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This is to certify that the
dissertation entitled

The Role of Mitochondria in the Hypovirulence of
Chestnut Blight Fungus Cryphonectria ParaSitica

presented by
Nibedita Mahanti
has been accepted towards fulfillment

of the requirements for

Doctoral degree in Botany & Plant Pathology

 

 

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Date A“? I]. [99/

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THE ROLE OF MITOCHONDRIA IN THE HYPOVIRULENCE OF
CHESTNUT BLIGHT FUNGUS CRYPHONECTRIA PARASITICA.

By

Nibedita Mahanti

A DISSERTATION
Submitted to Michigan State University in
partial fulfillment of the requirements
for the degree of

DOCI‘ OR OF PHILOSOPHY

Department of Botany and Plant Pathology

1991

ABSTRACT
THE ROLE OF MITOCHONDRIA IN THE HYPOVIRULENCE OF
CHESTNUT BLIGHT FUNGUS CRYPHONECI‘RIA PARASITICA.
by
Nibedita Mahanti

Hypovirulent strains of thonectria parasitica have been associated with the presence of
double-stranded ribonucleic acid (dsRNA). Hypovirulence can be cytoplasmically transferred to
virulent strains through hyphal anastomosis. Strain CL25 from Crystal Lake, Michigan, has many
Characteristics of dsRNA-associated hypovirulence but harbors no detectable amounts of dsRNA.
Genetic studies of CL25 showed that the hypovirulent phenotype is carried in 20% of the conidia,
maternally inherited in sexual crosses, and can be transferred to other virulent strains via hyphal
fusion. This suggests that there is a cytoplasmic "virulence inhibition factor” (VIF) other than dsRNA
responsible for hypovirulence in CL25.

Since hypovirulence in CL25 is maternally inherited, several studies were initiated to
investigate the possibility that mitochondria might be involved. Two low copy number plasmids of 6
and 10 kilobase pairs (kb) in size were discovered in the mitochondria of CL25. A portion of the 6
kb plasmid was cloned and was used to screen other strains. The plasmid was detected in all virulent
and hypovirulent strains of Q. parasitica screened, and thus, the 6 kb plasmid could not be directly
involved in the hypovirulence of CL25. Thus far, the function of this plasmid remains unknown.

Respiration studies of CL25 and other dsRNA-free hypovirulent strains revealed that the

cyanide-insensitive or alternate oxidase pathway accounted for as much as 85% of the total

Nibedita Mahanti

respiration in these strains, compared to 8% in virulent and 16% in dsRNA-associated hypovirulent
strains. This study suggests that a respiratory defect may be closely associated with dsRNA-free
hypovirulence.

Using RFLPs and chloramphenicol resistance as markers for the mitochondrial genome and
pigment production as a marker in the nuclear genome, it was possrble to demonstrate that
mitochondria can transfer from a dsRNA-free hypovirulent strain to a virulent strain. The converted
strain obtained the hypovirulence and chloramphenicol-resistance traits of CL25, but retained the
pigmentation phenotype of the virulent strain, and contained the mitochondrial RFLPs of both
strains. The alternate oxidase pathway accounted for 82% of total respiration in the converted strain
as compared to 8% before conversion indicating concerted cytoplasmic transfer of mitochondria, the
hypovirulent phenotype and alternate oxidase activity. This supports the hypothesis that mitochondria

are associated with the dsRNA-free hypovirulence phenotype.

Dedicated in the memory of my parents,
Mr. Nishamani Khuntia
and

Mrs. Shantisudha Khuntia

ACKNOWLEDGEMENTS

I would like to thank my major professor, Dr. Dennis W. Fulbright, for his guidance
and the opportunities he has provided. I would like to express my appreciation to all the
members of my guidance committee; Dr. Raymond Hammerschmidt, Dr. Barbara B. Sears and
Dr. C. Adinarayana Reddy for their guidance and useful suggestions. My sincere appreciation
goes to Dr. Helmut Bertrand who has made it possible to explore this new direction in
hypovirulence.

I would like to thank all the members of Fulbright’s lab for their help in the lab and
their concern and stimulating discussions throughout the course of this work.

I would like to thank my family, especially my sister Sanghamitra for her confidence
in me and all those encouraging letters that made me feel that I am still a part of their world
despite the distance separating us. I appreciate the patience and encouragement from my
extended family. '

My endless thanks goes to my husband, Nanak, who has made this possible and
contributed in every possible way. His support was more than I could ever have asked for.
My special thanks goes to my son, Abhishek, who has done his best in assuring me that I am
worth something at frustrating times and has helped me accomplish this understanding.

TABLE OF CONTENTS

Page

LIST OF TABLES ................................................... viii

LIST OF FIGURES ................................................... ix

LITERATURE REVIEW ............................................... 1

The history of chestnut blight ....................................... 1

Biology and Genetic organization of dsRNA ........................... 2

Mitochondrial plasmids of filamentous fungi ............................ 7

Mitochondrial plasmids of Neurosmra .......................... 8

Mitochondrial plasmids of Podosmra anserina .................... 9

"Ragged” mutants of Asxrgjllus .............................. 10

Mitochondrial plasmids of Claviceps purpurea .................... 11

Mitochondrial Plasmids in Cochliobolus heterostrophus ............ 11

Alternative oxidase pathway: cyanide-resistant respiration ................. 12

References .................................................... 17

CHAPTER 1. CRYPTIC MITOCHONDRIAL PLASMIDS IN

CRYPHONECI'RIA PflyfiITICA ...................................... 22

Abstract ...................................................... 22

Introduction ................................................... 23

Materials and methods ........................................... 24

Fungal strains and growth conditions ........................... 24

Mitochondrial DNA isolation ................................ 26

Electrophoresis of mitochondrial DNA ......................... 27

Isolation and cloning of the plasmids ........................... 27

DNA labelling and hybridization .............................. 28

Results and discussion ........................................... 28

References .................................................... 34
CHAPTER 2. TRANSFER OF MITOCHONDRIA DURING HYPHAL

ANASTOMOSIS IN CRYPHONECI'RIA PARASITICA ...................... 36

Abstract ...................................................... 36

Introduction ................................................... 37

Materials and methods ........................................... 39

Fungal strains and growth conditions ........................... 39

Sexual crosses ............................................ 41

Single conidial isolation .................................... 41

Pathogenicity test ......................................... 42

Mitochondrial DNA isolation ................................ 42

vi

Restriction enzyme analysis and electrophoresis .................. 43

Isolation and cloning of the RFLP fragment ..................... 44
DNA labelling and hybridization .............................. 44
Cytoplasmic fusion via strain pairing ........................... 44
Results ....................................................... 45
RFLP analysis of mtDNA ................................... 45
Isolation of chloramphenicol resistant strain of CL25 .............. 45
Transfer of mitochondria during hyphal anastomosis ............... 47
Discussion .................................................... 50
References .................................................... 53

CHAPTER 3. ASSOCIATION OF ELEVATED MITOCHONDRIAL
ALTERNATE OXIDASE ACTIVITY WITH THE HYPOVIRULENCE

PHENOTYPE OF CRYPHONECI'RIA PARASITICA ....................... 55
Abstract ...................................................... 55
Introduction ................................................... 56
Materials and methods ........................................... 57

Strains and growth conditions ................................ 57
Respiration .............................................. 60
Isolation of mitochondria ................................... 61
Determination of enzyme activities ............................ 61
Cytochrome oxidase activities ................................ 61
Succinate oxidase activity ................................... 62
NADH oxidase activity ..................................... 62
Determination of dry weight ................................. 62
Results ....................................................... 62
Respiration .............................................. 62
Enzyme assays ........................................... 66

Dry weight of different strains ............................... 66
Discussion .................................................... 66
References .................................................... 72
CONCLUSION ...................................................... 74

vii

LIST OF TABLES
Table Page
1.1. _C_. parasitica strains used in the plasmid screening study ..................... 25

2.1. Strains of Cyphonectria parasitica used in the study of mitochondrial
transfer during hyphal anastomosis .................................... 40

2.2. Pathogenicity test of virulent, hypovirulent and converted hypovirulent
strains of Cuphonectria parasitica ..................................... 48

2.3. Phenotypic and genotypic characteristics of single-conidial isolates of
strain 4-C(H). .................................................... 49

3.1. Alternate oxidase pathway as percentage of total respiration in different
strains of Cgphonectria parasitica ..................................... 58

3.2. Induction of alternate oxidase pathway with chloramphenicol in different
strains of Cgphonectria parasitica ..................................... 65

3.3. The activities of cytochrome c oxidase, succinate oxidase and NADH oxidase
in partially purified mitochondrial preparations of isogenic strains of CL25 ...... 67

3.4. Determination of dry weight of different strains of Q. parasitica grown
in Endothia minimal broth ........................................... 68

LIST OF FIGURES

Figure Page
1.1- Intact mtDNA of isogenic strains of Q. parasitica ......................... 30
1.2- Mitochondrial plasmids digested with different enzymes ..................... 31

1.3- Intact mtDNA of different isolates of _C. parasitig,
Endothia mosa and Colletotrichum lagenarium ........................... 32

2.1- _Sau3A digestion pattern of mtDNA of different isolates
of _C_. parasitica .................................................... 46

3.1- Oxygen electrode recordings of the respiration of
different strains of ngphonectria parasitica .............................. 64

LITERATUREREVIEW

Cgphonectria (= Endothia) parasitica (Murr) Barr. is the fungal phytopathogen
responsible for chestnut blight, the disease which has virtually eliminated the American
chestnut (Castanea glen—mtg (Marsh) Borkh) from the eastern deciduous forests of North
America. Hypovirulent strains of this fungus have resulted in a natural biocontrol of the
disease (MacDonald and Fulbright, 1991). Hypovirulence of Q. parasitica is described as a
cytoplasmically transmissible trait that results in attenuation of virulence of the pathogen (Van
Alfen et a1, 1975). Double-stranded RNA (dsRNA) genetic elements have been associated

with the phenomenon of ”transmissible hypovirulence".

The History of Chestnut blight

Chestnut blight remains among the most devastating plant diseases known. It has
eliminated the American chestnut as a major tree species in eastern North America. The first
infected trees were reported in 1904 in the Bronx zoo (Anagnostakis, 1982). The fungus was
apparently introduced into New York city and Long Island on nursery stock of oriental
chestnuts, possibly from Japan. Chinese chestnut (Q. mollissima) and Japanese chestnut (Q.
gm Sieb. and Zucc.) exhibit a high level of tolerance to the blight, but these chestnut
species can act as carriers of the fungus. The American chestnuts proved to be very

susceptible and the fungus spread quickly throughout the natural range of the chestnut.

2

The most obvious symptoms of chestnut blight induced by virulent strains of Q.
parasitica are yellowing and wilting of the foliage of a branch or main stem. The fungus
infects through wounds and induces cankers that girdle the trunk or branch, affecting the
vascular system. The root system remains alive, but sprouts growing from the base of the
stump are eventually killed by subsequent infection.

In 1938, Biraghi found the fungus on European chestnut trees (Q. sativa Mill) in

 

northern Italy and within a decade the disease spread and killed trees in many areas. In 1951,
Biraghi noted that some coppice trees were recovering from blight in an area where blight
was observed in 1938.

The phenotype of these newly discovered strains was termed ”hypovirulent” by a
French mycologist J. Grente in the early 1960’s. Hypovirulent strains of the fungus were
described as white in culture and reduced in virulence. It was found that these characteristics
could be cytoplasmically transmitted to virulent strains of Q. parasitica via hyphal fusion. Day
et a1 (1977) discovered the presence of double-stranded RNA (dsRNA) in hypovirulent
strains and differentiated hypovirulent strains from virulent strains by the lack of dsRNA in
virulent strains. When the hypovirulence trait was transmitted to a virulent isolate, the dsRNA
was also transmitted (Anagnostakis and Day, 1979; Day et a1. 1977). The discovery of
hypovirulence gave hope that biological control of blight with hypovirulence might result in

the recovery of both the American chestnut in eastern North America.

Biology and Genetic organization of dsRNA
The cytoplasmic nature of hypovirulence was first proposed by Grente and Sauret.
Van Alfen et a1 (1975) first genetically demonstrated the cytoplasmic mode of hypovirulence

transfer by pairing genetically marked, auxotrophic strains of Q. parasitica in chestnut bark

3

and in culture. A hypovirulent lysine auxotroph was paired with a virulent methionine
auxotroph in the trunks of American chestnut seedlings. Ninety days later, methionine
auxotrophs isolated from the canker were hypovirulent. Conversion of the methionine
requiring strain from virulent to hypovirulent indicated that the hypovirulent phenotype was
transferred from the lysine auxotrophs. More evidence for the cytoplasmic nature of
hypovirulence was obtained when heterokaryons were formed after pairing hypovirulent
methionine and virulent arginine auxotrophs. From single-conidial isolates obtained from the
heterokaryon, hypovirulent strains were obtained which required both methionine and
arginine.

The ability of hypovirulent isolates to transfer the factors responsible for
hypovirulence to virulent isolates has been described as transmissible hypovirulence. The
transfer of hypovirulence to virulent isolates occurs between compatible isolates of the fungus
through hyphal fusion (Newhouse et al. 1983). In a compatible reaction, the hyphae of two
isolates fuse and cytoplasmic exchange occurs. Hyphal fusion is controlled by vegetative
compatibility (V C) genes. Anagnostakis and Day (1979) have estimated that there are at least
five VC genes, and all alleles must be the same at these loci for stable fusion to occur.
Cytoplasmic transfer of dsRNA is possible if all the alleles are not the same, however,
cytoplasmic transfer of dsRN A has been shown to occur more frequently when the alleles are
homologous and is reduced in frequency if the alleles are heterologous. Therefore, it appears
that the more stable the hyphal fusion, the more likely the occurrence of cytoplasmic transfer.

Correlative evidence indicating the involvement of dsRN A in hypovirulence was
strengthened by a dsRNA curing experiment conducted by Fulbright (1984). Through the
application of cycloheximide, hypovirulent Q. parasitica isolates were cured of their dsRNA
and the cured isolates had increased virulence. These cured strains could be converted back

to the hypovirulent form by fusion with a hypovirulent strain.

4

Double-stranded RNAs associated with different hypovirulent strains vary considerably
with respect to size, number of species, concentration and sequence homology. (Dodds, 1980;
Elliston, 1985; Garrod et al, 1985; Paul and Fulbright, 1988). There is no consensus yet as to
what constitutes the minimum dsRNA genome of any of the hypovirulent strains. A study by
Paul and Fulbright (1988), using dsRNA from different geographical locations showed that
there was no homology between Michigan dsRNA and dsRNA from West Virginia and other
North American locations. Hypovirulent strains from Michigan, however, in spite of the
variety of banding patterns, shared sequence homology. The only exception was strain RC1
which showed no homology to the dsRNAs found in hypovirulent strains from Michigan,
other North American locations, or Europe.

