particle Chapter I INTRODUCTION History of chestnut blight The American chestnut tree {Castanea dentata [Marsh.] Borkh.) was once a major component of the hardwood forests in eastern North America. In the early part of this century, over 3 billion chestnut trees were killed by the disease called chestnut blight. In 1904, the fungal phytopathogen Cryphonectria parasitica (M urr.) Barr, the causal organism of chestnut blight, was isolated for the first time in North America from an American chestnut tree at the Bronx Zoo in New York (Anagnostakis, 1982). It is believed to have entered the U nited States on nursery stock of Oriental chestnuts (reviewed by M acDonald and Fulbright, 1991). Chestnut blight spread at epidemic rates throughout its natural range in eastern North America, killing over 3.5 billion chestnut trees by the mid-1950’s (Hepting, 1974). Early researchers found that C. parasitica enters its host through wounds, such as branch scars, and penetrates the host periderm , phloem, and cambium, forming a canker that expands and girdles the tree (Bramble, 1936). Attempts to control this ascomycetous pathogen by 1 applying fungicides were unsuccessful, as C. parasitica was either unaffected or could acquire resistance to all of the compounds tested (Jaynes and Van Alfen, 1978). Breeding programs were also started in an attem pt to introduce into C. dentata resistance genes from the more resistant oriental species, such as C. crenata (Siebold and Zucc.) and C. mollissima (Blume) (M acDonald and Fulbright, 1991). Unfortunately, this approach was not quick or effective enough to have any impact on the epidemic. In 1938, chestnut blight spread from North America to Europe, where C. parasitica attacked the European chestnut ( Castanea sativa [Mill.]) (Anagnostakis, 1982). Blighted trees were first observed in northern Italy, and the disease spread at epidemic rates, similar to what had occurred in North America. By the 1950’s, the disease had spread throughout Italy, however, unlike American chestnut trees, some European trees were surviving even though they were infected by the chestnut blight fungus. Italian plant pathologist Antonio Biraghi was the first to notice these surviving trees, and he characterized them as possibly being resistant to chestnut blight. He observed that cankers were not leading to lethal infections, and that the fungus rem ained in the outer layer of bark (Biraghi, 1953). It was later determ ined that the trees were not resistant. Instead, C, parasitica strains isolated from these trees by the French mycologist Jean Grente were less virulent than other strains and were term ed hypovirulent (Grente, 1965). O ther phenotypic characteristics originally associated with these European hypovirulent strains included a color change from orange to white and a reduction in sporulation (G rente, 1965). Surprisingly, existing lethal cankers could be converted to non-lethal by inoculating them with mycelia from the hypovirulent strains (G rente and Berthelay-Sauret, 1978). It was determined that this was due to transmission of a cytoplasmic factor from the hypovirulent strain to the virulent strain. The hyphae of the two strains would anastomose, or fuse together, forming a bridge through which cytoplasm could pass from one strain to another. As a result of this process, the virulent strain was converted to hypovirulent (G rente and Berthelay-Sauret, 1978). A converted strain would also assume the other phenotypic traits associated with hypovirulence, including white pigmentation and reduced sporulation (G rente and Berthelay-Sauret, 1978). Researchers in North America were excited by these findings, but at that time, no surviving or recovering trees had been observed on this continent. It was soon found that European hypovirulent strains could convert virulent, North American strains of C. parasitica to hypovirulent. When a canker on an A m erican chestnut tree was inoculated with a European hypovirulent strain, the rate of canker expansion was reduced compared to virulent strains, and swelling and the formation of callus tissue was seen at the canker margins (Van Alfen et aL, 1975). Although hypovirulence was known to be transmissible and, therefore, thought to be cytoplasmic in nature, the factor responsible for the reduction in virulence was still unknown. In 1977, Day et ai. reported that hypovirulent forms of C. parasitica contained double-stranded ribonucleic acid (dsRNA) molecules, while virulent strains did not. A causal relationship between the presence of dsRNA and hypovirulence was implied, and it was suggested that the dsRNA may be of viral origin, since most fungal viruses (mycoviruses) are composed of dsRNA. Hypovirulent strains were first found in North America in 1976 (Elliston et al, 1977). Surviving American chestnut trees in Michigan were tested for hypovirulent isolates as researchers noted the similarity between the Michigan and European situations (Fulbright et al., 1983). Review of fungal viruses Viruses, whose hosts are in the plant or animal kingdoms, have been studied since the late 19th century. In contrast, mycoviruses were not described until 1962. A disease of the cultivated edible mushroom Agaricus bisporus was first observed at a mushroom farm owned by La France Brothers in Pennsylvania and was therefore named La France disease (Buck, 1986). M ushrooms infected with this disease had long thin stipes and small caps. La France disease was proposed to be of viral origin in 1960 (Gandy) and this theory was confirmed through electron microscopy in 1962 (Hollings). The genome of this virus, like most mycoviruses discovered since then, was found to be composed of dsRNA (Buck, 1986). Mycoviruses, or unencapsidated dsRNA molecules, have been detected in all classes of fungi. The majority of virus infections of fungi, however, are symptomless (Hollings, 1978). Several of the exceptions are discussed below. 5 Tv elem ents in yeast One class of mycoviruses that has been studied extensively are the retrovirus-like transposable elements found in many species of yeast, including Saccharomyces cerevisiae. There are 30-35 copies of a Ty elem ent in the average yeast cell, and each has the ability to move or transpose within the genome of its host (Boeke and Garfinkel, 1988). In many cells, Ty elem ents are symptomless, however they can have a major effect on adjacent gene expression when inserted in the 5’ region of a gene (Winston, 1988). Transposition occurs through a cytoplasmic RNA intermediate enclosed within virus-like particles (VLPs) that are composed of sugars and lipids, rather than virally encoded proteins. Also contained within the VLP is a reverse transcriptase, which catalyzes the synthesis of a complementary DNA (cDNA) copy of the Ty elem ent (Boeke et ah, 1985). This cDNA copy is then exported from the VLP and is integrated into host chromosomal DNA (Liebman and Picologlou, 1988). New RNA intermediates are transcribed from these cDNA copies in the host chromosome and are encapsidated in VLPs (Boeke et al., 1985). Ty elements are related structurally and functionally to retroviruses, found in mammalian cells, and are members of a family of retrovirus-like transposons known as retrotransposons (Boeke and Garfinkel, 1988). The DNA form of both Ty elements and retroviruses is characterized by direct repeats at the 5’- and 3’- ends of the molecule, which are known as long terminal repeats (LTRs) and are -334 bp in length (Elder et al, 1983). The internal sequence of Ty elements contains two overlapping open reading frames (ORFs). The first, tya, encodes a protein that is homologous to DNA binding proteins and corresponds to the gag proteins of retroviruses. The second ORF, tyb, encodes a polypeptide with homology to the reverse transcriptase and endonuclease regions of the retroviral pol gene (Clare and Farabaugh, 1985). A third gene associated with retroviral genomes, env, encodes the coat protein that encapsidates the virus. This gene is not present in retrotransposons, and thus is not found in the Ty elem ent genome. An O R F corresponding to the env gene was not expected, because Ty elements are encapsidated in VLPs rather than a virally encoded coat protein and do not have an extracellular stage in their life cycle, which would require a coat protein (Clare and Farabaugh, 1985). The study of retrotransposons is greatly facilitated by the vast am ount of genetic information available for S. cerevisiae. A complete understanding of retrotransposition in yeast could lead to identification of pharmacological products that could affect the replication of mammalian retroviruses (Boeke and Garfinkel, 1988). Killer viruses in yeast A nother interesting group of mycoviruses found in yeast are the killer viruses. M embers of this group of viruses confer a selective advantage to their hosts by producing a toxin that is secreted from the host cell, killing cells of the same species that lack the particular virus or an immunity factor. This type of killer system has been identified in eight yeast genera, however, it was first 7 described in S. cerevisiae (Bruenn, 1986). S. cerevisiae killer virus particles are icosahedral, contain a dsRNA genome, and are approximately 30-40 nm in diam eter (Bruenn, 1986). Each cell contains 100-3000 virus particles, all of which are located in the cytoplasm, but do not appear to be associated with the nucleus or mitochondria (Bruenn, 1980). A dsRNA molecule of ~4.8 kb has been associated with all dsRNA viruses of S. cerevisiae and is known as L dsRNA. This is the largest size class of dsRNA molecules in yeast, and it encodes the capsid polypeptide as well a replicase (Diamond et al., 1989). Many of the viruses also contain a 1.9 kb dsRNA (M), which encodes the extracellular killer toxin as well as resistance to this toxin. M is encapsidated separately from L dsRNA (Leibowitz, 1988). There are several subgroups of M dsRNAs, each of which encodes a separate toxin. Two killer types, K1 and K2, have been extensively studied and have specific M dsRNAs known as M l and M2, respectively. While killer cells are resistant to their own toxin, K1 cells are sensitive to K2 toxin and vice versa. Interestingly, M l and M2 can not co-exist in the same cell. W hen a strain containing both M l and M2 was constructed through mating, the M l dsRNA had a competitive advantage over M2, which was not detected in progeny cells. This may be due to an advantage of M l over M2 in obtaining a specific cellular com ponent necessary for replication (Leibowitz, 1988). Similar constraints on co-existence have been postulated with strains of plant viruses and among similar plasmids in bacteria. The K1 toxin has been extensively studied and consists of two protein subunits linked by disulfide bonds (Zhu et al., 1987). Both subunits are encoded on one M dsRNA as a single prepropolypeptide, which is then further processed (Zhu et al., 1987). It appears to be this precursor protein that is responsible for conferring immunity to the infected cell (Boone et al., 1986). It is believed that the K1 toxin acts by binding to a cell wall /1-glucan receptor, then forming a lethal cation channel in the plasma membrane of sensitive cells (Bussey, 1981; Kagan,1983; Boone et al., 1990). The M2 dsRNA that encodes the K2 toxin, which has structural and functional properties similar to those of the K1 toxin, appears to be common in yeast strains used in wine making (Thom as et al., 1991). During wine fermentation, desirable strains which secrete K2 toxin prevent the growth of cells sensitive to the toxin. This would ensure a non-containinated, uniform yeast culture, which is optimal for the best flavor of the wine. Viruses of plant pathogenic fungi The killer system in Ustilaso mavdis Ustilago maydis, the causal organism of corn smut, is a Basidiomycete, which causes the formation of galls on leaves, stems and in corncobs (Koltin, 1988). U. maydis is also a host for three dsRNA mycoviruses, known as PI, P4 and P6 (Koltin, 1986). These viruses are responsible for the production of a toxin effective against sensitive strains within the Ustilaginales (Nuss and Koltin, 1990). This phenomenon is very similar to that of the killer system found in 9 yeast. A similar situation is found with U. maydis killer viruses as with S. cerevisiae killer viruses in that viruses PI and P4 can coexist and function together in a single cell, while neither PI or P4 can coexist with P6 (Koltin, 1988). The basic dsRNA banding patterns have been determ ined for each virus. PI contains 6 dsRNA segments, P4 has 7 segments, and P6 has 5 segments. These dsRNA segments have been divided into three size classes designated heavy (H), medium (M) and light (L), referring to the molecular weight of each class (Koltin, 1988). Class H molecules range from 4.5 - 6.7 kb and encode the capsid protein in all three viruses, while class M molecules are 0.9 - 1.7 kb and encode the toxin in the PI and P6 viruses (Koltin, 1988). The role of class L molecules, which are derived from one end of the M dsRNA, is much less clear (Field et al., 1983). The L dsRNA from PI has been sequenced and was determ ined to be 355 bp in length, but no open reading frames have been identified (Chang et al., 1988). Previously, L dsRNA was thought to be involved in immunity to the toxin (Peery, 1982), however, molecular evidence seems to contradict this hypothesis (Chang et a!., 1988). Each of the U. maydis viruses, PI, P4 and P6, differs in the specificity of its secreted toxin, KP1, KP4 and KP6, respectively. Killer secreting strains are resistant to the toxin they produce, but not to the other two toxins (Koltin, 1986). The majority of toxin research has been performed on KP6, which is composed of two independent protein subunits (Peery et al., 1987). These subunits are synthesized as a single preprotoxin that is further processed into two secreted polypeptides (Tao et al., 1990). The mode of action of this toxin 10 is unknown, however cell wall receptors appear to be involved (Steinlauf et al., 1988). There are also specific nuclear genes, P lr, P4r and P6r, responsible for resistance to each toxin, KP1, KP4 and KP6, respectively (Koltin, 1988). Disease-factors in Ophiostoma ulmi The ascomycete Ophiostoma ulmi, the causal organism of Dutch elm disease, is a devastating disease of elm species in Europe, North Am erica and Southwest Asia. There are two major subgroups of this fungus, one is a highly pathogenic, aggressive strain, which is quickly replacing the other, a nonaggressive strain (Rogers et al., 1988). Cytoplasmically transmissible disease factors (d-factors) have been identified in both of these subgroups of O. ulmi. Diseased isolates are characterized by sectors of weak or abnorm al growth, reduced viability of conidia, and a reduction in the ability to sexually reproduce (Brasier, 1983). While unencapsidated dsRNA segments have been identified in both diseased and healthy O. ulmi strains, transmission of the diseased state to a healthy strain was correlated with the transmission of 10 dsRNA segments (Rogers et al., 1988). dsRNA in diseased strains has also been found to copurify with the mitochondria, which are deficient in cytochrome aa3 (Rogers et al., 1987). It is not known if the dsRNA is directly responsible for this deficiency, but suppression of respiration may be a mechanism by which fungal growth is reduced (Rogers et al., 1987). The possible use of dsRNA d-factors as a biological control is very 11 exciting, however, there are at least two barriers to overcome. First, since the dsRNA is not transm itted through the sexual cycle of the fungus, a large num ber of uninfected ascospores capable of causing disease will be produced (Brasier, 1986). Also, the large number of vegetative compatibility groups within O. ulmi limits the spread of d-factors through hyphal anastomosis. Even with these limitations, the number of naturally occurring diseased strains is quite high (up to 40% in some locations in Europe), leading researchers to believe that biocontrol may be possible (Brasier, 1986). dsRNA-associated virulence in Rhizoctonia solani The soil-borne plant pathogen Rhizoctonia solani causes damping-off in over 100 plant species and is found throughout the world (Baker, 1970). Initial studies on the relationship between virulence and the presence of dsRNA molecules indicated that dsRNA may be associated with hypovirulence in R. solani (Castanho and Butler, 1978). Debilitated strains of R. solani contained dsRNA molecules while no dsRNA was recovered from healthy strains. The debilitation phenotype, as well as the dsRNA, was transm itted from hypovirulent to virulent strains upon hyphal anastomosis (Castanho and Butler, 1978). Recently, the relationship between dsRNA and virulence was re­ examined using R. solani strains isolated from various soil types and host plants in Israel (Ichielevich-Auster et al., 1985). Of 109 strains characterized, approximately one third were nonpathogenic on 11 host plants tested and were 12 term ed hypovirulent. Nine strains (5 virulent and 4 hypovirulent) were chosen for further study of the relationship between dsRNA and virulence. No difference in growth rate in culture was seen among virulent and hypovirulent strains (Ichielevich-Auster et al., 1985). dsRNA molecules were detected in both virulent and hypovirulent strains, however, virus particles were detected only in virulent strains (Finkler et al., 1988). Extraction of dsRNA from the virus particles revealed the presence of two dsRNA segments within the particles. O ther non-encapsidated dsRNA molecules, that did not crosshybridize with the dsRNA from virus particles, were detected in both virulent and hypovirulent R. solani strains (Finkler et al., 1988). Transmission experiments, using genetically-marked strains, revealed that a virulent strain can convert a hypovirulent strain to virulence by transmission of the viral dsRNA through hyphal anastomosis (Finkler et al., 1985). The correlation of the presence of a dsRNA virus to virulence in R. solani by Finkler et al. (1988) seems to contradict earlier results reported by Castanho and Butler (1978), in which the presence of dsRNA was correlated to hypovirulence. Upon re-examination of the virulent strain used in the earlier study, both unencapsidated dsRNA and viruses were identified. Since culture debilitation and hypovirulence are separate phenotypic traits, it has been proposed that the debilitation seen by Castanho and Butler (1978) is associated with an unencapsidated dsRNA, while virulence is correlated with virus particles (Nuss and Koltin, 1990). 