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This is to certify that the

dissertation entitled

THE CLONING, COMPARISON AND EXPRESSION OF THREE FAMILY G
ENDO B- 1, 4- XYLANASE GENES OF THE MAIZE FUNGAL PATHOGEN
COCHLIOBOLUS CARBONUM AND ANALYSIS OF THEIR IMPORTANCE

FOR PATHOGENICITY ON MAIZE

presented by
Patricia Carlene Ape]

has been accepted towards fulfillment
of the requirements for

Ph.D. degree“, Botany and Plant Pathology

 

 

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Major professor

 

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1198 Woes-p.14

THE CLONING, COMPARISON, AND EXPRESSION OF THREE FAMILY G
ENDO [HA-XYLANASE GENES OF THE MAIZE FUNGAL PATHOGEN
COCHLIOBOL US CARBONUM AND ANALYSIS OF THEIR IMPORTANCE
FOR PATHOGENICITY ON MAIZE
by

Patricia Carlene Apel

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1996

ABSTRACT

THE CLONING, COMPARISON, AND EXPRESSION OF THREE FAMILY G
ENDO B-l,4-XYLANASE GENES OF THE MAIZE FUNGAL PATHOGEN
COCHLIOBOL US CARBON UM AND ANALYSIS OF THEIR IMPORTANCE
FOR PATHOGENICITY ON MAIZE

By

Patricia Carlene Apel

The filamentous fungus Cochliobolus carbonum, a pathogen of maize,
makes three xylanases when grown in culture. A degenerate oligonucleotide
based on the sequence of a tryptic peptide of the major xylanase, Xyll, was used
to clone the corresponding gene, XYLI. The oligonucleotide also hybridized to
another xylanase gene, XYL3. A third xylanase gene, XYL2, was cloned by
using XYLI as a heterologous probe. All three xylanase genes encode family G
endo-B 1,4-xylanases with basic pI’s and predicted Mr’s of approximately 22,000.
At the amino acid level Xyl2 and Xy13 are 60% and 42% identical to Xyll. Xyl2
and Xy13 are 39% identical. XYLI and XYL2 but not XYL3 are expressed at the
mRNA level in fungus grown in culture. XYLI and XYL3 but not XYL2 are

expressed in infected plants. Transformation-mediated gene disruption was

used to create strains mutated in all three xylanase genes. In the XYLI mutant,
total xylanase activity decreased by 85% to 94% and two of the three previously
characterized xylanase enzymes were gone. The XYLZ mutant lacked XYLZ
mRNA but no enzyme activity or major protein disappeared. By immunoblotting
using an antibody raised against a 22-kDa xylanase from Trichoderma viride, a
minor protein of 22 kDa could be observed to disappear in the XYLZ mutant.
Since in all three xylanase mutants the third peak of xylanase activity remained
in culture filtrates, this third xylanase must be encoded by yet another xylanase
gene. The single xylanase mutants were crossed to each other to obtain multiple
xylanase disruptions within the same strain. Strains disrupted in combinations
of two and in all three xylanases were obtained. The triple mutant grew at the
same rate as the wild type on xylan and maize cell walls. Additionally, the triple
mutant was still fully pathogenic on maize as determined by lesion size,

morphology, and rate of lesion development.

To my husband, Craig Birkhold, and my parents Jane and Milt Apel for all of

their loving support during the completion of this dissertation.

iv

ACKNOWLEDGMENTS

Many thanks to Dr. Jonathan Walton for his guidance and patience. I
am grateful for the input and encouragement from my guidance committee,
Dr. Ray Hammerschmidt, Dr. John Ohlrogge, and Dr. Helmut Bertrand. It is
with great appreciation that I would like to thank Dr. John Pitkin, Dr. John
Scott-Craig, and Dr. Daniel Panaccione for their scientific assistance with
this work. I thank Dr. James Anderson in Beltsville, MD, for the use of his
22-kDa xylanase anti-serum. Additionally, I thank Joe Leykam at the MSU
Macromolecular Facility, Michigan State University, for the amino acid
sequencing and oligonucleotide synthesis, as well as Kurt Stepnitz for his
photographic expertise. This research was supported by grants from the US.
Department of Agriculture NRICGP and the US. Department of Energy,
Division of Energy Biosciences. Amino acid sequencing was funded by grant
2-SO7-RR07049-15 awarded by the Biomedical Research Support Grant
Program, Division of Research Resources, National Institutes of Health.

The tobacco project was funded by the MSU/REF Center for New Plant

Products.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................ ix
LIST OF FIGURES ...................................................................................... x
LIST OF SYMBOL ..................................................................................... xii
CHAPTER 1: INTRODUCTION ................................................................. 1
Cell wall structure ............................................................................. 2
The role of cell wall degrading enzymes in pathogenesis ................ 3
Previous studies on the role of cell wall degrading enzymes in
pathogenesis ............................................................................ 4
Xylanases ........................................................................................... 6
Xylanases from plant pathogens ....................................................... 6
The fungal pathogen Cochliobolus carbonum .................................. 7
CHAPTER 2: MATERIALS AND METHODS ......................................... 10
Culture media and growth conditions ............................................ 10
Nucleic acid manipulations ............................................................. 1 1
Sequence and analysis .................................................................... 13
Construction of the XYLI disruption vector ................................... 14
Construction of the XYL2 disruption vector ................................... 14
Construction of the XYL3 disruption vector ................................... 14
Transformation-mediated gene disruption ..................................... 15
Analysis of disruption mutants ...................................................... l5
Xylanase activity assay ................................................................... 16
Pathogenicity tests .......................................................................... l7
Immunoblot analysis ....................................................................... l 7
Fungal crosses .................................................................................. 17

vi

CHAPTER 3: CLONING AND TARGETED GENE DISRUPTION
OF XYLI, A BIA-XYLANASE GENE FROM THE
MAIZE PATHOGEN COCHLIOBOLUS CARBON UM 18

INTRODUCTION ............................................................................ 18
XYLI isolation and characterization .................................... 19
Transformation-mediated gene disruption .......................... 21
Growth and pathogenicity of a XYLI mutant ..................... 26
DISCUSSION .................................................................................. 29
CHAPTER 4: CLONING AND TARGETED GENE DISRUPTION
OF XYL2 AND XYL3 ......................................................... 32
INTRODUCTION ............................................................................ 32
RESULTS ......................................................................................... 32
XYL2 and XYL3 isolation ..................................................... 32
Comparison of the three xylanases ...................................... 35
Transformation-mediated gene disruption of
XYL2 and XYL3 .......................................................... 35
Creation of a double mutant ................................................. 39
Growth and pathogenicity of the XYL2 mutant, XYL3
mutant, and the XYLI/XYL2 double mutant ............ 42
mRN A expression in culture and in infected leaves ........... 46
DISCUSSION .................................................................................. 49
CHAPTER 5: TRIPLE XYLANASE MUTANT ........................................ 53
INTRODUCTION ............................................................................ 53
RESULTS ......................................................................................... 53
DISCUSSION .................................................................................. 54
CHAPTER 6: CONCLUSIONS ................................................................. 61
APPENDIX: EXPRESSION OF A FUNGAL ENDO-XYLANASE
GENE IN TOBACCO .......................................................... 65
ABSTRACT ............................................................................................. 65
INTRODUCTION ............................................................................ 66
MATERIALS AND METHODS ....................................................... 67
Gene constructions ................................................................ 67
Plant materials, growth and transformation ....................... 68
Nucleic acid manipulations .................................................. 68
Xylanase activity assays ....................................................... 70
Immunoblot analysis ............................................................ 7 1
RESULTS ......................................................................................... 7 1
DISCUSSION .................................................................................. 74
REFERENCES ................................................................................ 76

vii

LITERATURE CITED ................................................................................ 79

viii

LIST OF TABLES

CHAPTER 3:

Table 1. Comparison of peptide sequences from tryptic
digests of xylanase I and the corresponding peptide

sequences deduced from the DNA sequence of XYLI ......... 20
CHAPTER 4:
Table 2. C. carbonum strains ......................................................... 42
CHAPTER 5:
Table 3. Crosses to obtain triple xylanase mutant ....................... 55
APPENDIX:
Table 4. XYLI constructs used and transformation results ......... 72
Table 5. Codon usage comparison .................................................. 75

ix

LIST OF FIGURES

CHAPTER 3:

Figure 1. Nucleotide and deduced amino acid sequences

of XYLI .................................................................................. 22
Figure 2. Comparison of bacterial and fungal xylanases

to XYLI from C. carbonum ................................................... 23
Figure 3. DNA blot analysis of wild type and disruption

mutants ................................................................................. 24
Figure 4. RNA blot of wild type and disruption mutant T2-8 ...... 25

Figure 5. Growth and xylan-degrading activity in wild-type
and mutant T2-8 strains ....................................................... 27

Figure 6. Cation exchange HPLC analysis of extracellular

xylanase activity in the wild type and mutant .................... 28

Figure 7. Pathogenicity test of xylanase mutant T2-8 .................. 30
CHAPTER 4:

Figure 8. Nucleotide and deduced amino acid sequences

of XYL2 .................................................................................. 34
Figure 9. Nucleotide and deduced amino acid sequences

of XYL3 .................................................................................. 36
Figure 10. Genomic Southern of XYLI, XYLZ and XYL3 ............. 37

Figure l 1. Comparison of the deduced amino acid
sequences of three C. carbonum xylanases .......................... 38

X

Figure 12. DNA blot analysis of wild-type and XYL2
disruption mutant ................................................................. 40

Figure 13. DNA blot analysis of wild-type and XYL3
disruption mutant ................................................................. 41

Figure 14. HPLC protein profiles of the wild type and
XYLZ mutant ......................................................................... 44

Figure 15. Xylanase activity from cation exchange HPLC
of the XYL2 mutant .............................................................. 45

Figure 16. Immunoblot of concentrated culture filtrate
of the wild-type and the XYLI mutant, XYLZ
mutant, and XYLI/XYL2 double mutant ............................. 47

Figure 17. Pathogenicity test of xylanase mutants
447 -8a,(XYL1'/XYL2*)T446- l7A(XYL1"/XYL2‘), and
477.7a(XYL1‘/XYL2’), and wild type strain

CHAPTER 5:

APPENDIX

367 ~2A(XYL l*/XYL2*). .......................................................... 48
Figure 18. RNA blot hybridized with XYLI, XYLZ and XYL3 ..... 50
Figure 19. RNA blot of infected maize leaves ............................... 5 1
Figure 20. DNA blot analysis of wild-type and

disruption mutants ............................................................... 56
Figure 2 1. RNA blot of wild-type and disruption mutants ........... 57
Figure 22. Growth of wild-type and mutant strains ..................... 58
Figure 23. Pathogenicity test of xylanase mutants ....................... 59
Figure 24. RNA blot of XYLI transgenic plants ............................ 73

xi

cDNA
kb
kDa
MS
XYLI
Xyll
XYLI’
XYL2
Xy12
XYLZ
XYL3
Xy13

XYLI

LIST OF SYMBOLS

........................................................................................ Base pair
............................................................................................ Celsius
........................................ Complementary deoxyribonucleic acids
.......................................................................................... Kilobase
....................................................................................... Kilodalton
.................................................................... Murashige and Skoog
..................................................... Gene for protein encoding Xyll
...................................................... Protein encoded by XYLI gene
........................................................ Disrupted copy of XYLI gene
..................................................... Gene for protein encoding Xy12
....................................................... Protein encoded by XYL2 gene
........................................................ Disrupted copy of XYL2 gene
..................................................... Gene for protein encoding Xy13
..................................................... Protein encoded by XYL3 gene

........................................................ Disrupted copy of XYL3 gene

xii

CHAPTER 1

INTRODUCTION

For years, plant pathologists have been trying to determine what
makes an organism a plant pathogen and what distinguishes saprophytic
organisms from pathogenic organisms. During the process of infection, a
plant pathogen must enter the plant. Pathogens may enter via stomata,
wounds, directly by mechanical pressure from melanized appresoria, or
enzymatically by cell wall degrading enzymes (Misaghi, 1982). Once inside,
pathogens must have mechanisms to overcome plant defenses and to spread
within the plant. Pathogens must also obtain nutrients once inside the plant
(Misaghi, 1982).