Since most known mycoviruses have dsRNA as their genome, attempts were made to
look for viral particles in Q. parasitica. In the strains of Q. parasitica studied to date, none of
these dsRNA have been shown to be encapsidated within protein coats, unlike dsRNA of
other filamentous fungi. Double-stranded RNA from the most thoroughly studied
hypovimlent strain, EPll3, is thought to be packaged within fungal vesicles (Hansen et al,
1985). Electron microscopic studies indicated that these vesicles are associated with the
endoplasmic reticulum (Newhouse et al, 1983). Using this strain, Dodds (1980) isolated two
fractions of membrane-bound vesicles, which differ in their density. Hansen et al.(1985)
determined that one of the fractions contains most of the dsRNA. Although dsRNA of Q.
parasitica is not encapsidated within viral particles, the vesicles associated with the dsRNA
appear to have an RNA polymerase associated with them. The vesicles containing greater
amounts of dsRN A are able to incorporate nucleotide triphosphate into trichloroacetic acid
(ICA) precipitable products. No detectable polymerase activity was found in the fraction
containing little dsRNA and the similar vesicles from strains that do not have dsRNA

(Hansen et al, 1985).

5

The composition of fungal vesicles is similar in both hypovirulent and virulent strains
that do not contain dsRNA. The vesicles from the hypovirulent strain are much higher in lipid
components and lower in protein content than those from the virulent strain. The protein
composition is too low to account for the presence of a capsid. The neutral sugars that are
present (arabinose, mannose, galactose, glucose) in these particles are also present in the
fungal cell walls. This suggests that these vesicles are of fungal origin and are probably
involved in cell wall synthesis (Hansen et al, 1985).

Structural properties of hypovirulence-associated dsRNAs are most studied in GH2,
a North American strain and EP713, a European strain. These two dsRNA genomes reveal
some common structural properties. Double-stranded RNA in both strains consist of multiple
species, consisting of a large species (L—RNA) and other smaller ones. GH2 has two smaller
molecules, namely M-RNA and S-RNA and EP713 has a number of medium and small
dsRNAs. Hybridization studies and sequence analysis of some of the dsRNA shows that the
smaller dsRN As are derived by internal deletion of the LRNA (Shapira et al, 1991b), with
the exception of the GH2 S-RNA species. This was concluded because of the fact that the
smaller dsRNA species have the same terminal sequence as the L-RNA. Double-stranded
RNA of GH2 and EP713 contain a stretch of polyadenylic acid (poly A) at the 3’-terminus
which base pairs with a stretch of polyuridinic acid (poly U) at the 5’-terminus of the
complementary strand. This end is referred to as the homopolymer terminus and the other
terminus is referred to as the heteropolymer end (Hiremath et al, 1986, Tartaglia et al, 1986,
N uss and Koltin, 1990).

Recent reports (Choi et al, 1991, and Shapira et al, 1991A) have revealed the
complete nucleotide sequence and the genetic organization of the largest dsRNA (L—dsRN A)

of hypovirulent strain EP713. L-dsRNA consists of 12,712 bp excluding the poly (A): poly (U)

6

homopolymer domain. The strand which terminates with a 3’ poly (A) is referred to as the
positive strand and contains two open reading frames, designated as ORF A and ORF B.
ORF A encodes two polypeptides, p29 and p40, which are released from polyprotein, p69
(Choi et al, 1991). The cleavage is autocatalytic and is mediated by p29. The ORF A/ORF
B junction consists of the sequence 5’-UAAUG-3’, where the UAA of the pentanucleotide
serves as the termination codon of the ORF A, whereas the AUG portion serves as the 5’
proximal initiation codon of ORF B. This situation is similar to a pentanucleotide sequence
observed in influenza virus RNA segment 7. ORF B also appears to be involved in an
autocatalytic proteolysis event and encodes a 48 kd polypeptide, p48. Both polypeptides, p29
and p48, are presumably involved in catalyzing additional steps in the processing of the
predicted ORF B encoded polyprotein. A helicase motif has been identified within the
carboxyl terminal domain of ORF B (Shapira et al, 1991A).

Another report by Shapira et a1. (1991B) strengthened the evidence that the M-
dsRNA (810 kb) and S-dsRNA (0.6-1.7 kb) of EP713 are derived from the L-dsRNA by
internal deletion. Sequence analysis of cloned cDNA copies of M-dsRNA and S-dsRNA
species confirmed that these elements retained both heteropolymor and homopolymer
domains found in L-dsRNA.

In spite of these recent developments in characterization of dsRNA molecules, it is
still not clear how dsRNA affects the virulence of the fungus. A transformation system for
Q. parasitica has been developed (Churchill et a1. 1990) and the transformation and
expression of the cDNA clones of dsRNA into isogenic virulent strains may help in better
understanding the role of dsRNA in hypovirulence (Hillman, 1990).

Even though dsRNA is correlated with the hypovirulence of Q. parasitica in many

strains, there are exceptions. In Michigan, hypovirulent strains which do not harbor detectable

7
amounts of dsRNA have been isolated from Crystal Lake, (Frankfort) and Kellogg Forest,

(Augusta). Genetic studies on one of these dsRNA-free strains, CL25, showed that this type
of hypovirulence is carried in 20% of the total conidia, is maternally inherited and can be
cytoplasmically transferred to other virulent strains via hyphal anastomosis (Fulbright, 1985).
This suggests that there is a cytoplasmic "virulence inhibition factor" other than dsRNA
involved in the dsRNA-flee type of hypovirulence since dsRNA is not maternally inherited.
There are several reports on the debilitation of fungi which are associated with mitochondrial

plasmids. Some of those model systems are described below.

Mitochondrial plasmids of filamentous fungi

Mitochondrial plasmids of filamentous fungi have been classified into two groups.
"True" mitochondrial plasmids are defined as DNA or RNA species that are present in
mitochondria, but are not derived from the mtDNA (Lambowitz et al 1986). True
mitochondrial plasmids appear to have neither a positive nor a negative effect on the fungus.
Examples of such plasmid are seen in mitochondria of higher plants, mushrooms, and several
filamentous fungi, including Neurospgra and Claviceps purpurea. "Defective mitochondrial
DNAs" are circular DNAs found in the mitochondria and they are derived from the
mitochondrial genome by excision and amplification. They are often suppressive relative to
wild-type mitochondrial DNA (mtDNA) and accumulation of defective mtDNA is usually
associated with a respiration-deficient phenotype and vegetative senescence. The classical
examples are petite mutants in yeast (Locker et al, 1979), stopper mutants of Neurospgra
(Bertrand et a1. 1980), ragged mutants of Asmrgm us (Lazarus and Kuntzel, 1981) and

senescence in Podosmra (Wright 1982).

Mitochondrial plasmids of Neurosmra

Mitochondrial plasmids were first discovered and characterized in N eurosmra (Collins
et aL 1981). These are true mitochondrial plasmids since they are not derived from the
standard mtDNA. Three different circular plasmids, termed Fiji, LaBelle and Mauriceville,
are found in different strains of Neurosmra and they belong to three different homology
groups.

Mauriceville plasmid contains a long ORF and encodes a hydrophilic polypeptide of
710 amino acids or one or more smaller polypeptides beginning at internal AUG codons.
These polypeptides show homology to the proteins encoded by group II introns. The long
ORF of the plasmid uses distinctive codons that are characteristic of ORFs in introns. These
characteristics suggest that this plasmid is related to introns found in mtDN A. Others suggest
that Mauriceville is related to transposable elements. Michael and Lang (1985) showed that
proteins encoded by the Mauriceville plasmid contain short blocks of conserved amino acids
that are characteristic of reverse transcriptase and related proteins. Characteristics of the
plasmid suggest that it may belong to a class of mobile elements that are related to introns
of mtDNA.

Unlike Mauriceville, LaBelle plasmid does not produce any discrete transcript. The
ORF of LaBelle has no homology to reverse transcriptase. These findings suggest that
LaBelle plasmids belong to a different class of intron related genetic elements than does the
Mauriceville plasmid.

Several field isolates of N eurospgra from Hawaii and India contain linear
mitochondrial plasmids termed k_alil_o and maranhar respectively (Bertrand et al. 1989; Court
et al.1991). These linear plasmids induce senescence and are absent in long-lived strains. The

kalilo plasmid is about 9 kb long, whereas the maranhar plasmid has a length of 7.2 kb

9
(Bertrand and Griffith, 1989). Both these plasmids are present in high copy numbers in

presenescent, juvenile as well as the senescent hyphae of the senescence-prone strains. LaLilg
and maranhar plasmids have dissimilar nucleotide sequences, but they have very similar overall
characteristics. Both plasmids have covalently-linked 5’-terminal proteins (V ierula et al 1990)
and long inverted terminal repeats. DNA of both plasmids contain two long open reading
frames (Bertrand and Griffith, 1989), one coding for an RNA polymerase and the other
coding for a DNA polymerase. The two open reading frames are located on two opposite
strands of the DNA in both the plasmids. These plasmids cause senescence by inserting into
the mitochondrial genome which leads to the progressive accumulation of mutant forms of

mtDNA. This ultimately results in respiratory deficiencies.

Mitochondrial plasmids of Podosmra M

During senescence in Podospgra, specific regions of the mitochondrial genome excise
and replicate as autonomous plasmids. One plasmid, termed a senDNA is found more
frequently but others are also observed (B, e and 0 senDN A). In senescing cultures, the 94
kb mitochondrial DNA was shown to be replaced by multimeric sets of small circular DNA
(Cummings, 1979). These small circular molecules consist of head-to-tail tandem arrays of
specific regions of the mitochondrial genome (J amet-Vierney et al.1980). a senDNA is 2.6
kb long and the DNA sequence analysis shows that this 2.6 kb fragment is a complete group
II intron of the cytochrome oxidase subunit 1 (C01) gene (Cummings and Wright 1983).
Excision of a senDNA occurs precisely at the exon-intron junction within the mosaic gene
encoding COI. Like other group II introns, a senDNA codes for amino acids sequences that
have significant homology with retroviral reverse transcriptase (Matsura et al 1986). B
senDNA and e senDNA are 9.8 kb and 5.5 kb long, respectively. No introns have been found

in B senDNA whereas in 0 senDNA three group I introns have been found.

10

Long-lived mutants in Podosmra are defined as strains capable of growth renewal
after one or more senescent stoppages. (Cummings, 1986). These mutants lack the region
corresponding to a senDNA. Belcour and Vierney (1986) sequenced across the deleted
regions of a senDNA and found that a senDNA was not precisely excised. Long lived
mutants reveal the presence of a novel family of small, autonomously replicating elements
termed th-l, s-Mt2 and s-Mt3. DNA sequence analysis shows that s-Mtl is 368 bp, s-Mt2
is 114 bp and s-Mt3 is 67 bp. The s-Mt2 and s-Mt3 elements overlap and are completely
contained within the s-Mtl element. In early stages of growth, several of these long-lived
strains contain a senDNA but none of the th plasmid. Later in the vegetative growth the
th elements are observed. Gene rearrangement of the mitochondrial genome is seen as soon
as the th plasmids appear. It is believed that the senDNA and the th elements play an

antagonistic role in the determination of life span.

"Ranged" mutants of Aspgggiflus
Ragged mutants of Asmrgillus contain head-to-tail repeats of plasmid like sequences

which arise from different areas of the mtDNA. In one mutant strain (Rgd 1), the amplified
mtDNA segment (rgd 1 DNA, monomeric length 0.9kb) maps downstream of the large
subunit ribosomal gene whereas in other strains (Rgd 3), the amplified sequence (rgd 3-7
DNA) are located between genes coding for cytochrome b and ATPase subunit 6. These
ragged mutants display a number of physiological defects, including cytochrome abnormalities

and high levels of cyanide-insensitive respiration (Lazarus and Kuntzel, 1981).

11
Mitochondrial plasmids of Clavicem purpurea

Clavicep_§ purpurea is a fungal phytopathogen of cereals and nonagricultural grasses.
Mitochondrial plasmids were discovered and characterized in strain K1 from Germany and
other strains. There are three main mitochondrial plasmids in Claviceps. They are termed
pClKl, pC1K2 and pC1K3 and are 6.7, 5.5 and 1.1 kb, respectively. The smaller plasmids are
homologous to the larger plasmid. There is no homology between the plasmids and mtDNA.
These plasmids are linear and contain terminal inverted repeats (TIRs). In at least two strains,
plasmid-homologous segments can be found in the mtDNA. This is thought to be the result
of integration events like those found in Neurosmra with the kalil_o and maranhar plasmids
(Tudzynski and Esser 1986; Bertrand and Griffiths, 1989). In Q. purpurea, there appears to

be no deleterious effects on fungal metabolism of plasmid integration.

Mitochondrial Plasmids in Cochliobolus hetemstrophus

Cochliobolus heterostrophus, a fungal phytopathogen of maize, has two mitochondrial
plasmids called T40 and T21. They are both circular and exist as a series of head-to-tail
multimers. The mitochondrial genome is not noticeably affected by the presence of the
plasmid and the plasmid containing strain does not exhibit any distinct phenotype. Plasmids
T40 and T21 have the monomenic sizes of 1.8 and 2.2 kb, respectively. They share 1.5 kb in
common. Portions of both plasmids show high homology with the mitochondrial ATPase
subunit 6 gene. The gene is present in its entirety on the T40 plasmid, whereas the first 130
nucleotides at the 5’ end of the gene are missing from the T21 plasmid, suggesting that the
presence of an intact copy of ATPase submit 6 gene in the plasmid is not required either for

the maintenance of the plasmid or the growth of strain T21.

12
Alternative oxidase pathway: cyanide-resistant respiration

It is apparent that the aberrant growth phenomena encountered among some
filamentous fungi may be associated with grossly defective mtDNA. Physiological defects
leading to abnormal growth of the fungus in culture appears to be the result of cytochrome
abnormalities which are associated with high levels of cyanide-resistant respiration and
defective mtDN A.