13 The Reoviridae M embers of the largest family of dsRNA viruses, the Reoviridae, all have a dsRNA genome consisting of 10, 11 or 12 segments (Joklik, 1983). The type m em ber of this family, respiratory enteric orphan virus (reovirus), was isolated from humans, and is not associated with any known disease. O ther m em bers of this family, however, can cause very serious diseases of plants, animals and insects (Joklik, 1983). Viruses within this family are divided into six genera which vary in host range, and in the number and size of dsRNA molecules within a viral genome. Two genera of the Reoviridae family infect plants, the fijiviruses and the phytoreoviruses. The fijivirus genus is divided into three subgroups, including fiji disease virus (subgroup 1), which is the type member of the genus, maize rough dwarf virus (subgroup II), and oat sterile dwarf virus (subgroup III) (Nuss and Dali, 1990). Each subgroup is distinguished on the basis of serology and electrophoretic genome profile. All fijiviruses have 10 dsRNA genome segments, which are contained within spiked virions (Pereira, 1991). Fijiviruses are transm itted by planthoppers and only infect graminaceous plants (Francki and Boccardo, 1983). Phytoreoviruses are characterized by 12 dsRNA segments ranging in size from 851 bp - >3 kb (Nuss and Dali, 1990). The type mem ber of this genus, wound tumor virus (WTV), was only recently isolated from a naturally infected plant (Hillman et al., 1991). Previous to 1991, all WTV isolates studied originated from a single progenitor, which was isolated from a leafhopper 14 (Hillman et al., 1991). The virus was then transferred to (via leafhoppers) and maintained in many species of dicotyledonous plants. Variation of the nucleotide sequence between individual segments of the type strain and the newly isolated strain ranged from only 1 - 3% (Hillman et al., 1991). Each of the 12 dsRNA genome segments of WTV contains a single O R F that encodes a unique protein. The 5’- and 3’-terminai ends of each segment contain a 6 and 4 bp consensus sequence, respectively. Just internal to each consensus sequence is a 6-14 bp inverted repeat, the length and nucleic acid sequence of which varies between segments (Nuss and Dali, 1990). Every WTV virion contains one copy of each segment, and it is thought that each of the 12 segments interacts with the coat protein to insure proper virion assembly (Anzola et al., 1987). It was proposed that the 5’- and 3’-terminal consensus sequences and inverted repeats act as recognition signals during packaging (Anzola et al., 1987). Replication of WTV is similar to other members of the Reoviridae family. An RNA-dependent RNA polymerase is present within the virion. This RN A polymerase transcribes ssRNA copies from the viral dsRNA (Nuss and Peterson, 1980). Replication occurs in the cytoplasm within viroplasms, which are composed of viral proteins and dsRNA (Francki and Boccardo, 1983). A current hypothesis regarding replication suggests that viral RN A is synthesized in the viroplasm where m ature particles are assembled. These particles then move from the viroplasm into the cytoplasm of the host cell (Francki and Boccardo, 1983). Viroplasms have also been identified in the cytoplasm of 15 leafhopper cells (Chiu et al., 1970). As the name WTV implies, this virus has the ability to cause tumors in some host plants, including clover (Black, 1945). A nother m em ber of the phytoreoviridae, rice gall dwarf virus, also produces tumors in infected rice plants, however the mechanism of tumor induction is unknown (Nuss and Dali, 1990). G enera of the Reoviridae that infect animals include orthoreovirus, orbivirus, and rotavirus, which differ primarily in the number of dsRNA segments per genome and in disease severity (Joklik, 1983). These viruses enter their hosts through the gastrointestinal tract, where primary replication is thought to occur. The virus then spreads to other organs, where it enters host cells, uncoats, and is transcribed and translated (Sharpe and Feilds, 1983). In many cases, viroplasms similar to those seen in WTV appear to be formed. Some of these viruses appear to be asymptomatic, while others can cause very severe diseases (Pereira, 1991). Other dsRNA viruses found in plants A final group of dsRNA molecules found within plants are collectively known as cryptoviruses. These viruses are considered to be cryptic, because they cause no visible symptoms. The cryptoviruses are transmissible only through pollen and seeds and are present in low concentrations in the infected host plant (Boccardo et al., 1987). Studies have shown that the dsRNA of beet cryptic virus 1 (BCV1) can be translated in vitro, producing two polypeptides, 16 one from each of the two dsRNA segments present in BCV1. One of the proteins is thought to be involved in viral replication, while the other functions as the coat protein (Accotto et al., 1987). RNA-dependent RN A polymerase activity has been identified in all cryptovirus particles tested, and is thought to be responsible for replication (Boccardo and Accotto, 1988). There is also evidence that the dsRNA from Phaseolus vulgaris cv. Black Turtle specifically cross-hybridizes to chloroplast DNA (cpDNA) from P. vulgaris cv. Black Turtle and to mung bean cpDNA (W akarchuk and Hamilton, 1990). However, these dsRNA molecules have not been associated with virus particles and, therefore, may not be cryptoviruses (Boccardo et al., 1987). dsRNA from P. vulgaris cv. Black Turtle also cross-hybridized to DNA from plant species that do not contain dsRNA. Results of these studies may eventually point to the evolutionary origin of these dsRNA molecules. dsRNA in Cryphonectria parasitica dsRNA-containing hypovirulent isolates of C. parasitica have been collected from many locations throughout France and Italy, as well as in several locations in North America. The dsRNA molecules in C. parasitica have many viral properties, but should not be considered true viruses. For example, they do not have an extracellular state, are non-infectious, and are only transm itted through hyphal anastomosis (Nuss and Koltin, 1990). A coat protein that would encapsidate the dsRNA has not been identified, but virus-like particles (VLPs) have been reported to be associated with dsRNA in the cytoplasm of 17 hypovirulent C. parasitica strains (Dodds, 1980; Hansen et aL, 1985). Similar vesicles have been found in virulent strains, however they do not contain dsRNA. Fungal polysaccharides have been detected in vesicles from virulent and hypovirulent strains, suggesting that these vesicles could function in fungal cell wall synthesis (Hansen et al., 1985). An RN A-dependent RN A polymerase associated with dsRNA-containing-vesicles has also been identified and is thought to function in the replication of the dsRNA (Hansen et al., 1985). Hypovirulence-associated dsRNA molecules from Europe and North Am erica have many similarities, including: cytoplasmic transmissibility, correlation with reduction in virulence, association with m embrane-bound particles, and effects on fungal growth and morphology. However, major differences have been identified. The most striking differences were the lack of pigm entation and suppressed sporulation associated with European dsRNA, while North American hypovirulent strains were fully pigmented and sporulation was not as noticeably suppressed. dsRNA from several European strains cross-hybridized upon northern analysis, however, they did not crosshybridize to dsRNA isolated from North American strains of C. parasitica (L’Hostis et al., 1985). Furthermore, northern analysis perform ed on dsRNA from C. parasitica isolates from West Virginia, Michigan, France and Italy revealed several distinct homology groups (Paul and Fulbright, 1988). dsRNA isolated from West Virginia strains did not cross-hybridize to dsRNA from Michigan C. parasitica strains, and none of the North American strains tested cross-hybridized to dsRNA of French or Italian origin (Paul and Fulbright, 18 1988). Recently, a C. parasitica isolate collected in New Jersey was found to contain dsRNA that cross-hybridized to dsRNA of European origin (Hillman et al., 1992). This was the first report of homology between dsRNA from North Am erican and European isolates. Within the last five years, the basic genomic organization of the dsRNA associated with the French hypovirulent strain EP713 has been elucidated. U pon gel electrophoresis, dsRNA from strain EP713 can be grouped into three size classes, large (L), medium (M) and small(S). The L-dsRNA is a single band -12.7 kb in length. The M-dsRNA size class consists of one or more bands 8.0 - 10.0 kb in length while S-dsRNAs consist of two or more bands which range in size from 0.6 - 1.7 kb (Hiremath et al., 1986). The M- and SdsRNA size classes result from internal deletions of the L-dsRNA (Shapira et al., 1991b). The 3’-terminus of all dsRNA size classes is characterized by a stretch of polyadenylic acid (poly[A]) (Hiremath et al., 1986). The L-dsRNA, which has been cloned and sequenced, is 12,712 bp in length. The strand of L-dsRNA terminating with 3’ poly(A) is the coding strand and contains two large open reading frames designated O R F A and O R F B (Shapira et al., 1991a). O R F A, which has been studied extensively, is 1,869 nucleotides (nt) in length and encodes two polypeptides, p29 and p40 (Choi et al., 1991a). The protein p29 is a protease, which is autocatalytically released from the O R F A polyprotein during translation (Choi et al., 1991a). The amino acid sequence of this protease is similar to that of the potyvirus helper com ponent protease, known as HC-Pro (Choi et al., 1991b). A function 19 for p40 has not been determined. Virulent C. parasitica protoplasts were transform ed with a cDNA copy of O R F A, which was found to confer phenotypic traits associated with European hypovirulent isolates, but did not reduce virulence (Choi and Nuss, 1992a). These traits included loss of pigmentation, suppression of sporulation, and a reduction in the accumulation of the fungal enzyme laccase. Although the biological function of laccase in C. parasitica is unknown, the enzyme is down-regulated in European dsRNAcontaining hypovirulent isolates (Rigling et al., 1989; Hillman et al., 1990; Rigling and Van Alfen, 1991; Choi et al., 1992). O R F B has been partially characterized and is 9,498 nt in length. An autocatalytic protease similar to that seen in O R F A has been identified using in vitro translation of O RF B. This protease, a 48-kd polypeptide (p48), was cleaved from the amino-terminal portion of the O R F B polyprotein (Shapira et al., 1991a). While no other polypeptides have been identified, several regions of nucleic acid similarity between O RF B and other viruses have been suggested. Putative helicase and RNA-dependent RNA polymerase domains have been proposed using amino acid comparisons of O R F B to several other viruses, including the potyvirus tobacco vein mottling virus (Koonin et al., 1991). Recently, Choi and Nuss (1992b) have transformed virulent C. parasitica protoplasts with a full-length cDNA copy of the dsRNA from strain EP713. Transform ants were hypovirulent and possessed all of the phenotypic traits normally associated with dsRNA from EP713. This was the first time dsRNA was conclusively dem onstrated to be responsible for hypovirulence. 20 Significantly, full-length EP713 L-dsRNA molecules were isolated from the cytoplasm of transformants. These dsRNA molecules were produced from the cDNA copy, which was integrated into the chromosome of the fungus. These cytoplasmic dsRNAs could be transferred, via hyphal anastomosis, to virulent strains, converting them to hypovirulent. As a biological control, these genetically engineered hypovirulent strains appear to have a major advantage over naturally occurring C. parasitica isolates in that the cDNA copy of the dsRNA is integrated into the fungal genome. Upon mating these strains with virulent strains, ascospore (sexual) progeny contained the cDNA copy of LdsRNA in their genomes, produced L-dsRNA, and were hypovirulent (Choi and Nuss, 1992b). dsRNA is not transmitted to ascospore progeny in naturally occurring hypovirulent strains (Anagnostakis, 1982). Hypovirulence in Michigan Many blighted, but surviving, stands of American chestnut can be found within Michigan. Most of the state is not within the natural range of American chestnut trees, however, trees can be found throughout the state as pioneers moving west planted chestnut seedlings (Fulbright et al., 1988). In fact, all recovering chestnut stands within Michigan are located outside of the natural range (M acDonald and Fulbright, 1991). It is thought that these stands are recovering because of the presence of hypovirulent C parasitica isolates within the natural population (Fulbright et al., 1985). O f the isolates collected to date, all but two hypovirulent isolates contained dsRNA. One of these isolates 21 (CL25) is of great interest since it was the first non-dsRNA containing hypovirulent isolate of C. parasitica to be discovered (Fulbright, 1985) and further investigation of these strains is presently underway (M ahanti, 1991). When comparing dsRNA-containing Michigan hypovirulent isolates, obvious differences in culture morphology can be identified. Isolates have coloration ranging from dark to bright orange, have uneven to smooth margins, and have a slow to moderate growth rate (Fulbright et al., 1983). At the molecular level, isolation of dsRNA followed by gel electrophoresis reveals three general banding patterns among hypovirulent isolates. The most predom inant type was first identified in C. parasitica strain G rand Haven 2 (GH2), which contains three dsRNA segments of -9.0, 3.5 and 0.8 kb in length. Many strains containing GH2-like dsRNA possess only the large, -9.0 kb segment (Fulbright et al., 1983). A second dsRNA genome consists of two smaller dsRNA segments -2.8 and 1.6 kb in length. This genome has only been identified in C. parasitica isolates from Roscommon, Michigan and was first isolated from strain RC1 (Fulbright et al., 1983). The third class of dsRNA genomes in Michigan has only been identified in one isolate of C. parasitica, GNC, and contains a single dsRNA segment with a size of -1 0 kb. Upon northern analysis, dsRNA isolated from strains GH2, RC1, and GNC did not cross-hybridize (Paul and Fulbright, 1988; P. McManus, Michigan State University, personal communication). There appear to be two distinct homology groups within the dsRNA genome of strain G H Z The two larger segments cross-hybridize to each other, but not to the ~0.8-kb segment 22 (Tartaglia et al., 1986). By sequence analysis of cDNA clones, it was determ ined that the ~0.8-kb segment does not have the ability to produce any protein products (D. Nuss, Roche Institute of Molecular Biology, personal communication), and thus appears to be a satellite of the larger segments (D. Fulbright, Michigan State University, personal communication). Further analysis of the -9.0- and 3.5-kb segments of the GH2 dsRNA genome revealed that the 3.5-kb segment probably results from an internal deletion within the 9.0-kb segment, as both the 5’ and 3’ ends of the two segments are homologous (Tartaglia et al., 1986). Objectives of this study The overall objective of my dissertataion research was to further investigate the role of dsRNA molecules in the reduction of virulence in Michigan isolates of C. parasitica. Using isolates that were known to function in nature as biocontrols of chestnut blight, I was first able to multiply infect a virulent isolate with two dsRNA genomes, creating a super-debilitated strain. These experiments provided valuable information about the segregation and potential packaging of individual segments within a dsRNA genome. Second, I began a detailed molecular analysis of the ~9.0-kb dsRNA segment found within isolate GH2. By generating and sequencing cDNA clones of this dsRNA, the basic genomic organization has been elucidated. Using this information, it was possible to compare, at the nucleic acid and amino acid levels, North American and European hypovirulence-associated dsRNA 23 molecules. These comparisons are important in determining the similarity between these geographically distinct dsRNA genomes, which may reveal evolutionary relationships between them. By determining which regions are similar between vastly different dsRNA genomes, one may be able to identify regions that have functional significance and gain further insight into the mechanism of dsRNA-associated hypovirulence. The final phase of my project concerned the expression of the fungal enzyme laccase, which is thought to be down-regulated by dsRNA in European hypovirulent strains of C. parasitica and has been implicated as a virulence factor (Rigling et al., 1989). The specific region of dsRNA that must be present and expressed in the fungus for the down-regulation of the nuclearlyencoded enzyme laccase, has been identified in C. parasitica strain EP713 (Choi and Nuss, 1992a). Michigan dsRNA-containing isolates had never been assayed for the accumulation of laccase. Several biochemical analyses for the presence of the enzyme were employed to determine if dsRNA within Michigan isolates had the ability to down-regulate this fungal enzyme. The ability of dsRNA to down-regulate laccase is im portant in determining whether laccase is a virulence factor in Michigan isolates of C. parasitica and in identifying differences between dsRNA genomes isolated from European and Michigan strains of C. parasitica. The ultimate goal of this project is to obtain a complete understanding of how dsRNA within C. parasitica causes a reduction in fungal virulence and, thereby, serves as a biological control of chestnut blight in Michigan. The work 24 presented in this dissertation represents several of the steps necessary to attain this goal. 25 Literature cited Accotto,G.P., Brisco,M.J. and Hull,R. 1987. In vitro translation of the double­ stranded RN A genome from beet cryptic virus 1. J. Gen. Virol. 68:1417-1422. Anagnostakis,S.L. 1982. Biological control of chestnut blight. Science 215:466471. Anzola,J.V., Xu,Z. Asamizu,T. and Nuss,D.L. 1987. Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tum or virus genome and defective interfering RNAs. Proc. Natl. Acad. Sci. USA 84:83018305. Baker,K.F. 1970. Types of Rhizoctonia diseases and their occurrence, pp. 125148. In: Rhizoctonia solani: biology and pathology. J.R. Parm eter (ed.). University of California Press, Los Angeles. Biraghi,A. 1953. Possible active resistance to Endothia parasitica in Castanea sativa. Pages 643-645 in: Rep. Congr. Int. Union For. Res. Orf. 11th. Black,L.M. 1945. A virus tum or disease of plants. Am. J. Bot. 32:408-415. Boccardo,G. and Accotto,G.P. 1988. RNA-dependent RNA polymerase activity in two morphologically different white clover cryptic viruses. Virology 163:413419. Boccardo,G., Lisa,V., Lusioni,E. and Milne,R.G. 1987. Cryptic plant viruses. Ads. Virus Res. 32:171-214. Boeke,J.D., Garfinkel,D.J., Styles,C.A. and Fink,G.R. 1985. Ty elements transpose through an RNA intermediate. Cell 40:491-500. Boeke,J.D. and Garfinkel,D.J. 1988. Yeast Ty elements as retroviruses, pp. 1539. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. Boone,C., Bussey,H., Greene,D., Thomas,D.Y. and Vernet,T. 1986. Yeast killer toxin: site-directed mutations implicate the precursor protein as the immunity component. Cell 46:105-113. Boone,C., Sommer,S.S., Hensel,A. and Bussey,H. 1990. Yeast KRE genes provide evidence for a pathway of cell wall /?-glucan assembly. J. Cell Biol. 110:1833-1843. Bramble,W.C. 1936. Reaction of chestnut bark to invasion by Endothia 26 parasitica. Am. J. Bot. 23:89-94. Brasier,C.M. 1983. A cytoplasmically transmitted disease of Ceratocystis uimi. N ature 305:220-222. Brasier,C.M. 1986. The d-factor in Ceratocystis uimi - its biological characteristics and implications for Dutch elm, pp. 177-208. In: K.W. Buck (ed.), Fungal Virology. CRC Press, Boca Raton, FL. Bruenn,J. 1980. Virus-like particles of yeast. Annu. Rev. Microbiol. 