For both penetration and ramification, pathogens must deal with the
plant cell wall. The intact cell wall can be penetrated either by mechanical
pressure or by enzymatic degradation (Walton, 1994). Mechanical pressure
via melanized appresoria is required for penetration by two pathogens,
.Magnaporthe and Colletotrichum (V alent and Chumley, 1990; Kubo et al.,
1991). Other pathogens are thought to penetrate using cell wall degrading

enzymes, but proof of this is lacking (Cooper, 1984). The importance

2
of enzymatic penetration has been suggested by indirect evidence such as the

presence of cell wall degrading enzymes in infected tissue, by loss of host cell
wall polysaccharides during infection, and by ultrastructural studies (Cooper,
1984). A Colletotrichum lagenarium mutant that has melanized appressoria
is unable to form penetration hyphae (Katoh et al., 1988). The authors
propose that this may be due to the inability of the pathogen to secrete some
cell wall degrading enzymes (Katoh et al., 1988).
Cell wall structure

The plant cell wall is composed of many complex components which
are interlinked (Roberts, 1989; Darvill et al., 1980a). A major component of
the walls of all land plants is xylan. Xylan is a polymw of B-l,4-xylose with
substitutions of short chains of arabinose and glucuronic acid (W ada and
Ray, 1978; Kato and Nevins, 1984; Darvill et al., 1980a; Labavitch and Ray,
1978). In the angiosperm's secondary cell wall the most abundant
hemicelluloses are xylans (Aspinal, 1980; Burke et al., 1974; Wada and Ray,
1979). A small percentage of the primary cell wall of dicots is xylan, whereas
it is the major constituent of the secondary cell wall (Darvill et al., 1980b).
The monocot primary cell wall is at least 40% B-1,4 xylan (Burke et al., 1974;
Cooper, 1984; Darvill et al., 1980a). In maize, the host of the fungus
Cochliobolus carbonum, arabinoxylan is the major non-cellulosic component

of the primary cell wall (Kato and Nevins, 1984).

3
The role of cell wall degrading enzymes in pathogenesis

While the walls of plants contain many components such as xylan,
cellulose, and pectin, plant pathogens produce a large array of enzymes that
can degrade these components (Cooper, 1984). Some of the major groups of
cell wall degrading enzymes made by plant pathogens are xylanases,
cutinases, pectinases, cellulases and glucanases (Cooper, 1987). These
enzymes may be important for a variety of reasons. Enzymatic degradation
could be important for the plant pathogen to penetrate into the plant. It
could also be important for a pathogen to be able to ramify through the cell
walls as it moves through the tissues of the plant. The metabolism of the
degraded cell wall polymers could provide nutrients to support the growth
and development of the pathogen inside the plant (Cooper, 1984).
Additionally, certain cell wall degrading enzymes may not be necessary for
growth of the pathogen inside the plant, but may be necessary for nutrition
during over-wintering or saprophytic growth when the host plant is not
present.

Besides their possible role in pathogenesis, cell wall degrading
enzymes might also have a role in triggering host defenses. Some cell wall
degrading enzymes indirectly elicit host defense responses (Cervone et al.,
1987). For example, short oligogalacturonides produced by endo-
polygalacturonase (EC 3.2.1.15) cause phytoalexin accumulation in soybean

(Davis et al., 1984). In addition, superoxide radical formation, a putative

4
defense response, is stimulated by pectin lyase in rice protoplasts (Ishii,

1988).
Previous studies on the role of cell wall degrading enzymes in
pathogenesis

Some cell wall degrading enzymes may be involved in pathogenesis,
but others may not be necessary (Walton, 1994). Only by creating mutants
that specifically lack particular enzymes can the role of enzymes be
unequivocally tested (Cooper, 1987; Walton, 1994). Mutants have been used
to determine the relationship between specific cell wall degrading enzymes
and pathogenicity in both bacterial and fungal host-pathogen interactions
(Cooper, 1984; Payne et al., 1987 ; Reid and Collmer, 1988; Roberts et al.,
1988; Gough et al., 1988; Scott-Craig et al., 1990). After transposon
mutagenesis of the pectin methylesterase gene of Erwinia chrysanthemi
3937, the bacterium became non-invasive on Saintpaulia plants (Boccara and
Chatain, 1989). While some cell wall degrading enzymes have been shown to
be important for pathogenesis, others have been shown to be unimportant.
When all four pectate lyase genes in Erwinia chrysanthemi are deleted, the
bacterium's ability to degrade pectin is drastically reduced. However, the
bacterium can still grow on pectin as a carbon source and the mutant still
causes significant maceration of carrot, pepper, and potato tissue (Payne et

al., 1987; Reid and Collmer, 1988).

5
Pseudomonas solanacearum and Xanthomonas campestris are both

root pathogens and cause wilting by blocking water flow in the plant. Both of
these bacteria produce large amounts of endo-glucanase, which is thought to
contribute to maceration of the plant tissue during infection (Roberts et al.,
1988; Gough et al., 1988). Mutation of the endo-glucanase gene of P.
solanacearum reduces virulence on tomato (Roberts et al., 1988), but
disruption of the same enzyme in X. campestris does not reduce virulence on
radish and turnip (Gough et al., 1988).

While there are some examples of bacterial cell wall degrading
enzymes that are important in pathogenesis, there are no examples of fungal
cell wall degrading enzymes that are definitely important for pathogenesis.
The endo-polygalacturonase of Cochliobolus carbonum was demonstrated by
targeted gene disruption to be non-essential for the infection of maize (Scott-
Craig et al., 1990). Additionally, an alkaline protease, a cellobiohydrolase
and an exo-B 1,3-glucanase have been shown not to be required for
pathogenicity or contribute to virulence of C. carbonum on maize (Murphy
and Walton, 1996; Sposato et al., 1995; Schaefi‘er et al., 1994).

In Nectria haematococca (Fusarium solani f. sp. pisr), a cutinase has
been found to either contribute (Rogers et al., 1994) or not contribute (Stahl
et al., 1994) to virulence on pea. Disruption of a cutinase gene in
Magnaporthe grisea and in Alternaria brassicicola had no effect on

pathogenicity (Sweigard et al., 1992; Yao and Koller, 1995). However,

6
cutinase activity still remained in both cases (Sweigard et al., 1992 ; Yao and

Koller, 1995). A pectin lyase gene was disrupted in Glomerella cingulata but
the mutant retained pathogenicity on apple and Capsicum (Bowen et al.,
1995).
Xylanases

Many micro-organisms produce xylanases. Xylanases and cellulases
have been divided into families based on hydrophobic cluster analysis and
analysis of the catalytic sites (Henrissat et al., 1989). Xylanases are
currently divided into two categories, Family G and Family F (Henrissat et
al., 1989; Gilkes et al., 1991). Family G includes primarily low-molecular-
mass alkaline xylanases such as the 22-kDa Trichoderma viride xylanase
(Dean et al., 1989; Gilkes et al., 1991). Family F primarily includes high-
molecular-mass xylanases that can be either basic or acidic.
Xylanases from plant pathogens

Because such a large proportion of the monocotyledonous cell wall is
xylan, it is possible that enzymes that can degrade it play a major role in
pathogenesis of monocots. Many fungal pathogens make xylanases (Ii-1,4-
endo-xylanase, EC 3.2.1.8) including Cochliobolus heterostrophus
(Helminthosporium maydis), a species closely related to Cochliobolus
carbonum (Anderson, 1978; Cooper et al., 1988; Strobel, 1963; Bateman et al.,
1973, 1969; Reilly, 1981; Van Etten and Bateman, 1969; Mullen and

Bateman, 1975). Xylanases have been found in infected plant tissue (Mullen

7
and Bateman, 1975; Bateman et al., 1969; Cooper et al., 1988). When

monocot pathogens are grown on plant cell walls, xylanase is produced
earlier than other cell wall degrading enzymes (Mullen and Bateman, 197 5).
In lesions on wheat caused by Rhizoctonia cerealis, xylanase is the first
enzyme produced (Cooper et al., 1988).

While xylanases may be important for penetration of the plant cell
wall and ramification through the tissues, they may also play a role in the
elicitation of plant defense responses. The xylanase from Trichoderma viride
can elicit electrolyte leakage, ethylene biosynthesis, necrosis, and
pathogenesis-related proteins in tobacco (Dean and Anderson, 1981; Fuchs et
al., 1989; Bailey et al., 1990; Lotan and Fluhr, 1990; Raz and Fluhr, 1993).
The plant recognizes the xylanase and not the enzymatic products (Fuchs et
al., 1989). A single gene controls sensitivity of tobacco to the xylanase
(Bailey et al., 1993).

The fungal pathogen Cochliobolus carbonum

Cochliobolus carbonum causes Northern leaf spot of maize (Jennings
and Ullstrup, 1957). There are four races of the fungus. Race 1 produces a
host selective toxin, HC-toxin, a primary virulence factor, and causes large
oval lesions on lines of maize that are homozygous recessive at the Hm locus
(Walton, 1987). Resistant maize has a dominant allele which encodes an
enzyme that detoxifies the toxin (Meeley et al., 1992). Race 2 does not

produce a host selective toxin but causes small lesions on most inbred maize

8
lines (Jones and Dunkle, 1993). Both race 1 and race 2 cause small lesions

on maize resistant to the toxin. The remaining races of C. carbonum vary in
their morphological, pathogenic, and cultural characteristics (Jones and
Dunkle, 1993). Histological studies have shown that C. carbonum spores
germinate and penetrate resistant and susceptible leaves equally well.
Approximately 90% of the penetrations are direct while approximately 10%
of the infections are through the stomata (Jennings and Ullstrup, 1957).
Two lines of evidence suggest that cell wall degrading enzymes are
important in pathogenicity of C. carbonum. First, Cochliobolus appresoria
are only weakly melanized and melanin is not required for penetration
(Walton et al., 1995; Valent and Chumley, 1990; Kubo et al., 1991). Melanin
is not required for C. carbonum penetration into the host, suggesting that cell
wall degrading enzymes rather than mechanical pressure are involved in
penetration. Secondly, the mycelia of C. carbonum ramify both
intercellularly and intracellularly as the fungus infects the chlorenchyma.
The hyphae advance from cell to cell in advance of the necrotic lesions
(Jennings and Ullstrup, 1957). Cell wall degrading enzymes may be
important for growth through the walls of cells as the hyphae advance.
Because of the large amount of xylan in maize cell walls, xylanases may be
particularly important cell wall degrading enzymes during pathogenesis by

C. carbonum.

9
Previous work characterized three major endo-xylanases from culture

filtrates of C. carbonum grown on media containing xylan or isolated maize
cell walls (Holden and Walton, 1992). In order to determine if these
xylanases have any role, either as virulence factors or as triggers of plant
defense responses, xylanase-deficient mutants were made by cloning the
genes encoding xylanases and using transformation-mediated gene
disruption to create mutants. The mutants were then inoculated onto the
host and the lesion phenotype was observed. This was done for three
xylanase genes. The mutants were individually tested for pathogenicity on
maize. Additionally, the mutants were crossed to each other to obtain double
and triple mutants and their pathogenicity was tested on maize. The
expression of the three endo-xylanases was also examined during leaf

infection.

CHAPTER 2

MATERIALS AND METHODS

Culture media and growth conditions

Cochliobolus carbonum was maintained and grown as previously
described (Walton, 1987). Flask cultures were inoculated with two mycelia
plugs each approximately 1 cm2. For enzyme production, the fungus was
grown in still culture on mineral salts medium supplemented with trace
elements (Van Hoof et al., 1991), 0.2% yeast extract, and 0.2% sucrose. When
added, maize cell walls (English et al., 1971), oat spelt xylan (Fluka), or
additional sucrose were at final concentrations of 0.8%, 1.2%, and 2.0%,
respectively. For growth measurements, mycelia] mats were harvested,
washed briefly in distilled water, frozen, lyophilized to dryness, and weighed.