The presence of an alternate, cyanide-resistant pathway of mitochondrial respiration
has been reported in filamentous fungi, yeast, green algae, and higher plants. The alternate
oxidase pathway is of great interest in that energy is not generated in the form of ATP when
the electrons flow through it. The potential energy of the system is lost as heat. The electron
transport in these mitochondria is branched, with a classical cyanide-sensitive cytochrome
pathway that terminates with cytochrome oxidase and a hydroxamic acid-sensitive pathway
that terminates with an alternative oxidase. The alternative pathway was shown to branch
from the cytochrome chain at the ubiquinone pool (Storey, 1976). In both higher plants and
Neurosmra mitochondria, a remarkable feature of the branch point is its ability to regulate
electron flow so that the cytochrome system is used maximally and the alternate oxidase is
used only to accommodate surplus electron flow (Lambowitz et al.1972, Edwards et al. 1974).
Direct measurement of phosphate esterification in Neurosmra shows that the alternate
oxidase does not result in oxidative phosphorylation (Lambowitz et al. 1972), a feature
common with alternative pathways of higher plants and yeast mitochondria (Henry and Nyns,
1975).

M! (Mi-1) is perhaps the best known of the cytochrome deficient mutants of
N eurosmra. In wild type strains, more than 90% of total respiration is mediated by a

conventional cytochrome chain (sensitive to antimycin and cyanide). Poly mutants use both

13

the cytochrome chain and an alternate oxidase (resistant to antimycin and cyanide, but
sensitive to salicyl hydroxamic acid). The alternate oxidase pathway accounts for as much as
70% of the respiration in My mitochondria.

A comparative spectrophotometric study of the electron transport system in
mitochondria from wild-type and My strains of Neurosmra showed striking differences in
the concentrations of electron carriers. m mitochondria contain flavoproteins and
cytochrome c at concentration that are 30% higher than that of wild type mitochondria, but
My mitochondria are grossly deficient in b and a type cytochromes. The increased
concentration of fiavoproteins in pglgy mitochondria led to speculation that the alternate
oxidase might be a flavoprotein (Lambowitz et al. 1972).

In wild-type N eurosmra, alternate oxidase is not present constitutively but can be
induced by a variety of treatments that share the capacity to impair the cytochrome system.
The agents include antimycin, cyanide, oligomycin, CCCP, chloramphenicol (a specific
inhibitor of mitochondrial protein synthesis) and ethidium bromide (an inhibitor of
mitochondrial DNA transcription). Using actinomycin D, an inhibitor of nuclear transcription,
Edwards et al. (1973) reported that wild-type Neurospgra strains require de novo expression
of nuclear genes. These data are compatible with the model proposed by Barath and Kuntzel
(1972) for the regulation of nuclear genes specifying the mitochondrial genetic apparatus of
Neurosmra. This model proposes the synthesis of mitochondrially coded repressor proteins
on mitochondrial ribosomes. These proteins are then transported to the nuclear genes that
they repress. As long as mitochondrial protein synthesis continues to function normally,
repressor proteins are synthesized and the nuclear genes are repressed. Treatment with
chloramphenicol results in inhibition of mitochondrial protein synthesis with the resultant

depression of the repressed genes.

14

Edwards and co-workers studied six antimycin-sensitive (ANT) mutants which could
not synthesize functional alternate oxidase and found them to fall into two complementation
groups, alLl and E2 (Edwards et al. 1976). Bertrand et al. (1983) have characterized 27
antimycin-sensitive mutants of Neurosmra which are unable to induce alternate oxidase
activity. These mutants fall into two complementation groups, M and 99$; In response
to the different inducing conditions, the wild type strain and 19 out of 20 aod-l mutants were
found to accumulate a polypeptide that was tentatively identified as being associated with
alternate oxidase. The remaining M mutants and all four aod-2 mutants that were tested
did not accumulate this polypeptide. Based on those findings, Bertrand et a] (1983)
hypothesized that aod-l is the structural gene for alternate oxidase and that the aod-2 gene
encodes a component that is required for induction of alternate oxidase activity.

Elthon and McIntosh (1987) have raised polyclonal antibodies to the solubilized and
partially purified alternate oxidase from the thermogenic Voodoo lily, Sauratum guttatum, the
antiserum cross reacts with 37, 36 and 35 KD polypeptides. Monoclonal antibodies have now
have been prepared to these proteins and designated as AOA (binds to all three proteins of
the alternate oxidase cluster), AOU (binds to 37 KD protein) and AOL (binds to the lower
36 and 37 KD proteins). These polypeptides appear to be remarkably conserved and the
antibodies cross-react with similar proteins in nonthermogenic plant species and also
Neurosmra which makes these antibodies useful in identifying alternate oxidase proteins in
other systems.

Using the monoclonal antibody (AOA) and two alternate oxidase mutants (Led-_lgoi

2) described above, Lambowitz et al.( 1989) found that aod-l mutants produced inactive

 

polypeptides whereas aod-2 mutants gene failed to produce these polypeptides. These findings

 

support the previous hypothesis of Bertrand (1983) that aod-l is a structural gene for the

 

15

alternate oxidase and that the aod-2 gene encodes a component that is required for induction

 

of alternate oxidase activity. This work also shows that the induction of alternate oxidase in
Neurosmra results hour the synthesis of alternate oxidase polypeptides and not from the
activation of preexisting polypeptides.

The physiological role of alternate oxidase is best understood in thermogenic higher
plants, most of which are members of the m. In the flowers or inflorescence of these
plants, heat produced by way of electron flow through the alternate oxidase pathway is often
used to volatilize insect attractants facilitating pollination. In Neurosmra, cells that
predominantly use this pathway, (fly, and wild type grown in the presence of antimycin)
have conspicuously slow growth rates, which seem to be related to the residual activity of the
cytochrome system rather than to the level of the alternate oxidase (Lambowitz and Slayman.
1972). For most plants and fungi, the physiological role of alternate oxidase is obscure. One
hypothesis is that it serves as a method to rid the cells of excess carbohydrates (Lambowitz
et al. 1978, Lambers. 1982). Another possibility is that the alternate oxidase defends the
fungus or plant against respiratory inhibitors produced by competing organisms in the
environment (Lambowitz and Zannoni, 1978). The hypothesis is suggested by studies of the
fungus Stremphylium m (Rissler and Miller,1977), which as a pathogen of cyanogenic pants
is likely to encounter cyanide in its natural environment. Rissler and Miller noted that
induction of alternate oxidase in this organism following the addition of cyanide is correlated
with the induction of formamide hydrolase activity. This enzyme converts cyanide to nontoxic
formamide and is thought to be partially responsible for the tolerance of §. lg; to high
concentrations of cyanide in vitro. Fungi, bacteria and higher plants have the ability to
produce antibiotics and toxins. The presence of an insensitive alternate oxidase pathway may

attribute to an important selective advantage in such an environment.

16

My work in this thesis involves several recently discovered hypovirulent isolates of
Q. parasitica that do not contain detectable amounts of dsRNA. Genetic studies with one of
these dsRNA-free hypovirulent strains, C125, from Crystal Lake, MI. suggests that the
factor(s) responsible for the dsRNA-free hypovirulence phenotype is cytoplasmic in nature.
In this thesis I have asked the following questions to help understand the basis of this novel
form of hypovirulence.
"' Are plasmids involved in the hypovirulence of Q. parasitica?
* Are mitochondria associated with the dsRNA-free hypovirulence phenotype and if so, can
they be transferred to other virulent strains via hyphal fusion?

"‘ Are respiratory defects partially or totally responsible for hypovirulence of Q. parasitica?

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19

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20

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21

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selectively amplified during senescence in Podosp_(_)ra anserina? Curr. Genet. 7:45

CHAPTER-l

CRYPTIC MITOCHONDRIAL PLASMIDS IN

CRYPHONECI‘RIA PARASITICA.

ABSTRACT

Hypovirulent strains of Cryphonectria parasitica have been associated with the
presence of cytoplasmically transmissible double-stranded RNA (dsRNA). In Michigan, several
hypovirulent strains of Q. parasitica have been discovered that do not harbor detectable levels
of dsRNA. Genetic studies with a dsRNA-free hypovirulent isolate, CL25, suggests that there
is a transmissible, maternally inherited cytoplasmic ”virulence inhibition factor” other than
dsRNA that is responsible for the hypovirulence phenotype of these dsRNA-flee strains. A
study of cytoplasmic agents revealed the presence of two low copy number plasmids in the
mitochondria. These plasmids are about 6 and 10 kilobase pairs (kb) in size. Hybridization
studies with a cloned fragment of the 6 kb plasmid, indicated the presence of this plasmid in
all virulent and hypovirulent Q. parasitica strains screened regardless of geographical origin.
The cloned plasmid DNA also hybridized to plasmid-like elements of a different size in
Endothia gm, This result suggests that the 6 kb plasmid may not play a direct role in the
hypovirulence phenotype of CL25 and other strains. The cloned fragment of the plasmid does

not hybridize with the mitochondrial genome, and the function of the plasmid remains cryptic.

23
INTRODUCTION

The fungal phytopathogen Cgphonectria (=EMQ) mam has eliminated the
American chestnut |Castanea (_l_e_r;ta_ta (Marsh) Borkh] as a major tree species in the Eastern
deciduous forests of North America. Chestnut blight also invaded European chestnut forests
and orchards (Castanea saliva Mill.) and caused considerable damage. It is thought that this
fungus was introduced into North America on nursery stock of oriental chestnuts and the
fungus spread very quickly to the susceptible American chestnut trees. Hypovirulent strains
have been isolated fi'om recovering chestnut trees in Europe and North America where
chestnut trees survive infection. Hypovirulent strains are reduced in virulence, produce fewer
conidia and exhibit unusual culture morphology. Hypovirulence in Q. parasitica can be
transferred to other virulent strains via hyphal anastomosis and has been correlated with the
presence of dsRNA.

One fungal strain from Crystal Lake, Michigan, has many characteristies of dsRNA-
associated hypovirulence but harbors no detectable dsRNA. Genetic studies with CL25 reveal
that this type of hypovirulence is carried by 20% of the conidia (asexual spores), is maternally
inherited, and is cytoplasmically transferred to other virulent strains via hyphal anastomosis
(Fulbright, 1985). This suggests that there is a ”virulence inhibition factor" (VIF) other than
dsRNA responsible for the dsRNA-flee hypovirulent phenotype of CL25.

Few studies have made comparisons between the hypovirulence phenotype and
senescence, a phenomenon encountered in filamentous fungi where cellular death occurs after
prolonged asexual propagation (Bertrand, 1983). The symptoms of senescence include female
sterility, sterility of conidia, abnormal growth morphology and respiratory deficiencies.
Senescence is exhibited by several filamentous fungi such as "stopper” mutants of N. gr_a_ss_a,

where the fungus has an unusual "stop-start" growth phenotype on solid media (Bertrand et

24

al. 1980); Asmrgillus, which exhibits a characteristic ”ragged" appearance due to vegetative
death of the hyphal tips (Lazarus et al, 1980); and, Podosmra, where vegetative death of
hyphal tips occur (Smith and Rubenstein, 1973). In all cases of senescence described above,
there is evidence of the involvement of cytoplasmic factors within the mitochondria.
Mitochondria of senescing fungi invariably accumulate a diversity of plasmids which are
usually deletions of large segments of mtDNA resulting from intramolecular recombination
as seen in "stopper” mutants of N. grass; (Bertrand, 1983; Gross et al. 1984) and the ragged
mutants of Aspgrgjllus (Lazarus and Kuntzel, 1981). In _P. m plasmids result from
excision of small fragments of mtDNA rather than intramolecular recombination (Cummings,
1979). Based on the association of mitochondrial plasmids with senescence and similar
features shared by both hypovirulence and senescence, I decided to examine the possible
involvement of mitochondrial plasmids in the hypovirulence phenotype of Q. parasitig
Here, I present evidence of the existence of at least two plasmids in the mitochondria
and show that at least one of these elements may not be directly associated with

hypovirulence of Q. parasitica.

MATERIALS AND METHODS
Fungal strains and growth conditions
Cryphonectria parasitica strains used in this study are listed in Table-1.1. Endothia
mg and Colletotrichum lagenarium were used as controls (Table-1.1). Cultures of Q.
parasitica were maintained on potato dextrose agar (PDA, Difco) at 20°C under fluorescent
light. Cultures used for mitochondrial DNA isolation were grown for 10-14 days in the liquid

medium of Puhalla and Anagnostakis (1971) modified by the omission of glucose.

TABLE 1.1- Q. grasitica strains used in the plasmid screening study.

 

 

Strain Virulence Description

CL25 HV Isolated from a non lethal canker from Crystal Lake,
MI.; no dsRNA.

CL1-16 V Single spore isolate of a virulent strain from Crystal
Lake, MI.

EP155 V Virulent strain from Connecticut.

GHU4 HV A dsRNA containing hypovirulent strain from Grand
Haven, MI.

TR48 HV Strain from Italy Europe; dsRNA present.

NCF-B V Strain from Accokeek, MD.

JG HV Strain from Judy Gap, WV, dsRNA present.

 

HV= Hypovirulent, V= Virulent.

*Colletrichum lagenarium and Endothia gygosa cultures were kindly donated to us by Dr.
Alice Bonnen and Mark Double, respectively.

Mitochondrial DNA Isolation

Mitochondrial DNA was isolated following the procedure of Mackenzie et al (1988)
with several modifications. Twelve-day-old cultures of Q. parasitiga were strained through
Miracloth (Calbiochem) and pressed dry. Twenty to twenty-five grams (pressed weight) of
fungus was ground in liquid nitrogen with glass beads using a chilled mortar and pestle.
Ground mycelia were stirred in 400 mls of cold buffer A (0.5M sucrose, 0.05M Tris, 0.005M
EDTA, 0.1% BSA, pH 7.5 and 0.005M mercaptoethanol added just before use) for 10 min.
The homogenate was filtered through two layers of Miracloth and centrifuged for 15 min at
1,000Xg to pellet nuclei. The resulting supernatant was centrifuged at 15,900Xg for 20 min.
The pellet was resuspended with a paint brush in buffer G (0.03M sucrose, 0.05M Tris, pH
7.5) using 10 ml per 20 g starting material. After centrifuging for 10 min at 1,000Xg, the
supernatant was brought to 10 mM MgClz and 20 pg DNase (Sigma, Type IV)/ml or 200
Kunitz units/ml, and incubated with gentle mixing at 4°C for 60 min. The suspension was
underlaid with 20 ml shelf buffer (0.6M sucrose, 0.01M Tris, 0.02M NazEDTA, pH 7.5) per
5 ml and centrifuged for 25 min at 12,000Xg. The pellet was resuspended with the shelf
buffer (5 ml) and the mitochondria were pelleted by centrifuging for another 25 min at
15,900Xg. The mitochondria were then lysed in 5 ml lysis buffer (0.01M Tris, 0.05M EDTA,
0.1M Nacl, 1% SDS, pH 8.0)l20-25g of fungus and 100 pg proteinase K/ml at 65°C for 30
min. The protein-carbohydrate complex was precipitated by adding one third the volume 5M
potassium-acetate, incubating on ice for one hour with periodic shaking, and centrifuging for
20 min at 25,000Xg. The supernatant was poured into a siliconized corex tube and the
mtDNA precipitated with 1/20 volume of 5M ammonium acetate and 1/2 volume of ice cold
isopropanol at -20° C.