34:49-69. Bruenn,J. 1986. The killer systems of Saccharomyces cerevisiae and other yeasts, pp. 85-108. In: K.W. Buck (ed.), Fungal Virology. CRC Press, Boca Raton, FL. Buck,K.W. 1986. Fungal virology - an overview, pp. 1-84. In: K.W. Buck (ed.), Fungal Virology. CRC Press, Boca Raton, FL. Bussey,H. 1981. Proteases and the processing of precursors to secreted proteins in yeast. Yeast 4:93-122. Castanho,B. and Butler,E.E. 1978. Rhizoctonia decline: a degenerative disease of Rhizoctonia solani. Phytopathology 68:1505-1510. Chang,T.S., Banerjee,N. Bruenn,J.A., Held,W., Peery,T. and Koltin,Y. 1988. A very small viral double-stranded RNA. Virus Genes 2:195-206, Chiu,R.J., Zliu,H.Y., MacLeod,R. and Black,L.M. 1970. Potato yellow dwarf virus in leafhopper cell culture. Virology 40:387-396. Choi,G.H. and Nuss,D.L. 1992a. A viral gene confers hypovirulence-associated traits to the chestnut blight fungus. EMBO J 11:473-477. Choi,G.H. and Nuss,D.L. 1992b. Elypovirulence of the chestnut blight fungus conferred by an infectious viral cDNA. Science 257:800-803. Choi,G.FI., Shapira,R. and Nuss,D.L. 1991a. Cotranslational autoproteolysis involved in gene expression from a double-stranded RNA genetic elem ent associated with hypovirulence of the chestnut blight fungus. Proc. Natl. Acad. Sci. USA 88:1167-1171. Choi,G.H., Pawlyk,D.M. and Nuss D.L. 1991b. The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro. Virology 183:747-752. 27 Choi,G.H., Larson,T.G and Nuss,D.L. 1992. Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Mol. Plant-Microbe Inter. 5:119-128. Clare,J. and Farabaugh,P. 1985. Nucleotide sequence of a yeast Ty element: evidence for an unusual mechanism of gene expression. Proc. Natl. Acad. Sci. USA 82:2829-2833. Day,P.R., Dodds,J.A., Elliston,J.E., Jaynes,R.A. and Anagnostakis,S.L. 1977. Double-stranded RNA in Endothia parasitica. Phytopathology 67:1393-1396. Dodds,J.A. 1980. Association of type 1 viral-like dsRNA with club-shaped particles in hypovirulent strains of Endothia parasitica. Virology 107:1-7. Diamond,M .E., Dowhanick,J.J., Nemeroff,M.E., Pietras,D.F., Tu,C.L. and Bruenn,J.A. 1989. Overlapping genes in a yeast dsRNA virus. J. Virol. 63:39833990. Elder,R.T., Loh,E.Y. and Davis,R.W. 1983. RNA from the yeast transposable elem ent T yl has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc. Natl. Acad. Sci. USA 80:2432-2436. Elliston,J.E., Jaynes,R.A., Day,P.R. and Anagnostakis,S.L. 1977. A native Am erican hypovirulent strain of Endothia parasitica. Proc. Am. Phytopathol. Soc. 4:83-84. Field,L.J., Bruenn,J.A., Chang,T.H., Pinchasi,0., and Koltin,Y. 1983. Two Ustilago maydis viral dsRNAs of different size code for the same product. Nucleic Acids Res. 11:2765-2778. Finkler,A., Ben-Zvi,B.S. and Koltin,Y. 1988. dsRNA virus of Rhizoctonia solani, pp. 387-407. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. Finkler,A., Koltin,Y., Barash,!., Sneh,B. and Pozniak,D. 1985. Isolation of a virus from virulent strains of Rhizoctonia solani. J. Gen. Virol. 66:1221-1232. Francki,R.I.B. and Boccardo,G. 1983. The plant reoviridae. pp. 505-563. In: W.K. Joklik (ed.), The Reoviridae. Plenum Press, New York. Fulbright,D.W. 1985. A cytoplasmic hypovirulent strain of Endothia parasitica without double-stranded RNA (dsRNA). Phytopathology 75:1328. Fulbright,D.W , W eidlich,W .H, H aulfer,K .Z, Thomas,C.S, and Paul,C P. 1983. Chestnut blight and recovering American chestnut trees in Michigan. Can. J. 28 Bot. 61:3164-3171. Fulbright,D.W., Paul,C.P. and Garrod,S.W. 1988. Hypovirulence: a natural control of chestnut blight. In: Mukeiju,K.G. and Garg,K.L. (eds.), Biocontrol of plant diseases, vol. II. CRC Press, Boca Raton, FL pp. 122-139. Gandy,D.G. 1960. Watery stipe of cultivated mushrooms. Nature 185:482-483. G rente,J. 1965. Les formes hypovirulentes D 'Endothia parasitica it les espoirs de lutte contre le chancre du chataignier. C.R. Hebd. Seances Acad. Agric. France 51:1033-1037. G rente,J. and Berthelay-Sauret,S. 1987. Biological control of chestnut blight in France. Pages 30-34 in: Proc. Am. Chestnut Symp. W.L.MacDonald, F.C.Cech, J.Luchok and H.C. Smith, eds. West Virginia University Books, Morgantown. H ansen,D.R., Van Alfen,N.K., Gillies,K. and Powell,W.A. 1985. Naked dsRNA associated with hypovirulence of Endothia parasitica is packaged in fungal vesicles. J. Gen. Virol. 66:2605-2614. Hepting,G.H. 1974. Death of the American chestnut. J. For. History. 18:60-67. Hillman,B.I., Shapira,R. and Nuss,D.L. 1990. Hypovirulence-associated suppression of host functions in Cryphonectria parasitica can be partially relieved by high light intensity. Phytopathology 80:950-956. Hillman,B.I., Anzola,J.V., Halpern,B.Y., Cavileer,T.D. and Nuss,D.L. 1991. First field isolation of wound tumor virus from a plant host: minimal sequence divergence from the type strain isolated from an insect vector. Virology 185:896-900. Hillman,B.I., Tian,Y., Bedker,P.J. and Brown,M.P. 1992. A North American hypovirulent isolaate of the chestnut blight fungus with European isolate-related dsRNA. J. Gen. Virol. 73:681-686. Hiremath,S., L’Hostis,B., Ghabrial,S.A. and Rhoads,R.E. 1986. Terminal structure of hypovirulence-associated dsRNAs in the chestnut blight fungus Endothia parasitica. Nuc. Acids Res. 14:9877-9896. Hollings,M. 1962. Viruses associated with a die-back disease of cultivated mushroom. Nature 196: 962-965. Hollings,M. 1978. Mycoviruses: viruses that infect fungi. Adv. Virus Res. 22:353. 29 Ichielevich-Auster,M., Sneh,B., Koltin,Y. and Barash,I. 1985. Pathogenicity, host specificity and anastomosis groups of Rhizoctonia spp. isolated from soils in Israel. Phytopathology 75:1080-1084. Jaynes,R.A. and Van Alfen,N.K. 1978. Control of the chestnut blight fungus with infected methyl-2-benzimidazole. Plant Dis. Rep. 61:1032-1036. Joklik,W.K. 1983. The members of the family reoviridae, pp. 1-7. In: W.K. Joklik (ed.), The Reoviridae. Plenum Press, New York. Kagan,B. 1983. Mode of action of yeast killer toxins: channel form ation in lipid bilayer membranes. Nature 302:709-711. Koltin,Y. 1986. The killer systems of Ustilago maydis, pp. 109-141. In: Fungal Virology, ed. K.W.Buck. CRC Press, Boca Raton, FL. Koltin,Y. 1988. The killer system of Ustilago maydis: secreted polypeptides encoded by viruses, pp. 209-243. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. Koonin,E.V., Choi,G.H., Nuss,D.L., Shapira,R. and Carrington,J.C. 1991. Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses. Proc. Natl. Acad. Sci. USA 88:10647-10651. Leibowitz,M.J., Hussain,!, and Williams,T.L. 1988. Transcription and translation of the yeast killer virus genome, pp. 133-160. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. L ’Hostis,B., Hiremath,S.T., Rhoads,R.E. and Ghabrial,S.A. 1985. Lack of sequence homology between double-stranded RNA from European and Am erican hypovirulent strains of Endothia parasitica. J. gen. Virol. 66:351-355. Liebman,S.W. and Picologlou,S. 1988. Recombination associated with yeast retrotransposons, pp. 63-90. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. MacDonald,W.L. and Fulbright D.W. 1991. Bilogical control of chestnut blight: use and iimitaitons of transmissible hypovirulence. Plant Disease 75:656-661. Mahanti,N. 1991. The role of mitochondria in the hypovirulence of the chestnut blight fungus Cryphonectria parasitica. Ph.D. Thesis. Michigan State University. Nuss,D.L. and Dall,D.J. 1990. Structural and functional properties of plant reovirus genomes. Adv. Virus Res. 38:249-306. 30 Nuss,D.L. and Koltin,Y. 1990. Significance of dsRNA genetic elem ents in plant pathogenic fungi. Annu. Rev. Phytopathol. 28:37-58. Nuss,D.L. and Peterson,,A.J. 1980. Expression of wound tum or virus gene products in vivo and in vitro. J. Virol. 34:532-541. Paul,C P. and Fulbright,D.W. 1988. Double-stranded RNA molecules from Michigan hypovirulent isolates of Endothia parasitica vary in size and sequence homology. Phytopathology 78:751-755. Peery,T., Koltin,Y. and Tamarkin,A. 1982. Mapping the immunity of the Ustilago maydis PI virus. Plasmid 7:52-58. Peery,T., Shabat-Brand,T., Steinlauf,R., Koltin,Y. and Bruenn,J. 1987. The virus encoded toxin of Ustilago maydis - two polypeptides are essential for activity. Mol. Cell. Biol. 7:470-477. Pereira,H.G. 1991. Double-stranded RNA viruses. Seminars in Virology 2:39-53. Rigling,D. and Van Alfen,N.K. 1991. Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA. J. Bact. 173:8000-8003. Rigling,D. Heiniger,U. and Hohl,GH.R. 1989. Reduction of laccase activity in dsRNA-containing hypovirulent strains of Cryphonectria parasitica. Phytopathology 79:219-223. Rogers,H.J., Buck,K.W. and Brasier,C.M. 1987. A mitochondrial target for double-stranded RNA in diseased isolates of the fungus that causes Dutch elm disease. Nature 329:558-560. Rogers,H.J., Buck,K.W. and Brasier,C.M. 1988. Double-stranded RN A in diseased isolates of the aggressive subgroup of the Dutch elm fungus Ophiostoma uimi, pp. 327-351. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. Shapira,R., Choi,G.H. and Nuss,D.L. 1991a. Virus-like genetic organization and expression strategy for a double-stranded RNA genetic elem ent associated with biological control of chestnut blight. EMBO J. 10:731-739. Shapira,R., Choi,G.H., Hillman,B.I. and Nuss,D.L. 1991b. The contribution of defective RNAs to the complexity of viral-encoded double-stranded RNA populations present in hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica. EMBO J. 10:741-746. 31 Sharpe,A.H. and Fields,B.N. 1983. Pathogenesis of reovirus infection, pp. 229286. In: W.K. Joklik (ed.), The Reoviridae. Plenum Press, New York. Steinlauf,R., Peery,T., Koltin,Y. and Bruenn,J. 1988. The Ustilago maydis virus encoded toxin - effect of KP6 on cells and spheroplasts. Exp. Mycol. 12:264274. Tao,J., Ginsberg,!., Banerjee,N., Held,W., Koltin,Y. and Bruenn,J.A. 1990. Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Mol. Cell. Biol. 10:13731381. Tartaglia,J., Paul,C.P., Fulbright,D.W. and Nuss,D.L. 1986. Structural properties of double-stranded RNAs associated with biological control of chestnut blight fungus. Proc. Natl. Acad. Sci. USA 83:9109-9113. Thomas,D.Y., Whiteway,M and Dignard,D. 1991. Expression and mutagenesis of the K2 killer toxin gene. In: M. Leibowitz (ed.), International workshop on viruses of fungi and simple eukaryotes. Abstract. Van Alfen,N.K., Jaynes,R.A., Anagnosatkis,S.L. and Day,P.R. 1975. Chestnut blight: biological control by transmissible hypovirulence in Endothia parasitica. Science 189:890-891. W akarchuk and Hamilton. 1990. Partial nucleotide sequence from enigmatic dsRNAs in Phaseolus vulgaris. Plant Mol. Biol. 14:637-639. Winston,F. 1988. Transcriptional regulation of Ty elements in Saccharomyces cerevisiae, pp. 41-61. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. Zhu,H., Bussey,H., Thomas,D.Y., Gagnon,J. and Bell,A.W. 1987. D eterm ination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin. Requirem ent of a carboxypeptidase B-like activity for maturation. J.Biol. Chem. 262:10728-10732. Chapter II Characterization of a Cryphonectria parasitica strain infected with multiple dsRNA genomes Introduction Cryphonectria parasitica (Murr.) Barr, the fungal pathogen responsible for chestnut blight, destroyed the mature American chestnut forest ( Castanea dentata [Marsh] Borkh.) in the earlier part of this century. The European chestnut (C. sativa Miller) in Italy and France, and the American chestnut in a few locations in North America, including Michigan, are surviving despite repeated infection by C. parasitica (Anagnostakis, 1982; Fulbright et al., 1983). Trees within these stands are frequently found to be infected with hypovirulent forms of C. parasitica. Besides reduced virulence, hypovirulent strains often dem onstrate several phenotypic traits that distinguish them from virulent strains including changes in growth rate, culture morphology, sporulation and pigm entation (Van Alfen, 1982). Most hypovirulent strains of C. parasitica isolated from surviving chestnut trees in Europe and North America harbor double-stranded RNA (dsRNA) molecules (Dodds, 1980). One of these dsRNA molecules is known 32 33 to be responsible for the expression of the hypovirulence phenotype (Choi and Nuss, 1992), yet the molecular mechanism by which this occurs is unknown (Nuss and Koltin, 1990). The dsRNA molecules isolated from different hypovirulent strains frequently vary in size, number of size classes, concentration and homology (L’Hostis et al., 1985; Paul and Fulbright, 1988). The dsRNA purified from Michigan isolates GH2 and RC1 do not crosshybridize and include 3 and 2 segments respectively, representing different size classes of dsRNA (Paul and Fulbright, 1988). Hypovirulence-associated dsRNA genomes are thought to be packaged in membrane vesicles (Van Alfen, 1982; Newhouse et al., 1990). It is not known if all of the size classes of dsRNA in a particular genome are always packaged together in a single vesicle, or how the molecules in different size classes are transmitted to asexual progeny. Based on changes in dsRNA banding patterns after asexual segregation, it has been postulated that a hypovirulent strain of C. parasitica (EP-60) harbors more than one dsRNA genome (Elliston, 1985). This is the only observed case of infection of C. parasitica with multiple dsRNA genomes. It is not known if the various dsRNA molecules of a single genome found within the cytoplasm of C. parasitica interact with one another, or if they can act independently. There may be limitations in C. parasitica to maintaining and/or transmitting more than one dsRNA genome, since only one multiply-infected strain has been isolated (Elliston, 1985). The goal of this study was to construct strains of C. parasitica infected with multiple dsRNA genomes, then to study the transmission of different size 34 classes of molecules from this mixture of dsRNA genomes. A multiply-infected hypovirulent strain was constructed by transferring the two non-homologous dsRNA genomes (GH2 and RC1) to a virulent strain (CL1-16). In this paper we report on the segregation patterns of these dsRNA molecules, as well as their interaction and effect on fungal virulence. Materials and methods Cultures and growth conditions The C. parasitica cultures used in this study are listed in Table 2.1 and are shown in Figure 2.1. These strains are vegetatively compatible and all additional strains used for this work were derived from these cultures. The fungus was grown on potato-dextrose agar (PDA; Difco,Detroit, MI) at room tem perature under cool white fluorescent lights with a 16-h photoperiod (Garrod et al., 1985). Cultures were stored on PD A slants at 4°C. Cultures used for dsRNA isolation were grown on cellophane-covered PDA plates for 7 days or in stationary culture in Endothia complete broth without glucose (Puhalla and Anagnostakis, 1971) for 14-21 days. Strain construction To reduce the potential for interference resulting from different nuclear backgrounds, the dsRNA molecules from the two hypovirulent isolates (G H 2 and RC1) were transferred to the common nuclear background of CL1-16, a virulent single-conidial isolate of the bark isolate CL1 (Fulbright et al., 1983). Transfer of dsRNA from each of the hypovirulent isolates to CL1-16 35 Table 2.1. Isolates of C. parasitica used in this study. Isolate designation dsRNA8 Virulenceb CL1-16 - V Paul and Fulbright, 1988 GH2 + H Fulbright et al., 1983 Paul and Fulbright, 1988 RC1 + H Fulbright et al., 1983 Paul and Fulbright, 1988 a+ , dsRNA is present; Reference dsRNA is not detectable. bV, determ ined virulent in virulence assays; H, determ ined hypovirulent in virulence assays. 36 Figure 2.1. Culture morphology of C. parasitica strains used in this study. 37 was accomplished by pairing a hypovirulent isolate with CL1-16 on PDA as previously described (Anagnostakis and Day, 1979). The construction of a multiply-infected strain from two singly-infected strains was perform ed as described above, except the two hypovirulent strains, CL1-16(GH2) and CL116(RC1), were paired and conversion of either strain was deduced by noting a significant decrease in growth of one or both isolates at the margin of the two colonies. Subcultures of the newly constructed strains were taken from the margin of the colony and placed on PDA. In all cases, transfer of dsRN A was confirmed by polyacrylamide gel electrophoresis of the dsRNA molecules. Also, culture morphology changes were noted and virulence assays performed. Converted strains were given designations listing the source of the dsRNA in parentheses (Table 2.2). Single-conidial isolation and virulence assays Single pycnidiospores (conidia) were isolated from the multiply-infected strain CL1-16(GH2/RC1) by removing 10 mm plugs from 10-day-old C. parasitica cultures and placing them in sterile distilled water. Plates containing PDA were inoculated with ten-fold serial dilutions of the spore suspensions. After three days, individual germinating spores were identified, cut from the agar surface and subcultured on PDA. Virulence was determ ined by inoculating Golden Delicious apple fruit with mycelial plugs taken from PDA plates and measuring the discolored area resulting after three weeks of growth at room tem perature (Fulbright, 1984). 38 Double-stranded RNA isolation and cDNA cloning Double-stranded RN A was isolated as described (Morris and Dodds, 1979). Electrophoresis was perform ed using 5% polyacrylamide gels and stained with ethidium bromide. The large (9.0 kb) dsRNA band of strain GH2 (Tartaglia et al., 1986) was cut from polyacrylamide gels and eluted with the Elutrap system as described by the m anufacturer (Schleicher and Schuell, Keene, NH). A cDNA library was generated from the electroeluted 9.0 kb GH2 dsRNA segment as described by Rae et al. (1989). The RC1 cDNA library was made as described for GH2 except that cDNA clones were generated from total RC1 dsRNA (not a single segment). Northern blot analysis Electrophoresis of dsRNA for northern blot analysis was perform ed using a 1.2% (w/v) agarose gel. The dsRNA was then denatured by treating the gel as described by Shapira et al. (1991) and transferred to nylon m em brane (MSI, Westboro, MA) by capillary blotting using 20X SSC. Prehybridization was performed in 50% formamide (v/v), 5 X SSC, sonicated denatured salmon sperm DNA (250 ng!m\), 5 X D enhardt’s at 42°C for a minimum of two hours (Ausubel et al., 1987). One of two cDNA clones (pGH234 and pR49), generated from GH2 and RC1 dsRNA, respectively, was labeled with [a-32P]dCTP by the random primer method (Feinberg and Vogelstein, 1983) and added to the prehybridization mix. Hybridizations were perform ed at 42°C for a minimum of 6 hours (Paul and Fulbright, 1988). 