Mycelia for protoplast preparations or DNA extractions were obtained
from germinating conidia. Conidia were collected with 0.1% Tween 20 from
fungal cultures grown on V8 juice agar plates. The conidia were inoculated
into modified Fries' medium (Walton and Cervone, 1990) and grown for 14 hr
at room temperature in a gyratory shaker at 125 rpm. Protoplasts were

prepared from the resultant mycelia as described by Panaccione et al. (1989).

10

l 1
Nucleic acid manipulations

A genomic library of DNA from C. carbonum race 1, isolate SBl 1 1,
(ATCC 90305) in lambda EMBL3 vector with an average insert size of 15-20
kb (Scott-Craig et al., 1990) was screened with a 32-fold degenerate 17mer
oligonucleotide of sequence CAYTTYGAYGCNTGGGC. This sequence was
based on an amino acid sequence, HFDAWA, from a tryptic fragment of the
purified endo-B 1,4-xylanase of C. carbonum (Holden and Walton, 1992). The
oligonucleotide was labeled at the 5' end with T4 polynucleotide kinase
(Sambrook et al., 1989). Hybridizations with the oligonucleotide were done
overnight at 50°C in 5X SSPE (1X SSPE = 0.15M NaCl, 50 mM sodium
phosphate, 1 mM EDTA, pH 7.7), 5% SDS, 0.5% nonfat dry milk, and 0.1 mg
of denatured salmon sperm DNA per ml (Sambrook et al., 1989).
Nitrocellulose and Zeta-probe (BioRad, Richmond, CA) membrane blots were
washed three times in 2X SSPE and 0.1% SDS at 50°C for 20 min.

A cDNA library constructed from poly At mRNA from C. carbonum
grown on corn cell walls for 8 days was made in the phagemid vector Uni-Zap
XR with the Zap cDNA Synthesis Kit from Stratagene (Pitkin et al., 1996).
This library was screened with a sub-cloned fragment of XYLI DNA labeled
with 32P-dCTP by random prime labeling (Sambrook et al., 1989). The XYLI
and XYL2 cDNAs were isolated from this library.

DNA was transferred to Zeta-probe nylon membranes with 0.4 M

NaOH; RNA was transferred with 20X SSPE. Nucleic acids were fixed to the

12
membrane using a UV Stratalinker 1800 (Stratagene) at 1200 mjoule

according to the manufacturer’s instructions. Routine hybridizations with
sub-cloned DNA fragments labeled with 32P-dCTP by random prime labeling
(Sambrook et al., 1989) were done in 5X SSPE, 7% (w/v) SDS, 0.5% non-fat
dry milk, and 0.1 mg of denatured salmon sperm DNA per ml at 65°C
overnight. The blots were washed in 0.1X SSPE and 0.1% SDS with the final
wash at 65°C for 1 hr.

DNA and RNA were isolated from C. carbonum mycelium by the
method of Yoder (1988) except cresol and glass beads were omitted from the
RNA isolation protocol. For xylanase expression studies in infected leaves,
mRNA was isolated from infected lesions seven days post-inoculation.
mRNA was isolated using the mini-oligo (dT) cellulose Spin Column Kit (5'-3'
Inc., Boulder, CO) according to the manufacturer's instructions. For the
genomic DNA blots, 4 ug of DNA were loaded per lane of a 0.7% agarose gel
run for 17 hours at 17 V prior to blotting. For the RNA blots, 10 pg of total
RNA or 4 pg of poly A+ mRNA was loaded per lane of a 1.2 % denaturing
agarose gel (Sambrook et al., 1989). RNA electrophoresis was done in the
presence of formaldehyde at 70 V for 4 hours (Selden, 1987). GIBCO-BRL
RNA standards (0.24 — 9.5 kb) were used for transcript size estimation. The
clone of the glyceraldehyde-3-phosphate dehydrogenase gene from C.
heterostrophus was used as a reference for constitutive expression (Van Wert

and Yoder, 1992).

13
Sequence and analysis

The complete nucleotide sequences of both strands of a genomic clone
of XYLI were obtained by the dideoxynucleotide method using 32S-dATP
(Sanger et al., 1979). Sequencing reactions were performed with a
Sequenase Kit (United States Biochemicals, Cleveland, OH) and double-
stranded templates according to the manufacturer’s instructions. Sequencing
reactions were electrophoresed on 8% and 6% denaturing polyacrylamide
gels, soaked in 12% acetic acid and 12% methanol, dried, and exposed to X-
ray film according to Sambrook et al. (1989). When necessary, all restriction
site junctions created by sub-cloning were sequenced using the appropriate
template and sequence-specific primers. Sequence data were analyzed with
DNASIS and PROSIS programs (Hitachi Software Engineering Co., San
Bruno, CA), and the University of Wisconsin GCG software package
(Devereux et al., 1984).

The sequences of both strands of all of the cDNA clones and the XYL2
and XYL3 genomic clones were determined by automated fluorescence
sequencing by the MSU-DOE-PRL Plant Biochemistry facility using an ABI
Catalyst 800 (Foster City, CA) for Taq cycle sequencing and an ABI 373A
Sequencer (Foster City, CA) for analysis of the products. Sequence data were
analyzed with DNASIS and PROSIS programs (Hitachi Software

Engineering Co., San Bruno, CA).

14
Construction of the X YLI disruption vector

The transformation vector was created by first sub-cloning an internal
241-bp ScaI/BalI fragment of the XYLI gene into the SmaI site of pBluescript
H KS to create pCC 166. This plasmid was then digested with XhoI and
HindIII and ligated with the 2.5-kb Sail/HindIII fragment of pUCHl, which
contains a gene conferring hygromycin resistance (Schafer et al., 1989). The
product of these manipulations (pCC 167) was linearized at a unique SalI site
contained within the XYLI sequence prior to transformation of C. carbonum
isolate 164R10.

Construction of the X YL2 disruption vector

pHYGl was created by sub-cloning the SalI/HindIII fragment of
pUCHl, which contains a gene conferring hygromycin resistance, into
pBlueScript SK II. The transformation vector was created by sub-cloning a
532-bp HindII fragment of the XYL2 gene into the SmaI site of pHY G1,
which contains a gene conferring hygromycin resistance. The product of these
manipulations (pXLB37-2) was linearized at a unique BamHI site prior to
transformation of C. carbonum isolate 367 -2A.

Construction of the X YL3 disruption vector

pHYG2 was created by subcloning the SalI/HindIII fragment of
pUCHl, which contains a gene conferring hygromycin resistance, into pSP7 2.
The transformation vector was created by sub-cloning a 323-bp KpnI/XbaI

fragment of the XYL3 gene into the KpnI/Xbal fragment of pHYG2. The

15
product of these manipulations (pXLC42-l) was linearized at the unique

KpnI site prior to transformation of C. carbonum isolate 367 -2A.
Transformation-mediated gene disruption

Protoplasts for transformation were prepared by the method of Yoder
(1988) except Driselase (10 mg/ml) (Sigma D-9515) and Novozyme 234 (10
mg/ml) (N ovo Laboratories, Wilton, CT) were the only enzymes used. The
transformation protocol was derived from those developed for C.
heterostrophus ('I‘urgeon et al., 1987) and Aspergillus nidulans (Oakley et al.,
1987). Transformation of C. carbonum to hygromycin resistance was as
described by Scott-Craig et al. (1990).
Analysis of disruption mutants

The transformants were single-spared twice to purify them to nuclear
homogeneity. Xylanase was purified from 200 ml of culture filtrate of the
wild-type and mutants grown on HMT supplemented with 0.8% maize cell
walls for 8 days. The culture filtrate was filtered through cheesecloth and
Whatman #1 filter paper and then diluted two-fold with 25 mM sodium
acetate buffer, pH 5.0, and loaded onto a column of DEAE-cellulose (D-3764,
Sigma) with a bed volume of 33 ml. Following the DEAE-cellulose was a
column of CM-cellulose (C-4146, Sigma) with a bed volume of 33 ml. The
CM-cellulose was eluted with a 7 5-ml linear gradient from 25 mM sodium
acetate, pH 5.0, to 25 mM sodium acetate plus 0.4 M KCl, pH 5.0. The eluant

from the CM-cellulose was dialyzed (Spectrapor 6,000 to 8,000 MW cutoff, 32

16
mm diameter) overnight in 12.5 mM sodium acetate, pH 5.0. DEAE-cellulose

and CM-cellulose chromatography were followed by cation exchange HPLC.
Ion exchange HPLC was done on a 200 X 4.6 mm polysulfoethyl asp artamide
cation exchange column (The Nest Group, Inc., Southboro, Massachusetts)
using a linear gradient from 0-100% B in 30 min (Holden and Walton, 1992).
Buffer A was 25 mM sodium acetate, pH 5.0, and buffer B was buffer A plus
0.4 M KCl. One ml fractions were collected.
Xylanase activity assay

Xylanase activity was assayed by a reducing sugar assay (Lever,
1972). Thirty pl per HPLC cation exchange fi'action or 2 pg of protein fiom
total culture filtrate (protein was measured by the method of Bradford
(1976)) was assayed in a 300 pl reaction volume containing 1% oat spelt
xylan (Fluka), 50 mM sodium acetate, pH 5.0, at 37°C for 30 min. Twenty
five pl aliquots from the reactions were taken at 0 min and 30 min and added
to 1.5 ml of p-hydroxybenzoic acid hydrazide working solution which was
heated to 100 °C for 10 min prior to measuring the absorption at 410 nm.
The p-hydroxybenzoic acid hydrazide working solution was prepared from a
5% (w/v) stock solution in 0.5 N HCl by diluting 1:4 with 0.5 N NaOH. The 0
min sample absorbance was subtracted from the 30 min absorbance and

xylose was used as a standard.

17
Pathogenicity tests

Pathogenicity was tested by inoculating leaves of maize inbred K6 1
(genotype hm/hm) with a suspension of conidia (104/ml) in 0.1% Tween 20
harvested from fungal cultures grown on V8 juice agar plates as previously
described (Walton, 1987). All plants were inoculated at the 3 to 4 leaf stage
and the development of symptoms was visually recorded.

Immunoblot analysis

Twenty ml of culture filtrates of C. carbonum grown for 8 days on
maize cell walls was passed through a column of DEAE-cellulose
chromatography and then concentrated by precipitation with one-tenth
volume 100% (w/v) trichloroacetic acid. Eighty ug of protein of each sample
was fractionated on a 15% acrylamide SDS-PAGE gel. The gel was blotted to
nitrocellulose (0.2a pore size) and hybridized with anti-serum raised in
rabbit against the 22-kDa xylanase of Trichoderma viride (Dean et al., 1989)
at a 1:1000 dilution. The antibody was a gift from Dr. James Anderson
(USDA Beltsville, MD). The protein was visualized by cross reacting with a
goat anti-rabbit antibody conjugated with alkaline phosphatase (Cooper
Biomedical, Inc., Malvern, PA). Markers correspond to the prestained low
molecular weight standards from Gibco-BRL.

Fungal crosses
Fungal crosses were done as previously described (Y oder, 1988; Pitkin

et al., 1996).