The mtDNA was pelleted by centrifuging at 10,000Xg for 45 min. The pellets were

air dried, dissolved in 0.7 ml T50E10 buffer (50mM Tris, 10mM EDTA, pH 8.0). The solution

27

was transferred to a microfuge tube and microfuged for 10 min. The supernatant was
transferred to a clean tube and the mtDNA was precipitated as above with ammonium acetate
and isopropanol. The mtDNA was washed twice with 70% ethanol, vacuum dried for 10-15

min, and allowed to dissolve in TE (10mM Tris, lmM EDTA, pH 8.0) overnight at 4° C.

Electrophoresis of mitochondrial DNA

The mtDNA was electrophoresed in 0.7% agarose gels using TBE buffer (Maniatis
et al, 1982). Molecular size markers were obtained by digesting Lambda DNA with @111
Gels were electrophoresed at room temperature for 8 hours at a constant voltage of 50-60
volts. The gels were stained with ethidium bromide (0.5 rig/ml) for 15-20 min and
photographed. The mtDNA in the gel was transferred to nylon hybridization membranes
(MSI, Westboro, MA.) as described by Maniatis et al (1982). The DNA was fixed on the

membrane by UV crosslinking for 3-5 min.

Isolation and cloning of the plasmids

Mitochondrial DNA of CL25 was electrophoresed on an agarose gel under the same
conditions as described above. After staining the gel with ethidium bromide, the agarose
containing the two plasmid bands was cut out fi'om several lanes and the DNA was
electroeluted with an electroeluter using the procedure suggested by the manufacturer
(Schleicher and Schuell). The buffer with the electroeluted DNA was precipitated overnight
with 2 volumes of absolute EtOH and 1/10 volume of 3 M NaOAc at 0°C. The DNA was
collected by spinning at 15K for 50 min.

The electroeluted DNA was digested with restriction enzyme Bglll and was cloned

into the BamHI site of the polylinker of cloning vector pRL498 (Elhai and Wolk, 1988). The

28
boiling lysis miniprep method described by Maniatis et al (1982) was used to identify and

prepare the resulting recombinant plasmid, pNK40.

DNA labelling and hybridization

The recombinant plasmid was digested with restriction enzyme H_irLdIII and
electrophoresed in an agarose gel. The insert was cut out of the gel, electroeluted and used
for probes after radiolabelling. Probes were radiolabelled using the random primer method

of Feinberg and Volgenstein (1983).

RESULTS AND DISCUSSION
The mitochondria of filamentous fungi contain a wide range of plasmids and plasmid-
like elements. These plasmids show many different characteristics in a wide range of fungal
species. Many of these plasmids do not have a phenotype or a physiological role associated
with them. Examples of these are the Mauriceville, Fiji and LaBelle plasmids of Neurosmra
(Collins et al, 1981); T40, T21 of Cochliobolus (Garber et al,1986); and pClKl, pC1K2 and
pC1K3 of Clavicem purpurea (Tudzynski et al, 1986). Other mitochondrial plasmids affect

the mitochondrial genome, as seen with kalilo and maranhar plasmids of Neurosmra

 

(Bertrand et al. 1986, Court et al. 1991). The plasmids of Podospgra min; are believed
to be derived from the mtDNA by deletion and amplification. In both events, the
mitochondria become progressively deficient in cytochrome M3 and b, and begin to
accumulate excess cytochrome c and flavoprotein (Lambowitz et al. 1972, Tissiers et al. 1953,
Lambowitz and Zannoni. 1978, Bertrand et al. 1976). It is thought that these changes lead
to senescence in those fungal strains.

When undigested mtDNA of Q. parasitica is electrophoresed in an agarose gel, two

DNA bands of approximately 6 and 10 kb are seen in addition to the mitochondrial genome

29

(Fig 1.1). These plasmids are present in low copy number and are often difficult to visualize
on ethidium bromide stained gels. Restriction digest of these plasmids after electroelution
from the agarose gel showed that the 10 kb plasmid band can be digested with restriction
enzyme le whereas the 6 kb plasmid cannot be digested with _EC_QR1 (Fig 1.2). These
results indicated that the two plasmids are in fact two different plasmids and not different
forms of the same plasmid. This was again confirmed with DNA hybridization studies (Fig
1.3). A 2.5 kb fragment clone, pNK40, of the 6 kb plasmid was used to screen several
virulent and hypovirulent strains from different geographical locations. This clone hybridized
to all the Q. parasitica strains assayed. The clone pNK40 frequently hybridized to three
different bands which suggested that the 6 kb plasmid may be a circular plasmid and that the
three bands corresponded to different forms of the same plasmid. There was also
hybridization to larger DNA bands which are probably multimeric forms of the plasmid. This
clone did not hybridize to the 10 kb plasmid, substantiating the earlier interpretation that
these plasmids are different. Hybridization of pNK40 also occurred to plasmid-like elements
in Endothia M (Fig 1.3-B, lane 9). This is a species that is believed to be related to Q.
parasitica (previously known as Endothia parasitica). Not only was the plasmid-like band in
E. gym of a different size, but the _E_Z. ma strain also had a different mitochondrial DNA
banding pattern (data not shown). Mitochondrial DNA of Colletotrichum lagenarium was
used as a negative control (Fig 1.3-B, lane 8).

Since the 6 kb plasmid is present in all hypovirulent and virulent strains screened, the
direct involvement of this plasmid in hypovirulence of Q. parasitica is doubtful. An indirect
role of the 6 and 10 kb plasmids in the hypovirulence phenotype can not yet be ruled out as

they might be involved in mtDNA aberration.

30

 

 

 

Fig 1.1- Intact mtDNA of isogenic strains of Q. parasitica.

Lane 1- CL25#9 (Hypovirulent)

Lane 2- CL25#4 (virulent)

M- A DNA digested with MIR used as molecular size marker. The sizes of the bands from
top to bottom are 23.13, 9.41, 6.55, 4.36, 2.32, 2.02 kilo bases, respectively.

31

 

Fig 1.2- Mitochondrial plasmids digested with different enzymes.

Lane 1- _BglII, 2- B_amH1, 3- _I__Iin_dIII, 4- SASA, 5- 32R].

M- 1. DNA digested with H_in_dIII used as molecular size marker. The sizes of the bands from
top to bottom are 23.13, 9.41, 6.55, 4.36, 2.32, 2.02 kilo bases, respectively.

32

A B
M123456789 123456789

23.13

9.41
. 6.55

4.36

2.32
2.02

 

Fig 1.3- Intact mtDNA of different isolates of Q. parasitica, Endothia mesa and
Colletotrichum lagenarium.

Lane 1- GHU4, 2- JG, 3- TR48, 4- NCF-B, 5- EP155, 6— CL1-16, 7- CL25,

8- Q. lagenarium, 9- E. mesa.

A- Ethidium bromide stained gel, M- A DNA digested with HindIII used as molecular size
marker.

B- Southern blot hybridized with a clone if a fragment of 6kb plasmid.

 

33

We did not observe the accumulation of a great diversity of plasmids in the dsRNA-
free hypovirulence strains as has been observed in senescing strains of other fungal species.
However, one strain of Q. parasitica, NCF, showed the presence of unique very abundant
DNA band other than the mitochondrial genome and the 6 and 10 kb plasmids observed in
other strains (Fig 1.3-A, lane 4).

The 6 kb plasmid does not have any homology with the mitochondrial genome.
Therefore it can be categorized as a ”true mitochondrial plasmid", as defined by Lambowitz
et al (1986). Like many other mitochondrial plasmids in other fungal species, no function has

been attributed to this plasmid in Q. parasitica.

34
REFERENCES

Bertrand H., N. A. Szakacs, N. A., Nargang, F. E., Zagozeski, C. A., Collins, R. A. and
Harrigan, J. C. (1976). The function of mitochondrial genes in Neurosmra crassa.
Can. J. Genet. Cytol. 18. 397-409.

Bertrand H., Collins R.A., Stohl L.L., Goewert RR. and Lambowitz AM. (1980). Deletion
mutants of Neurosmra crassa mitochondrial DNA and their relationship to the ”stop-
start" growth phenotype. Proc. Natl. Acad. Sci., USA 77 :6032-6036.

 

Bertrand H (1986). The kalilo senescence factor of Neurosmra intermedia. In: Wickner R.B.,
Hinnebusch A., Lambowitz A.M., Gunsalus LC. and Hollaender A. (eds)
Extrachromosomal elements in lower Eykaryotes, Basic life science, vol. 40. Plenum
press, New York London, 93-103.

Bertrand H. (1983). Aging and senescence in fungi. In, Intervening in the aging process,
Edited by W. Rogelson and F. M. Sinex. Alan R. Liss, New York. 233-251.

Collins R.A., Stohl L.L., Cole MD. and Lambowitz AM. (1981). Characterization of a novel
plasmid DNA found in mitochondria of Neurosmra crassa. Cell 24:443-452.

Court D.A., Griffiths A.J.F., Kraus S.R., Russell P]. and Bertrand H. (1991). A new
senescence-inducing mitochondrial linear plasmid in field-isolated Neurosmra crassa
strains from India. Curr. Genet. 19: 129-137.

Cummings DJ ., Belcour I... and Grandchamp C. (1979). Mitochondrial DNA from Podosmra
anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent
cultures. Mol. Gen. Genet. 171:239-250.

Elhai J. and Wolk, CP. (1988). A versatile class of positive-selection vectors based on the
nonviability of palindrome-containing plasmid that allows cloning into long polylinkers.
Gene 68: 119-138.

Feinberg and Volgenstein. (1983) Radiolabelling by random priming method. Analytical
Biochemistry. 132:6-13.

Fulbrigt, D.W. 1985. A cytoplasmic hypovirulent strain of Endothia pagasitica without
double-stranded RNA (dsRNA). (Abstr.) Phytopathology 75:1328.

Garber, R. C., Lin, J. J. and Yoder O. C.(1986). Mitochondrial plasmids of in Cochliobolus
heterostrophus. In, Extrachromosomal elements in lower eukaryotes. (Wikner, R.B.,
A. Hinnebusch, A.M. Lambowitz, I.C. Gunsalus and A. Hollaender, editors) 105-118.

Gross, S. R., Hsieh, T., and Levine, P. H. (1984). Intramolecular recombination as a source
of mitochondrial chromosome heteromorphism in Neurosmra. Cell, 38: 233-239.

Lambowitz A.M., Slayman CW. and Slayman CL. (1972) The electron transport components
of wild type and fly strains of Neurospgra crassa. J. Bacteriol. 247:1536-1547.

 

35

Lambowitz AM. and Zannoni D. (1978). Cyanide-Insensitive Respiration in Neurosmra.
Genetic and Biophysical Approaches. Elsevier/North-Holland Biomedical Press Plant
Mitochondria.

Lambowitz, A.M., Akins, R.A., Kelly, R.L., Pande, S. and Nargang, RE. (1986). Mitochondrial
plasmids of Neurospgra and other filamentous fungi. In, Extrachromosomal elements
in lower eukaryotes. (Wikner, RE, A. Hinnebusch, A.M. Lambowitz, I.C. Gunsalus
and A. Hollaender, editors) 83-92.

Lazarus, C. M., Earl A. J., Turner, G. and Kuntzel, H. (1980) Amplification of a
mitochondrial DNA sequence in the cytoplasmically inherited "ragged” mutant of
Asmrgillus amstelodami.

Lazarus, C. M., and Kuntzel, H. 1981. Anatomy of amplified mitochondria in "ragged"
mutants of Asxrgillus amstelodami. Excision points within protein genes and a

common 215 bp segment containing a possible origin of replication. Curr. Genet. 4:
99-107.

Mackenzie, S.A., Pring, D.R., Bassett, MJ., Chase, CD. 1988. Mitochondrial DNA
rearrangement associated with fertility restoration and cytoplasmic reversion to fertility

in cytoplasmic male sterility in cytoplasmic male sterile Phaseolus vulgaris L. Proc.
Natl. Acad. Sci. USA 85:2714-2717.

Maniatis, T., Fritsch, ER, and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor. NY.

Puhalla, J .E. and Anagnostakis, S.L.(1971).Genetic and nutritional requirements of Endothia
parasitica. Phytopathology 61. 169-173.

Smith, J. R., Rubenstein, I. (1973). Cytoplasmic inheritance of the timing of "senescence" in
Podospgra anserina. J. Gen. Microbiol. 76, 297-304.

Tissiers, A., Mitchell H.K. and Haskins EA. (1953) Studies on the respiratory system of the
fly strain of Neurosp_ora crassa. J. Biol. Chem. 205:423- 433.

Tudzynski, P., Duvell, A. and Oeser B.(1986). Linear plasmids in the plant pathogenic fungus
Clavicefi purpurea. In, Extrachromosomal elements in lower eukaryotes. (Wikner,
RE, A. Hinnebusch, A.M. Lambowitz, I.C. Gunsalus and A. Hollaender, editors).

TRANSFER OF MITOCHONDRIA DURING HYPHAL ANASTOMOSIS

IN CRYPHONECI‘RIA PARASITICA.