39 Results Multiple infection Transfer of dsRNA into CL1-16 from hypovirulent isolates G H 2 and RC1 was verified by a significant reduction in virulence (Table 2.2) and similar dsRNA electrophoretic banding patterns (Figure 2.2, Figure 2.3 step 1). Pairing the resulting hypovirulent strains CL1-16(GH2) and CL1-16(RC1) resulted in strain CL1-16(GH2/RC1), which contained dsRNA that co-migrated with the dsRNA from both sources (Figure 2.3 step 2) and was more debilitated in culture than either parent. Multiple infection was verified by examinations of dsRNA electrophoretic banding pattern profiles and northern analysis in which dsRNA of both parents was observed (Figures 2.2, 2.4 and 2.5). Although strain RC1 is characterized by 2 dsRNA bands upon gel electrophoresis (Figure 2.2), the larger 2.8-kb band is sometimes lost or degraded during the isolation procedure and therefore the 1.6-kb molecule would be the only band visualized by northern analysis (Figure 2.4). CL116(GH2/RC1) was less virulent than either parent in virulence assays perform ed on apple fruit (Table 2.2). Identification of dsRNA molecules present in smgle-conidial isolates To determ ine if dsRNA segments from two different hypovirulent strains segregate independently, single-conidial isolates were obtained from CL1-16(GH2/RC1) (Figure 2.3, step 3). These isolates were grouped according to 40 Table 2.2. Characteristics of C. parasitica strain CL1-16 after infection with dsRN A genomes. Strain8 Virulence testsb dsRNA molecules detected (mm2) GH2 RC1 CL1-16 3125 a - - CL1-16(GH2) 1062 b + - CL1-16(RC1) 831 b - + CL1-16(GH2/RC1) 224 c + + aStrains in parentheses served as the source dsRNA, which was transferred into the virulent strain CL1-16. bVirulence data are derived from mean lesion area on inoculated Golden Delicious apple fruit of three replicates per strain. Means followed by the same letter do not differ significantly (P=0.05) according to Tukey’s honestly significant test. Figure 2.2. Banding patterns of dsRNA from converted strains of C. parasitica. dsRNA was electrophoresed in a 5% polyacrylamide gel and stained with ethidium bromide. Lanes: 1, CL1-16(GH2); 2, CL1-16(RC1); 3, CL116(GH2/RC1). Sizes of dsRNA molecules present in strain GH2 are indicated in kilobases (kb). Arrow indicates a dsRNA band which is sometimes present in strain G H Z 42 CL1-16(RC1) CL1-16(GH2) CL1 -16(GH2/RC1) sci15 scil sci6 sci7 Figure 2.3. Diagrammatic representation of dsRNA banding patterns in ethidium bromide stained polyacrylamide gels. Thick horizontal lines represent dsRNA bands in a gel. Numbers designate steps referred to in text. Arrow indicates a dsRNA band which is sometimes present in strain GH2 and can be seen in lane 1 of Figure 2.2. This band is only included in step one of the diagram. 43 morphological phenotype and 2 - 5 representatives of each group were chosen for virulence and dsRNA assays. Single-conidial isolates from CL116(GH2/RC1) exhibited segregation of the dsRNA segments, resulting in strains without dsRNA or containing dsRNA electrophoretic banding patterns similar to RC1, GH 2 and GH2/RC1 (Figures 2.2, 2.4 and 2.5). No exchange of dsRNA segments was observed to occur between genomes. Virulence of the various single-conidial isolates showing specific dsRNA electrophoretic banding patterns was comparable to the parental hypovirulent strains with similar banding patterns (Table 2.3). Discussion The primary goal of this study was to analyze the segregation and m aintenance of two non-homologous dsRNA genomes introduced into a single nuclear background. The dsRNA genomes associated with isolates G H 2 and RC1 segregated independently without any mixture of individual segments from the two genomes occurring in transmission to asexual progeny. When dsRNA was present in single-conidial isolates, the banding pattern profiles always included both or either complete genome, suggesting that both size classes of dsRNA molecules of RC1 are packaged together and separately from the GH2 dsRNA genome. The strain CL1-16(GH2/RC1) was found to be less virulent than strains CL1-16(GH2) and CL1-16(RC1), suggesting that these two dsRNA genomes act additively. It is possible that the RC1 and GH2 dsRNA genomes use a 44 A 1 2 B 34 5 6 kb 2.8 1.6 - Figure 2.4. Northern blot analysis of dsRNA. dsRNA was isolated from the multiply-infected strain CL1-16(GH2/RC1) and several single-conidial isolates (sci) from this strain. The blot was probed with the 32P-labeled cDNA clone pR49, which was generated from RC1 dsRNA. The 2.8-kb band is sometimes lost during isolation of dsRNA. Panel A lanes: 1, GH2; 2, RC1. Panel B lanes: 3, CL1-16; 4, CL1-16(GH2/RC1); 5, CLl-16(GH2/RCl)sci6; 6, CL116(G H 2/RC l)scil. 45 1 2 3 4 5 6 kb 9. 0 3.5 - Figure 2.5. Northern blot analysis of dsRNA probed with the cDNA clone pGH234. This cDNA clone was generated from GH2 dsRNA. Lanes: 1, CL116; 2, GH2; 3, RC1; 4, CL1-16(GH2/RC1); 5, CLl-16(GH2/RCl)sci6; 6, CL116(G H 2/RC l)scil. 46 Table 2.3. dsRNA content of single-conidial isolates of strain CL116(GH2/RC1). Strain dsRNA molecules detected3 GH2 RC1 sc 1 + - sc 2 + - sc 3 + + sc 6 - + sc 7 - - sc 11 - + sc 12 - + sc 19 + + sc 20 - - sc 21 + - sc 26 - - sc 36 + - sc 38 + + sc 42 + + sc 48 + - sc 50 - - sc 100 + + sc 103 - - sc 115 - + sc 116 - - sc 122 - - sc 130 - - sc 133 - + sc 137 _ _ 47 Table 2.3 cont. sci 140 - - sci 144 + - sci 145 + + sci B1 - - sci B4 - - sci B5 - - sci B8 - - sci B9 - + sci B l l + + sci B12 + - sci B16 + - sci B22 + + sci B30 - - sci B31 + - sci B35 - - sci B40 + + sci B42 - - sci B50 - - sci B51 + + sci B52 + - sci B73 + - sci B77 - - sci B81 - - sci B82 + - sci B85 + + sci B96 - - sci B102 _ - a+, dsRNA is present; dsRNA is not detectable. 48 different mechanism for reduction of fungal virulence and function independently of one another. This hypothesis is substantiated by morphological characteristics of the three cultures, as the multiply-infected strain possesses the phenotypic characteristics associated with each dsRNA genome (Figure 2.1). A nother explanation, however, is that the two genomes do act by a similar mechanism, and the combination of the two amplifies their effects. The results of our study concur with those of Elliston (1985), who observed a distinct culture morphology and level of virulence that could be identified with an individual dsRNA genome upon isolating single conidia from each multiply-infected strain. An additive effect conferred by the presence of two dsRNA genomes was not observed by Elliston (1985) in contrast to my studies. It is unknown if there was any sequence similarity between the two dsRNA genomes in strain Elliston’s experiment. It is possible that the two dsRNA genomes used similar mechanisms for reduction of fungal virulence, and therefore did not additively affect virulence. The lack of hybridization analysis of single-conidial isolates from those studies makes interpretation of dsRNA segregation patterns more difficult. The use of northern analysis to detect the presence of dsRNA in single-conidial isolates of strain CL116(GH2/RC1) conclusively demonstrated that the GH2 and RC1 dsRNA genomes segregate independently from one another among asexual progeny. 49 Acknowledgements I thank Donald Nuss and Dennis Fulbright for generating the cDNA clone from G H 2 dsRNA. I am also grateful to Alvin Ravenscroft for his technical support. 50 Literature cited Anagnostakis,S.L. 1982. Biological control of chestnut blight. Science 215:466471. Anagnostakis,S.L., and Day,P.R. 1979. Hypovirulence conversion in Endothia parasitica. Phytopathology 69:1226-1229. Ausubel,F.M., Brent,R. Kingston,R.E., Moore,D.D., Seidman,J.G., Smith J.A. and Struhl,K., eds. 1987. Current Protocols in Molecular Biology. J. Wiley & Sons, New York. Choi,G.H. and Nuss,D.L. 1992. Hypovirulence of the chestnut blight fungus conferred by an infectious viral cDNA. Science 257:800-803. Dodds,J.A. 1980. Revised estimates of the molecular weights of dsRNA segments in hypovirulent strains of Endothia parasitica. Phytopathology 70:1217-1220. Elliston,J.E. 1985. Further evidence for two cytoplasmic hypovirulence agents in a strain of Endothia parasitica from Western Michigan. Phytopathology 75:1405-1413. Feinberg,A.P., and Vogelstein,B. 1983. Radio labelling by random prim er method. Analytical Biochemistry 132:6-13. Fulbright,D.W. 1984. Effect of eliminating dsRNA in hypovirulent Endothia parasitica. Phytopathology 74:722-724. Fulbright,D.W., Weidlich,W.H., Haulfer,K.Z., Thomas,C.S., and Paul,C P. 1983. Chestnut blight and recovering American chestnut trees in Michigan. Can. J. Bot. 61:3164-3171. Garrod,S.W ., Fulbright,D.W. and Ravenscroft,A.V. 1985. The dissemination of virulent and hypovirulent forms of a marked strain of Endothia parasitica in Michigan. Phytopathology 75:533-538. L ’Hostis,B., Hiremath,S.T., Rhoads,R.E. and Ghabrial,S.A. 1985. Lack of sequence homology between double-stranded RNA from European and Am erican hypovirulent strains of Endothia parasitica. J. gen. Virol. 66:351-355. Morris,T.J. and Dodds,J.A. 1979. Isolation and analysis of Double-stranded RN A from virus-infected plant and fungal tissue. Phytopathology 69:854-858. Newhouse,J.R., MacDonald,W.L. and Hoch,H.C. 1990. Virus-like particles in 51 hyphae and conidia of European hypovirulent (dsRNA-containing) strains of Cryphonectria parasitica. Can. J. Bot. 68:90-101. Nuss,D.L., and Koltin,Y. 1990. Significance of dsRNA genetic elem ents in plant pathogenic fungi. Annu. Rev. Phytopathol. 28:37-58. Paul,C.P. and Fulbright,D.W. 1988. Double-stranded RNA molecules from Michigan hypovirulent isolates of Endothia parasitica vary in size and sequence homology. Phytopathology 78:751-755. Puhalla,J.E. and Anagnostakis,S.L. 1971. Genetics and nutritional requirem ents of Endothia parasitica. Phytopathology 61:169-173. Rae,B.P., Hillman,B.I., Tartaglia,J. and Nuss,D.L. 1989. Characterization of double-stranded RNA genetic elements associated with biological control of chestnut blight fungus. EM BO J. 8:657-663. Shapira,R., Choi,G.H. and Nuss,D.L. 1991. Virus-like genetic organization and expression strategy for a double-stranded RNA genetic elem ent associated with biological control of chestnut blight. EMBO J. 10:731-739. Tartaglia,J., Paul,C.P., Fulbright,D.W. and Nuss,D.L. 1986. Structural properties of double-stranded RNAs associated with biological control of chestnut blight fungus. Proc. Natl. Acad. Sci. USA 83:9109-9113. Van Alfen,N.K. 1982. Biology and potential for disease control of hypovirulence of Endothia parasitica. Annu. Rev. Phytopathol. 20:349-362. Chapter IQ Molecular characterization of a dsRNA molecule from the Michigan Cryphonectria parasitica isolate GH2 To be submitted for publication as C. Durbahn Smart, D.L. Nuss and D.W. Fulbright Introduction Some isolates of the fungal pathogen Cryphonectria parasitica ([Murr.] Barr), the causal agent of chestnut blight, have been found to contain virus-like molecules of dsRNA. There is a correlative relationship between the presence of dsRNA in the cytoplasm of C. parasitica and a reduction in fungal virulence (hypovirulence) (Fulbright, 1984). Recently, a dsRNA molecule from a European isolate of C. parasitica has been shown to be directly responsible for hypovirulence (Choi and Nuss, 1992b). Hypovirulent strains have been studied for their involvement in the biological control of chestnut blight (M acDonald and Fulbright, 1991). These strains also offer an intriguing model system in which to study fungal-viral interactions, such as viral effects on fungal 52 53 metabolism, virus replication and transfer among fungal strains, and origins of the dsRNA molecules. Studies on the European hypovirulent strain of C. parasitica, EP713, have shown that the largest dsRNA segment within this strain (L-dsRNA) is 12,712 base pairs in length and contains two large open reading frames (ORFs), known as O R F A and O R F B (Shapira e t al., 1991a). The junction between O R F A and O R F B consists of a one base pair overlap of the two ORFs, suggesting that a -1 frameshift may occur following the termination of O R F A and prior to the initiation of O R F B (Shapira e t al., 1991a). O R F A produces a polyprotein from which an autocatalytic protease is cleaved producing two smaller polypeptides (Choi e t al., 1991a). This protease is similar at the amino acid level to those found in the helper component of several members of the potyvirus family (Choi e t a l, 1991b). O R F B, which is ~ 9.5 kb in length, encodes a polyprotein that includes a protease domain (48 kd) which contains amino acid motifs similar to those found in O R F A (Shapira e t al., 1991a). Within the deduced amino acid sequence of O R F B, two other putative domains have been identified, which would encode an RN A -dependent RNA polymerase and an RNA helicase (Koonin e t al., 1991). It has been hypothesized that the dsRNA viruses of C. parasitica originated from ssRNA viruses (Shapira e t al., 1991a; Koonin e t al., 1991), as the putative domains were reported to be similar to those of ssRNA viruses, including barley yellow mosaic virus (BaYMV) which is a poty-like virus that is transmitted through a fungal vector (Koonin e t al., 1991). Recently, virulent C. parasitica protoplasts 54 were transform ed with full-length cDNA clones of the L-dsRNA (Choi and Nuss, 1992b). Transformants were hypovirulent, confirming that in C. parasitica strain EP713 the dsRNA is responsible for the reduction in fungal virulence (Choi and Nuss, 1992b). Sequence analysis has also been perform ed on cDNA clones spanning the 5 ’ and 3’ termini of dsRNA isolated from the New Jersey hypovirulent C parasitica strain NB58 (Hillman e t al., 1992). The dsRNA from strain NB58 cross-hybridized with that of EP713, and there was nucleotide sequence identity of 65-70% at the 3’-termini of the molecules. Strain NB58 contains a single segment of dsRNA ~ 12.5 kb in length, which is slightly smaller than the LdsRNA of strain EP713 (Hillman et al., 1992). This was the first report of cross-hybridization of dsRNA isolated from a North American strain of C. parasitica to dsRNA from a European strain. The complete nucleotide sequence of the dsRNA molecule isolated from strain NB58 is now known (B. Hillman, Rutgers University, personal communication). Hypovirulent forms of C. parasitica have been isolated from recovering American chestnut stands throughout Michigan (Fulbright e t al., 1983). The culture morphology of the Michigan hypovirulent strains, which are orange in color and sporulate abundantly, differs from that of isolates from Europe which are white with suppressed sporulation (Fulbright e t al., 1983; Anagnostakis, 1982). One hypovirulent C. parasitica isolate, GH2, was collected in 1980 from an American chestnut ( Castanea dentata [Marsh] Borkh.) grove located in G rand Haven, Michigan, where the trees have been surviving chestnut blight 55 (Fulbright e t al., 1983). This hypovirulent strain has been used successfully as a biological control of chestnut blight in Michigan (Fulbright e t al., 1983; G arrod e t al., 1985). GH2 contains three dsRNA segments approximately 9.0 kb, 3.5 kb, and 0.8 kb in size (Tartaglia e t al., 1986). Upon northern analysis, the 9.0and 3.5-kb segments were found to share sequence homology, while the 0.8-kb segment did not cross-hybridize with either of the larger segments (Tartaglia e t al., 1986; Paul and Fulbright, 1988). It is speculated that the 3.5-kb segment occurs as the result of an internal deletion of the largest dsRNA segment in isolate GH2, as the dsRNA molecules within each size class are 3’polyadenylated (Tartaglia e t al., 1986; Shapira e t al., 1991b). N orthern analysis also revealed that dsRNA from isolate GH2 did not cross-hybridize with dsRNA genomes of European origin (Paul and Fulbright, 1988). In this study, we report on sequence analysis of cDNA clones generated from the ~ 9.0-kb dsRNA segment of isolate GH2 and on the comparison of the G H 2 dsRNA sequence to that of the other dsRNA molecules isolated from C. parasitica. Materials and methods Cultures and growth conditions C. parasitica isolate GH2 was collected from a non-lethal canker on an American chestnut tree in Grand Haven, Michigan (Fulbright e t al., 1983). C. parasitica cultures were grown on potato dextrose agar (PDA; Difco, Detroit, MI) at room tem perature under cool-white, 56 fluorescent lights with a 16 h photoperiod (Garrod et al., 1985). Cultures were stored on PD A slants at 4°C. Cultures used for dsRNA isolation were grown in stationary culture in Endothia complete broth without glucose (Puhalla and Anagnostakis, 1971) for 14-21 days or on cellophane-covered PDA plates for 510 days. Double-stranded RNA isolation and cDNA cloning Standard molecular genetic techniques were performed as described by Sambrook et al. (1989). D ouble­ stranded RN A was isolated using CF-11 cellulose (W hatman) column chrom atography as described by Morris and Dodds (1979). Electrophoresis was perform ed using 5% polyacrylamide gels, which were stained with ethidium bromide following electrophoresis. The largest dsRNA segment of strain GH2 was cut from polyacrylamide gels and eluted from the gel using the Elutrap system as described by the manufacturer (Schleicher and Schuell, Keene, NH). Using oligo (dT )12.i8 as a prim er for first strand synthesis, a cDNA library was generated from the electroeluted dsRNA segment, as described by Hillman et al. (1992). Double-stranded cDNA was ligated into the cloning vector pUC9, which had 5’ poly(dG) extensions (Pharmacia, Piscataway, NJ), and recom binant plasmids were used to transform Escherichia coli strain HB-101 (BRL, Gaithersburg, MD). This cDNA library did not contain dsRNA sequences from the 5’ region of the GH2 dsRNA molecule. Therefore, a second cDNA library was constructed using a specific oligonucleotide prim er for first strand synthesis. This primer was generated using a sequence known to 57 be near the 5’ end of the G H 2 dsRNA molecule. The cloning vector was again pUC9, however E. coli strain DH5-a (BRL) was transformed with the recom binant plasmids. D ot blot and Southern analysis of cDNA clones was perform ed using 32P-labeled GH2 dsRNA as a probe, to ensure that each cDNA clone originated from the GH2 dsRNA. The location of each cDNA clone, with respect to the 3’ poly(A) tail of the dsRNA, was determined using Southern hybridization, and a map of the cDNA clones was generated. Sequence analysis of cD N A clones DNA sequence was obtained from double­ stranded DNA plasmid tem plates containing cDNA inserts. The plasmids were first denatured using the method of Zhang et al. (1988), followed by dideoxy term inator sequencing reactions using Sequenase (United States Biochemical, Cleveland, OH). The ends of each insert were sequenced using the -20 universal prim er (United States Biochemical) and the M13 reverse prim er (Promega, Madison, WI). Additional sequence was obtained using synthetic oligonucleotide primers specific for each cDNA clone. Nucleotide sequence analysis was perform ed using Editbase software (Neils Neilsen, Purdue University). Deduced amino acid sequence comparisons were made using the program s of the University of Wisconsin Genetics Com puter Group. 58 Results Cloning and sequence analysis of dsRNA from isolate GH2 dsRNA was isolated from C. parasitica strain GH2, and three segments were identified. The largest segment, > 9.0 kb, was purified from polyacrylamide gels, and a partial cDNA library was constructed in the plasmid vector pUC9 (Figure 3.1). cDNA clones spanned the entire length of the dsRNA molecule, with the exception of the extreme 5’ end. At least two independent clones were identified for much of the length of the dsRNA molecule. Cloned inserts were sequenced and were found to extend 9,608 nucleotides from the 3’ end of the dsRNA molecule (Figure 3.2). The 3’ end was polyadenylated, confirming the results of Tartaglia et al. (1986). The strand of dsRNA that terminates with this 3’ poly(A) appears to be the coding strand, as sequence analysis revealed a single, long ORF, of 8,625 nucleotides, ending 857 base pairs (bp) from the 3’ terminus of the dsRNA molecule (Figures 3.1 and 3.2). No other large ORFs were identified in any of the remaining five reading frames (Figure 3.3). Computer alignments of deduced amino acid sequences A comparison of the deduced amino acid sequence of cDNA clones from GH2 dsRNA to amino acid sequences deduced from DNA sequences in the GenBank/EM BL database revealed similarity with only one sequence. This was the sequence of the dsRNA from C. parasitica strain EP713, however, the sequence of the dsRNA from strain NB58 has not yet been entered into the database (B. Hillman, 59 GH2 dsRNA 3’ 5’ n >9.6 Kb (A) 18 2.0 Kb (A) 36 2.3 Kb 2.3 Kb cDNA clones 4.5 Kb 2.5 Kb 2.5 Kb 1.8 Kb 5’ 'PR ORF ■ ■ i i ! POL • I ..T i i 3’ ■HEli Figure 3.1. Map of cDNA clones of dsRNA from C. parasitica strain GH2. The dsRNA is represented as a hatched box. Individual cDNA clones are shown as solid bars. The open reading frame (ORF) is shown as a rectangle below the cDNA clones. Putative functional domains are delineated with dashed lines: PR, protease; POL, RNA-dependent RNA polymerase; HEL, RN A Helicase. cDNA clones marked with an asterisk were generated by D. Nuss and D. Fulbright. 