CHAPTER 3*
CLONING AND TARGETED GENE DISRUPTION OF XYLI , A [31,4-

XYLANASE GENE FROM THE MAIZE PATHOGEN COCHLIOBOLUS
CARBON UM

INTRODUCTION
Because of the abundance of xylan in the cell walls of monocotyledons,
xylanase might be an important pathogenicity factor in diseases of this group
of plants. Holden and Walton (1992) identified three xylanases, xylanase I,
xylanase II, and xylanase IH, from the culture filtrate of C. carbonum. The
protein, xylanase I (Xyl 1), associated with the major xylan degrading
activity, was purified to homogeneity (Holden and Walton, 1992). This
xylanase was shown to be a basic, 22 kDa protein (Holden and Walton, 1992).
The protein was digested with trypsin and several peptide fragments were
sequenced (Holden and Walton, 1992).
*The majority of these results were originally published in Molecular Plant-
Microbe Interactions
©1993, The American Phytopathological Society
Apel, P.C., Panaccione, D.G., Holden, RR, and Walton, RD, (1993) Cloning
and targeted gene disruption of XYLI, a BIA-Xylanase gene from the

maize pathogen Cochliobolus carbonum. Mol. Plant-Microbe
Interactions 6(4):467 -47 3.

18

19
RESULTS

X YLI isolation and characterization.

Six tryptic peptides were isolated from xylanase I (Xyll) and
sequenced (Table 1) (Holden and Walton, 1992). A completely degenerate 17 -
mer oligonucleotide coding for the amino acid sequence HFDAWA was used
to screen a lambda EMBL3 genomic library from C. carbonum (Scott-Craig et
al., 1990). The xylanase-encoding sequences were subcloned into pBluescript
and pUCl8. Figure 1 shows the genomic sequence of XYLI and the deduced
amino acid sequence. The region identified by the probe is overlined. The
sequenced peptides corresponding to those in Table 1 are double underlined.
A 53-bp intron, identified by sequencing a corresponding cDNA, is indicated
by italics in Figure 1. The intron contains two stop codons and a frame shift,
as well as 5' GivGTATGG (consensus GivGTAN GT) and 3' ACAG (consensus
ACAG) intron splice sites and an internal CACTAAC (consensus TACTAAC)
sequence (Ballance, 1986).

A possible translational start site at nucleotide 226 (Figure 1) has a
proper consensus context of CAAAA_TGGT (consensus CAMMA__T_GNC, where
M = A or C) (Ballance, 1991). Since the N-terminus of the enzyme was
blocked (Holden and Walton, 1992) the exact start of the mature protein is
not known. Seventeen of the first 30 amino acids, including the putative
start methionine, are hydrophobic amino acid residues (Leu, Phe, Val, He,

and Ala). The known signal peptide of the endo-polygalacturonase from C.

20

Table 1. Comparison of peptide sequences from tryptic digests of xylanase
I and the corresponding peptide sequences deduced from the DNA
sequence of X YLI.

 

Tryptic Peptide Deduced Amino Acid

Se quencea Se quenceb
ATYTNGAGGSYSVSWGSGGNNV ATYTNGAGGSYSVSWGSGGNIV
GIN PGTAR GwNPGTAR
LAV(Y)A LAVYg
NPLVEYYVVENFGTY DP(P/S)SQWQN K N PLVEYYVVEN FGyYD PSSQsQN K
TFQQYWSVR TFQQYWSVR
THFDAWA(S)(A/K) TH FDAWASK

.Amino acids in parentheses indicate uncertainties in the peptide sequencing
(Holden and Walton, 1992).

bLower case letters denote mismatches between the protein sequence of the tryptic
digests and the deduced amino acids from the DNA sequence (Apel et al., 1993).

carbonum (Scott-Craig et al., 1990) ends in LDAR; XYLI contains LVAR
starting at amino acid 27 (Figure 1). Therefore, the mature xylanase protein
probably begins at amino acid 31. Based on the cDNA sequence, the
polyadenylation site is at nucleotide 1 130, 184 bp past the stop codon (Figure
1). There is no apparent polyadenylation consensus sequence (Ballance,
1986). The predicted mature protein encoded by XYLI has 191 amino acids,
a M of 20,869, and a pI of 9.1. These figures are close to the size (22,000 to
24,000) and pI (greater than 9.3) measured for the enzyme (Holden and
Walton, 1992).

XYLI has a high degree of similarity to several prokaryotic and
eukaryotic xylanases (Figure 2). Between amino acid 30 and 209 of the C.

carbonum xylanase, the identity ranges from 38% to 68%. The amino acid

2 1
sequence TFXQYWSVR obtained from the enzyme (Holden and Walton,

1992) is one of the most highly conserved motifs in these xylanases (Figure
2).
Transformation-mediated gene disruption.

In order to disrupt XYLI, the linearized transformation plasmid,
pCC 167, was used to transform C. carbonum to hygromycin resistance.
Nineteen hygromycin resistant transformants were obtained and nine of
these were purified by single-sparing. Five of the single-spored isolates were
analyzed at the molecular level by digestion of their DNA with Apal and
blotting; the KpnI/KpnI and KpnI/SacI (nucleotides 342-685 and 686-1634;
Figure 1) fragments of XYLI were used together as a hybridization probe.
Based on the hybridization pattern, one transformant ('I‘2-4) was a single
insertional mutant, two transformants (one of which is T2-8) were multiple
insertional mutants, and the remaining two transformants that were
analyzed appeared to have ectopic integration. The pattern of hybridization
of transformant T2-4 with hybridizing band sizes of 4.8 kb and 8.1 kb, and no
band at 6.9 kb, is consistent with homologous integration of a single copy of
pCC 167 at XYLI (Figure 3). The pattern obtained with DNA from
transformant T2-8 is consistent with multiple tandem integrations at XYLI,
_ as evidenced by bands at 4.8 kb and 8.1 kb and a strong band at 5.7 kb
(Figure 3). The XYLI mutant T2-8 lacks a 1.0-kb XYLI RNA that hybridizes

to XYLI (Figure 4).

22

l GATCTACACCCTAGCTAATATTACTCTAGCAGATGGAATACCGCTATCATGACGAGGATG

61 TTTGGCTTGGCTGTGCACAACATCCGGTCTTGTTTCCACTCACATGCCATGCGAGGGCGA

121 GTATAAAGCTATCCTGAGTCGGCTCCATGGCACACTTTCCTCACCAACCATCACCACAAC

181 TACCACTTCCCTTCGCTTCATTCACTCAACAACAACACCATCAAAATGGTTTCTTTCACC
M V S F T

241 TCCATCATCACCGCTGCTGTTGCGGCTACCGGCGCTCTTGCCGCCCCCGCCACTGATGTG

6 S I I T A A V A A T G A L A A P A T D V
KEnI

301 TCTCTCGTTGCCCGTCAGAACACCCCCAACGGCGAGGGTACCCACAACGGCTGCTTCTGG

26 S L V A R Q N T P N G E G T H N G C P w

361 TCTTGGTGGTCTGATGGCGGTGCCCGCGCTACCTACACCAACGGTGCCGGTGGTAGCTAC
46 S W W S D G G A R A T Y T N G A G G S Y

421 AGCGTAAGCTGGGGAAGCGGTGGCAACCTCGTCGGTGGAAAGGGATGGAACCCAGGAACT
66 S V S W G S G G N L V G G K G W N P G T

481 GCCCGGTATGGACTGTCTTACTCAACTCTGATAGACTACATACACTAACATTGATACAGT
86 AR

 

541 ACCATCACCTACTCTGGTACTTACAACTACAACGGCAACTCCTACCTTGCCGTCTACGGC
88 T I T Y S G T Y N Y N G N S Y L A V Y G
Seal
601 TGGACCCGCAACCCCCTTGTCGAGTACTACGTCGTTGAGAACTTCGGCACCTACGACCCC
108 W T R N P L V E Y Y V V E N F G Y Y D P
KEnI
661 TCTTCCCAGTCCCAGAACAAGGGTACCGTCACCTCTGATGGATCTTCCTACAAGATCGCT
128 S S Q S Q N K G T V T S D G S S Y K I A
SalI
721 CAGTCGACCCGTACCAACCAGCCCTCCATCGATGGCACCAGGACCTTTCAGCAGTATTGG
148 Q S T R T N Q P S I D G T R T P Q Q Y W
01192
781 TCTGTTCGTCAGAACAAGCGCTCTTCCGGCTCCGTCAATATGAAGACTCACTTTGACGCC
168 S V R Q N K R S S G S V N M K T H E D A
#1 Ball
841 TGGGCCAGCAAGGGCATGAACCTTGGCCAGCACTACTACCAGATTGTTGCCACCGAGGGT
188 W A S K G M N L G Q H Y Y Q I V A T E G

901 TACTTCTCCACTGGTAACGCCCAGATCACCGTCAACTGCCCATAAATTCTTCACCACGAA
208 Y F S T G N A Q I T V N C P *

961 GATTAAACGAATGGCCCTGATGGCTCTCTGAACGACTGGAAGATGACAGGCGTGGTTTTT
1021 GGTTACGTGCTGTGCTAGCTGAGCTGGTGGATTCTTTTCTGTATATACTATCTTTGTCAA
1081 CCCGATTTGTCTATAGCTTAACAATGTCAAAATCTTGGCATTACTCACAACGCGTCTTTT
1141 CATTTTTGCCTCTAAGTGAATTGTGATGGTCTTGACGCATGATGACGTCTACTACGAGCA
1201 TTCTCTCCGGTGTCCTTCTAATGATAAAAATAAACAAACTTCAATACTCGCAATATGAAA
1261 CTCCGCAGTACCAAATTTAGATGTTTGACTTCACAATAGGCTGGCCTATCTACGCTGCCT
1321 CATCTATTACATACAACGGCATCGTACCAGGAGTCATCCTCAGTGCCTCCTCAATGATTC
1381 CCAATGCAACATGCAACTCCTCTTCCATAACAGTAATCGGGTGTGCAATTCTGAAAACAC
1441 CACCAAGCGACGCCATTGTCGCCAACTGCGCCCACAAACCCATTTTCTCCATCTTTTGCT
1501 ACACAGCAGCTCCGAAATCAGGAGTCCCAACCGTAAGAAGGTGCAGCCTTCAGTGTTACT
1561 GGAGGCACCGCTAGTGTGGTGGTGCTTTCCCAATATAGCTTAGTGCAAATAGTAGGTGGG

SacI
1621 TAGCATCTCGCTGAGAGCTC

Figure 1. Nucleotide and deduced amino acid sequences of X YLI.
The double underlined regions correspond to the sequences of tryptic
fragments obtained from the purified protein (Table 1). The overlined region
indicates the location of the oligonucleotide used to clone the gene. The
italicized sequence indicates the intron. The endonuclease restriction sites
KpnI, ScaI, SalI, Ball, and SacI are indicated.