ABSTRACT

Hypovirulent strains of the chestnut blight fungal phytopathogen, Cryphonectria
parasitica have been shown to be associated with the presence of transmissible double-
stranded RNA (dsRNA). Recently, hypovirulent strains have been isolated which do not
harbor detectable dsRNA. The dsRNA-free hypovirulence phenotype is maternally inherited
and transmissible to other Q. parasitica strains during hyphal fusion indicating that the
dsRNA-flee hypovirulence phenotype is cytoplasmic in nature. Therefore, the involvement
of mitochondria in dsRNA-free hypovirulence was investigated. To determine whether or not
mitochondria can transfer during hyphal anastomosis, both genetic and molecular
mitochondrial markers were constructed. First, a chloramphenicol resistant variant was
isolated from the dsRNA-free hypovirulent strain CL25. The resistance phenotype was shown
to be maternally inherited, indicating that the resistance was conferred by a mitochondrial
gene. Secondly, a restriction fragment length polymorphism of 2.5 kb was found that was
unique to CL25 mitochondrial DNA (mtDNA). The naturally occurring light pigmentation
phenotype of CL25 was used as a nuclear genetic marker. Vegetative pairings were made

between the light pigmented, chloramphenicol-resistant hypovirulent strain of CL25 and the

36

37

virulent, orange pigmented, chloramphenicol-sensitive strain, 4-C. After vegetative fusion, an
orange pigmented subculture removed from the 4-C side of the pairing was shown to be
chloramphenicol resistant and it contained the mitochondrial RFLP of both strains. The
subculture containing mtDNA from both 4-C and C125 produced only single-conidial isolates
with orange pigmentation containing either one or the other mtDNA, indicating that the
subculture was not contaminated by a hyphae or nuclei from CL25. In this chapter, I report
on the use of nuclear and mitochondrial markers to provide evidence that mitochondria can
transfer between strains of Q. parasitica during hyphal anastomosis. This finding links the

dsRNA-free hypovirulence phenotype of Q. parasitica with its mitochondrial genome.

INTRODUCTION

Cgphonectria (= Endothia) parasitica (Murr.)Barr, the causal agent of chestnut blight
was responsible for the demise of the once prevalent American chestnut (Castanea m
[Marsh] Borkh) in North America and European chestnut (Q. sa_ti_va Mill) in Europe. Isolates
with reduced virulence (hypovirulent) have been found in Europe and North America where
chestnut trees survive infection. Well-characterized hypovirulent strains of Q. parasitica have
been found to be infected with cytoplasmically transmissible double-stranded RNA (dsRNA)
molecules which appear to be correlated with changes in virulence, growth rate, sporulation
and/or culture morphology.

One strain of Q. parasitica, CL25 from northern (Crystal Lake) Michigan, has all the
characteristics of dsRNA-associated hypovirulence but harbors no detectable dsRNA
(Fulbright, 1985). We have used 30 times the quantity of mycelium of CL25 for isolation of
dsRNA, and still have been unable to detect dsRNA in this strain (unpublished data).

Genetic studies of CL25 showed that hypovirulent and virulent phenotypes segregate in the

38

conidia of C125, with hypovirulence being found in only 20% of the conidia. Furthermore,
the hypovirulence phenotype is maternally-inherited and cytoplasmically-transferred via hyphal
anastomosis (Fulbright, 1985). These studies suggest that there is a cytoplasmic “virulence
inhibition factor” (VIF) other than dsRNA involved in the hypovirulence phenotype, since
dsRNA-associated hypovirulence has not been found to be maternally inherited. Therefore,
we initiated a study on the possible involvement of the mitochondrial genome in the dsRNA-
free hypovirulence phenotype. If mitochondria are involved in dsRNA-free hypovirulence,
mitochondria or genetic factors associated with mtDNA would have to transfer to other
strains, along with the hypovirulence trait.

Since hypovirulence is a cytoplasmically transmissible trait, hyphal fusion must occur
in order for the determinants for hypovirulence to be transferred (Anagnostakis, 1984). It has
been demonstrated that dsRNA can successfully transfer between the strains that are
vegetatively compatible and to some extent between vegetatively incompatible strains. The
ability of dsRNA to spread between strains of different vegetative compatibility (v_c) groups
depends on the number of ye loci at which the groups differ (Anagnostakis, 1984).
Information regarding the movement of mitochondria during hyphal fusion of compatible as
well as between incompatible strains is limited. Some reports indicate that mitochondria do
not move during hyphal fusion in filamentous fungi (Brasier, 1986; Casselton and Economou,
1985). Gobbi et al. (1990) reported the absence of mitochondrial transfer in Q. parasitig, but
pointed out that low levels of mitochondrial transmission could have evaded their detection.
These reports on apparent lack of mitochondrial transfer during vegetative fusion contrast
with the reports on mitochondrial transmission in other filamentous fungi. In the

Basidiomycete, Coprinus cinereus recombination of the mitochondrial genome has been

 

shown to occur in the region of hyphal anastomosis (Baptists-Ferreha et al, 1983). In

39

Schizophyllum commune, another Basidiomycete, transfer of mitochondria was visualized by
means of phase-contrast microscopy (Watrud and Ellingboe, 1973). Collins and Seville (1990)
have provided evidence for the transfer of the mitochondrial chromosome and plasmids during
unstable vegetative fusion in Neurosmra. In similar studies by Griffith et al (1990), the
transmission of a mitochondrial plasmid in a transient fusion between N. intermedia and N.
gr_a_ss_a_ cells was reported. Due to the conflicting reports about mtDNA transfer in fungi, we
decided to reexamine the question of mitochondrial transmission in Q. parasitica. Using
mitochondrial and nuclear markers, we present evidence that mitochondria in Q. parasitica

can transfer between strains during hyphal anastomosis.

MATERIALS AND METHODS

Fungal strains and growth conditions

Strains used in this study are listed in Table 2.1. CL25 is a bark isolate of Q. parasitica
from Crystal Lake, Michigan (Fulbright, 1985). This is a light pigmented hypovirulent isolate
without detectable amounts of dsRN A. Strain 4-C is a dark pigmented virulent isolate without
dsRNA. Single conidial isolates from these cultures consistently maintained the pigmentation
trait of the parent.

A chloramphenicol resistant strain was isolated using the procedure of Brasier and
Kirk (1986). A range of eight concentrations of chloramphenicol (2-10 mg/ml) in potato
dextrose agar (PDA) were inoculated with C125 to determine the inhibitory concentrations
of chloramphenicol suitable for selection of mutants. Marginal outgrowths from mycelium
growing on 8 mg chloramphenicol/ml were subcultured on PDA plates supplemented with 8

mg chloramphenicol/ml.

TABLE 2.1. Strains of thonectria Erasitica used in the study of mitochondrial transfer

 

 

during hyphal anastomosis.

Strain Virulence Description

C125 HV Isolated from a healing canker at Crys-
tal Lake, MI.

C125 capR HV A light pigmented chloramphenicol
resistant strain of C125.

4-C V An orange pigmented strain derived

from GHU4 by curing of dsRNA
(Fulbright, 1984).

 

HV= Hypovirulent, V= Virulent
CL25- a hypovirulent strain without any detectable amounts of dsRNA.
GHU4- a hypovirulent strain with dsRNA.

41
Cultures used in this study were maintained on PDA at 20°C under fluorescent light.

Cultures for mtDN A isolation were grown for 10—14 days in the liquid medium of Puhalla and
Anagnostakis (1971) modified by the omission of glucose. Liquid medium was amended by
the addition of 2-2.5 mg chloramphenicol/ml as appropriate. The chloramphenicol resistant

isolates were maintained on plates with 8 mg chloramphenicol/ml.

Sexual crosses

Sexual crosses were performed between fungal strains using the procedure of
Anagnostakis (1979). A chloramphenicol-resistant strain of CL25 and a chloramphenicol-
sensitive virulent strain were crossed by pouring 4% water agar around an autoclaved chestnut
twig in a petri dish (Difco). Mycelial plugs of the maternal strain were placed on the agar
surface on either side of chestnut wood and the fungal strain was allowed to grow on the
wood for about 20 days. When stromata formed on the bark, a conidial suspension of the
paternal parent, collected from a culture growing on PDA, was added to the plate and shaken
to cover the stroma. Perithecial necks were visible above the stromata 7-8 weeks later.
Perithecia were removed and aswspores collected and grown on PDA and PDA

supplemented with 8 mg chloramphenicol/ml.

Single-conidial isolation

To harvest conidia, 1 ml sterile distilled water was poured on the mycelial mat of 10-
day—old cultures grown on PDA. The plate was shaken gently to free the conidia from the
mat. The conidial suspension was collected in a sterile tube and 10-fold serial dilutions (up
to five) were made in distilled water. One hundred pl of each dilution was placed on PDA

plates and spread with a sterile glass rod. These plates were placed under fluorescent lights

42
for 1-2 days and germinating spores were visualized with a dissecting microscope. Single-
conidial isolates were obtained by transferring small pieces of agar with the germinating spore

to PDA plates.

Pathogenicity test

Fungal isolates were tested for virulence by placing 7 mm mycelial plugs from 10-day-
old PDA cultures into 9 x 7 mm-deep holes on the side of Golden Delicious apples
(Fulbright, 1984). The inoculation holes were covered with tape to prevent desiccation and

the apples were placed in open plastic bags for 3 weeks.

Mitochondrial DNA Isolation

Mitochondrial DNA was isolated following the procedure by Mackenzie et al (1988)
with several modifications. Twelve-day-old cultures of Q. parasitica were strained through
Miracloth (Calbiochem) and pressed dry. Twenty to twenty-five grams (pressed weight) of
fungus was ground in liquid nitrogen with glass beads using a chilled mortar and pestle.
Ground mycelia were stirred in 400 mls of cold buffer A (0.5M sucrose, 0.05M Tris, 0.005M
EDTA, 0.1% BSA, pH 7.5 and 0.005M mercaptoethanol added just before use) for 10 min.
The homogenate was filtered through two layers of Miracloth and centrifuged for 15 min at
1,000Xg to pellet nuclei. The resulting supernatant was centrifuged at 15,900Xg for 20 min.
The pellet was resuspended with a paint brush in 10 ml buffer G (0.03M sucrose, 0.05M Tris,
pH 7.5) per 20 g starting material. After centrifuging for 10 min at 1,000Xg the supernatant
was brought to 10 mM MgC12 and 20 pg DNase (Sigma; Type IV)/ml or 200 Kunitz units/ml,
and incubated with gentle mixing at 4°C for 60 min. The suspension was underlaid with 20

ml shelf buffer (0.6M sucrose, 0.01M Tris, 0.02M Na ZEDTA, pH 7.5) per 5 ml and

43
centrifuged for 25 min at 12,000Xg. The pellet was resuspended with the shelf buffer (5m!)

and the mitochondria were pelleted by centrifuging for another 25 min at 15,900Xg. The
mitochondria were then lysed in 5 ml lysis buffer (0.01M Tris, 0.05M EDTA, 0.1M Nacl, 1%
SDS, pH 8.0)/20-25g of fungus and 100 pg proteinase K/rnl at 65°C for 30 min. The protein-
carbohydrate complex was precipitated by adding one third the volume 5M potassium-acetate,
incubating on ice for one hour with periodic shaking, and centrifuging for 20 min at 25,000Xg.
The supernatant was poured into a siliconized corex tube and the mtDNA precipitated with
1/20 volume of 5M ammonium acetate and 1/2 volume of ice cold isopropanol at -20° C.
The mtDNA was pelleted by centrifuging at 10,000Xg for 45 min. The pellets were
air dried, dissolved in 0.7 ml T50E10 buffer (50mM Tris, 10mM EDTA, pH 8.0). The solution
was transferred to a microfuge tube and centrifuged for 10 min. The supernatant was
transferred to a clean tube and the mtDNA was precipitated as above with ammonium acetate
and isopropanol. The mtDNA was washed twice with 70% ethanol, vacuum dried for 10-15

min, and allowed to dissolve in TE (10mM Tris, lmM EDTA, pH 8.0) overnight at 4° C.

Restriction enzyme analysis and electrophoresis

The mtDNA was digested with restriction enzyme S_au3A or BglII for 2-3 hours at 37°
C using the buffers recommended by the manufacturers (Bethesda Research lab, Inc.
Gaithersburg, MD., [BRL]) The fragments were separated by electrophoresis in 0.7% agarose
gels using TBE buffer (Maniatis et al, 1982). Gels were electrophoresed at room temperature
for 18 hours at 35-40 volts. The gels were stained with ethidium bromide (0.5 p g/ml) for 15-20
min and photographed. The restriction fragments on the gel were transferred to nylon
hybridization membranes (MSI. Westboro, MA.) as described by Maniatis et al (1982). The

DNA was fixed on the membrane by UV crosslinking for 3-5 min.

44
Isolation and cloning of the RFLP fragment

Mitochondrial DNA of CL25 was digested with restriction enzyme wA and
electrophoresed in agarose gel as described above. After staining, agar containing the 2.5 kb
RFLP fragment was cut out from the gel and the DNA was electroeluted with an
electroeluter using the procedure described by the manufacturer (Schleicher and Schuell) in
TBE buffer. The bufi'er with the electroeluted DNA was precipitated overnight with 2
volumes of absolute EtOH and 1/10 volume of NaOAc at 0°C. The DNA fragment was
collected by spinning at 15K for 50 min.

The 2.5 Kb MA fragment was cloned into the M1 site of polylinker of the
cloning vector pUC118. The recombinant plasmid pSK51 was isolated using a boiling miniprep

method described by Maniatis et al (1982).

DNA labelling and hybridization

To release the cloned mtDNA insert, the recombinant plasmid was digested with
restriction enzymes H_indIII and E_ch1 and electrophoresed in an agarose gel. Agarose
containing the portion of the recombinant plasmid insert was cut out of the gel, electroeluted
with an electroeluter using the procedure suggested by the manufacturer (Schleicher and
Schuell). The electroeluted DNA was used for probes after radiolabelling. Probes were

radiolabelled using the random primer method (Feinberg and Volgenstein, 1983).

Cytoplasmic fusion via strain pairing
Resistance to 8 mg chloramphenicol/ml and a light pigment phenotype were used as
mitochondrial and nuclear markers, respectively, for strain C125. This strain was paired with

4-C, a chloramphenicol-sensitive, virulent isolate with orange pigmentation. Cytoplasmic

45

fusion was performed by placing inoculum plugs of the two isolates side by side approximately
5mm apart on cellophane layered over PDA. The isolates were allowed to grow and fuse for
5 days. The cellophane and fusing cultures were lifted off the PDA and placed on PDA
supplemented with 8 mg chloramphenicol/ml. After 5-7 days, plugs of mycelia from the side
of the fused strains containing the orange pigmented, virulent strain were removed and
subcultured on PDA supplemented with 8 mg chlorampheniconl. Subcultures that were
orange and able to grow on PDA with chloramphenicol were grown in liquid media with and
without chloramphenicol. The mtDNA from these subcultures was extracted to determine the

presence or absence of the RFLP associated with CL25 mtDNA.