60 Figure 3.2. Nucleotide and deduced amino acid sequences of G H 2 dsRNA cDNA clones. Nucleotide sequence is numbered on the left, and deduced amino acid sequence is num bered on the right. 61 1 CTTTGGAGGAACTTTAAATTGTATTAATTTTGCATTTGCAAGAGTTAGCCCATGTCTAAAATGAGGCTATCGCTGTGAACAGCCTTAAAGACCCTCTGGG M Q E E L Q N N Q P G S G CAAACTGATTATGAATTGAGAGTTAGACTTCTCAGAAAAAGTCTGGTCTTGTCCTAGATTATGCAGGAGGAACTCCAAAACAACCAGCCGGGATCTGGCT 13 101 S S R S G R D T S V N K V A P T R S G S V P D D T R V I T Q S R P S CTTCCCGCAGTGGCCGCGACACATCTGTGAATAAAGTCGCTCCGACACGGTCTGGGTCTGTGCCGGATGACACCAGGGTGATTACCCAATCCAGGCCTTC 47 201 T S S G D K K K S V G D D G F I P T L S P R E F L D C T S Y G G A TACTTCTTCAGGCGACAAGAAGAAGTCCGTTGGTGATGACGGTTTCATCCCAACCCTGTCTCCTCGGGAGTTTCTTGACTGCACATCCTATGGAGGTGCA 80 301 R V G G E P P L A K F T E N P C Y P V P L G E Q L K L S P D F T V CGGGTGGGTGGTGAACCCCCGTTAGCCAAATTCACTGAGAACCCCTGTTACCCTGTACCACTTGGGGAACAGTTAAAGCTAAGTCCAGACTTCACAGTCT 113 401 C R C Q V H I R P A S Y C L T D L K G C A I , K Q V G N H P V I Q A A GCAGATGTCAGGTGCATATCCGACCTGCATCTTACTGCCTAACGGATCTGAAAGGGTGTGCTCTGAAGCAGGTTGGGAACCATCCGGTTATCCAAGCTGC 147 501 A K K Q N T F C H A S E D V R R L D R T F V E L V C N S L P N Q P TGCAAAGAAACAGAACACATTTTGTCATGCTTCTGAAGATGTTAGGAGATTAGATAGGACTTTCGTGGAACTTGTTTGCAATTCCTTACCTAATCAGCCT 180 601 A F S V F E G N P L A N R L A S S H S A D V K D F G M G Y C A L S GCTTTTTCAGTGTTTGAAGGAAATCCATTGGCTAATAGGTTGGCATCATCACATTCTGCCGACGTCAAGGATTTCGGCATGGGTTATTGTGCATTGTCTG 213 701 V L R P K L R W R S A R V L G P D C L L G D F D K V F N W A G L K Q TCCTGCGTCCTAAACTCAGGTGGCGGTCTGCCAGAGTTCTTGGACCCGATTGTCTCCTTGGCGACTTTGATAAGGTCTTTAATTGGGCAGGGCTTAAACA 247 801 F K A M T F V E V Y R G F Y H L V T V P G A K G V D L E A G E N L ATTTAAAGCGATGACGTTTGTCGAAGTCTATCGTGGATTCTATCATCTGGTGACTGTGCCTGGCGCGAAGGGGGTGGACCTTGAGGCCGGTGAGAACTTG 280 901 K N E I S K I L E K N P D A R V G T G S G V D N G T Y L T P D Y F AAGAATGAAATCTCAAAAATCCTTGAGAAGAACCCTGATGCAAGGGTAGGTACTGGGTCGGGTGTTGACAATGGCACCTACTTGACTCCCGACTATTTTG 313 1001 E E L G A E A E Y L E E E D L P L D L C W K E M F P P S Y G Y S A L AAGAGTTAGGTGCTGAGGCTGAGTACTTGGAGGAGGAAGATCTTCCGCTCGACCTCTGCTGGAAGGAGATGTTCCCACCATCTTATGGGTACTCTGCACT 347 1101 F G M H V G D F G D E M D V F K F L A N I D L M V Y E Y V N F G I GTTTGGTATGCACGTAGGTGACTTTGGTGACGAGATGGACGTCTTCAAGTTCCTAGCAAATATAGATCTGATGGTGTATGAGTACGTTAATTTTGGCATT 380 1201 G D G F C H V A E G S Q T K D S E L H L D K A T L R A G L H R M I GGGGATGGTTTTTGTCATGTCGCCGAGGGTTCCCAGACCAAGGACAGTGAGCTTCATCTAGATAAAGCTACGCTGAAGGCTGGCTTGCACAGGATGATTG 413 1301 E A D W N F G L L P V E L T T A V V M G T I G D V E P T L D V V A T AGGCTGACTGGAATTTCGGTTTGCTTCCTGTTGAGCTAACCACGGCTGTTGTTATGGGCACAATTGGCGATGTGGAGCCAACGTTGGATGTCGTTGCAAC 447 1401 N Y F A A Q M E E Q P E P I P V E E Q Y K F T V G E N G W M S S D GAATTACTTCGCTGCGCAGATGGAAGAACAACCTGAGCCTATCCCCGTGGAGGAACAATATAAATTCACGGTGGGTGAAAATGGTTGGATGTCTTCTGAC 480 1501 F F G I C D G F A K S L F D S F G S G K G Q L E V S P A L W N E I TTCTTCGGCATCTGCGACGGTTTCGCTAAATCTTTGTTCGATTCTTTTGGTTCTGGTAAAGGGCAATTGGAGGTATCTCCAGCCCTATGGAATGAAATAC 513 1601 R A A W D G E P I I G D P D E T N Y P V P G S F L V E L S D D S E W GCGCTGCCTGGGACGGTGAAGCAATCATCGGCGACCCAGATGAGACAAACTACCCTGTACCTGGGTCATTTCTTGTGGAACTTTCAGATGACTCAGAATG 547 1701 L K V P D R P V G T H K R V M P P D F V L V T H P D H V A R F A H GCTCAAAGTTCCTGATAGGCCAGTGGGCACACATAAGAGGGTTATGCCCCCTGATTTTGTATTGGTTACGCATCCGGACCATGTTGCAAGATTTGCACAT 580 1801 E G W E T V S L P M D S K A F I S L G Q A I L E K G L Y A A L D M GAAGGATGGGAGACTGTCTCCCTCCCAATGGACAGCAAAGCCTTTATATCTTTAGGGCAGGCGATTCTTGAGAAAGGCTTGTATGCAGCTCTAGACATGA 613 1901 K A M H E T L F E H I K A A M P I C E Q S D L I Y L V S G T T F H Y AGGCTATGCATGAGACACTCTTCGAGCATATCAAGGCAGCAATGCCTATATGCGAACAGAGTGATCTCATCTACCTTGTGTCCGGGACGACGTTCCATTA 647 2001 F L A S V F P E K Q V F E V C P V P R E D N G V C P E F Y L G H Y CTTCCTTGCCTCTGTGTTCCCTGAAAAACAAGTCTTTGAGGTGTGTCCAGTTCCACGCGAGGACAATGGTGTTTGTCCTGAGTTCTATCTTGGACACTAC 680 2101 F K D V T V D P G F G F G V G R L Y N K W L T A P K Y L N E L E W TTCAAAGATGTCACTGTCGATCCTGGTTTTGGTTTTGGAGTTGGAAGACTATATAACAAATGGTTAACCGCTCCGAAATATCTGAATGAGTTAGAGTGGG 713 2201 E G E F R Y I E P P K F H S I A P W A A D R W Q I S S P S I G F K P AAGGTGAGTTCAGGTACATTGAACCACCTAAGTTTCACTCTATAGCACCGTGGGCGGCTGATAGATGGCAGATTTCCTCTCCATCAATTGGCTTTAAACC 747 2301 Y D D F V K R T S F Q E N E T I K G Y F S L G S C E S I T K E T R CTACGATGACTTTGTCAAGAAAACTTCATTTCAGGAAAATGAAACCATCAAAGGTTATTTCTCTCTTGGTTCTTGTGAGTCAATAACGAAAGAAACTCGT 780 2401 S A L A W L R S L P V K W E V D K R W T Y L F E G T D Y V E S P F TCGGCTCTTGCTTGGTTACGCTGT1TGCCTGTGAAATGGGAGGTTGACAAAAGGTGGACGTACCTTTTTGAGGGTACTGACTACGTGGAGTCTCCTTTCA 813 2501 T N H A T Y L H K F D W V V H H G G S G V T N T C C A V R V P Q T I CGAACCATGCTACCTATCTCCACAAATTTGATTGGGTAGTCCATCACGGAGGATCGGGTGTGACCAATACGTGTTGTGCGGTTCGTGTCCCTCAAACCAT 847 2601 L P Q V G D Q F V W E D A L K E H T I P L H V P E D E L R A L L F CTTGCCTCAGGTTGGCGACCAATTTGTCTGGGAAGATGCACTGAAGGAACACACAATTCCACTACATGTGCCAGAGGATGAGTTGCGTGCTCTCCTCTTC 880 2701 H D R S K P I T K P D W A K V V K D G P Q W W V D A L V D N D A S CATGATAGGAGCAAACCTATAACTAAACGCGACTGGGCCAAAGTCGTTAAAGATGGTCCTCAATGGTGGGTAGATGCTCTTGTGGACAATGATGCTTCTC 913 2801 P V Q P F R I L N H C C H D W D A Y G W K P T D L V V G Q N H D W W CCGTTCAACCGTTTAGGATTCTGAACCACTGCTGCCATGATTGGGATGCATATGGTTGGAAGCCGACGGATCTAGTAGTTGGGCAGAATCATGATTGGTG 947 2901 F T L F N S A W E R G E F E L T L K F V R K V C K P T G S P L V G GTTCACTCTGTTCAACTCTGCCTGGGAACGTGGAGAGTTTGAGTTGACGTTAAAATTTGTCCGTAAGGTGTGCAAGCCAACTGGCTCACCACTCGTTGGG 980 3001 W S G F S L T Q T Y P E H A H G W L T S E R L E F E P F A K K A F TGGTCTGGGTTTAGCCTTACCCAGACATACCCTGAACACGCTCATGGCTGGCTGACCTCCGAAAGGTTAGAGTTCGAGCCAtTCGCCAAAAAGGCTTTTT 1013 3101 S A R Y I A V N E E T K Y R E L G S R A L T S R S V H N P Y V H M N CTGCACGGTATATTGCCGTGAATGAAGAAACGAAGTACAGAGAGCTAGGTTCTAGAGCTCTGACTTCGCGGTCTGTGCACAACCCTTATGTGCACATGAA 1047 3201 P R N I D C P C G G R G Y D F G G K C E R C F L Q G L D N G K L D TCCACGCAATATCGACTGCCCTTGCGGGGGTAGAGGCTACGATTTCGGTGGAAAATGTGAACGTTGTTTTCTTCAGGGTTTAGATAATGGCAAGCTTGAT 1080 3301 F ig u re 3. 2 62 A I D L E A I T T T V Y R G F E R K N K P C R K D V T F R K S T A GCCATTGACCTGGAGGCCATTACAACTACGGTGTACAGGGGTTTCGAACGTAAAAACAAGCCCTGTCGAAAAGATGTCACATTCAGGAAGAGCACGGCGA 1113 3401 S F W V R S R K R Y Y Q L D S R V V K N T A S P T L A R A L L D Q I GTTTCTGGGTGAGATCACGAAAGAGGTACTATCAGTTGGATTCACGTGTTGTCAAAAACACTGCATCACCAACACTTGCTAGAGCCCTCTTGGATCAAAT 1147 3501 Q D M S D V A N V E A F W E Y T N S K P P H Y T Y K T S R H M L N CCAGGACATGTCTGATGTGGCAAACGTCGAAGCTTTCTGGGAGTACACAAACTCAAAACCTCCCCACTACACCTATAAGACGAGTAGGCATATGCTGAAC 1180 3601 H L I S E Q A R L H V D V G M S N I A T D V V K A L G G V L S V G CACTTGATTAGTGAGCAGGCTCGTCTTCATGTTGATGTGGGTATGTCTAATATCGCAACGGATGTTGTGAAGGCACTTGGTGGGGTTCTTTCTGTTGGTT 1213 3701 S F G A L Y S R L A N A N C F S S S G R D F M K K R W W V L V D A L CTTTCGGGGCACTATACTCACGTCTCGCAAATGCCAATTGTTTCTCGTCGTCCGGTCGGGATTTCATGAAGAAGCGTTGGTGGGTTCTGGTTGATGCTTT 1247 3801 R H F D D L T R S V G I K A M P E L V R G I F P E L P Q A S V T S GAGGCACTTCGATGATCTGACTAGAAGTGTTGGTATAAAAGCTATGCCAGAATTGGTGCGTGGTATATTTCCTGAGCTCCCTCAAGCTTCAGTCACCTCT 1280 3901 V I P A S F A L S R P L R T R V A G K L W M D Q M R A Q P A S V R GTTATCCCAGCTTCCTTCGCTTTGTCAAGACCATTGAGGACAAGGGTGGCAGGTAAACTCTGGATGGATCAGATGCGAGCACAACCAGCGTCGGTTCGGG 1313 4001 V H L F N I R L P V L G K D F G V F H A F V E F D G W F W E L Q Q I TGCACTTGTTCAACATACGGTTACCGGTGCTTGGTAAAGATTTTGGTGTCTTCCATGCATTTGTCGAATTTGATGGCTGGTTCTGGGAATTGCAACAAAT 1347 4101 S Q E K C R I N R S K F P P E A S S D R P L A K T I L V K S P I V TTCTCAAGAAAAATGCCGTATCAACCGTAGCAAGTTCCCTCCTGAAGCTAGCTCTGATCGTCCTTTGGCAAAAACTATCCTGGTTAAATCGCCCATTGTT 1380 4201 G S L D L R R I S R E F D G I G Y K I L G D N C L V F A N M L V Y GGTTCTCTGGACCTAAGGAGAATCTCACGCGAATTTGACGGGATAGGATACAAGATCTTGGGTGACAATTGTCTCGTTTTTGCGAATATGTTGGTGTATC 1413 4301 L L T G T V I P W R H F G I F G K D L S L N I Q K E L M Q W A S S H TTCTGACTGGGACAGTGATACCTTGGAGACACTrTGGGATCTTCGGTAAAGATTTGTCACTTAACATTCAGAAGGAACTAATGCAGTGGGCCTCATCACA 1447 4401 F F L H E D E Q R L Q T R D N N A A G L T H T G Q T V S S I K S W TTTCTTCCTTCATGAGGATGAGCAACGATTGCAGACCCGTGACAATAATGCTGCTGGTCTGACGCACACTGGTCAAACGGTTTCCTCAATAAAATCTTGG 1480 4501 T G P K R F I R D Y G L T C V Q K L E A S L E A Y S D D P D I D C ACAGGGCCAAAACGGTTCATAAGAGATTACGGTCTTACCTGTGTTCAGAAACTTGAAGCCTCTTTGGAGGCCTACAGTGATGATCCAGATATAGACTGTC 1513 4601 P Q E R D H M L D F M Q F A I T K F G L S G A I V S R A I L S R R V CCCAGGAACGTGATCACATGTTGGACTTCATGCAATTCGCAATTACAAAGTTTGGTTTGTCTGGTGCCATAGTGTCAAGAGCGATCTTGTCTCGTCGTGT 1547 4701 R R M A T S G R K W K F M H H L L V L F K Q V V Q T R L G E D A M CCGTAGGATGGCGACCTCGGGTCGGAAATGGAAATTTATGCACCATCTGCTTGTTCTTTTCAAGCAGGTGGTACAAACACGACTGGGAGAGGATGCTATG 1580 4801 G V L T A T S N L R G G L R G G K K V G W T P I L S I S V P R H W GGTGTTTTAACTGCAACCTCGAACCTACGCGGTGGCCTCCGCGGAGGTAAGAAGGTTGGTTGGACTCCCATACTTAGCATAAGTGTCCCTAGGCATTGGT 1613 4901 F R S G N R L V T V D H L P E N L N M R T K K R V Q L D L P Q I A K TTCGCTCTGGTAATCGGTTGGTCACAGTTGACCATCTCCCTGAGAATTTGAACATGAGGACAAAGAAACGTGTTCAACTGGATTTGCCCCAAATAGCGAA 1647 5001 R Y Q H Y F G V D P P P L G F K W I R P G E Y E I G V K V P V R T GAGGTATCAACACTATTTTGGGGTGGATCCTCCGCCACTAGGTTTCAAGTGGATTCGACCTGGAGAATACGAGATTGGCGTGAAGGTTCCTGTGAGAACT 1680 5101 N L P K M D S L T Q E L C H E L Q E L H P F E L G V F S L R F G T AACTTGCCAAAAATGGATTCTCTTACGCAAGAGCTCTGTCATGAGCTCCAAGAATTACACCCTTTTGAGCTTGGGGTGTTTTCCTTGCGCTTCGGAACAG 1713 5201 5301 A Q M A E E V T N R Y F A G G F K E G T L I P E Q D Q E E L A Q A I 1 7 4 CTCAGATGGCCGAGGAAGTCACAAATCGGTACTTCGCTGGTGGATTCAAAGAGGGGACACTCATACCTGAGCAAGATCAGGAGGAACTTGCTCAGGCGAT F E N E S H L F S D T Q L I S P E E V W K K W H R N Y S A G F P F ATTTGAAAATGAGTCTCACCTTTTCTCTGACACCCAACTCATATCTCCGGAAGAGGTATGGAAGAAGTGGCATAGAAACTATTCTGCTGGTTTCCCGTTC 1780 5401 R F T D R G N S S R Q K L I D A V G G K E R F L Q C V R D Y I E S CGTTTCACTGACCGTGGAAATTCAAGTCGTCAAAAGTTGATTGATGCTGTTGGCGGGAAGGAAAGGTTTCTTCAGTGTGTTAGAGATTATATAGAGTCTC 1813 5501 P E A F P T V S H A F I R D E V L P K S Y V E R E K I R T I I A Q D CTGAAGCATTTCCGAGCGTCAGCCATGCGTTTATTAAGGATGAGGTTTTACCAAAATCTTATGTTGAACGTGAGAAAATTCGCACAATCATTGCACAAGA 1847 5601 P L N Y Y L S M A V Q G D A A K R L D P S S F S A V G V S R S H G TCCTTTGAACTACTATCTATCAATGGCAGTTCAGGGAGACGCTGCAAAAAGACTTGACCCATCTTCATTCTCGGGTGTTGGTGTATCTCGATCTCATGGA 1880 5701 E M S A L A E K H L A Y K H H T A M D V T A M D S T A S I D A V G GAGATGTCCGCTTTGGCTGAAAAACATCTTGCTTACAAGCATCATACCGCAATGGATGTGACAGCCATGGATTCCACTGCATCCATTGACGCAGTGGGGG 1913 5801 5901 V I K K L R R K G F Q K H S Q R D A I E S A I D A T Y D N L V A S W1947 TAATCAAGAAGCTACGGAAAAAAGGTTTCCAAAAACACTCTCAACGTGATGCTATCGAGAGTGCTATTGATGCAACCTATGACAATCTCGTTGCTTCTTG I I D I H S G R A R F K R Q G L S T G H A T T T P S N T E Y M R V GATTATTGACATACACTCTGGGAGGGCGCGTTTCAAACGCCAAGGTCTTTCTACCGGTCATGCTACAACGACACCATCGAACACTGAGTATATGCGTGTG 1980 6001 L M L Y S W K Q I T G R P Y S E F Y D C V K F S S F S D D N F W S TTGATGTTGTATTCGTGGAAGCAGATAACCGGAAGACCCTACTCTGAATTCTATGATTGTGTAAAATTCAGTTCCTTCTCAGATGACAATTTTTGGTCTA 2013 6101 T N L D E N V F S G R L V S D F W L S R G V Q V R V E G V S D S L S CTAATCTCGATGAAAATGTGTTTTCAGGTAGATTGGTATCTGACTTCTGGTTGAGTCGTGGTGTGCAAGTTCGTGTCGAAGGTGTTTCAGACAGTCTTTC 2047 6201 D L S F L A K K F S F E Q K H L D E V A S L T G A H P K V A I V H TGAGTTATCTTTTTTGGCGAAGAAGTTTTCTTTTGAACAAAAGGACTTAGATGAGGTTGCCTCTTTGACAGGTGCACACCCTAAGGTTGCTATTGTCCAT 2080 6301 D I N R L L T K F S D Y K K K N T L R Y R W E K F T A L Q L N C A GACATCAATCGATTACTCACAAAATTCTCTGACTATAAGAAGAAGAATACACTTCGGTATAGGTGGGAGAAGTTCACTGCTCTTCAGCTGAACTGTGCCC 2113 6401 6501 H Y P E V H E K V G E Y L D V M E K L L L K R K S G R L F M K Q H P 2 1 4 ACTACCCAGAAGTGCATGAAAAAGTGGGTGAGTATCTTGATGTTATGGAAAAACTTCTGTTGAAGAGGAAATCGGGAAGGTTGTTTATGAAACAACACCC R T S Y D D V L R L M Y L P T D K T R Q S L L V S T H E P D L M E TAGGACAAGTTATGATGACGTCTTGAGGTTGATGTACCTGCCAACTGATAAGACTCGCCAGAGTCTATTAGTTTCAACACATGAACCTGATCTCATGGAG 2180 6601 K V H D W W N T L Q V D I M T F D S T A N T Y G R I L S Q F A G L AAGGTTCATGATTGGTGGAACACTCTTCAGGTCGACATTATGACATTCGACTCAACGGCGAACACATATGGTCGTATTCTTTCACAATTCGCAGGCCTGT 2213 6701 F ig u re 3. 2 ( c o n t . ) 7 7 63 L E I G G L N V E D P G L F L K G P G E Y P H D P E F T L E H H I Y TGGAGATAGGTGGTCTAAATGTTGAGGACCCAGGGTTGTTCTTGAAAGGCCCTGGGGAATATCCTCATGACCCAGAGTTCACTCTAGAGCATCATATCTA 2247 6801 L L N G C P E T Y E K M E I L A Q K T P F S T F M D I P G F W A R CCTGTTGAATGGATGTCCAGAGACCTATGAAAAAATGGAGATTCTTGCTCAGAAGACTCCATTTTCAACATTCATGGACATTCCTGGTTTCTGGGCCAGG 2280 6901 R E Y Y D I S E S M A N A L R V K V S L L L G I Y T L V A W L E Q CGGGAATACTACGATATCAGTGAGAGCATGGCTAATGCCCTCCGTGTCAAAGTCAGTTTGCTTCTTGGTATATACACTTTGGTTGCTTGGTTGGAACAGG 2313 7001 G L M T V P I I G P A Y R L L A T A K Y V S E K A Y S R L N S L Y Y GCTTAATGACTGTTCCAATAATTGGACCGGCTTACAGGTTGCTGGCGACAGCAAAGTATGTTAGCGAGAAAGCTTATTCGAGGCTCAATTCACTATACTA 2347 7101 A V F G D S S A I I S A M M P K D R Y L M L K V T . A Y R V W M V T TGCAGTTTTTGGTGACTCATCTGCGATCATATCAGCGATGATGCCAAAGGATCGTTACCTTATGCTTAAGGTGATTGCTTATCGTGTGTGGATGGTGACA 2380 7201 T P I D C F A F D G G I E R A Q E W V D A C L K F G Q D I H Q I A ACTCCGATCGACTGCTTTGCTTTCGATGGTGGTATCGAGAGAGCTCAAGAATGGGTGGACGCGTGTCTTAAGTTTGGGCAAGACATACATCAAATTGCTC 2413 7301 L D F D I S S I L P T P G T G E R E K Q G T P T G W T G I D H A D S TAGACTTTGACATAAGTTCTATATTGCCTACACCTGGCACAGGCGAGCGTGAAAAACAGGGTACACCAACGGGATGGACTGGAATTGATCATGCAGATTC 2447 7401 V C S V Q E A L L E E K T P M V T G V P G A G K S T D F V I S L K TGTGTGTAGTGTTCAAGAGGCTCTGCTTGAGGAAAAGACACCCATGGTCACAGGTGTCCCTGGTGGTGGGAAATCTACGGATTTTGTCATCAGCCTGAAG 2480 7501 Q K Y E T V I V A C P R Q I L V K N N P V A Q T K L Y S G C E D N CAGAAATATGAAACGGTGATCGTTGCCTGCCCCCGTCAAATTCTAGTTAAAAACAACCCTGTAGCGCAGACAAAACTATATTCGGGTTGTGAGGACAATT 2513 7601 L I R G Y I N F G T A G Y L R R T L A D L P E S T I L C L D E F H E TGATAAGGGGCTACATCAACTTTGGTACTGCTGGCTACTTGCGTAGAACACTTGCAGATTTACCTGAAAGCACAATACTATGTCTGGATGAATTCGATGA 2547 7701 M D E D S L W L L D R Y R G Q C V V I T A T P D F Y G S Q R F S E GATGGATGAGGACAGTTTATGGCTGTTAGACAGATATAGAGGACAATGTGTTGTTATCACAGCAACACCTGACTTTTATGGTAGCCAAAGGTTCTCTGAG 2580 7801 V R L S K G R N S A W T I Q D D F R D T P G K L E D G W N C L M E GTACGTCTTTCAAAAGGTAGGAATTCTGCCTGGACAATACAAGACGACTTCAGAGACACTCCCGGAAAGCTAGAAGATGGATGGAATTGCCTAATGGAAA 2613 7901 S A K T N D R V L M I V P S I Q D V E T C K R H A A Q L V T N K R V GTGCAAAGACGAACGATCGTGTACTGATGATCGTCCCAAGTATTCAAGATGTGGAGACATGCAAGCGACATGCAGCACAACTTGTTACGAACAAACGAGT 2647 8001 C G L Y R G Q N T V T E A D W Y F A T S I V D A G L T I P G L T K CTGTGGATTGTACAGAGGGCAAAACACTGTTACAGAAGCTGATTGGTATTTCGCAACATCAATTGTTGACGCAGGTTTGACTATACCAGGGTTGACGAAA 2680 8101 I I D L G W S L G Y K H G K F I K R P S S R N I S A Q R R G R T G ATCATAGATCTTGGTTGGTCGTTGGGTTACAAGCATGGCAAGTTCATCAAGCGTCCTTCTTCTCGCAACATATCTGCTCAGAGAAGGGGAAGAACGGGAC 2713 8201 R T C A G Q Y I R L I K D Y D D S N W D F S T Q F Q C F S W S T A K GGACTTGTGCAGGCCAGTACATACGACTAATCAAAGATTACGATGATTCCAACTGGGACTTTTCGACACAGTTCCAATGCTTCAGTTGGTCGACTGCTAA 2747 8301 K W D P N F R R G K C R T P G M I E A L P G G Y E S V F G E G D W AAAGTGGGATCCAAACTTCAGACGTGGTAAGTGTCGCACACCAGGTATGATAGAAGCTCTACCTGGGGGTTACGAATCTGTCTTCGGTGAAGGCGATTGG 2780 8401 S M V L Y A V F H Y D A R L D V N R A R A S Y Q A M R K F P E R K TCAATGGTGCTTTATGCAGTTTTTATGTATGATGCACGCTTAGATGTGAACCGTGCGCGAGCCAGTTACCAGGCTATGCGTAAATTCCCTGAACGTAAAG 2813 8501 E F S H L T G R I E N F H F D D L F M V E D K L K R F Q L P G Q N G AATTCAGTCATCTTACTGGGAGGATCGAAAACTTCCACTTTGATGACCTGTTCATGGTTGAAGACAAACTTAAACGGTTCCAACTTCCTGGGCAGAACGG 2847 8601 N F W S W D L H S C V Q V D F E Q K C P S H L L D V D * GAACTTCTGGTCATGGGATTTGCATTCCTGTGTCCAGGTGGACTTCGAACAAAAATGCCCATCTCATTTACTAGACGTTGACrAGTACAATAACAACGGT 2874 8701 8801 ACATTTCTTTTGTCATTTCTTTTGTCAGTGTATCCACAACCTGTGCGAGCCCGCGAAGGCCTAAAACTAGCAATTGGGGAGAACTTTGCCGTAATCAATG 8901 GCCTCTGAGGCTGAAACTGGGGTAAACATTTTGGGACAACCTCCTATGGCATTTAGAGGTTTTTGCCCAATTTCCCAGAAGTAGGAAGGCATGATGGTGC 9001 CCTCACACCTTGTGGGTTACGCCAAAGTTGGACAAGGACTGCGGGTCGCTTGAAGTCGAATCTCCCATCACCAGTATCTGGAGTAGACCAGACTGGGACC 9101 TTTTTAGGTTGCTAAAGGTCCTACGGGGCTTAGTAAACCGTCGTTCTCACAATCTAGGAGTGGAAAAGCAGACCTCAGTCTCGCGCTGAGATGGCGCTTA 9201 GTACGCCTAGTGTATTGGGTGGTTCCCAAGCACACGACGAAAATAAACCAAACAGGTTGTTTTXTTTGCTTTATATTTAAAGCGTTCCTTAGTTCTCTGG 9301 AAAATGAGACCAGAGCTGGACGATATTTAAAATTCAAAAATATCTTTCTTCCCGTTAAAAGAAGATAAAGATCAACTTGGATTAACCTTTGGCCAAAGGG 9401 TAATTGTTCAATGGAGATTGAGACCATTGCTACCCAACTGGAAAAGGTGCTAGGAAGCTTTGTTCCGTGTGACGCACTGCAGGTACGTTCATGTTCAAAT 9501 TAACCAGATAGTAATAACGGGCTGGTAACAAAGTGGAGCAATCCTTTTGCGTTTTAGCGGCGGCACCGTATATACGGGCGTCGCTTTCGCAAAAAAAAAA 9601 AAAAAAAA F ig u r e 3 .2 ( c o o t . ) 64 4 ; 5• 5 '3 M i # ' j ti 4 I M 1 J - |i 4 # # 4 = i— 3• — |t— • - ■ . 1 . . * ■ 1 2.000 . . 1 . . < ■ ■ . ■ 1 4. 000 . . 4 1 4 4 - 4 ------------ 4— M - i l f U H i f ii i [4 4 rt - f 4 — 4— i ± r t = L ( ± M - - - l-tl I p l l^i i [11 • \\ t t i i t)— i- - - - - - - - - - - - - - i [ij— ]M L 4 j— u_4- - - - - - - - - - |LpM — P -i= M — 4 4 — 4 t W — ■ f] 4 4 4 1 4 4 \ h h i - - h ■ 3 ■ i^JiW J ~ i - 4 - 1 4 — 4 - - - - - - - J-IM 4 4 -I— I.. - I - - - - - - - - 1— )l i; ! rtl fM _f I 5• I {I M -liI 1 p l - 4 fl l l l l f l !_ F f - M - — p . . . 1 6 .000 . . . . , a. , . , , 000 Figure 3.3. Open reading frames in the six reading frames of the cDNA sequence. Rectangles represent O R F ’s. Hatch marks above the rectangle represent the start of an ORF, hatch marks below the rectangle represent the end of an ORF. The DNA sequence is num bered in base pairs. 