23

CCl 1 HVSFTS IITAAVAAT. CALAAPAT.. ..DVS... ................. LVA
BC 3 KFKKNFLVG. ..LSAAIMSI SLFSATASAA S .......................... TD.
Sp 4 RKLRLLFVMC IGLTLILTAV .................................... PAHA
Bs 3 KFKKNFLVG. ..LSAALHSI SLFSATASAA S .......................... TD.
Ca 1 HLRRXVIFTV LATLVMTSLT .IVDNTAFAA TNLNTTESTF SKEVLSTQKT YSAFNTQAAP
Rf ? KIKKVLSGTV SAIMIASAAP VV .....................................
Sb 20 RSAHA ..... .VAIAR.SPL .HLPGTAQAD T .......................... VV.
SC 22 SRRGFLGGAG TLAIATASGL .LLPGTAHAA T .......................... TI.
An 1 M KVTAAFAGLL VTAFAAPVPE PV ......................... LVS
Tr 1 HVSFTS L ......... .LAASPPSR. ASCRPAAEVE .......... SVAVEK
H1 1 HVSlKS VLAAATAVSS .AIAAPFDFV .PR.DNSTA. ................. LQA
Mg 1 HVSFTS IVTAVVALAG SALAIPAPDG NMTGFPFEQ. ................. LHR
CCl 30 RQNTPNGEGT HNGCFWSWWS DGGARATYTN GAGGSYSVSW GSCGNLVGGK GWNPGTA.RT
BC .......... ....YHQNWT DGGGIVNAVN GSGGNYSVNH SNTGNFVVGK GWTTGSPFRT
Bp RTITNNEMGN HSGYDYELWK DYGHTSHTLN NG.GAFSAGH NNIGNALFRK GKKFDS.TRT
BS .......... ....YWONWT DGGGIVNAVN GSGGNYSVNN SNTGNFVVGK GWTTGSPFRT
Ca KTITSNEIGV NGGYDYELHK DYGNTSMTLK NG.GAFSCQW SNIGNALFRK GKKFND.TQT
Rf SAADQOTRGN VGGYDYEHWN QNGQGQASHN PGAGSFTCSW SNIENFLARM GKNYDSQKKN
Sb ...TTNQEGT NNGYYYSFWT DSQGTVSMNH GSGGOYSTSW RNTGNFVAGK CWANGGR.RT
SC ...TTNQTGT .DGMYYSFWT DGGCSVSMTL NGGGSYSTQH TNCGNFVAGK GWSTGD..GN
An RS ..... AGI .N.YVQNYNG NLGDFTYDES AFTFSMYWED GVSSDFVVGL GWTTGSSKA.
Tr RQTIQPGTGY NNGYFYSYWN DGHGGVTYTN GPGGQFSVNH SNSGNFVGGK GWQPGTHNKV
Hi RQVTPNAEGW HNGYFYSWWS DCGGOVQYTN LEGSRYQVRH RNTGNFVGGK GWNPGTG.RT
Hg RQSTPSSTGR HNGYYYSWWT DGASPVQYON GNGGSYSVQH QSGGNFVGGK GWHPGGSKS.
Ccl 89 ........ IT YS.GTYNYNC NSYLAV.YGW TRN.PLVEYY VVENFGTYDP SSQSQ.NKGT
BC ........ IN YNAGVWAPNG NCYLT.LYGH TR.SPLIEYY VVDSWGTYRP T...GTYKGT
Bp HHQLCNISIN YNAS.FNPSG NSYLCV.YGH TQ.SPLAEYY IVDSHGTYRP TC..AY.KGS
BS ........ IN YNAGVHAPNG NGYLT.LYGH TR.SPLIEYY VVDSWGTYRP T...GTYKGT
Ca YKQLGNISVN YDCN.YQPYG NSYLCV.YGH TS.SPLVEYY IVDSWGSWRP PG..GTSKGT
Rf YKAFGNIVLT YDVE.YTPRG NSYMCV.YGW TRN.PLHEYY IVEGWGDWRP PGNDGEVKGT
Sb ........ VQ YS.GSFNPSG NAYLA.LYGW TSN.PLVEYY IVDNHCTYRP T...GEYKGT
SC ........ VR YN.GYFNPVG NGYCC.LYGW TSN.PLVEYY IVDNWGSYRP T...GTYKGT
An ........ IT YSAEYSASGS SSYLAV.YGW V.NYPOAEYY IVEDYGDYNP CS.SATSLGT
Tr IN FS.GSYNPNG NSYLSV.YGW SRN.PL1EYY IVENFGTYNP STGAT.KLGE
Hi ........ IN YG.GYFNPQG NGYLAV.YGH TRN.PLVEYY VIESYGTYNP GSQAQ.YKGT
Hg ....... ITY SGTFNPVNNG NAYLCI.YGH TQN.PLVEYY ILENYGEYNP GNSAQ.SRGT
Ccl 137 VTS DGSSYK IAOSTRTNQP SIDGTR.TFQ QYNSVRQNKR ...S ..... S GSVNHKTHPD
BC VKS DGGTYD IYTTTRYNAP SIDGDRTTFT QYNSVRQSKR PTGS ..... N ATITFTNHVN
Bp FYA DGGTYD IYETTRVNOP SIIGI.ATFK QYHSVRQTKR .T.S ...... CTVSVSAHFR
BS VKS.DGGTYD IYTTTRYNAP SIDGDRTTFT QYWSVRQSKR PTGS ..... N ATITFSNHVN
Ca ITV.DGGIYD IYETTRINQP SIQGN.TTFK QYHSVRRTKR .T.S ...... GTISVSFHFA
Rf VSAN.GNTYD IRKTMRYNQP SLDGT.ATFP QYHSVRQTSG SANNQTNYHK GTIDVTHHFD
Sb VTS.DGGTYD IYKTTRVNKP SVEGTR.TFD QYHSVRQSKR .T ....... G CTITTGHHPD
SC VSS.DGGTYD IYQTTRYNAP SVEGTK.TFQ QYHSVROSKV .T.SG S GTITTGNHFD
An VYS.DGSTYQ VCTDTRTNEP SITGTS.TFT QYISVRESTR .T.S ...... GTVTVANHFN
Tr VTS.DGSVYD IYRTQRVNQP SIIGTA.TFY QYWSVRRNHR ...S ..... S GSVNTANHFN
Hi FYT.DGDQYD IFVSTRYNQP SIDGTR.TFQ QYWSIRKNKR V.G ....... GSVNHONHFN
Hg LQA.AGGTYT LHESTRVNQP SIEGTR.TFQ QYWAIRQQKR ..NS ...... GTVNTGEFFQ
Ccl 187 AWASKGHNLG .QH.YYQIVA TEGYFSTGNA QITVNCHKFF TTKINEHPDR LSERLEDDRL
BC AHKSHGMNLG SNWA.YQVMA TEGYQSSGSS NVTVWO

Bp KHESIBMPMG KMYETAFTV. .EGYOSSGSA NVMTNQLFIG N.

BS AHKSHGHNLG SNHA.YQVMA TEGYQSSGSS NVTVH'

Ca ANLSKGHPLG KMHETAFNI. .EGYQSSGKA DVNSMSINIG K‘

Rf AWSAAGLDHS GTL.YEVSLN IEGYRSNGSA NVKSVSVTQG CSSDNGGQQQ NNDWNQQNNN
Sb AWAPAGMPLC .NFSYYHIHA TEGYQSSGTS SINVGGTGGG DSGCCDNCGG GGGCTRRCPP
SC AWARAGMNMG .QFRYYMIMA TEGYQSSGSS NITVSG'

An FHAOHGFGNS .DF.NYQVHA VEAHSGAGSA SVTISS'

Tr AWAQQGLTLG .TM.DYQIVA VEGYFSSGSA SITVS'

Hi AWQQHCHPLG .QH.YYQVVA TEGYQSSGES DIYVOTH'

Hg AWERAGHRMG NHN.Y.HIVA TEGYRSAGNS NINVQTPA*

Ccl 145 GF‘

Figure 2. Comparison of bacterial and fungal xylanases to X YLI
from C. carbonum. Shading denotes identity between XYLI and at least
one other xylanase. Cc=Cochliobolus carbonum; Bc=Bacillus circulans
(GenBank accession number X07723), Bp=B. pumilus (X00660), Bs=B.
subtilis (M36648), Ca=Clostridium acetobutylicum (M31726),
Rf=Ruminococcus flavefaciens (Z1 1 127), Sb=Streptomyces lividans xylanase
B (MG4552), Sc=S. lividans xylanase C (MG4553), An=Aspergillus niger
(A19535), Tr=Trichoderma reesei xylanase I(S5 197 3), Hi=Humicola insolens
(X76047), and Mg=Magnaporthe grisea (1137529).

Numbering is fiom the known or putative translational start sites.
The sequence for the R. flavefaciens xylanase gene in GenBank is partial.
Alignment was done using BESTFIT.

24

A w: '1‘2-4'1‘2-8 kb

-I4.I

— - 8.4

- - - 7.2
- 5.7

— - 4.3

‘ - 3.6

- 2.3

GENOMlC RESTRICTION MAP - WILD TYPE

A —.

6.9 kb
GENOMlC RESTRICTION MAP - MUTANTS
Single Insertion (T2—4)
A

._ A o A
4.5 kb 8.] kb
Multiple Insertions (T2-8)
A — A — A - A
'— ‘——1 —r——
”45—147; 8.1 kb
5.7 kb

Figure 3. DNA blot analysis of wild type and disruption mutants.

A. Genomic DNA from C. carbonum wild type (Wt) and the putative XYLI
disruption mutants T2-4 and T2-8 was digested with Apal, fractionated,
blotted, and hybridized with the KpnI/KpnI and KpnI/SacI fragments of
XYLI.

B. Predicted restriction map. The black shading indicates XYLI and the
lightly shaded box denotes the gene for hygromycin phosphotransferase.
The hybridization seen in part A is consistent with the predicted single
and multiple integration patterns. The arrow indicates the direction of
transcription of XYLI. A=ApaI endonuclease restriction sites.

25

A Wt T2-8
V
Y
. ' ' - 1.01:1:
B

g ' -l.4kb

Figure 4. RNA blot of wild type and disruption mutant T2-8.

A. Approximately 30ug total RNA, isolated from fungal mats of the wild type
and T2-8 grown for 6 days on maize cell walls, was fractionated on a 1.2%
agarose gel containing formaldehyde and blotted to a nylon membrane.
The blot was probed with the KpnI/SacI genomic DNA fragment (pCC 157)
corresponding to nucleotides 686-1639 of XYL1 (Figure 1).

B. The blot was stripped and rehybridized with the glyceraldehyde-3-
phosphate dehydrogenase gene.

26
Equivalent loading of RNA in each lane was confirmed by stripping and

probing the same blot with the glyceraldehyde-3-phosphate dehydrogenase
gene of C. heterostrophus (Figure 43).
Growth and pathogenicity of a XYL1 mutant.

Wild type and mutant T2-8 strains were grown on a mineral salts
medium supplemented with trace elements and yeast extract and containing
either maize cell walls, oat spelt xylan, or sucrose as the sole carbon source.
There was no significant difference in growth of the wild type and the
disruption mutant T2-8 when grown on the same substrate (Figure 5A).

Culture filtrates from the growth studies in Figure 5A were assayed
for xylanase activity. When grown on maize cell walls for 8-16 days, the
mutant had a 85%-94% reduction in extracellular xylanase activity compared
to the wild type (Figure 5B). Xylanase activity in mutant T2-4 was decreased
by a similar amount (data not shown).

Xylanase activities from mutant T2-8 and wild type were purified in
parallel through cation exchange HPLC (Holden and Walton, 1992). The
XYL1 mutant specifically lacked the major peak of UV absorption,
corresponding to the major xylanase, xylanase I, in fraction 7 (Figure 6)
(Holden and Walton, 1992). The transformant also lacked the second peak of
activity (xylanase ID, in fraction 9, but maintained the third peak of activity
(xylanase III), in fraction 12 (Holden and Walton, 1992). Barring any

epistatic effects of XYLI expression on the expression of xylanase II, we

27

   
 

 

I O A
SUCFOSG
A 0.8
E .
3
E) l
.3 _ / xylan
3 0.4
5‘
O . A
O '7 ‘
‘- _ - _ _ _ — — a
o—‘I’J__—_1—‘_-§
r I
cell wells
0 0 ~
5.0 - I I
B. R
’ \
’ \
/
C‘ l \
{I 4.0 ' ’ \
Q) I \
8 ’ \
3. ’ \
: P ‘
0 3.0 - ’ \
E ’ ‘
v / \
>‘
:3 I, b
g 2.0 . ’
a) I
8 /
C i
O
3‘ /
x 1.0 . ’
/
I
I ”no -
oo- . r“ 4"" f fi‘ '
O 5 IO 15

Age of Culture (doys)

Figure 5. Growth and xylan-degrading activity in wild-type and

mutant T2-8 strains.

A. Dry weights of fungal mats. .

B. Xylanase activity in the culture filtrates from the same experiment.
A = Wild type grown on sucrose; A = Mutant grown on sucrose; D = Wild
type grown on oat spelt xylan; I = Mutant grown on oat spelt xylan; o =
Wild type grown on maize cell walls; 0 = Mutant grown on maize cell
walls. All treatments were done in duplicate and the range was less than
10% of the average.