RESULTS

RFLP analysis of mtDNA

Mitochondrial DNA of C125 digested with restriction enzyme Say3A contained a
unique fragment of about 2.5 kb, while all other Q. parasitica strains contained two smaller
bands not observed in CL25. The presence of this fragment did not seem to correlate with
the hypovirulence phenotype in CL25 since a virulent single conidial isolate of strain CL25
also contained this band. A clone of the 2.5 kb fragment of CL25 (pSK51) was used to screen
CL25 and other strains. Clone pSK51 hybridized to the 2.5 kb fragment in CL25 and to the
two smaller bands in 4-C (Fig 2.1-B, lane 1 and 4) and other strains. This RFLP pattern was

subsequently used to detect C125 and 4-C mtDNA after hyphal fusion.

Isolation of chloramphenicol resistant strain of CL25
Growth of C125 (and other strains) was greatly reduced at 6 mg chloramphenicol/ml,

but a better discrimination between chloramphenicol resistant and sensitive strains could be

 

Fig 2.1 - WA digestion pattern of mtDNA of different isolates of Q. parasitica. Lane 1,
CL25. Lane 2, 4-C(H) grown in broth (without chloramphenicol). Lane 3, 4-C(H) grown in
broth supplemented with chloramphenicol. Lane 4, 4-C.

A - Ethidium bromide stained gel, M: A DNA digested with HindIII used as molecular size
marker.

B - Southern blot hybridized with a 2.5 kb fragment clone of CL25 mtDNA. Polymorphic
bands in the gel are indicated with arrows.

C - A longer exposure of the blot B.

 

47
obtained at 8 mg chloramphenicol/ml or greater concentration. A C125 strain resistant to 8
mg chloramphenicol/ml was isolated on PDA plates containing 8 mg chloramphenicol/ml. The
chloramphenicol resistant isolates (C125 capR) tolerated chloramphenicol at high
concentrations when compared to sensitive isolates. The chloramphenicol resistant isolate
grew at a normal rate on PDA when compared to the sensitive strains.

To demonstrate that chloramphenicol resistance was maternally inherited and
therefore mitochondrial-associated in Q. parasitig reciprocal crosses using C125 capR and
4-C capS were made and ascospores were collected fiom each cross. These ascospores were
grown on PDA and PDA supplemented with 8 mg chloramphenicol/ml. Cultures from
ascospores collected from 8 perithecia in which the maternal parent was chloramphenicol
resistant were able to grow in the presence of chloramphenicol, whereas when C125 capR

was the paternal parent, none of the ascospore cultures from 16 perithecia grew on media

supplemented with chloramphenicol.

Transfer of mitochondria during hyphal anastomosis

To determine if mtDNA from C125 capR could transfer via anastomosis to 4-C caps,
the isolates were paired as described. After pairing, subcultures from the 4-C capS side of the
pairing that grew on media amended with chloramphenicol were selected. Each subculture
was divided and grown in broth with and without the addition of chloramphenicol. The
mitochondrial DNA of one subculture 4-C(H), selected on chloramphenicol, showed the
RFLP of 4-C when it was grown without chloramphenicol (Fig 2.1-B, lane 2). However, when
grown in the liquid media supplemented with chloramphenicol, it showed the RFLP pattern

of both C125 and 4-C (Fig 2.1-B, lane 3). Upon longer exposure of the same blot, the

mtRFLP of C125 appeared as a faint band in the mtDNA of 4-C(H) grown in the liquid

TABLE 2.2. Pathogenicity test of virulent, hypovirulent and converted hypovirulent strains

of erphonectrla Larasitica.

 

Pathogenicjty tests“
)

 

Strain (mm Description
CL1-16 89 Virulent strain without dsRNA.
C125 capR 10 A light pigmented chloram-

phenicol resistant C125 strain.

4-C 76 An orange pigmented strain
derived from GHU4 by curing
of dsRNA (Fulbright, 1985).

4-C(H) 12 Strain 4-C converted with
C125 hypovirulence.

 

“Apple fruit were inoculated by removing a 9 x 7-mm-deep plug of apple tissue and inserting
mycelium of strain to be tested. Three weeks later the area of the infected discolored region
was calculated (Fulbright, 1984).

49

Table 2.3. Phenotypic and genotypic characteristics of single-conidial isolates of strain

 

 

 

4-C(I-I).

SCI Vir_ry_lgr_r_ce“ RFLJP Cflk 0’ S Pigmentation
#1 4—C S Orange
#2 V 4-C S Orange
#3 V 4-C S Orange
#4 V 4-C S Orange
#5 V 4-C S Orange
#6 H 4-C S Orange
#7 H 4-C S Orange
#8 H CL25 8 Orange
#9 H 4-C S Orange
#10 V 4-C S Orange
#11 H 4—C S Orange
#12 V C125 S Orange
CL1-16 V NA S White
CL25 CapR H C125 white
4-C(H) H 4-C and CL25 R Orange

 

SCI = Single-conidial isolates, S= Caps, R= CapR, H= Hypovirulent, V= Virulent,

NA= Not applicable.

“Apple fruit were inoculated by removing a 9 x 7-mm-deep plug of apple tissue and inserting
mycelium of strain to be tested. Three weeks later the area of the infected discolored region

was calculated (Fulbright, 1984).

50

broth without chloramphenicol (Fig 2.1-C, lane 2), indicating either a mixture of cultures or
a mixture of mitochondria within the same thallus. To determine if the light-pigmented CL25
hyphae might be contaminating the orange-colored 4-C(H) thallus, single-conidial isolates
were obtained from the subculture. All single-conidial isolates recovered were orange
indicating that they had the 4-C nuclear type. Twelve of these single-spore isolates were
analyzed for their RFLP pattern. Ten single-spore isolates showed the mitochondrial RFLP
banding pattern of 4-C and two showed the mitochondrial banding pattern of CL25 (Table-
2.3). A virulence assay performed with 4-C(H) demonstrated that this strain was reduced in

virulence similar to C125 capR (Table-2.2).

DISCUSSION

Nuclear and mitochondrial markers were required to determine whether or not
mitochondria could transfer during vegetative fusion. Pigmentation of Q. parasitica has been
described as a nuclear trait by Anagnostakis (1982) and therefore, the naturally light
pigmentation phenotype of C125 and the orange pigmentation phenotype of 4-C were used
as nuclear markers.

Both genetic and molecular mitochondrial markers were constructed. Chloramphenicol
resistance has been shown to be a genetically stable extrachromosomal trait associated with
the mitochondria of many other fungi such as Ophiostoma glgyi (Brasier and Kirk, 1986),

Coprinus cinereus (Baptists-Ferreha et 31,1983), Saccharomyces cerevisiae (Borst and Grivell,

 

1978; Dujon B., 1980), Asmrgillus nidulans (Gunatilleke et al, 1975), and Podosmra anserina

 

(Belcour and Begel, 1977). Chloramphenicol resistance in C125 also was shown to be a
cytoplasmic trait in the study described above. These data lend strong support to the concept

that capR is an allele on the mtDNA. Restriction digest analysis with MA revealed a

51
mitochondrial RFLP of a 2.5 kb band which was unique to CL25. Although this RFLP serves

as a molecular marker for C125 mitochondrial DNA, it is not likely to have a role in
hypovirulence since a virulent single spore isolate of C125 also contains this fragment. After
vegetative pairing between CL25 capR and 4-C, a subculture, 4-C(H), was selected that
showed the nuclear trait of 4-C and the mitochondrial RFLP of both C125 and 4-C (Fig 2.1-
C, lane 2). These results strongly suggest that mitochondria have moved from C125 to 4-C
via hyphal fusion. The increased intensity of the mitochondrial RFLP unique to C125 when
4-C(I-I) was grown continuously in the presence of chloramphenicol is clearly due to the fact
that chloramphenicol enhances the selection of the capR mitochondrial population within the
4-C(H) thallus.

Virulence tests with 4-C(H) indicated that this heteroplasmic strain became
hypovirulent after vegetative fusion. Thus, the mitochondrial genome and the factor
responsible for the hypovirulence phenotype are capable of transfer during hyphal fusion.
This supports the hypothesis that mitochondria are associated with the dsRNA-free
hypovirulence phenotype found in C125 and perhaps other strains showing the same
phenotype.

To eliminate the possibility that CL25 hyphae might be contaminating the pigmented
strain, single-conidial isolates of 4-C(H) were selected. All single spore cultures contained the
pigment nuclear marker of 4-C. Out of twelve single-spore isolates that were screened for
their RFLP pattern, two of those contained the mitochondrial RFLP marker of CL25, thus
eliminating the possibility of hyphal contamination. Virulence analysis with the twelve single-
conidial isolates of 4-C(H) demonstrated that five of these isolates were hypovirulent and
seven of them were virulent. All twelve of these isolates did not grow in the presence of
chloramphenicol, and thus are capS (Table- 2.3). These data suggest that the strain 4-C(H)

is a heteroplasmon and the single conidial isolates are the result of non-reciprocal

 

52
recombination between DNAs of the two different types of mitochondria in 4-C(H) and their

subsequent vegetative segregation. If all the loci are carried by the mitochondrial genome,
then these results are reminiscent of polar recombination between mtDNAs of other fungi.

Genetic studies of Neurosmra and yeast have shown that recombination between
mtDNA occurs frequently in heteroplasmons. Ultimately a single mitochondrial type
predominates, be it the either a parental type or recombinant type. When a recombinant type
predominates, it often results from unidirectional gene conversion (Manella and Lambowitz,
1978; Manella and Lambowitz, 1979; Dujon, 1980). Examples of gene conversion or non-
reciprocal recombination are encountered in Saccharomyces cerevisiae crosses where the
polarity of recombination of mitochondrial alleles is determined by the o locus on the
mitochondrial DNA. 0+ differs from o' by the presence of an intron (1143 bp) in the 21S
rRNA gene; polarity of recombination originates at the o locus (Borst et al, 1977; J acq et al,
1977, Dujon, 1980), with gene conversion favoring the alleles that are linked to co +. Although
more studies are needed to show conclusively that the C125 hypovirulence is truly linked to
the other markers on the mitochondrial genome, we believe that our genetic evidence

supports this data.

 

 

 

 

53
REFERENCES

Anagnostakis, S.L (1979). Sexual reproduction of Endothia parasitica in the laboratory.
Mycologia. 71: 213-214.

Anagnogtakis, S.L (1982). Genetic analysis of Endothia parasitica: linkage data for four single
genes and three vegetative compatibility types. Genetics. 102:25-28.

Anagnostakis, S.L (1984). The mycelial biology of Endothia parasitica. II. Vegetative
incompatibility. In The Ecology and Physiology of the Fungal Mycelium, British
Mycological Society Symposium Volume 8, ed. Jennings, D.H. & Rayner, A.D.M.,
pp.4999-507. Cambridge University Press.

Bapista-Ferreira, J .LC., Economou, A. and Casselton, LA. (1983). Mitochondrial Genetics
of Coprinus: Recombination of mitochondrial genomes. Curr. Genet. 7:405-407.

Belcour L, Begel O (1977). Mitochondrial genes in Podosmra anserina: Recombination and
linkage. Mol Gen Genet 153:11-21.

 

Borst P, Grivell LA. The mitochondrial genome of yeast.(1978) Cell 15:705-723.

Borst P, Bos J. L, Grivell L A., Groot G. S. P., Heyting C., Moorman A. F. M., Sanders J.
P. M., Talen J. L Van Kreijl C. F. and Van Ommen G. J. B. (1977). The physical
map of yeast mitochondrial DNA anno 1977. In Mitochondria, W. Bandlow, R,
Schweyen, T. Y. Thomas, K. Wolf and F. Kaudewitz, eds. (Berlin: DeGruyter). pp.
213-266.

Brasier, CM. (1984). Inter-mycelial recognition system in Ceratgggtis gl_r_m_;: their physiological
properties and ecological importance. In The Ecology and Physiology of Fungal
Mycelium, British Mycological Society Symposium Volume 8, ed. Jennings, D.H. &
Rayner, A.D.M., pp.451-497. Cambridge University Press.

Brasier, C. W. (1986). The d-factor in Cerat_o_cgtis _ulmi- Its biological characterization and
implications for Dutch elm disease. Pages 177-220 in: Fungal virology. K. W. Buck,
ed. CRC Press, Inc. Boca Raton, FL

Brasier, CM. and Kirk, S. A. (1986). Maternal inheritance of chloramphenicol tolerance in
Ophiostoma £111.1- Trans. Br. Mycol. Soc. 1987 (3):460-462.

Casselton, L A. and Economou, A. (1985). Pages 213-229 in: Developmental biology of
higher fungi. BMS symposium 10. D. Moore, LA. Casselton, D. A. Wood, and J. C.
Frankland, eds. Cambridge university press, Cambridge.

Collins, RA. and Saville, B. J. (1990). Independent transfer of mitochondrial chromosomes
and plasmids during unstable vegetative fusion in N eurosmra. Nature. 345:177-179.

Dujon, B. (1980). Sequence of the intron and flanking exons of the mitochondrial 218 rRNA
gene of yeast strains having different alleles at the to and rib-1 loci. Cell. 20:185-197

1b. kilos a.
. r'

 

 

 

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Feinberg and Volgenstein. (1983) Radiolabelling by random priming 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. (1985). A cytoplasmic hypovirulent strain of Endothia parasitica without
double-stranded RNA (dsRNA). (Abstract) Phytopathology. 75:1328.

Gobbi, E., Wang, Y., Martin, R.M., Powell, WA and Van Alfen,N.K. (1990). Mitochondrial
DNA of (_hyphonectria parasitica: Lack of migration between vegetatively compatible
strains. Mol. Plant-Microbe Interact. 3:66-71.

Griffith, A.J.F., Kraus,S.R., Barton, R., Court, D.A., Myer, CJ., and Bertrand, H. (1990).
Heterokaryotic transmission of senescence plasmid DNA in Neurosmra. Curr. Genet.
17:139-145.

Gunatilleke, I. A. U. N., Scazzoccio, C. and Arst, Jr. H. N. (1975). Cytoplasmic and nuclear
mutations to chloramphenicol resistance in Asxrgm us 91M. Mol. Gen. Genet.
137:269-276.

J acq C. Kujawa C. Grandchamp C. and Netter P. (1977). Physical characterization of the
difference between yeast mitochondrial DNA alleles in + and (0'. 255—270. In
Mitochondria, W. Bandlow, R, Schweyen, T. Y. Thomas, K. Wolf and F. Kaudewitz,
eds. (Berlin: DeGruyter). pp. 213—266.

Mackenzie, S.A., Pring, D.R., Bassett, MJ., Chase, CD. (1988). Mitochondrial DNA
rearrangement associated with fertility restoration and cytoplasmic reversion to fertility
in cytoplasmic male sterility in cytoplasmic male sterile Phaseolus vulgaris L Proc.
Natl. Acad. Sci. USA 85:2714-2717.