65 Rutgers University, personal communication). The GH2 nucleotide sequence is 38% identical to both those from EP713 and NB58; the deduced amino acid sequence of the GH2 O R F is 19% identical to that of EP713 O R F B and 18% identical to that from NB58 O RF B. Putative protease, RN A -dependent RNA polymerase, and RNA helicase domains have been identified in dsRNA molecules isolated from C. parasitica strain EP713 (Koonin et al., 1991). These domains appear to be conserved in GF12 dsRNA, as well. The deduced amino acid sequence of dsRNA from strains EP713, NB58 and GH2 were aligned, and some similarity was identified within these putative domains (Figures 3.4-3.6). Within the protease domain, alignments were made between the GH2 O R F and putative proteases identified in O R F A and O R F B of EP713, O R F B of NB58, and RNA 1 of BaYMV, a ssRNA plant virus. A putative protease domain was identified in the GH2 O R F and appeared to be most similar to the domain identified in O RF A of strain EP713 (Figure 3.4). A putative domain encoding an RNA-dependent RN A polymerase, which would be involved in viral replication, has also been identified in the G H2 ORF. This domain is interesting, because the overall structure is more similar to BaYMV than to either dsRNA from C. parasitica. There is a 153 amino acid insertion in the center of the domain in both EP713 and NB58, however in BaYMV and GH2, this region does not exist (Figure 3.5). The final putative domain, an RNA helicase, which would also be involved in viral replication, can be identified in all four deduced am ino acid sequences (Figure 3.6). This domain is located in what would be the carboxy- 66 GH2 EP713-A EP713-B NB58 BaYMV GH2 EP713-A EP713-B NB58 BaYMV 104 24 223 256 29 QFDAYG GH2 EP713-A EP713-B NB58 BaYMV GH2 EP713-A EP713-B NB58 BaYMV jjOEPlSPD |YG| |S A R V G G E p fe AK Fffl........................... E 0P C Y S m v SV gT E E V V pjA GCIgjLW EYRD S C g (d |VPG | D g S V P T S L L S N R QHARFW NE. . KDIPTGLK)L|£ LR^LRTP :fa Q D |GAR|rSL L F N R Q H K R F W D E S S EGVjEfTVACLT REGFiMfcL RD V M D E H I S S T W N A V I R R H M L A PNAgjAETlBG R D G L P . . . S A FfT vC R CQV GH2 EP713-A EP713-B NB58 BaYMV GH2 EP713-A EP713-B NB58 BaYMV aa aa aa aa aa PA gY C 0 ELP. . . . HMA R S ifcL S H E A A RLA RS l b l S @ R S 0 P S F ID A L N A P T VRiRLD RT FVE L V S N S L P LD S R IFLQ LVPGGfLl? T (t V E AK^JTFEhA W93C®LK|_ MU,K|SGSTGT F IG R P R R g p L ME EA^jEf................. v r a t r v RI e VRVTRD0 ........................ g^0IS@PLS T g S Ip :A A|F ASS EG 0 . . AR P . . PS APRQQ 0TK PH LTPM S SH A P0LY M A N . C A Y EA T k: WRSRRV ?d tfTTjG^C L|FNN. . .Q V . illF D K K DFjKjPi. . . S @ _i_ i’lR P T K L S 0 F IP J L S B 0 IT P E N A R S F S ^Mvc. s H E E E V E ID T L EPGDLELPEE R V PV E E EVPVKE] ............... If! NWAGLK| QWf7| jMfTFVE^YlRgF F Q IE K S A V D H eJTen^ ptmds E I S H S ..D Q D IT R E . _ T .V T N V T R D S . .Ig a &t p t l a EI iTKTMjLFjAIR YHL tK * V Y H 7i/|V p| SY CVH ^A G ETF LVH CEPG DNY L FPE V 02§PI DA. ISM) CSS VPRRgTEVC E A A R G ir a r jv B k p v ^ 1 ? ia e |e cb: VR| P| PSP R FgE| G f/D t EN0 RLF RN Y d Je IK A V B TG Yt i=i i v a r l ) P g J lA K R P G v jy q s |e YQ, LftEK JE JL E N E P D ,___ |r e , : e g i s e @ p Q FHlV^D . ARG DtSRVGfTGS 0 V D N G lIr k i g g ................... IL\ C\ _ LfjPSW FPM K C SVRS Figure 3.4. Alignment of putative protease domains. The distance between the N terminus of each polyprotein and the beginning of this domain is indicated. Amino acid residues identical to those in GH2 O R F are boxed and shaded, while similar residues are boxed. Residues in bold type were determ ined to be necessary for autocatalytic processing in EP713-A (Choi et al, 1991b). Similar amino acid residues were determined as follows: G and A; S and T; D, E, N, and Q; K and R; L, I, V, and M; F, Y, and W. 67 GH2 EP713 NB58 BaYMV 1784 1786 1918 159 <--------j — ErKoBvE BElSjPEAFPfTVl si ISHJGKYPTQF YHAiFWKlS VIRKNALE IjK NG VYPTQ F YRftgjAWS KALD FL E G KSlTlGVWNG|SL| k a e l r t i H r f l H c v r JBTy .FYK SR LCAH LSD D ^V yH L A > GH2 a®rpfeRE EP713 NB58 BaYMV PjGQPL DGgiAjL EAE < GH2 EP713 NB58 BaYMV GH2 EP713 NB58 BaYMV KL K RjSjHjGjEM^IjLA L p b sM A R IW D LjSjgjgMARIWD _ INK.FGF{G |W E K! AYKHHT RKRE RLRE DKLNRPG WjLHG DATpDjN KRI KRI YiRVfL| iFjYDCV. KQlfG ARA jC S N . ARAjTG J F K T N . RFScN DDfrVW IHKI R E L A L L ^ p R F T VC N G b b PSN T EP713 NB58 BaYMV WDNT A TFt A TF VpiTAF^jft L@E®V.......... GH2 EP713 NB58 BaYMV VA a L T ITEVteiTLS KL ITEVS YL S KV L T gpK T PFG V P gF jD pE F G H D GjVgfK KLRKg ...... T AY D SN ftp D k! a y D SK RFDSS D P 0 F F D W n IDS ..0 aa.. . 153 aa. . 152 aa. ..0 aa. . GH2 GH2 EP713 NB58 BaYMV KKjFYA gjN L <------ III — S Q R D A I E S g j. CKPALFHG. . CKPALFLG. . FLPSE H H K g. LDP |SJS ITW E ITW E SMAV [V D Q IF M D Q IF AMKFY VDl RM K F S ROS DFW LSR G V Q V R w S KDfc LS S .............................AE w s KDFLT S .............................AE TYEFp F s P EyV jELG L -VIII|§AH{PlKjVjAI|VjH PRR P T A E D S A PRQ P IK E D S E @ F S L E @ E R |I|I — W ffJljDIHSGRA RF| . . IS N V h : . . LSN I h: . LANGM V I | G|VSD|S[LpD LS p i AKKF RFKQ L E IG E IG RFK2 D liiT s q n > DljNRL “lYRAW ifKAW AgjMQW Figure 3.5. Alignment of putative RNA-dependent RN A polymerase domains. The distance between the N terminus of each polyprotein and the beginning of this domain is indicated. Amino acid residues identical to those in G H 2 O R F are boxed and shaded, while similar residues are boxed. Similar amino acid residues are as described in Figure 3.4. Conserved motifs found in all positivestrand RN A viral RN A-dependent RNA polymerases (Koonin et al., 1991) are indicated by arrows above the sequence. 68 < GH2 EP713 NB58 BaYMV 2465 2664 2790 482 x- < ------ — > > -Ia- CPRQILV Kb. |N||p]^CTkLgb K KLWI VM RKI LRDNWEIPFD as WAERKNL K KIWI W Rill SqijFGgfeP|Fb 3 S GKSTJYLP0 QY SDR RQQI L I DEPT |gAAT@jNVCAG A GRSTb F|Vg]S aa aa aa aa as GKSTF FPAA GKSTF FPSA < --- GH2 EP713 NB58 BaYMV g c e |d IRS VPS NL !0 fgS[A3 KTLDP SADI Y V T I Y 5 VTLNP NADIY V T I Y G RG 0 . . . . VK. RG R. RG v a a n LGRAVY GRHEqwjSRMG - I I DHCgQjV^jYjG O. PESTILC LjVPpDNLVF DLRENjIVF .HFLTR SALQCHAMDP SFISTFDAgjF > GH2 EP713 NB58 BaYMV [LDl'FHfiMOHD |sH. . .WjLLDR] FfDEFHIMDGF a , . . .QD ‘ FpEFHEMEpF ML...QG fCDE^DV[K |SL}VFES|I GH2 EP713 NB58 BaYMV RNSAWfl • QD D|FRE|rPGKjL]. :pME S AgrgDjRlVLMI KRFNL EV YKV SDDVLEMW. QFAiD2 PELLARSMII KRFPL I V YKV SDDVLEMW. QFAjDQ PDILA» MVI R|KYPLH]V|3TS VCDSYRKFgjA AQGf3GDpLLqI S@H@TAtVjF|L W VRKFY GIPF DIPF ffl m PEAg QDVlETC KKT VKKT KAA < -III-> GH2 EP713 NB5S BaYMV VTg [KRjVCGlLlEYRGgl.................. DRS ITWHE fSSNS .................. ia g l e I *SRKjNl.................. N A W N fp V T g HI | | 9 a FS4 JssdIn| fa td fsm lte KRH IA G LE i NlppjEADWYgf PLVPKTGGLV PKJVPMTGGMV rlk@hktii@ A C C t Lffl Vf2[IG r Gvg -VI- GH2 EP713 NB58 BaYMV 3 YKHGK F f j K R P S . . g g g l S / RpEfrfjpT. VI VHK RL ’PH PYTDEKTNEt VNjRV3RT1VV IHKG RM [PH PYTDDKTNEC gHgjMRPCllD LNQKALRLDR RRVfiKffeRQlQ R L3 W Figure 3.6. Alignment of putative RNA helicase domains. The distance between the N terminus of each polyprotein and the beginning of this domain is indicated. Amino acid residues identical to those in GH2 O R F are boxed and shaded, while similar residues are boxed. Similar amino acid residues are as described in Figure 3.4. Conserved motifs found in the helicase domain of many positive-strand RNA viruses (Hodgman, 1988) are indicated by arrows above the sequence. 69 term inal portion of the G H 2 O R F and in O R F B of EP713 and of NB58 (Figure 3.7). Discussion The dsRNA from the Michigan hypovirulent C. parasitica isolate G H 2 is intriguing, because it is presumably responsible for the reduction of fungal virulence and alteration of colony morphology. However, it does not alter fungal pigmentation or sporulation as is seen in many European hypovirulent isolates (Fulbright et al., 1988). Also, dsRNA from isolate GH2 does not crosshybridize with dsRNA from any other hypovirulent strain of C. parasitica outside of Michigan (Paul and Fulbright, 1988) yet it is homologous with 80% of the dsRNA molecules found in Michigan hypovirulent strains (Fulbright, 1990). I began a molecular analysis of the dsRNA associated with hypovirulence in isolate GH2, in order to identify the similarities, as well as the differences, between the dsRNA genomes of GH2 and other dsRNAs from C. parasitica. The overall genomic organization of the largest segment of dsRNA from isolate GH2 is shown in Figures 3.1 and 3.3. I have identified a single, large O RF, 2,875 codons in length which may encode a polyprotein similar to that encoded by O R F B of EP713 dsRNA (Shapira et al., 1991a). In the dsRNA characterized from EP713 and NB58, two ORFs were identified which are contiguous but distinguished by a reading frame shift. The characterization of 70 EP713 dsRNA 12,712 bp ORFA PR ORF B POL PR HEL 3’ An NB58 dsRNA 12,500 bp 5’ ORF A “■1 •PR i i ORF B POL HEL 3’ An GH2 dsRNA >9,608 bp 3’ 5' PR ORF POL •HEL' Figure 3.7. Genomic organization of protein coding regions of dsRN A from EP713, NB58 and GH2. Rectangles represent ORFs. Functional domains are delineated by dashed lines: PR, protease; POL, RN A -dependent RN A polymerase; HEL, RNA helicase. 71 cD NA clones of dsRNA from GHZ indicates that this dsRNA only encodes a single O R F and lacks the second O R F (O R F A) found in strain EP713. O R F A in EP713 dsRNA has been shown to be responsible for the phenotypic traits associated with European hypovirulent strains, such as reduction of pigm entation and sporulation, as well as the down-regulation of the fungal enzyme laccase (Choi and Nuss, 1992a). The fact that O R F A or an "ORF Alike" region is lacking on the GH2 dsRNA may be reflective of the differences in pigmentation, sporulation, and accumulation of laccase observed between Michigan and European hypovirulent strains (see Chapter IV). The deduced amino acid sequences of ORFs identified in dsRNA from C. parasitica strains GH2, EP713 and NB58 were aligned and compared. The amino acid sequence of the potyvirus-like BaYMV was included in the sequence comparisons since previous studies have shown a relationship between this ssRNA virus and the dsRNA in strain EP713 (Koonin et al., 1991). Two protease domains have been identified in the dsRNA of strain EP713. One domain was identified in ORF A by Choi et al. (1991a), while the second resides in the amino-terminal portion of O R F B (Shapira et al., 1991a). The deduced amino acid sequence of the GH2 O R F appears to be most closely related to the domain identified in O RF A of strain EP713. However, the putative domain from GH2 differs from EP713 O R F A since it does not contain cysteine and histidine residues or the glycine dipeptide cleavage site, all of which are essential for autocatalytic processing (Choi et al., 1991b). The presence of the amino acid sequence GYCALS within the GH2 ORF, which 72 aligns with the GYCYLS motif of EP713 O R F A and BaYMV (Figure 3.4), suggests that this region of the polyprotein acts as a protease. The overall level of similarity between the GH2 O R F and any other amino acid sequence of the putative RNA-dependent RN A polymerase domain was very low. The presence of the amino acid sequence SDD, which is also found in the deduced amino acid sequences of dsRNA from EP713 and NB58, is similar to the GDD sequence found in plant viral RN A -dependent RNA polymerases, suggesting that this region of the polyprotein acts as a polymerase. Interestingly, a large insertion (153 aa), which is present in the middle of the EP713 polymerase domain (Koonin et al., 1991), is not present in the GH2 domain (Figure 3.5). This insertion is not present in the RN A -dependent RN A polymerase domains of BaYMV or several other ssRNA viruses within the potyvirus family (Koonin et al., 1991). The RNA helicase domain of the GH2 O R F deduced amino acid sequence represents the region that is most similar to deduced amino acid sequences of O R F B from either EP713 or NB58 (Figure 3.6). The sequence of seven exactly conserved residues within motif II are especially intriguing as they may represent an essential portion of the RNA helicase peptide. The overall similarity of the genomic organization of the three dsRNA molecules that have been characterized at a molecular level, suggests that there is an evolutionary relationship between them (Figure 3.7). cDNA clones of the extrem e 5’ end of the GH2 dsRNA have not been obtained. This region may include sequences im portant for regulation of expression and/or replication of 73 this virus-like molecule. The comparison between C. parasitica dsRNA sequences and the plant ssRNA virus BaYMV, which has a fungal vector, suggests that these dsRNA molecules may have originated from ssRNA viruses. The similarities which have been discovered between dsRNA from GH2, EP713 and NB58, provide insight into the mechanism of dsRNA replication and protein processing, but do not provide, at this time, further elucidation of the mechanism involved in the reduction of fungal virulence. Use of these cDNA clones in in vitro translation experiments and in the transformation of virulent C. parasitica strains may lead to a further understanding of the molecular mechanisms of dsRNA-associated hypovirulence. Acknowledgements The complete nucleotide sequence cDNA clones generated from the dsRNA of C. parasitica strain NB58 was the kind gift of Dr. Bradley Hillman, Rutgers University. cDNA clones representing the 3’ end of GH2 dsRNA were generated by Donald Nuss, Roche Institute of Molecular biology, and Dennis Fulbright, Michigan State University. I gratefully acknowledge the technical assistance of Alvin Ravenscroft, Michigan State University, and Diane Pawlyk, Roche Institute of Molecular Biology. 74 Literature cited Anagnostakis,S-L. 1982. Biological control of chestnut blight. Science 215:466471. Choi,G.H. and Nuss,D.L. 1992a. A viral gene confers hypovirulence-associated traits to the chestnut blight fungus. EMBO J 11:473-477. Choi,G.H. and Nuss,D.L. 1992b. Hypovirulence of the chestnut blight fungus conferred by an infectious viral cDNA. Science 257:800-803. Choi,G.H., Shapira,R. and Muss,D.L. 1991a. Cotranslational autoproteolysis involved in gene expression from a double-stranded RNA genetic elem ent associated with hypovirulence of the chestnut blight fungus. Proc. Natl. Acad. Sci. USA 88:1167-1171. Choi,G.H., Pawlyk,D.M. and Nuss D.L. 1991b. The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro. Virology 183:747-752. Fulbright, D.W. 1990. In Baker,T.L. arnd Dunn,P. (eds), New directions in biological control. UCLA symposium on molecular and cellular biology, new series, Alan R. Liss, Inc., New York, Vol. 112. Fulbright,D.W. 1984. Effect of eliminating dsRNA in hypovirulent Endothia parasitica. Phytopathology 74:722-724. Fulbright,D.W., Weidlich,W.H., Haulfer,K.Z., Thomas,C.S., and Paul,C P. 1983. Chestnut blight and recovering American chestnut trees in Michigan. Can. J. Bot. 61:3164-3171. Fulbright,D.W., Paul,C P. and Garrod,S.W. 1988. Hypovirulence: a natural control of chestnut blight. In: Mukeiju,K.G. and Garg,K.L. (eds.), Biocontrol of plant diseases, vol. II. CRC Press, Boca Raton, FL pp. 122-139. Garrod,S.W., Fulbright,D.W. and Ravenscroft,A.V. 1985. The dissemination of virulent and hypovirulent forms of a marked strain of Endothia parasitica in Michigan. Phytopathology 75:533-538. Hillman,B.I., Tian,Y., Bedker,P.J. and Brown,M.P. 1992. A North American hypovirulent isolate of the chestnut blight fungus with European isolate-related dsRNA. J. Gen. Virol. 73:681-686. Koonin,E.V., Choi,G.H., Nuss,D.L., Shapira,R. and Carrington,J.C. 1991. Evidence for common ancestry of a chestnut blight hypovirulence-associated 75 double-stranded RNA and a group of positive-strand RNA plant viruses. Proc. Natl. Acad. Sci. USA 88:10647-10651. MacDonald,W .L. and Fulbright D.W. 1991. Bilogical control of chestnut blight: use and limitaitons of transmissible hypovirulence. Plant Disease 75:656-661. M orris,T.J. and Dodds,J.A. 1979. Isolation and analysis of D ouble-stranded R N A from virus-infected plant and fungal tissue. Phytopathology 69:854-858. Nuss,D.L. and Koltin,Y. 1990. Significance of dsRNA genetic elem ents in plant pathogenic fungi. Annu. Rev. Phytopathol. 28:37-58. Paul,C.P. and Fulbright,D.W. 1988. Double-stranded RNA molecules from Michigan hypovirulent isolates of Endothia parasitica vary in size and sequence homology. Phytopathology 78:751-755. Puhalla,J.E. and Anagnostakis,S.L. 1971. Genetics and nutritional requirem ents of Endothia parasitica. Phytopathology 61:169-173. Shapira,R., Choi,G.H. and Nuss,D.L. 1991a. Virus-like genetic organization and expression strategy for a double-stranded RNA genetic elem ent associated with biological control of chestnut blight. EMBO J. 10:731-739. Shapira,R., Choi,G.H., Hillman,B.I. and Nuss,D.L. 1991b. The contribution of defective RNAs to the complexity of viral-encoded double-stranded RNA populations present in hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica. EMBO J. 10:741-746. Tartaglia,J., Paul,C.P., Fulbright,D.W. and Nuss,D.L. 1986. Structural properties of double-stranded RNAs associated with biological control of chestnut blight fungus. Proc. Natl. Acad. Sci. USA 83:9109-9113. Sambrook,J., Fritsch,E.F. and Maniatis,T. 1989. Molecular cloning: a laboratory manual, 2nd edn. New York: Cold spring harbor laboratory. Zhang,H., Scholl,R., Browse,J. and Somerville,C. 1988. Double-stranded DNA sequencing as a choice for DNA sequencing. Nucl. Acids Res. 16:1220. Chapter IV Presence of laccase in hypovirulent strains of Cryphonectria parasitica recovered from Michigan Introduction Cryphonectria parasitica (Murr.) Barr, the causal organism of chestnut blight, was responsible for killing over three billion American chestnut ( Castanea dentata [Marsh] Borkh.) trees in the Eastern hardwood forests of North America (Fulbright et al., 1988). Chestnut trees are surviving in several localized areas in North America because of the presence of hypovirulent or less virulent forms of the fungus (MacDonald and Fulbright, 1991). Many strains of the fungus with the hypovirulent phenotype contain double-stranded RN A (dsRNA) molecules, which vary in size, homology and concentration among the different strains isolated (Paul and Fulbright, 1988). The molecular mechanism by which dsRNA causes the reduced virulence phenotype is unknown. However, the presence of dsRNA in C. parasitica isolates recovered from E uropean chestnut trees (C sativa Mill.) has been correlated with reduced accumulation of the enzyme laccase in these strains (Rigling et al., 76 77 1989; Hillman et al., 1990). Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) is a m ulticopper oxidase, which catalyzes the oxidation of organic substrates, such as mono-, di- and polyphenols, aminophenols, and diamines (G erm ann et al., 1988). This enzyme is common throughout the plant and fungal kingdoms and has been purified from several sources, however, the exact biological function of laccase is still unknown. In Aspergillus nidulans, laccase is involved in pigmentation (Clutterbuck, 1972), while in Botrytis cinerea it has been shown to be involved in pathogenicity (Bar Nun et al., 1988). It is also known that in many wood decaying fungi, including Heterobasidium annoses, laccase is involved in lignin degradation (Mayer, 1987). The genes encoding laccase have been cloned and sequenced from nuclear DNA of the fungi A. nidulans (Aramayo and Timberlake, 1990), Coriolus hirsutus (Kojima et al., 1990), Neurospora crassa (Germ ann et al., 1988) and, recently, C parasitica (Choi et al., 1992; Rigling and Van Alfen, 1991). The dsRNA from the hypovirulent C. parasitica strain EP713, which is of European origin, contains two contiguous open reading frames (ORFs) (Shapira et al., 1991). The first ORF, designated O R F A, encodes a polyprotein 622 amino acids in length, which is processed into two polypeptides. O ne of the polypeptides is an autocatalytic protease, but the function of the other is unknown (Choi et al., 1991). The second O R F (O R F B), is 3165 amino acids in length and also encodes a polyprotein, which is processed into at least two polypeptides, one of which is an autocatalytic protease (Shapira et al., 78 1991). Although several domains have been identified within O R F B, only the protease has been positively identified. The dsRNA from the European strain EP713 does not cross-hybridize to dsRNA from Michigan strains, so it is interesting to compare phenotypes caused by the presence of these dsRNA molecules. The presence of hypovirulence-associated dsRNA molecules in the cytoplasm of C. parasitica causes not only a reduction in fungal virulence, but also alters other phenotypic traits. By comparing the associated phenotypes induced by different dsRNA genomes, we hope to gain a better understanding of dsRNA function. European hypovirulent strains, such as EP713, are white, sporulation is suppressed, and laccase activity is reduced (Anagnostakis, 1982), while Michigan hypovirulent strains exhibit normal orange pigmentation and continue to sporulate. O R F A, encoded by dsRNA isolated from strain EP713, was found to be responsible for the morphological phenotypic characteristics and the reduction in laccase accumulation (Choi and Nuss, 1991). When virulent C. parasitica protoplasts were transformed with a cDNA clone encoding O R F A, pigmentation, sporulation and laccase activity were reduced to levels similar to those of EP713 (Choi and Nuss, 1991). It has recently been determ ined that the EP713 dsRNA suppresses the accumulation of laccase by down-regulating laccase mRNA accumulation (Choi et al., 1992; Rigling and Van Alfen, 1991). Although a great deal is known about the regulation of laccase in European hypovirulent strains of C. parasitica, this enzyme has not been 79 examined in Michigan hypovirulent strains containing dsRNA. In this study, both virulent and hypovirulent strains of C. parasitica isolated in Michigan were analyzed to determ ine if a relationship exists between the presence of dsRNA and a reduction in the fungal enzyme laccase. The correlation between hypovirulence and laccase activity was also examined in a hypovirulent C. parasitica strain (CL25), which does not contain dsRNA (Fulbright, 1985). Materials and methods Cultures and growth conditions Cultures used in this study are listed in Table 4.1. C parasitica cultures were grown on potato dextrose agar (PDA, Difco, Detroit, MI) in Petri dishes at 25 °C with a 16 h photoperiod under cool white fluorescent lights (G arrod et al, 1985). Cultures were stored on PD A slants at 4°C. Bavendamm assay for phenol oxidase activity To test for phenol oxidase activity, C. parasitica strains were grown on cellophane-covered Bavendamm medium containing 2% agar (Difco), 1.5% malt extract (Difco) and 0.5% tannic acid (Fluka, Switzerland) (Bavendamm, 1928). Petri dishes containing Bavendamm medium were inoculated with a portion of fungal mycelia 4 mm in diameter. Cultures were stored at 25 °C in the dark for 5 days. Presence of brown pigment in the medium indicated a positive reaction for laccase activity. 80 Table 4.1. Strains used in this study. Virulence8 dsRNAb CL1-16 V - Paul and Fulbright, 1988 GH 2 H + Fulbright et al., 1983 Paul and Fulbright, 1988 CL1-16 (GH2/RC1) H + Durbahn Smart and Fulbright, unpublished (see Chap. II) CL25 H - Fulbright, 1985 EP155 V - Anagnostakis, 1981 EP713 H + Anagnostakis, 1981 Strain aV, virulent; H, hypovirulent b+ , contains dsRNA; no detectable dsRNA Reference 81 Assay for laccase activity Fungal cultures were grown in 10 ml of Endothia minimal media (EM M ) (Puhalla and Anagnostakis, 1979) in 25 ml Erlenmeyer flasks, three flasks per strain. Culture flasks were inoculated with a mycelial plug (5 mm in diam eter) and shaken (225 RPM ) at 25 °C in the dark. The cultures were maintained in the dark to induce laccase expression (Hillman et al, 1990). After 7 days of growth, mycelia were pelleted by centrifugation at 10,000 RPM in a Sorvall SS-34 rotor for 5 min. The supernatant fluid was used for protein determ ination and for laccase enzyme assays (Rigling et al, 1989). Protein assays were performed using the Bio Rad Bradford solution according to the m anufacturer (Bio Rad). Laccase activity was measured with 2,6-dimethoxy-phenol (DMOP, Fluka, Switzerland) as a substrate (Bollag, 1979). Culture supernatant (0.2 ml) was added to 0.8 ml 2.5mM DM OP in 80 mM sodium tartrate buffer pH 3.0. Absorbance at 468 nm (O D 468) was measured in a disposable, 10 mm pathlength cuvette at 0 and 5 min using a Gilford Response spectrophotom eter. Native polyacrylamide gel electrophoresis Culture supernatant proteins (0.5 Hg ) were separated by electrophoresis using anionic polyacrylamide gels run at 50 V for -15 h as described (Keleti and Lederer 1974). After electrophoresis, gels were incubated for at least 30 min in 80mM sodium tartrate buffer (pH 3.0) with 2.5mM 2,2’-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), a substrate of laccase, until a green precipitate was formed as a product of laccase activity. 82 Isolation of dsRNA C. parasitica strains were grown for 7 days on cellophanecovered PDA medium. The fungus was removed from the cellophane, and 3 g (fresh weight) samples were used for dsRNA purification. dsRNA was isolated by CF-11 cellulose (W hatman) column chromatography as described by Morris and Dodds (1979). dsRNA was resolved using 5% PAGE and was visualized by ethidium bromide staining. Results and discussion To characterize the expression of the extracellular laccase in Michigan C. parasitica strains, laccase activity was assayed by growth on Bavendamm medium, by a spectrophotometric assay, and by native gel activity staining. W hen grown on Bavendamm medium, all strains tested except EP713 caused the media to turn brown due to the oxidation of tannic acid, indicating the presence of a phenol oxidase (Figure 4.1). These results indicate that Michigan hypovirulent strains do accumulate laccase enzyme, while EP713 has reduced laccase activity, as reported (Flillman et al., 1990; Rigling et aL, 1989). To quantify the relative amount of laccase in each sample, the spectrophotom etric assay was used. Each strain was grown in liquid Endothia minimal medium in the dark for seven days. Minimal medium, which contained no protein, was 83 EP155 CL1-16 CL25 EP713 GH2 CL1“16(GH2/RC1) Figure 4.1. Virulent and hypovirulent isolates of C parasitica grown on Bavendamm medium. Brown pigment in the medium indicates laccase activity. 84 used so that protein concentrations of the culture supernatant could be accurately determined. The absorbance change at 468 nm due to laccase activity using DM OP as a substrate was measured after 5 min with a spectrophotom eter. The increase in OD 468 appeared to be similar for culture supernatants from all isolates assayed except for strain EP713. It has been reported that laccase activity in EP713 is down-regulated (Hillman et al., 1990; Rigling et al., 1989), but in our assays specific activity in the culture supernatant from EP713 was the highest of the strains tested (Table 4.2). R epeated assays using a single culture supernatant were consistent, but activity varied considerably between different flasks of the same strains and between replicate experiments. O ther researchers have also noted inconsistency in laccase expression in culture by northern analysis (Choi et al., 1992). This variability was partially corrected by the addition of low levels of the protein synthesis inhibitor cycloheximide (Choi et al., 1992). A recent report describing the regulation of laccase biosynthesis in C. parasitica, Rigling and Van Alfen (1991) presented spectrophotom etric data on laccase activity from only one of three replicate flasks assayed rather than taking the average of all replicates in each experiment, as is presented here. Results of native polyacrylamide gel electrophoresis followed by staining with the substrate ABTS were similar to those of spectrophotom etric assays. Culture supernatants containing 0.5 /xg protein from each strain produced a diffuse band, staining with approximately equal intensity (Figure 4.2). In each gel stained, one or two samples had a band which was lighter in intensity than the others (Figure 4.2, lane 2), however 85 Table 4.2. Laccase activity assays. Activity was measured using three replicates per strain. Strain Activity® EP713 0.427±0.079 CL1-16 0.342±0.233 CL1-16 (GH2/RC1) 0.148± 0.047 GH2 0.134±0.074 CL25 0.240±0.029 EP155 0.250b aAOD468 after 5 min / mg protein bOnly one m easurem ent performed 86 1 2 3 4 5 6 IpPlilljl fS mm Figure 4.2. Laccase activity in a native protein polyacrylamide gel. Culture supernatant proteins (0.5 ng) were run in a 7.5% anionic gel system. Following electrophoresis, the gel was incubated in the presence of ABTS, a substrate of laccase. Coloration in the gel indicates laccase activity. Lanes: 1, EP713; 2, EP155; 3, CL25; 4, CL1-16; 5, CL1-16(GH2/RC1); 6, GH2. 87 the strain which produced a lighter band varied from gel to gel. As with the spectrophotom etric assays, culture supernatant from strain EP713 appeared to have as much laccase activity as those from the other strains used. All C. parasitica strains tested to date have some extracellular laccase activity (Rigling et al., 1989; Choi et al., 1992). Even strains in which laccase activity is known to be reduced, such as EP713, contained measurable activity (Rigling and Van Alfen, 1991). Perhaps the spectrophotometric assays and native gel staining used in this study were too sensitive for the amounts of laccase present in culture supernatant from each strain. A nother possibility is that since neither of the substrates used was specific for laccase, the assays detected the activity of other phenoloxidases. For this reason, the Bavendamm assay is the best test for the presence of laccase in C parasitica, as the results are consistent and indicate a gross, but measurable change in the ability to oxidize tannic acid. The most significant observation of this study was that both virulent and hypovirulent Michigan C. parasitica strains produced laccase when grown on Bavendamm medium. The Michigan dsRNA-containing hypovirulent isolate GH 2 had laccase activity as did strain CL1-16(GH2/RC1), in which the dsRNA from two hypovirulent strains was transferred into the nuclear background of the virulent CL1-16 (see Chapter II). Therefore, it appears that these dsRNA genomes do not have the ability, either individually or in combination, to downregulate laccase. It is also interesting to note that CL25, a hypovirulent nondsRNA containing Michigan isolate (Fulbright, 1985), also shows laccase 88 activity. Thus, there is no association between hypovirulence and a reduction in laccase in Michigan C. parasitica isolates. dsRNA molecules were detected in four of the seven C. parasitica strains used (Figure 4.3). There was no correlation between the presence of dsRNA molecules in a strain and a positive reaction on Bavendam m’s agar. This is in contrast to the European strains tested in which the virulent, nondsRNA-containing strain had high laccase activity, while activity in the hypovirulent, dsRNA-containing strain was reduced (Figure 4.2). This finding is significant, since this is the first report of a hypovirulent dsRNA-containing C. parasitica strain which does not down regulate the fungal enzyme laccase. Because the overall genomic organization of dsRNA isolated from C. parasitica strains EP713 (European origin) and GH2 (Michigan origin) is similar (see C hapter III), we find it most interesting that GH2 does not down regulate laccase. The O R F A region of the dsRNA from strain EP713 appears to be responsible for the reduction in laccase activity (Choi and Nuss, 1992). Correspondingly, sequences similar to O RF A that are necessary for the downregulation of laccase in EP713 appear to be absent in strain GH2 (see C hapter III). The ability of laccase to degrade lignin (Lewis and Yamamoto, 1990), which reportedly can act as a defense mechanism of the chestnut tree (H ebard et al., 1984), and the close correlation between a reduction in laccase and hypovirulence in European C. parasitica strains, has lead to the suggestion that reduction of laccase accumulation may be fully or partially responsible for 89 1 2 3 4 5 6 kb 9.0 3.5 0.8 - Figure 4.3. Banding patterns of dsRNA from Michigan and European C. parasitica isolates. The dsRNA was electrophoresed in a 5% polyacrylamide gel and stained with ethidium bromide. Strain CL25 is a Michigan hypovirulent strain, which does not contain dsRNA. Strains CL1-16(GH2/RC1) and GH2 are Michigan hypovirulent strains, which contain dsRNA. CL1-16 and EP155 are virulent, non-dsRNA-containing strains from Michigan and Connecticut, respectively. EP713 is a hypovirulent strain containing European dsRNA. Lanes: 1, CL25; 2, CL1-16(GH2/RC1); 3, GH2; 4, CL1-16; 5, EP713; 6, EP155. Approximate sizes of dsRNA molecules present in strain GH2 are indicated in kilobases (kb). 90 hypovirulence (Rigling et al., 1989; Choi et al., 1992). Results of this study clearly indicate that reduction in laccase accumulation is not responsible for hypovirulence in Michigan strains of C. parasitica. Conclusions Based on the Bavendamm assay for laccase activity, I have found no correlation between laccase activity and the hypovirulence phenotype in Michigan isolates of C. parasitica. Results from other laboratories, when basing their work on European-derived dsRNA have found that a correlation does exist. This is indicative of the diversity found among C. parasitica dsRNA genomes (Paul and Fulbright, 1988; L’Hostis et al., 1985; Fulbright et al., 1988). Finally, use of previously described assays for laccase activity with the substrates DM OP or ABTS yielded inconsistent data within a single experiment, as well as between experiments. Therefore, I conclude that the Bavendamm assay is the most reliable test for laccase activity at the present time. 91 Literature Cited Anagnostakis,S.L. 1981. Stability of dsRNA components of Endothia parasitica through transfer and subculture. Exp. Mycol. 5:236-242. Aramayo,R. and Timberlake,W.E. 1990. Sequence and molecular structure of the Aspergillus nidulans yA (laccase I) gene. Nucleic Acid Res. 18:3415. Bar Nun,N., Tal Lev,A., H arel,E and Mayer,A.M. 1988. Repression of laccase form ation in Botrytis cinerea and its possible relation to phytopathogenicity. Phytochemistry 27:2505-2509. Bavendamm,W. 1928. U eber das vorkommen und den nachweis von oxydasen bei holzzerstorenden pilzen. Z. Pflanzenkrank. Pflanzenschutz. 38:257-276. Bollag,J.M., Sjobland,R.D., Liu,S.Y. 1979. Characterization of an enzyme from Rhizoctonia praticola which polymerizes phenolic compounds. Can. J. Microbiol. 25:229-233. Choi,G.H. and Nuss,D.L. 1992. A viral gene confers hypovirulence-associated traits to the chestnut blight fungus. EMBO J 11:473-477. Choi,G.H., Larson,T.G and Nuss,D.L. 1992. Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Mol. Plant-Microbe Inter. 5:119-128. Choi,G.H., Shapira,R. and Nuss,D.L. 1991. Cotranslational autoproteolysis involved in gene expression from a double-stranded RNA genetic elem ent associated with hypovirulence of the chestnut blight fungus. Proc. Natl. Acad Sci. USA 88:1167-1171. Clutterbuck,A.J. 1972. Absence of laccase from yellow-spored mutants of Aspergillus nidulans. J. Gen. Microbiol. 70:423-435. Fulbright,D.W. 1985. A cytoplasmic hypovirulent strain of Endothia parasitica without double-stranded RNA (dsRNA). Phytopathology 75:1328. Fulbright,D.W., Paul,C.P. and Garrod,S.W. 1988. Hypovirulence: a natural control of chestnut blight. In Mukeiju,K.G. and Garg,K.L. (eds.), Biocontrol of plant diseases, vol. II. CRC Press, Boca Raton, FL pp. 122-139. Garrod,S.W ., Fulbright,D.W. and Ravenscroft,A.V. 1985. The dissemination of virulent and hypovirulent forms of a marked strain of Endothia parasitica in Michigan. Phytopathology 75:533-538. 92 Germann,U.A., Muller,G., Hunziker,P.E. and Lerch,K. 1988. Characterization of two allelic forms of Neurospora crassa laccase. J. Biol. Chem. 263:885-896. Hebard,F.V., Griffin,G.J. and Elkins,J.R. 1984. Developmental histopathology of cankers incited by hypovirulent and virulent Endothia parasitica on susceptible and resistant chestnut trees. Phytopathology 74:140-149. Hillman,B.I., Shapira,R. and Nuss,D.L. 1990. Hypovirulence-associated suppression of host functions in Cryphonectria parasitica can be partially relieved by high light intensity. Phytopathology 80:950-956. Keleti,G. and Lederer,W .H. 1974. Handbook of micro-methods for the biological sciences. Van Nostrand Reinhold, New York. 166pp. Kojima,Y., Tsukuda,Y., Kawai,Y., Tsukamoto,A., Sugiura,J., Sakaino,M. and Kita,Y. 1990. Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus. J. Biol. Chem. 265:15224-15230. Lewis,N.G. and Yamamoto,E. 1990. Lignin:occurrence, biogenesis and biodegradation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41:455-496. L ’Hostis,B., Hiremath,S.T., Rhoads,R.E. and Ghabrial,S.A. 1985. Lack of sequence homology between double-stranded RNA from European and American hypovirulent strains of Endothia parasitica. J. gen. Virol. 66:351-355. MacDonald,W.L. and Fulbright D.W. 1991. Biological control of chestnut blight: use and limitations of transmissible hypovirulence. Plant Disease 75:656-661. Mayer,A.M. 1987. Polyphenol oxidases in plants - recent progress. Phytochemistry. 26:11 -20. Morris,T.J. and Dodds,J.A. 1979. Isolation and analysis of Double-stranded RN A from virus-infected plant and fungal tissue. Phytopathology 69:854-858. Paul,C.P. and Fulbright,D.W. 1988. Double-stranded RNA molecules from Michigan hypovirulent isolates of Endothia parasitica vary in size and sequence homology. Phytopathology 78:751-755. Puhalla,J.E. and Anagnostakis,S.L. 1971. Genetics and nutritional requirem ents of Endothia parasitica. Phytopathology 61:169-173. Rigling,D. and Van Alfen,N.K. 1991. Regulation of laccase biosynthesis in the plant-pathogenic fungus Cryphonectria parasitica by double-stranded RNA. J. Bact. 173:8000-8003. 