X,ln<e released, nrwol

 

e"), V‘MOI

SP relens

 

 

Figure 6. Cation exchange HPLC analysis of extracellular xylanase
activity in the wild type and mutant. One mg of total protein (Bradford,
1976) from partially purified culture filtrate of each was loaded on the
column. The solid line is the absorbance at 280nm and circles are enzyme
activity. The peaks of activity for xylanases I, II, and III appear in fractions
7, 9, and 12, respectively (Walton and Holden, 1992). A. wild-type B.
mutant T2-8.

29
conclude that xylanases I and II are both encoded by XYLI, but that xylanase

IE is encoded by a different gene.

The rate of development, size, and morphology of the lesions produced
by the wild type and mutant T2-8 on susceptible maize appeared the same up
to seven days after inoculation (Figure 7).

DISCUSSION

XYL1, the gene for the major xylan-degrading enzyme in C. carbonum,
was cloned and sequenced. Transformation-mediated gene disruption
created a mutant specifically lacking a functional copy of this gene, as shown
by DNA and RNA blotting and biochemical characterization.

XYLI appears to encode two forms of endo-xylanase activity (xylanase
I and II, Holden and Walton, 1992), which together are responsible for 85-
94% of the extracellular xylan-degrading activity of C. carbonum when it is
grown on maize cell walls (Figure 5B). However, growth of the mutant strain
was indistinguishable from the wild type in media containing maize cell
walls or xylan as the sole carbon source. The residual xylan-degrading
activity in the mutant is apparently sufficient to support wild-type growth
rates on xylan. The pathogenicity tests indicate that XYLI is dispensable for
pathogenicity on maize. Whether xylan degradation is important or not in
pathogenicity is still an open question, however, due to the low but

significant amount of xylanase remaining when XYL1 is disrupted (Figure 5).

 

30

164R II)

 

Figure 7. Pathogenicity test of xylanase mutant T2-8. Maize leaves
were inoculated with 104 spores per ml of the wild-type strain 164R10 and
the disruption mutant T2-8. Photograph was taken seven days after
inoculation.

 

 

“LE.

31
Some studies allude to the possible importance in pathogenicity of

enzymes such as a-arabinosidase and arabinanase that can at least partially
degrade native plant xylans (Howell, 1975; Cooper et al., 1988). The oat spelt
xylan used in our growth studies and enzyme assays to contains only 60%
xylose, with the remainder being mainly arabinose and glucose (W eimer,
1985). Therefore, metabolism of the non-xylan components of oat spelt
"xylan" might account for both the growth of the XYLI mutant on xylan and
for at least some of the apparent residual "xylanase" activity in culture
filtrates of the mutant. Xylanase I, Xyll, is clearly a xylanase by its
similarity to other fungal and bacterial xylanases, but xylanase III, and any
other xylanases made by C. carbonum might actually catalyze the

depolymerization of substrates other than B 1,4-1inked xylose.

CHAPTER 4

CLONING AND TARGETED GENE DISRUPTION OF XYLZ AND XYL3

INTRODUCTION

Cochliobolus carbonum makes three extracellular xylanases in culture
(Holden and Walton, 1992; Apel et al., 1993). XYLI, the gene encoding the
major xylan degrading activity, was cloned and disrupted in order to test its
involvement in pathogenicity (Apel et al., 1993). XYL1 is not required for
pathogenicity nor does it contribute to virulence of Cochliobolus carbonum on
maize. However, xylanase may still be important for C. carbonum infection
of maize because the XYL1 mutant still has xylan degrading activity (Apel et
al., 1993). The purpose of this work was to clone and disrupt two additional
xylanase genes that are related to XYL1.
RESULTS
X YL2 and X YL3 isolation

XYL2 was cloned using the XYL1 genomic clone to screen a cDNA
library constructed from mRNA from C. carbonum race 1 (isolate SBl 1 1)
grown on maize cell walls for 8 days. A genomic clone for XYL2 was obtained

by screening a lambda EMBL3 genomic DNA library with the XYLZ cDNA.

32

 

 

 

33
A XYL3 genomic clone was obtained by screening the EMBL3 genomic

library with the same 17-mer oligonucleotide that was used to clone the
genomic XYLI clone.

Figure 8 shows the nucleotide sequence and the deduced amino acid
sequence of XYLZ. The region identified by the oligonucleotide is overlined.
There are two introns, of 53-bp and 56-bp, based on a comparison of the
genomic DNA and cDNA sequences of XYL2. The first intron contains five
stop codons and a frame shift as well as a 5’ GlGTAGGT (consensus
GIGTAN GT), 3’ GTAG (consensus ACAG) intron splice sites and an internal
TACTAAT (consensus TACTAAC) sequence (Ballance, 1986). The second
intron has six stop codons and a frame shift as well as 5’ GIGTAAGT
(consensus GIGTAN GT), 3’ ATAG consensus ACAG) intron splice sites. A
possible translational start site (Figure 8) has a consensus context of
CAAGA’LGGT (consensus CAMMA_TG_rNC, where M = A or C)(Ballance, 1991).
Twenty two of the first 40 amino acids including the putative start
methionine are hydrophobic amino acid residues (Met, Leu, Phe, Val, Ile, and
Ala). Based on similarity to XYLI (Apel et al., 1993) and the known signal
peptide of endo-polygalacturonase from C. carbonum (Scott-Craig et al.,

1990) the mature xylanase protein probably begins at amino acid 41 (Figure
8). There is one potential glycosylation site at amino acid 32 (Figure 8).
The DNA and deduced amino acid sequences of XYL3 are shown in

Figure 9. XYL3 has a putative intron of 60 bp that occurs in the same

34

1 CCCGGGTAGAGCGATGTTCTTTTAATTATGTTATGCAGATTTACGCAAGGGCTACAGATC
6O TTTTTGATCACCCCGTCGTGGCCGTTGCTACCGAAACTGGATGCCGATCGGTGCATGTCC
121 CCTATGTGTCTCGACTTGGTCATTGTGGCCGCCGCTTAGGGTGAGCTTGTCGAAGTGAGA
181 GGCCTTAACCTACCACCAATGTGTACACCATCACGAATAGAGACAGCACGCGAGATGAGA
241 CCTCATGACCACCAGTGTCATTTCATCCTGCGATTCAGGCTCAACTATTACTCGGCGTAG
301 AGATATAAGTCGATAGCTGCTCCCAGTCAGTAGCATCACTGCATCCAACACAGATCAGCA
361 ACACTCAACACAAGATGGTTTCCTTCAAGTCTCTGCTTCTCGCCGCTGTGGCTACCACCA

M V S F K S L L L A A V A T T S

421 GCGTCCTCGCTGCTCCCTTCGATTTCCTTCGTGAGCGCGACGATGTCAACGCGACTGCTC
17 V L A A P F D F L R E R D D V N A T A L
481 TCCTTGAGAAGCGTCAGTCTACTCCCAGCGCCGAGGGATACCACAATGGATACTTCTACT
37 L E K R Q S T P S A E G Y H N G Y E Y S
541 CGTGGTGGACTGATGGCGGTGGCTCTGCCCAGTACACTATGGGTGAGGGCAGCAGGTACT
57 W W T D G G G S A Q Y T M G E G S R Y S
601 CTGTGACCTGGAGGAACACTGGCAACTTTGTTGGTGGAAAGGGGTGGAACCCTGGAAGCG
77 V T W R N T G N F V G G K G W N P G S G
661 GCCGGTAGGTTGCGAAGACTGTTTGGTGATGAGAATCTTACTAATGTGGATTCGTAGTGT
97 R V
721 CATCAACTACGGCGGAGCCTTCAACCCCCAGGGCAACGGATACCTCGCTGTGTACGGATG
99 I N Y G G A F N P Q G N G Y L A V Y G W
781 GACCCGCAACCCGCTTGTCGAGTACTACGTGATTGAATCCTACGGAACCTACAACCCCAG
119 T R N P L V E Y Y V I E S Y G T Y N P S
841 CAGTGGAGCCCAAATCAAGGGCAGCTTCCAGACCGACGGTGGTACTTACAACGTTGCCGT
139 S G A Q I K G S E Q T D G G T Y N V A V
901 CTCCACCCGTTACAACCAGCCCTCCATTGACGGAACAAGGACCTTTCAGCAGTACTGGTA
159 S T R Y N Q P S I D G T R T E Q Q Y W
961 AGTCATTTCGGTGTTTAATCAGACAGAAGCGTAGATGTTGACTGTTATGATAGGTCTGTC
178 S V
1021 CGCACCCAGAAGCGTGTCGGTGGAAGCGTGAACATGCAGAACCACTTCAACGCTTGGTCT
180 R T Q K R V G G S V N M Q N H F N A W S
1081 CGCTATGGCTTGAACCTTGGTCAACACTACTACCAGATCGTCGCCACTGAGGGTTACCAG
200 R Y G L N L G Q H Y Y Q I V A T E G Y Q
1141 TCCTCTGGAAGCTCTGACATCTATGTGCAGACTCAGTAGAGTAGTTGTTGTCTGTGAAGC
220 S S G S S D I Y V Q T Q *
1201 GAGCAACTAGTAGACGAGATTAGACAAGAATAGTGTCTCGGATAGTTGTAAGCAGGTTGG
1261 AGAAAGAGCGATGTGTTCGATTTCCTGTACATAGAAACATGTCAATTCACTCGCTATAAA
1321 ACCTTCCGTCCTCGAATGTGTTCTCTTTTGTATTAGCTGAGCATTGTATCGTGGTTCGGC
1381 AAAAGCAGGTAAAGTTTAGGTGAGGGCATTTGTTATAAACAGTCATAGTCTCGTAGCCAT
1441 CGAATGCTGGTGTGTGAATTC

Figure 8. Nucleotide and deduced amino acid sequences of X YLZ.
The italicized sequences indicate the introns.

35
position as the intron of XYL1 and the first intron of XYLZ. The XYL3

putative intron contains three termination codons as well as 5’ GIGTATGC
(consensus Gl-GTAN GT), 3’ ACCG (consensus ACAG) intron splice sites and
an internal TACTAAC (consensus TACTAAC) sequence (Ballance, 1986). A
possible translational start site (Figure 9) has a consensus context of
CAAAflGT (consensus CAMMALGNC, where M = A or C)(Ballance, 1991).
Twenty two of the first 39 amino acids including the putative start
methionine are hydrophobic amino acid residues (Met, Leu, Phe, Val, Ile, and
Ala). Based on similarity to XYL1 (Apel et al., 1993) and the known signal
peptide of endo-polygalacturonase from C. carbonum (Scott-Craig et al.,
1990) the mature Xy13 protein probably begins at amino acid 40 (Figure 9).
Comparison of the three xylanases

In order to be certain that the three xylanase genes could be identified
individually, a Southern blot of genomic DNA digested with HindIII was
sequentially hybridized with XYL1, XYLZ, and XYL3. Figure 10 shows that
the three genes can be identified from each other on genomic Southern blots
despite their high degree of similarity to each other (Figure 1 1). Xyll is 75%
similar and 60% identical to Xy12. Xyll is 60% similar and 42% identical to
Xy13. Xy12 is 59% similar and 39% identical to Xy13.
Transformation-mediated gene disruption of X YL2 and X YL3