Manella C.A. And Lambowitz AM. (1978). Interaction of wild type and my mitochondrial
DNA in heterokaryons of Neurosmra. Biochem. Biophys. Res. Commun. 80:673-679.

Manella C.A. And Lambowitz AM. (1979). Unidirectional gene conversion associated with
two insertions in Neurospgra crassa mitochondrial DNA. Genetics 93 :645-654.

Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor. NY.

Puhalla, J .E. and Anagnostakis, S.L(1971). Genetic and nutritional requirements of Endothia
parasitica. Phytopathology 61.169-173.

Watrud, LS. and Ellingboe, A. H. (1973). Use of cobalt as a mitochondrial vital stain to study
cytoplasmic exchange in matings of the basidiomycete Schizophyllum commune. J.
Bacteriol. 115:1151-1159.

ASSOCIATION OF ELEVATED MITOCHONDRIAL ALTERNATE OXIDASE
ACTIVITY WITH THE HYPOVIRULENCE PHENOTYPE OF

CRYPHONECI'RIA PARASITICA.

ABSTRACT

Hypovirulent strains of ngphonectria parasitica, the causal agent of chestnut blight
have been associated with the presence of cytoplasmically transmissible double stranded RNA
(dsRNA). Recently, several hypovirulent strains have been found that are not associated with
detectable levels of dsRNA. Respiration studies of dsRNA-free hypovirulent strains revealed
the presence of an induced alternate oxidase pathway and possibly defects in the electron
transport system. We present evidence that alternate oxidase can be induced in other Q.
parasitica strains after they undergo hyphal anastomosis with a dsRNA-free hypovirulent
strain. Some of these dsRNA-free hypovirulent strains had a slower growth pattern compared
to virulent strains. These results suggest that the hypovirulence phenotype, altered growth
pattern and the induction of the alternate oxidase result from a defective cytochrome
mediated electron-transport. This work indicates that the phenotype known as hypovirulence
may have more than one cause and that the mitochondrial genome may, in some cases, be

responsible for hypovirulence in Q. parasitica.

55

56
INTRODUCTION

The mitochondria of higher plants and fungi have a branched electron transport chain
with a cyanide-sensitive cytochrome pathway that terminates with cytochrome oxidase and a
cyanide-resistant, hydroxamic acid sensitive alternative pathway that terminates with an
alternative oxidase (Lambowitz and Zannoni, 1978). Both oxidases reduce oxygen to water
as the end product of electron transport (Solomos T. 1977). When electrons flow through the
cytochrome pathway, energy is conserved in the form of ATP, but when the electrons flow
through the alternate oxidase pathway, the potential energy of the system is lost as heat. In
Sauratum ggttatum (voodoo lily), a member of family Araceae, the alternate pathway is most
highly expressed in thermogenic tissue, where its function is to generate heat to attract
insects, thereby facilitating pollination. This is probably the best understood physiological role
of alternate oxidase (Elthon and McIntosh, 1987). In fungi, characterization of the alternative
pathway has been described best in the my mutant of Neurosmg, which has a
conspicuously slow growth rate. In this mutant the alternate pathway accounts for 70% of the
total respiration (Mitchell et al,1952). The mitochondria of my contain abnormally low
amounts of cytochrome aa3, b and 30% more soluble cytochrome c and flavoproteins
(T issieres et al.1953, Lambowitz et al. 1972; Jagow et al, 1973; Bertrand et al. 1976).
Alternate oxidase can be induced in wild-type cells by growing the mycelium in the presence
of chloramphenicol, antimycin A (Lambowitz and Slayman, 1971) or oligomycin (Szakacs and
Bertrand, 1976; Bertrand et al, 1976), which are inhibitors of mitochondrial protein synthesis,
electron transport, and ATP synthesis, respectively.

In this chapter, I investigated the possibility that respiratory deficiencies could be a
factor responsible for the reduced virulence (hypovirulence) phenotype of ngphonectria

(Endothia) parasitica (Mum) Barr., the causal agent of chestnut blight. Chestnut blight was

57

responsible for the demise of the once prevalent American chestnut (Castanea m
[Marsh] Borkh) in North America. Isolates with reduced virulence (hypovirulent) have been
isolated where chestnut trees survive infection. The well-characterized hypovirulent strains
of Q. parasitica have been found to be infected with cytoplasmically transmissible, double-
stranded RNA (dsRNA) molecules which are correlated with changes in virulence, growth
rate, sporulation and/or culture morphology. Several isolates of Q. parasitig, including C125
from Crystal Lake, Michigan, have the same characteristics of dsRNA-associated
hypovirulence but harbor no detectable dsRNA. Genetic studies of CL25 show that the
hypovirulence phenotype is carried in 20% of the conidia, is maternally inherited and is
cytoplasmically transferred through hyphal anastomoses (Fulbright, 1985). These studies
suggested that there is a cytoplasmic ”virulence inhibition factor” (VIF, Fulbright (1990))
other than dsRNA that might be involved in the hypovirulence phenotype. Because of the
cytoplasmic nature of the hypovirulence phenotype, I investigated the possibility of
mitochondrial defects in the dsRNA-free strains of Q. parasitica. Evidence is presented that
supports a positive correlation between the presence of high levels of alternate oxidase and

the dsRNA-free type of hypovirulence.

MATERIALS AND METHODS
Strains and Growth Condition
The strains or isolates of Q. parasitica used in this study are listed in Table 3-1.
Cultures were maintained on potato dextrose agar (PDA, Difco) at 20°C under fluorescent
light. Fungal cultures were grown at room temperature for 5 days in the Endothia complete
liquid medium (ECM) of Puhalla and Anagnostakis (1971) modified by the omission of

glucose. Chloramphenicol (Sigma) was added to the cultures, when needed, fiorn a stock

58

TABLE 3.1. Alternate oxidase pathway as percentage of total respiration in different

strains of thonectria grasitica.

 

 

Presence Alt. oxd. as 79 of

Strains Description Phenom of dsRNA total respiration
EP155“ From Connecticut V - 9.0
CL1-16“ SCI from a standard

virulent strain from V - 10.5

Frankfort, MI.
14B-N1 SCI from a strain from

Kellogg forest, MI. V - 17.5
4-C"' dsRN A cured GHU4

(Fulbright,1984). V - 8
C125 SS#4"‘ SCI from a strain from

Crystal Lake, MI. V - 13.5
CL25 SS#9"' SCI from a strain from

Crystal Lake, MI. HV - 71.5
14B-D6“ SCI from a strain from

Kellogg forest, MI. HV - 84.5
14B-N6* " HV - 71.0
4-C(H)"' Strain 4-C converted with

C125 type of hypovirulence, HV - 82.0

contains the mitochondria
of both strains.

 

 

59
TABLE 3.1. (cont)

 

Presence Alt. oxd. as % of
Strains Qes_c__ription Phenom of dsRNA total respiration
GHU4" From Grand Haven, MI. HV + 53.0
61.121: " HV + 16.5
CL1-16(GH2) Strain CL1-16 converted
with GH2 type of HV + 15.0
hypovirulence.
R1 From Roscommon, MI. HV + 6.5

 

V = previously determined to be virulent (unpublished)

HV = previously determined to be hypovirulent (unpublished)
SCI= Single conidial isolate.

Strains with " represent averages of multiple samples.

60

solution in 95% ethanol to a final concentration of 2 mg/ml. The final concentration of
ethanol was always less than 1% (v/v) and this concentration did not affect respiration in Q.

arasitica.

Respiration

The respiration of whole mycelium was measured polarographically using a Clark
electrode and a YSI Model 53 biological oxygen monitor (Yellow Springs Instruments Co.)
connected to a Gould recorder. The respiration measurements were made using several
modifications of the basic techniques previously described for Neurosppra by Lambowitz and
Slayman (1971). Four-to-five day-old-cultures grown in ECM were homogenized for 15-20
seconds using a Polytron homogenizer. The reaction was begun by pipetting 3 ml of the
homogenized mycelium into a reaction chamber. The sample was aerated for about 1 min to
a final oxygen concentration of about 240 ppm. The electrode was quickly inserted into the
reaction chamber and the oxygen consumption recorded. The reaction chamber was
maintained at 25°C and the mycelial suspension was constantly mixed with a magnetic stirring
bar.

Potassium cyanide (KCN; Aldrich) and salicylhydroxamic acid (SHAM; AnalaR),
respiratory inhibitors of the cytochrome chain and the alternate oxidase pathways,
respectively, were used to analyze the capacities of the cultures to respire through each
pathway. The inhibitors were added separately to the reaction chamber during measurements
of oxygen consumption. SHAM was added from a 50 mg/ml solution in 95% ethanol to a final
concentration of 2.7 mM, and KCN was added to a final concentration of 0.8 mM from a 0.1
M solution in 10 mM Tris-HCl, 5 mM EDTA, pH 7.2. Both inhibitors were freshly prepared

prior to each experiment.

61
Isolation of mitochondria

For the determination of enzyme activities, mitochondria were isolated by a
modification of the procedure of Bertrand and Pittenger (1969). Five-to-seven day-old-
cultures grown in ECM were harvested by suction filtration. The mycelial mat was rinsed once
with deionized water, followed by a wash with sucrose-EDTA isolation buffer (0.44 M
sucrose, 5 mM EDTA, 10 mM Tris-HCl; pH 7.4). All the following steps were performed at
4°C. Cells were disrupted by grinding the mycelial mat with acid-washed sea sand in a cold
mortar and pestle. The mycelial paste was suspended in 20 to 30 ml of isolation buffer. This
was followed by two successive 10 min centrifugations at 2500Xg (4,500 rpm, Sorvall 55-34
rotor) to pellet all debris, nuclei and sand. Mitochondria were pelleted by centrifugation at

25,000Xg for 45 min (14,500 rpm, Sorvall SS-34 rotor).

Determination of enzyme activities
Mitochondria isolated as described above were used to determine enzyme activities.
In all cases, mitochondrial pellets were resuspended in a buffer containing 5 mM EDTA, 10

mM Tris-HCI; pH 7.2.

Cytochrome oxidase activity

To determine cytochrome oxidase activity, the oxidation of reduced cytochrome c was
followed spectrophotometrically at 550 nm, as described by Smith (1955) and modified by
Nargang (1978). Reduced cytochrome c was obtained by adding a few crystals of sodium
dithionite to a 10 mg/ml solution of cytochrome c. Excess dithionite was removed by bubbling
air through the mixture for 1 min 50 pl of the reduced cytochrome c solution was added to

900 pl of TE buffer (5 mM EDTA, 10 mM Tris-HCl; pH 7.6) and the reaction was started

62
by addition of 10 pl of the mitochondrial suspension. This was mixed quickly and the

absorbance was determined as above.

Succinate oxidase activity

Succinate oxidase was determined by a modification of the procedure described by
Stotz (1955). One ml of the reaction mixture contained 50 pl of 10 mg oxidized cytochrome
c/ml, 880 pl of TE buffer (5 mM EDTA, 10 mM Tris-HCl; pH 7.6), 50 pl of 35 mg
succinate/ml, 10 pl of 0.1M KCN and 10 pl of the mitochondrial suspension. The activity was

determined by spectrophotometrically following the reduction of cytochrome c at 550 um.

NADH oxidase activity
N ADH oxidase activity was determined in the same manner as described for succinate

oxidase with NADH substituted for the succinate.

Determination of dry weight

Cryphonectria parasitica strains were grown in Endothia minimal media (Puhalla and
Anagnostakis (1971). After 10 days of growth, the liquid medium was poured off the fungus
and the mycelial mass was air dried. When the fungal strains dried completely, their weight

was determined by scraping the mycelial fragments into preweighed weightboats.

RESULTS
Respiration
To determine if Q. parasitica isolates with the hypovirulence phenotype were affected

by mitochondrial defects, respiration was measured and compared between hypovirulent and

63

virulent strains. The isolates studied could be placed into one of four different respiration
patterns (Fig 3-1, A-D). In all cases, the initial slope of the curve in figure 3-1 represents
100% respiration. When an inhibitor of the cytochrome pathway, cyanide, is added, the
electron flow is directed towards the alternate pathway. Subsequently, when SHAM, the
alternate oxidase inhibitor, is added, respiration through the alternate pathway is inhibited,
and there is very little residual respiration. As seen in the altered rate of oxygen utilization
in the reactions, all virulent strains and most dsRNA-associated hypovirulent strains were
inhibited by the addition of cyanide (Fig 3-1, A and C), with the exception of GHU4 (Fig 3-1,
D). In contrast, respiration was only moderately inhibited by the addition of cyanide in the
dsRNA-free hypovirulent strains (Fig 3-1, B), since the curves representing the rate of oxygen
utilization did not change to a large extent as compared to the virulent and dsRNA-associated
type of hypovirulent strains (Fig 3-1, A and C). The alternate oxidase pathway represented
a significantly high percentage of the total respiration in these isolates (Table 3-1). These
results indicate that there is an association of increased alternate oxidase activity with the
hypovirulent phenotype, and a correlation of this phenotype with the dsRNA-free
hypovirulence state. To determine if the alternative oxidase pathway was inducible in strains
showing low levels of alternative oxidase, some of these isolates were grown in the presence
of chloramphenicol. The alternate oxidase was induced up to about 8 fold over the normal
amount (Table 3.2).

When virulent strain 4-C was paired with hypovirulent strain C125, the converted
strain 4-C(H) had respiration characteristics that resembled CL25 with the alternate oxidase
pathway accounting for 82% of the total respiration. Before conversion only 8% of the total

respiration of 4-C occurred through the alternate oxidase pathway (T able-3.1). The increase

64

Fig 3.1- Oxygen electrode recordings of the respiration of different strains of Cryphonectria
parasitica. (A) EP155 [virulent] (B) CL25 #9 [hypovirulent without dsRNA] (C) GH2
[hypovirulent with dsRNA] (D) GHU4 [hypovirulent with dsRNA] (E) Control- Respiration
of strain C125 without any addition of respiratory inhibitors. KCN= Potassium cyanide,
SHAM= Salicylhydroxamic acid.

A-EP1 55
B'CL25 E-CONTROL
100% . 100%
3 MIN
9% 0% 3 MIN
KCN A "
KCN 789‘» :3 MIN
0%
SHAM

 

C—GH2

 

Table 32- Induction of alternate oxidase pathway with chloramphenicol in different

strains of ngphonectria mitica.