93 Rigling,D. Heiniger,U. and Hohl,GH.R. 1989. Reduction of laccase activity in dsRNA-containing hypovirulent strains of Cryphonectria parasitica. Phytopathology 79:219-223. Shapira,R., Choi,G.H. and Nuss,D.L. 1991. Virus-like genetic organization and expression strategy for a double-stranded RNA genetic elem ent associated with biological control of chestnut blight. EMBO J. 10:731-739. Chapter V Discussion A great deal of knowledge has been gained about mycoviruses, since their discovery thirty years ago. The majority of mycoviruses have genomes composed of double-stranded RNA (Buck, 1986). While most mycovirus infections are symptomless (Hollings, 1978), others have a dramatic effect on their fungal host (see Chapter I). The dsRNA virus-like molecules in C. parasitica are unique among mycoviruses in that they cause a reduction in virulence, but do not kill their host. The disease-factors in Ophiostoma ulmi have also been observed to reduce virulence in infected strains, but this phenom enon has been correlated with the simultaneous presence of ten dsRNA segments (Rogers et al., 1987). In contrast, only one dsRNA segment is necessary for hypovirulence in C, parasitica (see Appendix A). M ost mycoviruses are encapsidated by protein coats that are virally encoded, while a few are contained within vesicles of fungal origin (Buck, 1986). The dsRNA genomes found within C. parasitica appear to be sequestered within vesicles which are located in the cytoplasm of the fungus (Dodds, 1980; Hansen et al., 1985). When the dsRNA genomes of GH2 and 94 95 RC1 were combined into a common nuclear background, and single conidial isolates were collected, no mixing of dsRNA segments between genomes was observed. This suggested that all three dsRNA segments found in the Michigan hypovirulent isolate GH2 are packaged together, as are the two segments of the Michigan hypovirulent isolate RC1 (see Chapter II). In contrast, the individual dsRNA segments of multi-segmented genomes of some mycoviruses, such as the killer viruses in yeast, are packaged individually (Leibowitz et al., 1988). The dsRNA genomes found in C. parasitica strains GH2 and RC1 also differ from those of the killer systems of yeast and Ustiiago maydis, in that when two genomes are combined into a single nuclear background, both of the individual genomes can be maintained (see Chapter II). In the killer systems, one dsRNA genome appears to out compete the second dsRNA genome (Bruenn, 1986). In C parasitica, the effect on the fungal host when harboring both dsRNA genomes of GH2 and RC1 appeared to be additive, as the multiplyinfected strain was less virulent than strains that contained either dsRNA genome individually (see Chapter II). This additive effect has not been reported for any other mycoviruses, and I hypothesize that the dsRNA genomes of isolates G H2 and RC1 have different modes of action in the reduction of fungal virulence. Future experiments could include sequencing the cDNA clones of RC1 dsRNA to determine if this dsRNA encodes a polypeptide. A comparison of the cDNA sequences obtained from GH2 and RC1 dsRNAs could provide information on the different factors responsible for the varied phenotypes associated with each genome, as well as the mechanism for 96 reduction of virulence. The RC1 dsRNA genome is much smaller than that of G H 2 and may represent the minimum complement of dsRNA necessary for reduction in virulence. A comparison of the deduced amino acid sequences of the two open reading frames (O RFs) from dsRNA from C parasitica strain EP713 with those of other mycoviruses, as well as plant viruses, revealed that EP713 was most closely related to positive-strand ssRNA viruses (Koonin, 1991). The O R F identified in the largest dsRNA segment from isolate GH2 has a similar genomic organization as those of dsRNA genomes from Europe and New Jersey (see C hapter III). GH2 is unique, however, in that only a single open reading fram e was identified, rather than the two overlapping ORFs that were identified in the European and New Jersey C. parasitica isolates (see Chapter III). Now that the complete sequence of the GH2 O R F is known, in vitro translation studies could be performed to determine if proteolytic cleavage occurs, producing smaller peptides from this putative polyprotein. These proteins could be isolated, and antibodies raised to them, which could be used to determ ine when and where these proteins are expressed in vivo. W estern blot analysis could also be performed to determine if antibodies raised to proteins produced by GH2 dsRNA were antigenically related to proteins produced by other C. parasitica hypovirulence-associated dsRNA molecules, as well as to those produced by other mycoviruses and plant viruses. The enzyme laccase is produced by many plants and fungi, however, the exact biological function of this enzyme is, in many cases, unknown (Mayer, 97 1987). The down-regulation of laccase by the dsRNA associated with European strains of C. parasitica is the only report of a virus that is responsible for a reduction in laccase. No other plant or fungal virus has been identified that down-regulates laccase. Michigan hypovirulent strains, including GH2, once again appear to be unique among hypovirulent strains of C. parasitica by not down-regulating laccase. A biological and molecular characterization of the dsRNA genome found in the Michigan hypovirulent C. parasitica strain GH2 has revealed that this viral-like genome appears to be quite different from other mycoviruses that have been described, 'while GH2 has a genomic organization that is similar to those of other dsRNA molecules purified from geographically distinct C. parasitica strains, it has several biological properties that are quite different. Those traits involve the effect of the dsRNA on pigmentation, sporulation, growth in culture, and accumulation of laccase. An argum ent can be made that GH2 and EP713 dsRNAs are evolutionarily distinct. There is only 18% deduced amino acid sequence homology, no cross-hybridization upon northern analysis, and only one O R F exists in GH2 dsRNA, while two are present in EP713 dsRNA. dsRNA from strain EP713 results in a down-regulation of laccase, pigmentation, and sporulation, and while GH2 has a slower growth rate than uninfected strains, it has normal pigmentation, sporulation, and laccase production. On the other hand, it seems easier to support the argument that dsRNA from strains EP713 and GH 2 are similar and perhaps evolutionarily related. Both reduce virulence, 98 contain large dsRNA viruses that lack protein capsules and are contained in vesicles. The dsRNA molecules in each strain are 3’ polyadenylated and contain a large open reading frame which is presumed to produce a polyprotein that contains putative RNA helicase, RNA-dependent RNA polymerase and protease domains. While a direct lineage between the dsRNA molecules isolated from strains EP713 and GH2 is doubtful, the similarities between these molecules suggest that an evolutionary relationship does exist. The working hypothesis concerning the mechanism of dsRNA-associated hypovirulence is that a polypeptide, encoded for by dsRNA, down-regulates a C. parasitica gene or genes necessary for fungal virulence. The specific C. parasitica genes that may be down-regulated, reducing fungal virulence, are unknown. A nother possibility is that the dsRNA itself, rather than a dsRNA encoded polypeptide, disrupts fungal processes, and thereby reduces virulence. Until a specific viral protein responsible for reduction in virulence is identified, both of these hypotheses are valid. However, since all of the dsRNA molecules sequenced to date contain open reading frames, the assumption is that the putative protein products would interact with the fungal host. A survey of the research performed on the host-parasite (fungal-viral) interactions involved in hypovirulence reveals that the viral side of the interaction has been studied much more thoroughly than the fungal biology. It is possible that fungal factors are involved in viral maintenance within the cytoplasm of C, parasitica. This phenomenon has been observed in the yeast killer system (Bruenn, 1986). If this is the case in C. parasitica, strain GH2G3 99 may be the ideal strain in which to study these interactions, since all singleconidial isolates (asexual progeny) from strain GH2G3 contain dsRNA (see Appendix B). A simple experiment, transferring the dsRNA into a new nuclear background, collecting asexual progeny, and assaying for the presence of dsRNA, would reveal if the nucleus was involved. The outlook for the survival of the American chestnut in North America is positive on several fronts. First, within Michigan, small stands of chestnut trees, which were declining only 5 years ago, now appear to be recovering and contain naturally occurring hypovirulent strains of C. parasitica. Second, artificial inoculation of hypovirulent strains around the margins of a potentially lethal canker can prevent it from killing the tree. This method is rather labor intensive, however, since each canker on a tree must be treated individually. The most exciting prospect for biological control of chestnut blight was elucidated through recent transformation experiments performed by Choi and Nuss (1992). In these experiments, the full-length cDNA clone of dsRNA from the European C. parasitica strain EP713 conferred hypovirulence to transform ants containing the cDNA, as well as to both ascospores and conidia produced by the transformants. If these transformants are found to be stable over long periods of time and can induce hypovirulence in field situations, they have the potential to be effective biological controls, because they would not be inhibited by vegetative compatibility, as are natural isolates. Because sporulation is suppressed by the dsRNA in strain EP713, dsRNA molecules from other strains, such as GH2, may be more effective biocontrols and should 100 continue to be studied. Also, the smaller dsRNA genome size found in C. parasitica strain RC1 may be more stable than larger genomes when integrated into the fungal genome, and may represent another effective biocontrol. While hypovirulence will prevent the American chestnut from going extinct, I do not believe that the eastern forests of North America will ever again be predominantly composed of chestnut trees. Various oak species, which are now well established, have filled the void left by the death of the chestnut trees. Chestnut orchards, however, are gaining popularity among nut growers, and hypovirulent strains of C, parasitica could no doubt be effective as a biological control in an orchard situation. I am confident that future research will not only elucidate the mechanism responsible for hypovirulence, but will also enable us to have American chestnuts roasted over an open fire. 101 literature cited Buck,K.W. 1986. Fungal virology - an overview, pp. 1-84. In: K.W. Buck (ed.), Fungal Virology. CRC Press, Boca Raton, FL. Bruenn,J. 1986. The killer systems of Saccharomyces cerevisiae and other yeasts, pp. 85-108. In: K.W. Buck (ed.), Fungal Virology. CRC Press, Boca Raton, FL. Choi,G.H. and Nuss,D.L. 1992. Hypovirulence of the chestnut blight fungus conferred by an infectious viral cDNA. Science 257:800-803. Dodds,J.A. 1980. Association of type 1 viral-like dsRNA with club-shaped particles in hypovirulent strains of Endothia parasitica. Virology 107:1-7. Hansen,D.R., Van Alfen,N.K., Gillies,K. and Powell,W.A. 1985. Naked dsRNA associated with hypovirulence of Endothia parasitica is packaged in fungal vesicles. J. Gen. Virol. 66:2605-2614. Hollings,M. 1978. Mycoviruses: viruses that infect fungi. Advan. Virus Res. 22:3-53. Koonin,E.V., Choi,G.H., Nuss,D.L., Shapira,R. and Carrington,J.C. 1991. Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RN A plant viruses. Proc. Natl. Acad. Sci. USA 88:10647-10651. Leibowitz,M.J., Hussain,I. and Williams,T.L. 1988. Transcription and translation of the yeast killer virus genome, pp. 133-160. In: Y. Koltin and M. Leibowitz (ed.), Viruses of fungi and simple eukaryotes. Marcel Dekker, Inc., New York. Mayer,A.M. 1987. Polyphenol oxidases in plants - recent progress. Phytochemistry. 26:11-20. Rogers,H.J., Buck,K.W. and Brasier,C.M. 1987. A mitochondrial target for double-stranded RN A in diseased isolates of the fungus that causes Dutch elm disease. Nature 329:558-560. APPENDICES APPENDIX A Virulence of Cryphonectria parasitica strains with various combinations of dsRNA molecules Introduction Chestnut blight, caused by Cryphonectria parasitica, is naturally controlled in several locations in Michigan by hypovirulent, or less virulent forms of the fungus (Fulbright et al. 1983). American chestnut trees ( Castanea dentata [Marsh.] Borkh.) in one such location (Grand Haven) have been infected with chestnut blight since the 1940’s, but are surviving due to protection by hypovirulent, double-stranded RNA containing strains of C. parasitica. A study of this site was performed by Fulbright et al., (1983), and a brief summary of their results is presented below. Two hypovirulent isolates containing different dsRNA banding patterns upon polyacrylamide gel electrophoresis (PAGE) were collected at the Grand Haven site and were designated GH2 and GHU4. Isolate GHU4 had an extremely slow growth rate and did not produce pycnidia or conidia on chestnut stems. Isolate GH2 was more interesting in that, while reduced in virulence, it was more aggressive than G H U 4 and produced swollen cankers that sporulated, demonstrating that 102 103 hypovirulence may have disseminated through asexual spores (Fulbright et al., 1983). It was later determ ined that dsRNA from C. parasitica isolates GH2 and G H U 4 cross-hybridized upon northern analysis, although their dsRNA banding pattern and the molecular weights of individual dsRNA segments were different (Paul and Fulbright, 1988). We have chosen isolate GH2 as the focus of the present study. C. parasitica strain G H 2 is characterized by a dsRNA genome that contains three size classes of dsRNA molecules (Dodds, 1980; Tartaglia et al., 1986). The largest is approximately 9.0 kb, while the smaller two are approximately 3.5 kb and 0.8 kb. The two largest molecules cross-hybridize upon northern analysis, while the smallest does not hybridize to either of the larger molecules (Tartaglia et al., 1986). We have identified C. parasitica isolates which contain one, two or all three of the dsRNA molecules characteristic of the GH2 dsRNA genome. Interestingly, a C. parasitica strain containing only the smaller two dsRNA molecules has never been reported. My goal was to determine if the variability observed in the dsRNA banding pattern could be associated with changes in virulence and, therefore, if the num ber of segments or any one specific segment accounted for changes in aggressiveness of the isolates. 104 Materials and methods Cultures and growth conditions The C parasitica isolates CL1-16 and GH2 were recovered from Crystal Lake and Grand Haven, Michigan, respectively, in 1980 (Fulbright et al., 1983). Isolates GNC, TG, M3, and F5 were recovered from an experimental plot at Crystal Lake in 1985 (G arrod e t al., 1985) in which G H 2 was released in 1983. Cultures were maintained on PDA (Difco, D etroit, MI) at 25 °C under cool white, fluorescent lights with a 16 h photoperiod (G arrod et al., 1985). dsRNA analysis and culture conversions dsRNA isolation by column chromatography over CF-11 cellulose (W hatman) was perform ed as described by M orris and Dodds (1979). dsRNA from each strain was transferred into the nuclear background of the virulent, non-dsRNA-containing isolate CL1-16 via hyphal anastomosis as described by Anagnostakis and Day (1979). Proper transfer was confirmed by dsRNA isolation, which was separated on 5% polyacrylamide gels and stained with ethidium bromide. Strains were designated with the name of the recipient isolate (CL1-16) followed by the nam e of the dsRNA donor strain in parentheses (eg. CL1-16(GH2)). The conversion of the virulent isolate CL1-16 to hypovirulent, due to the transfer of dsRNA molecules from a donor hypovirulent strain, was confirmed by virulence assays in which Golden Delicious apple fruit were inoculated with the converted CL1-16 strain. Inoculation with the virulent isolate CL1-16 served as a control. 105 The virulence of each converted strain was determ ined based on the size of the lesion produced on the apple fruit when compared to the size of the lesion produced by the virulent strain, CL1-16. Stem inoculations The 5 dsRNA-containing strains were inoculated on each of 5 American chestnut tree stems in a research plot in Frankfort, MI. Duplicate inoculations were perform ed in a separate plot at Russ Forest in southern Michigan. Stem inoculations were made using a sterile 5 mm cork borer and were approximately 3 mm deep, penetrating the bark and cambial layers of the stem. A fungal mycelial plug, 5 mm in diameter, was placed in the wound on the stem and was covered with tape to prevent desiccation. The virulent C parasitica isolate, CL1-16, was not tested, because such inoculations would rapidly kill the small trees. Instead, canker areas were com pared with those of CL1-16(GH2). The area of the canker caused by each strain was m easured on each stem at 1, 2 and 3 months after inoculation. The mean and standard deviation of the canker areas were calculated. After 3 months, C parasitica was re-isolated from each canker as described by G arrod e t al. (1985). Bark samples were placed on PDA, and the resultant C. parasitica isolates subcultured. dsRNA isolation was perform ed on the subcultures to verify that the dsRNA content of each strain had not changed. 106 Results and discussion Characterization of culture morphology and dsRNA content The unique culture morphology for each strain used in this study is shown in Figure A l. A fter dsRNA isolation and analysis on PAGE (Figure A2), it was observed that various culture morphologies were reflective of differing dsRNA genomes. Each hypovirulent strain contained dsRNA molecules with mobility in PA G E similar to 1, 2, or all 3 dsRNA size classes generally associated with strain GH2 (Figure A2). dsRNA was transferred from the hypovirulent strains GH2, M3, F5, TG, and GNC into the common nuclear background of the virulent strain CL1-16. The dsRNA banding pattern of the resulting strains were verified by PA G E to be consistent with those of the donor strains. Culture morphologies of the converted strains were also similar to those of the donor, hypovirulent strains. Strains CL1-16(M3) and CL1-16(GNC) each had only one dsRNA band observable after gel electrophoresis. The single dsRNA band in CL116(M3) appeared to be identical in size to that in strain CL1-16(GH2) (approximately 9 kb), however the band in strain CL1-16(GNC) was larger (approximately 10 kb). Strain CL1-16(F5) contained two bands of dsRNA identical in mobility to the two largest dsRNA segments found in CL1-16(GH2) (approximately 9.0 kb and 3.5 kb). Strain CL1-16(TG) contained three dsRNA bands similar in mobility to those in CL1-16(GH2), except that the middle band in CL1-16(TG) appeared to be smaller than the middle band in CL1-16(GH2) Figure A l. Culture morphology of isolates used in this study. 108 CM o X z 0 0 CO T“ I T— -J O to ST 1 ST 2E Sf in LL, & CO CO 1 B 1 1 r* I] HI I] Zj mJ O o o o o kb 9. 0 3. 5 - 0.8 Figure A2. Ethidium bromide-stained dsRNA isolated from each strain inoculated into chestnut stems. dsRNA was separated by 5% PAGE. M olecular size is indicated in kilobases (kb). 109 (Figure A2). Effect of dsRNA genomes on virulence To determine which dsRNA molecules in C. parasitica isolate GH2 might be required for the observed reduction in virulence, we obtained strains with various GH2 dsRNA banding patterns. The m ean canker areas for each strain at each plot is shown in Figures A3 and A4. Strain CL1-16(M3) was clearly the most virulent of the isolates used in this study, while strain CL1-16(GNC) was the least virulent. The other three isolates were approximately equal in virulence, although strain CL1-16(TG) was slightly more virulent than strains CL1-16(F5) and CL1-16(GH2) (Figure A3). My results indicate that a strain which contained only the largest molecule of the GH2 dsRNA genome was more virulent than strains which contained either two or all three molecules (Figures A2, A3, and A4). Strain CL1-16(M3), which has one dsRNA band, caused significantly larger cankers than strains CL1-16(F5), CL1-16(TG) and CL1-16(GH2), all of which had two or three bands. No clear differences were seen between strains CL1-16(F5), with two dsRNA bands and CL1-16(TG) and CL1-16(GH2), with three bands each. Perhaps if the study could have been continued for two years, small differences in virulence, which may exist between these strains would be observed. Unfortunately, a majority of the test trees died before further analysis could be made. Some of the trees died because of cankers of strain CL1-16(M3), and others died of natural cankers, which 110 6,000 5,000 tr 4,ooo n | 3,000 O 2,000 1,000 C L 1-16(G H 2) C L 1-16(M 3) C L 1-16(F 5) C L1-16(T G ) C L 1-16(G N C ) Strain 1 Month ■ 2 Months □ 3 Months Figure A3. Mean canker area produced by each strain at the Frankfort plot. Bars represent an average of 5 inoculations. Standard deviation is indicated as a line above each bar. Ill 6,000 5,000 c