Transformation-mediated gene disruption was used to create XYL2

and XYL3 mutants. The linearized transformation plasmid, pXLB37-2, was

36

1 TGCATGACTGGACCACTCAGCGCGCGGCTTCCCCACAAAAGTCTATATGAAGTATCCACC
6O GCCGTGATGGAGCTTGATTAACTGTCGGGATGGCGACTTGGCACGCGCCTTGAAATGCAG
121 GATAGCATCACGCCTAGGGGCAGGGCCGATTGCGCTCATTGGATACGAATACATCAAGAC
181 ACTAGTGTAGAACGATTCAGACGATTCATCCATGGGCAACGAGGGGCGAGAGGTGGCCCC
241 ATGACACAGGGAGGAAGACAGCGGGTGAAACAACCGCAATATGTTTCCTGTGGTGTGCAC
301 CCACGCTCTTTAACCGTCTCTAAGTATATAGGTCAAGACTTCCAAGACCGAATAAGCTCA
361 CCACCAAGCCTTTCATCTTCAACACTCAAGCAGTCTTTACTTCACTCACCCTCTTTTCAA
421 TTCACTCCTAGCGAGAACCAGCTTCTTTTCACACAAACACAAACAAATCTTCAATCTATC
481 AAAATGGTTGCCTTCACCTCCGTCCTCCTCGGCCTCTCCGCCATTGGCTCTGCCTTCGCC
M V A F T S V L L G L S A I G S A F A

541 GCCCCCGTTGCCGATGTCCCTGACTTCGAGTTCTCCGGCCCCAAGCACTTGGCTGCCCGC
20 A P V A D V P D F E F S G P K H L A A R

601 CAGGACTACAACCAGAACTACAAGACTGGTGGTAACATCCAGTACAACCCCACCAGCAAC
40 Q D Y N Q N Y K T G G N I Q Y N P T S N

661 GGTTACTCCGTCACCTTCTCTGGCGCCCAGGACTTCGTCCTTGGCAAGGGCTGGAAGCAA
60 G Y S V T F S G A Q D E V L G K G W K Q

721 GGTACTACCAGGTAAGCCACACACAGTGTTCTAGACCCAAATATCAATCAATACTAACTC
80 G T T R

781 CTTTCCTTCAGGACCGTCAAGTACACTGGTTCCACCCAGGCCCAGGCCGGTACTGTCCTC
84 T V K Y T G S T Q A Q A G T V L

841 GTCGCTCTCTACGGCTGGACCAGGGGCAGCAAGCTCGTCGAGTACTACATCCAGGACTTC
100 V A L Y G W T R G S K L V E Y Y I Q D F

901 ACCTCTGGCGGCTCTGGCTCCGCCCAGGGCCAGAAGATGGGCCAAGTCACCTGCGACGGC
120 T S G G S G S A Q G Q K M G Q V T C D G

961 TCCGTCTACGACATCTGGCAGCACACCCAGGTGAACCAGCCTTCCATCGTCGGCACGACC
140 S V Y D I W Q H T Q V N Q P S I V G T T

1021 ACCTTCGTCCAGTACATCAGCAACCGCGTCAGCAAGCGCTCCACCGGCGGTACCATCACC

160 T F V Q Y I S N R V S K R S T G G T I T
01190 #1

1081 ACCAAGTGCCACTTCGACGCCTGGGCCAAGCTCGGCATGAACCTTGGTAACCAGTGGGAC

180 T K C H E D A W A K L G M N L G N Q W D

 

1141 TACCAGACCATCTCCACTGAGGGTTGGGGCAACGCTGCTGGAAAGTCCCAGTACACCGTC
200 Y Q T I S T E G W G N A A G K S Q Y T V

1201 TCCGCTGCTTAAATTGTGGGCGCGGTCCGTTCTTTGGCTCAAGGGGAATGAACCAGGGGT
220 S A A *

1261 TCGAGAGATTTGTGAAACATTGGCTTGCGCAATCATCAATTCCTCTCCAGAGGAGAGGAA
1321 ATATGATAGCTTCAAGGGCTTTGTAGGGGTGTTGATTTAGGGGATGCTGCCGCTGCTGTT
1381 GCTTGATTCATTGGTGTTTATCTTTCTTTCTTCTTGATTTGTTCAGGGCCTTGACACACC
1441 CTGGCTTGTACATACTTTTTTTACTCGTTTCTTGTAGAAACGGGTAAATCCTGAATATAC
1501 ACGTTGCCTTGTTTCTCCCTCGTTTGGATGACAATAGCAATTGCCCGGTTATGAACCGAA
1561 AAAAAAACTCCTCATCTTCTTCTGTGTTTCTTATCATTGTGACAAGTGACTTTTTTTTCT
1621 TTTCTTACTTAAATTTATTTTTCTAGGTTTTCACAAAAGTATATATATTCTTAGCCATGC
1681 TGGCTGCCCCATCTTACTTAGTAAACAGGTGAAACTTCCAATTATCGCTGCCAAACGCAG
1741 GCTCGGATGTCGGCGGCGGCGCGTGGATGGAGATACATAACATTGTGTTGCTTAAAACAA
1801 GGCTAGGTTGGCTGGGCTGGGGCTGAACAGCCGGTGGTAGGATGATTTATCGGGCAAATA
1861 TCAAGACACACACACATCTTCTTAAAACGCCCTTTTGGTGGCGAAGGTTCTTAACCAAGT
1921 GGGTACCCGGGGGATCCACTAGTTCTAGA

Figure 9. Nucleotide and deduced amino acid sequences of X YL3.
The overlined region indicates the location of the oligonucleotide used to
clone XYLI and XYL3. The italicized sequence indicates the putative intron.

37

X

XYL2 XYL3

’. -8.60

 

' . -2.90

Figure 10. Genomic Southern of XYL1, X YL2 and X YL3.

Genomic DNA from C. carbonum wild type was digested with HindIII,
fractionated, blotted, and hybridized with the cDNA of XYLI, stripped and
hybridized with the cDNA of XYL2, and then stripped and hybridized with
the KpnI/XbaI fragment of XYL3.

38

XYL1 MVSFTSIITA.AVAATGALAA PAT......D VS.. ...... .LVARQNTPN
XYL2 MVSFKSLLLA AVATTSVLAA PFDFLRERDD VNATAL... .LEKRQSTPS
XYL3 MVAFTSVLLG LSAIGSAFAA PVA ...... D VPDFEFSGPK HLAARQDYNQ
XYL1 GEGTHNGCFW SWWSDGGARA TYTNGAGGSY SVSWGSGGNL VGGKGWNPGT
O. 0.. O O. O ‘ I O O .........
XYLZ AEGYHNGYFY SWWTDGGGSA QYTMGEGSRY SVTWRNTGNF VGGKGWNPGS
XYL3 .......... .NYKTGG. NI QY.NPTSNGY SVTFSGAQDF VLGKGWKQGT
XYL1 ARTITYSGTY NYNGNSYLAV .YGWTR. NPL VEYYVVENFG TYDPSSQSQN
XYLZ GRVINYGGAF NPQGNGYLAV .YGWTR. NPL VEYYVIESYG TYNPSSGAQI
XYL3 TRTVKYTGST QAQAGTVLVA LYGWTRGSKL VEYYIQDFTS GGSGSAQGQK
XYL1 KGTVTSDGSS YKIAQSTRTN QPSIDGTRTF QQYWSVRQNK RS. SGSVNMK
XYLZ KGSFQTDGGT YNVAVSTRYN QPSIDGTRTF QQYWSVRTQK RV. GGSVNMQ
XYL3 MGQVTCDGSV YDIWQHTQVN QPSIVGTTTF VQYISNRVSK RSTGGTITTK
Oligol
XYL1 THFDAWASKG MNLG.QHYYQ IVATEGYFST .GNAQITVNC HKFFTTKINE
XYL2 NHFNAWSRYG LNLG. QHYYQ IVATEGYQSS .GSSDIYVQT Q*
XYL3 CHFDAWAKLG MNLGNQWDYQ TISTEGWGNA AGKSQYTVSA A*
XYLl- WPDRLSERLE DDRLGF*

Figure 11. Comparison of the deduced amino acid sequences of three
C. carbonum xylanases. Double point marks indicate identity of XYL2 or
XYL3 to XYL1. Single point marks indicate similarity.

39

used to disrupt XYLZ. The isolates were analyzed by digestion of their DNA
with BamHI and blotting. Two out of five transformants were single
insertion mutants. The remaining three transformants

had ectopic integration of the vector. Based on the hybridization pattern
with a XYL2 cDNA, transformant T446-17A is a single insertion mutant as
seen in Figure 12.

The linearized transformation plasmid, pXLC42-1, was used to disrupt
XYL3. The isolates were analyzed by digestion of their DNA with BamHI
and blotting. The pattern of hybridization in T448-4A with the XbaI/KpnI
fragment of XYL3 to a band of 16.3 kb and no band at 1 1 kb is consistent
with homologous recombination of a single copy of pXLC42-l at XYL3 (Figure
13).

Creation of a double XYL1/XYL2 mutant

Strain 312-6A (XYL1‘/XYL2+)-was crossed with 367 -1a (XYL 1+/XYL2*)
to obtain a strain, 447 -8a (XYLI'IXYL2*), with the XYL1 mutation in a strain
of mating type "a". Strain 447 -8a was then crossed with the XYL2 mutant to

obtain the double mutants 47 7-3a and 47 7-7a (XYLI'IXYLZ') (Table 2).

 

 

14.1—

8.5—
6.4— ' -

4.8— I
3.7—

2.3—
1.9—

14- -

123—

0.7—

B.

XYLZGonomicResttictimMap-WiidTypa

 

B B
I..__—_—.|
l 3 kb J
XYL2 Genomic Restriction Map - Mutant
B B B
l._.d~ l
I 1.5 Id) I
l 7.5 kb I

 

Figure 12. DNA blot analysis of wild-type and X YL2 disruption

mutant.

A. Genomic DNA from Cochliobolus carbonum wild type (367-2A) and the
XYL2 mutant (T446-17A) was digested with BamHI, fractionated,
blotted, and hybridized with the XYL2 cDNA.

B. Predicted restriction map. The black shading indicates XYL2 and the
lightly shaded box denote the disruption vector. The hybridization seen
in part A is consistent with the predicted single integration pattern.
B=BamHI endonuclease restriction sites.

 

A. kb °§° $3”
-
14.1-
8 5_ .
6.4—
4.8..
3.7-
2.3—
1.9-
1.4—
1.3-
0.7-
XYL3 Genomtc Restriction Map - Wild Type
B B
L” 11 kb I

 

meenomicaestrtcuonmp-Mutam

B B
L]! ———-::I' .:=: ‘ ;'-:.-:t -_—.l
L_ II 16.3 kb J

 

Figure 13. DNA blot analysis of wild-type and X YL3 disruption

mutant.

A. Genomic DNA from Cochliobolus carbonum wild type (367 -2A) and the
XYL3 mutant (T448-4A) was digested with BamHI, fractionated, blotted,
and hybridized with the XYL3 KpnI/XbaI fragment.

B. Predicted restriction map. The black shading indicates XYL3 and the
lightly shaded box denote the disruption vector. The hybridization seen
in part A is consistent with the predicted single integration pattern.
B=BamHI endonuclease restriction sites.

42

Table 2. C. carbonum strains.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Transformed Cross X YL Antibiotic
Strain # Strain Parents Genotype Resistance

367-2A XYL1’. XYL2’. XYL3+

164R10 XYL1*,XYL2’,XYL3+

164R10 T2-4 164R10 XYL1‘, XYL2’, XYL3+ HYG
164R10T2-8 164R10 XYL1" XYL2‘, XYL3‘ HYG
T2164a 164R10T2-4 XYL1‘, XYL2’, XYL3’ HYG
312—6A T2164a X 164R17A XYL1', XYL2‘, XYL3‘ HYG
T446-17A 367-2A XYL1 1 XYL2 ', X Yin? * HY G
447-8a 312—6A X 367-1a XYL1 ', XYL2 ‘, X YL3 ‘ HYG
477-3a T446-17A x447-8a XYL1', xngz-vagy HYG
477-7a T4464 7A x 447-8a XYL1‘, XYL2', XYL3 * HYG
T4484A 367-2A XYL1 ’, X YL2 ’, X YL3’ HYG
I556-1 1 T4484A X 477-3a XYL1 ‘, XYL2 ’, XYL3 ‘ HYG
556-8 T4484A X 477-3a XYL1 ‘, X YL2 ', XYL3 ’ HYG
556-1 T4484A X 477-3a XYL1 *, XYL2 ‘, XYL3‘ HYG
556-16 T4484A X 477-3a XYL1 ', XYL2 ', XYL3 ‘ HY G
556-4 ' T4484A x 477-3a XYLJ ', XYL2 *, XYL3' HYG
556-12 T4484A X 477-3a XYL1 ', XYL2', XYL3 ' HYG
[556-2 T4484Ax 477-3a XYL1’, XYL_2*, XYL3’

[556-13 T448-4AX477-3a xw_1'I xw_2'I XYL3’ HYG

 

 

 

Growth and pathogenicity of the X YL2 mutant, X YL3 mutant,
and the XYL1/XYL2 double mutant

The wild-type and XYL2 mutant were grown on mineral salts medium
supplemented with trace elements and yeast extract and either maize cell
walls or oat spelt xylan as the sole carbon source. There was no significant
difference in growth between the wild type and the disruption mutant.
Culture filtrates from the growth studies were assayed for xylanase activity,
and no difference was seen between the wild type and the XYL2 disruption

mutant.