 

 

Alt. ox. as % of total respiration
Strains “-chIoramphenicol -chIoramphenicol
CL1-16 10.5 80
GH2 16.5 42
CL1-16(GH2) 15 62

 

" Cultures were grown in Endothia complete media amended with 2 mg/ml chloramphenicol
for 5 days.

 

66

in respiration through the alternate pathway in strain 4-C(H) implies that the induction of
alternative pathway may be a manifestation of mitochondrial disfunction and that this

disfunction is transferred to other strains through hyphal anastomosis.

Enzyme 353839

The enzymatic activities of cytochrome c oxidase, succinate oxidase and NADH
oxidase were determined in isogenic strains of C125 (Table 3.3). The ratio of cytochrome c
oxidase to NADH dehydrogenase was found to be the same in both virulent and hypovirulent
strains. The ratio of cytochrome c oxidase to succinate oxidase was two-fold higher in the
virulent isogenic single-conidial isolate CI25#4 than in the dsRNA-free hypovirulent isogenic
single-conidial isolate CL25#9. These results suggest that the hypovirulent strain CL25#9

may be deficient in cytochrome oxidase activity.

Dry weight of diflerent strains

Determination of dry weight of different strains after growth in broth for 10 days
showed that the converted hypovirulent strain 4-C(H) had a dry mycelial mass of about 43
mg as compared to about 133 mg in virulent strain 4-C (Table 3.4). Hypovirulent single-
conidial isolate, CL25#9, had a dry weight of 80 mg and the dry weight of a virulent control

strain CL1-16 was similar to virulent strain 4-C (Table 3.4).

DISCUSSION
Clonal deterioration and cellular death occurs in filamentous fungi after prolonged
asexual propagation. This phenomenon is described as senescence, and examples of some

fungi that experience vegetative senescence are Podosmra, N eurosmra and Asgrgillus.

67

Table 3.3- The activities of cytochrome c oxidase, succinate oxidase and NADH oxidase in

partially purified mitochondrial preparations of isogenic strains of C125.

 

 

 

Strains CCO SO NADH oxd. CCO/ CCO/SO
min-1 OD/min OD/min NADH oxd.

C125#9 0.154 0.092 0.216 0.428 1.67

CL25#4 0.088 0.024 0.059 0.404 3.6

Abbreviations-

CCO= cytochrome c oxidase

SO= succinate oxidase
NADH oxd= NADH oxidase

Table 3.4- Determination of dry weight of different strains of Q. mitica grown in

 

 

 

Endothia minimal broth.
Dry weight. of different strains in mg.
Replicates 4-C 4-C(H) CL25#9 CLl-l6
1 120 40 80 150
2 130 40 80 160
3 150 50 80 150
Mean 133.3 43.3 80 153.3

 

*Dry weight of different Q. parasitica strains were determined by growing two 9mm plugs of
mycelium (cut with a cork borer) in Endothia minimal media. After 10 days, the broth was
poured off the mycelial mass, air dried completely and weighed.

69

Some of the common symptoms of senescence are female sterility, respiratory abnormalities,
and cytochrome aa3 and b deficiencies in the mitochondria (Bertrand, 1983). There is
conclusive evidence that senescence in fungi is caused by mutations affecting the
mitochondrial genome.

”Diseased” isolates of Ophiostoma M, the pathogen of Dutch elm disease, display
a slow growth rate, reduced viability of conidia and impairment of sexual reproduction. The
diseased state is transmitted by extrachromosomal elements, referred to as d-factor, and is
correlated with the transmission of dsRNA (Brasier, 1983). A recent study suggests that the
diseased state is related to a deficiency in cytochrome M3 in dsRNA infected isolates. The
dsRNA in these strains copurifies with the mitochondria. It has been postulated that the
debilitation of growth may be due to suppression of respiration in diseased strains (Rogers
et al. 1987).

Our results indicate that Q. parasitica utilizes two oxidase systems, one sensitive to
SHAM and the other sensitive to cyanide. We showed that alternate oxidase accounted for
a high percentage (up to 85%) of the total respiration in dsRNA-flee hypovirulent strains as
opposed to 6-20 % of total respiration in the virulent and dsRNA-associated types. The
presence of higher percentage of alternate oxidase in dsRNA-free hypovirulent strains is
reminiscent of that found in senescing strains of Neurosmra and other fungi, in which there
is a branched electron transport system. The correlation of dsRNA-flee hypovirulence with
a reduced growth rate is consistent with the altered rate of alternate oxidase and cytochrome
oxidase activities. To determine if respiratory defects are affecting the grth of Q. parasitica,
some of the strains were grown in Endothia minimal media, where the growth of the fungus
is restricted by the concentration of the materials. Therefore, the fungus depends on its own

metabolism for energy and if there is any defect in the respiration, it should become apparent.

70

Dry weight measurements of different strains showed that the growth of converted strain
4-C(H) is much slower than that of virulent strain 4-C. Also the hypovirulent single-spore
isolate of C125 had a slower growth rate than either of the two virulent strains tested. This
study suggests that the induction of the alternate oxidase pathway may have an effect on the
growth of the fungus. Furthermore, assays with isogenic strains of C125 differing in virulence
and the amounts of alternate oxidase show that the hypovirulent strain C125#9 may be
deficient in cytochrome oxidase activity. Thus it appears that hypovirulence, altered growth
phenotype and the induction of the alternate oxidase result from a defective cytochrome-
mediated electron transport.

In Podosmra, Asmrgjllus, and Neurosmra, the extranuclear factors responsible for
respiratory defects and subsequent senescence of the culture spread to normal cultures after
hyphal fusion between senescent and normal hyphae (Bertrand, 1983). This conversion of
normal hyphae to a senescent state reveals that these factors are invasive or "suppressive" in
nature. A similar process may function in Q. parasitica, since hypovirulence is determined by
cytoplasmically transmissible factors that can convert virulent strains to hypovirulent via
hyphal anastomosis. Respiration studies of a converted hypovirulent strain 4-C(H) revealed
that the alternate oxidase pathway accounted for 82% of the total respiration compared to
8% before conversion. The strain 4-C(I-I), was found to contain a mixture of mitochondria
from both 4-C and C125 (chapter 2). The original CL25 is also probably a heteroplasmon
since only 20 % of the single-conidial isolates of CL25 inherit hypovirulence (Fulbright, 1985).
From these data, we assume that only a minority of the total mitochondria are defective. Our
speculation at this point is that these defective mitochondria are "suppressive" in nature and
if they exceed a certain proportion of the population (about 20%), it is lethal to the fungus.

Our observation regarding mitochondrial DNA recombination in the single-conidial analysis

71

of the heteroplasmon 4-C(I-I) (chapter 2) supports this theory since many rounds of non-
reciprocal polar recombination could account for the suppressivity of the hypovirulent
mitochondrial genome. These observations suggest that the induction of alternate oxidase may
be associated with the hypovirulence phenotype in dsRNA-free strains.

Since altered growth morphology and debilitation are common factors associated with
hypovirulence in Q. parasitigp, the "diseased” state of Q. u_lmi, and senescence in N eurosmra
and other fungi, it is important to also note that these phenotypes may share a common
mitochondrial disfunction. Thus studies on one system may help us understand the other. This

is a new direction to explore in connection with the phenomenon of hypovirulence.

72
REFERENCES

Bertrand H. and Pittenger TH. (1969). Cytoplasmic mutants selected from continuously
growing cultures of Neurosmra crassa. Genetics 61:643-659.

 

Bertrand H. (1983). Aging and senescence in fungi. In, Intervening in the aging process,
Edited by W. Rogelson and F. M. Sinex. Alan R. Liss, New York. pp. 233-251.

Bertrand H., Szakacs N. A., Nargang F. E., Zagozeski C. A., Collins R. A. and Harrigan J.
C. (1976). The function of mitochondrial genes in Neurospgra crassa. Can. J. Genet.
Cytol. 18: 397-409.

Brasier, CM. (1983) A cytoplasmically transmitted disease of Ceratgstis ul_rni. Nature
305:220-222.

Elthon TE. and McIntosh L (1987). Identification of the alternative terminal oxidase of
higher plant mitochondria. Proc. Natl. Acad. Sci. 84:8399-8403.

Fulbright D.W. (1984). Effect of eliminating dsRNA in hypovirulent Endothia parasitica.
Phytopathology 74:722-724.

Fulbright, D.W. (1985). A cytoplasmic hypovirulent strain of Endothia parasitica without
double-stranded RNA (dsRNA). (Abstract) Phytopathology. 75:1328.

Fulbright D.W. (1990). Molecular basis for hypovirulence and its ecological relationships. In,
New directions in biological control. Baker T.L and Dunn P., eds. UCLA Symp. Mol.
Cell. Biol. New Sen, Vol. 112, Alan R. Liss, Inc., New York. pp 693-702.

J agow, G. V., Weiss, H., and Klingenberg, M. (1973). Comparison of the respiratory chain of
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Lambowitz AM. and Slayman CW. (1971). Cyanide-Resistant Respiration in Neurosmra
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Mitchell MB. and Mitchell H.K. (1952) A case of ”maternal” inheritance in Neurosmra
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Puhalla J .E. and Anagnostakis S.L (1971). Genetic and nutritional requirements of Endothia
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Rogers H.J., Buck, K., Brasier. CM. (1987) A mitochondrial target for double-stranded RNA
in diseased isolates of the fungus that causes Dutch elm disease. Nature 329:558-5560.

Szakacs, N.A. and Bertrand, H. (1976). Selection of mutants of Neurosmra crassa defective
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CONCLUSION

Hypovirulent strains of Cgphonectria parasitica have been correlated with the
presence of double-stranded RNA (dsRNA). Recently, several hypovirulent strains have been
discovered that do not have detectable amounts of dsRNA. Due to the cytoplasmic nature
of hypovirulence, I decided to study the role of extrachromosomal factors that might be
involved in dsRNA-free hypovirulence. The presence of a 6 kb plasmid in the mitochondria
of all virulent and hypovirulent strains of Q. parasitica assayed reduced the possibility that this
plasmid might have a role in the dsRNA-free hypovirulence phenotype. However, this finding
does not eliminate the possibility of other mitochondrial-borne plasmids or other plasmids
causing hypovirulence.

Due to the low level of virulence and the maternally inherited nature of dsRNA-free
hypovirulent strains, a mutation in the mitochondrial genome was suspected. Mitochondrial
mutations may involve respiratory defects, such as those which are responsible for the
debilitation and senescence of Neurosmra, Podosmra and Asmrgfllus and possibly
Qphiostoma plpij. Respiration studies of the dsRNA-free hypovirulent strains revealed that
cyanide-insensitive respiration (the alternate oxidase pathway) accounted for as much as 85%
of the total respiration. Virulent strains and dsRNA-associated hypovirulent strains (except
one) had a very low percentage of total respiration occurring through alternate oxidase
pathway (up to 16%) when compared to the dsRNA-free hypovirulent strains. A virulent
strain having only 8% of its total respiration through the alternate oxidase pathway was
converted to the hypovirulence phenotype by a dsRNA-free hypovirulent strain. After
conversion, the alternate oxidase pathway accounted for 82% of the total respiration
indicating that the factor responsible for inducing hypovirulence and cyanide insensitive

respiration co-transferred during hyphal fusion. Determination of dry weight of this strain

74

75

before and after conversion showed that the growth rate of the converted strain was much
slower in minimal media after conversion. We believe that the growth of the fungus is
restricted due to a mitochondrial defect and this growth debilitation makes the fungus less
capable of infecting its host plants.

The converted strain has been shown to be a heteroplasmon with a mixture of
mitochondria from both the virulent and the hypovirulent strain used in the hyphal fusion
process. Even though the mitochondria of the hypovirulent strain appear to be a minority of
the total mitochondrial population within the converted strain, the defect in respiration and
the hypovirulence trait appear to be dominant. This may be fully or partially explained
through the phenomenon of ”suppressivity" observed in other senescing fungi such as
Neurosmrp, Podosmra and Asgrgiilus. In suppressivity, defective mitochondria replace or
outcompete normal mitochondria and spread throughout the hyphae. In these well studied
fungal species, senescence is associated with the rearrangement of the mitochondrial genome,
with suppressivity resulting in the gradual replacement of the wild-type mitochondrial genome
by the defective genome. It has also been proposed that the suppressivity may be due to
unidirectional gene conversion, and examples of gene conversion can be found in Neurosmra
and yeast.

In Q. parasitica, the mitochondrial defect and the associated hypovirulence may spread
by a similar process of suppressivity, but they appear to not totally replace the resident
mitochondria, because CL25 and all its derivatives are heteroplasmons. Conceivably, the
mitochondrial defect is lethal if homoplasmic. My preliminary data with single-conidial isolates
from the converted heteroplasmon showed that chloramphenicol resistance appears to be lost
during mitochondrial mixing. The unequal gene frequencies for capR/caps,

hypovirulence/virulence, and the mtRFLPs point to a polar unidirectional gene conversion.

76

The hypovirulence phenomenon has previously been described as a trait that can be
transferred to other strains via hyphal fusion. 1 have demonstrated a strong correlation
between the presence of a high percentage of alternate oxidase and the dsRNA-free
hypovirulence phenotype. We also have demonstrated that this trait could be transferred to
other strains via hyphal fusion. These results, when taken with the finding of a deficiency in
the cytochrome c oxidase activity in a hypovirulent strain makes the dsRNA-free
hypovirulence an interesting phenotype requiring further exploration to implicate alternate
oxidase in the occurrence of hypovirulence.

In the future, some of these results need to be confirmed using more samples of
virulent and hypovirulent strains. I have been able to see different patterns of respiration
using several virulent and hypovirulent strains (Table 1). The enzyme activities of cytochrome
oxidase, succinate oxidase and NADH oxidase should be determined in all strains listed in
Table 3.1 to see if the deficiency of cytochrome c oxidase is specific to hypovirulent strains.
The next obvious step is to perform cytochrome spectra of virulent and hypovirulent strains.
These experiments will enable us to determine the nature of the deficiency in the cytochrome
system that might be causing the respiratory defect.

Another direction will be to determine if the induction of alternate oxidase is
correlated with the presence of a higher percentage of cyanide-insensitive respiration out of
the total respiration. Immunoblotting experiments using monoclonal antrhodies developed
against alternate oxidase of Sauromatum gpttatum, which seems to be conserved in a variety
of different organisms, may help us determine if there is induction of alternate oxidase in
respiratory deficient strains.

If the correlation holds well between the presence of alternate oxidase and the
hypovirulence phenotype, it will be worthwhile to try to induce alternate oxidase deficient

mutations to examine their effect on the hypovirulence phenotype.