43
The XYL3 mutant also did not show any visible difference in growth on

xylan or maize cell walls when compared to the wild type. When compared to
the wild type, the XYL3 mutant did not have any loss of xylan degrading
activity. This was expected since XYL3 mRNA is not present when C.
carbonum is grown in culture.

Xylanase activities from the wild type, the XYL1 mutant, the XYL2
mutant, and the XYL1/m2 double mutant were purified in parallel through
cation exchange HPLC (Apel et al., 1993). There were no reproducible
differences in the protein profiles of 367 -2A and T447 -17A (Figure 14).
Xylanase activity was assayed in each fraction from the cation exchange
HPLC. As shown in Figure 15, the wild-type gives the characteristic two
peaks of xylan degrading activity in fractions 21 and 27. The second peak
corresponding to xylanase II, which is missing in this sample, is encoded by
XYL1 and is unstable (Apel et al., 1993). The XYL1 mutant lacks the two
peaks of activity in fraction 21 and 23 which correspond to xylanase I and
xylanase II, as found previously (Apel et al., 1993). The XYL2 mutant has all
three peaks of xylanase activity and does not appear to be missing any peaks
of xylan degrading activity. The XYL1/XYL2 double mutant is missing two
peaks of activity in fractions 21 and 23, like the XYL1 mutant. Because the
third peak, called xylanase III (Holden and Walton, 1992), in fraction 27 does
not disappear in the XYL1, XYL2, or XYL3 mutants, it is probably encoded by

a different gene.

44

 

Absorbance 280 nm (mAU)

 

 

 

'Irimer(min)r

Figure 14. HPLC protein profiles of the wild type and X YL2 mutant.
Four milligrams of total protein (Bradford, 1976) from partially purified
culture filtrate were loaded onto a cation exchange HPLC column. The solid
lines are absorbance profiles at 280 nm. The bottom line corresponds to the

wild type (367-2A) and the top line corresponds to the XYL2 mutant (T447-
17A).

45

 

 

 

 

’E
a
16 ‘
A M
a)
r:
c 19
a i .' } —°—th 1+
>‘ 'i I XY‘ 2*
r: i Q_
E , '(_' i —-a— Xyl t-
0 ’5' ". Xyl 2-
< It‘ '\
o; ‘
w L if " --O>-Xy|i*
g 7.: l Xyl 2.
2 a P2] ;
;‘ 0: .’ I ~ — -x— th 1»
/E i W 2-
‘ Q I, ',
0‘ 2 (Cl
C _A_ ELK/A i
4.»... ,‘y- K .
t '4' \u . 4(- X' :xc’ / _
OM’O'.—. '~.x.-" ‘4 x.-
14 ‘5 15 i7 '5 19 20 2‘ 22 a 24 25 2‘3 Z7 25 29 3O 31 32

43
Retention Time (Min)

Figure 15. Xylanase activity from cation exchange HPLC of the X YL2
mutant. The peaks of xylanase I, II, and III appear in fractions 21, 23, and
27 respectively. Xylanase I and H are encoded by XYL1.

46
Because the cation exchange HPLC protein profile for the XYL2

mutant did not appear to lack a protein peak when compared to the wild
type, an immunoblot of culture filtrate was done to see if any Xy12 protein
was produced. When the immunoblot was reacted with anti-serum to a 22-
kDa xylanase from Trichoderma viride (gift of Dr. James Anderson, USDA,
Beltsville, MD) many bands are seen but a 22-kDa band is the most
prominent (Figure 16). This 22-kDa band is missing in the XYLI mutant and
therefore we conclude that it is Xyll. A faint 22-kDa band is present in the
XYL1 mutant. This faint band is seen in the XYL1'IXYL2+ mutant but not
the XYL1‘IXYL2’ double mutant (Figure 16). Therefore, it appears that
XYL2 is expressed at the protein level but that Xy12 does not accumulate to
levels as high as Xyll. The protein encoded by XYL2 may not be active or its
activity may not be detected by the reducing sugar assay.

The rate of development, size, and morphology of the lesions produced
by the wild-type, XYL1 mutant, XYL2 mutant, and XYLI/XYL2 double
mutant appear to be the same (Figure 17). Pathogenicity of the XYL3
mutant was also tested on susceptible maize and no differences were seen in
lesion size, rate of development, or morphology.
mRNA expression in culture and in infected leaves

The mRNA expression of XYL1, XYL2, and XYL3 was studied in
culture and in infected plant leaves. XYL2 RNA is present in the wild type

as well as in the XYL1 mutant strain (Figure 18). The same RNA blot was

47

22kDa- - “‘5' .9

Figure 16. Immunoblot of concentrated culture filtrate of the wild-
type and the XYL1 mutant, X YL2 mutant, and XYL1/XYL2 double
mutant. Partially purified and concentrated culture filtrate of Cochliobolus
carbonum was loaded in each lane. The blot was hybridized with the anti-
serum raised against a 22 kDa xylanase from Trichoderma viride (gift from
Dr. James Anderson, USDA Beltsville, MD).

48

 

Figure 17. Pathogenicity test of xylanase mutants 447-8a (XYLl‘
IXYL2“), T446—17A (XYLl*/XYL2‘), and 477-7a (XYLI‘IXYLZ’) and wild
type strain 367-2A (XYLl*/XYL2*). Maize leaves were inoculated with 104
spores per milliliter. Photograph was taken 5 days after inoculation.

49
hybridized with XYL3 and no signal was present. To ensure that the blot

was intact, the blot was then hybridized with the glyceraldehyde-B-
phosphate dehydrogenase gene from C. heterostrophus. Each lane of the gel
was loaded with the same amount of RNA but the expression levels of
glyceraldehyde-3-phosphate dehydrogenase decline over time and under
difl‘erent growing conditions (Figure 18). This is probably due to the growth
state of the culture. To date, XYL3 mRNA has not been seen in culture when
C. carbonum is grown on xylan or maize cell walls.

mRNA was isolated from 7 -day old infected plant leaves, fractionated
on 3 formaldehyde gel, blotted to Zeta-Probe, and hybridized with XYLI. The
blot was then stripped and hybridized sequentially with XYL2, XYL3, and
the glyceraldehyde-3-phosphate dehydrogenase gene (CPD 1) from C.
heterostrophus. As seen in Figure 19, XYL1 mRNA is present, XYL2 mRNA
is not present, and XYL3 mRNA is present.
DISCUSSION

XYL2 and XYL3 were cloned and sequenced. Transformation-
mediated gene disruption created mutants specifically lacking intact copies of
XYL2 and XYL3. Immunoblot analysis showed that XYL2 produces a low
level of protein product, but this protein could not be detected enzymatically.

Pathogenicity tests showed that both XYL2 and XYL3 individually as
well as XYL1 and XYL2 in combination are dispensable for pathogenicity on

maize. This does not mean that xylanases are not important for

50

 

 

Wild Type XYL1 Mutant

 

Days4'812848128

 

SucroseLLLHLLLH

 

 

 

 

 

 

 

 

 

XYLI ..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 18. RNA blot hybridized with XYL1, X YLZ and X YL3. Total
RNA was isolated from fungal mats of the wild type (367 -2A) and the XYL1
mutant (447-8a) grown on maize cell walls for 4, 8, or 12 days with low or
high levels of sucrose (L=0.2% sucrose, H=2.0% sucrose). Approximately 10
pg of RNA per lane was fractionated on a 1.2% agarose gel containing
formaldehyde (Selden, 1987) and blotted to a nylon membrane. The blot was
sequentially hybridized with the XYL3 KpnI/Xbal fragment, the XYL2 cDNA,
the XYLI cDNA, and the glyceraldehyde-3-phosphate dehydrogenase gene
(GPD1)fiom C. heterostrophus.

51

 

Figure 19. RNA blot of infected maize leaves. mRNA was isolated from
infected maize lesions 7 days after inoculation with wild-type strain 367-2A.
Approximately 10 ug of the mRNA was loaded per lane on a 1.2% agarose gel
containing formaldehyde (Selden, 1987) and blotted to Zeta-Probe. The blot
was sequentially hybridized with the XYL1 cDNA, the XYL2 cDNA, the XYL3
KpnI/Xbal fragment, and the glyru ‘ ’ ‘, ie-3 -L -L ‘ dehydrogenase
gene (GPDI) from C. heterostrophus.

- = mRNA isolated from uninfected leaves. + = mRNA isolated from infected
leaves.

 

52
pathogenicity since C. carbonum produces other xylan degrading enzymes

such as Xyl4 (xylanase III) and B-xylosidases.

Not only does C. carbonum produce several xylan-degrading enzymes,
it appears that they are induced by different factors or may be produced
during different stages of the life cycle. XYLI is expressed in culture as well
as in infected leaves. XYL2 is expressed in culture but not in 7 -day infected
leaves, and XYL3 is not expressed in culture but is expressed in infected
leaves. Because of the different patterns of expression of these three genes,

their promoters will be interesting to study.

CHAPTER 5

TRIPLE XYLANASE MUTAN T

INTRODUCTION

Three genes, XYL1, XYL2, and XYL3, encoding endo-B 1,4-xylanases
were cloned, sequenced, and disrupted. In order to obtain a strain with
mutations in all three xylanase genes, strains containing single disruptions
were crossed to each other and the resulting double-disrupted (XYLI/XYL2
IXYL3’) strain was crossed with the third single-disruptant strain
(XYLI‘IXYLZ‘IXYLJ). The triple mutant was analyzed for ability to grow on
xylan or maize cell walls. mRNA expression levels of the three xylanases and
the ability of the triple mutant to infect maize were also studied.
RESULTS

A triple xylanase mutant was obtained by crossing strains 47 7 -3a
(XYLI/XYLYIXYLS") and T448-4A(XYL1*/XYL2*/XYL3*). When the
progeny were isolated no selection was used so that all eight progeny classes
were obtained from the cross. The eight strains from cross 556 are

representative of the eight progeny classes from cross #1 (Table 2; Chapter

53

54
4). The number of progeny obtained in each progeny class is shown in Table

3. Because no selection was used when the progeny were isolated, all of the
progeny were analyzed by Southern analysis to determine their xylanase
genotype. A Southern blot showing the representative progeny from each
class is shown in Figure 20.

The expression levels of the progeny from the cross were studied by
RNA blotting (Figure 2 l). XYL3 message is not present as expected from
previous experiments (Figure 21). XYL2 and XYLI appear to be slightly up-
regulated in mutants of the opposite genotype (Figure 2 1).

Growth on maize cell walls or xylan was measured by weighing the
mats. As seen in Figure 22, no significant difi'erence in growth was found
between the wild type (556-2), a XYLI mutant (556-1 1), a XYL2 mutant (556-
8), a XYL3 mutant (556-1), and a XYL1/XYLZ/XYL3 triple mutant (556-13)
as determined by dry weight at 3 or 8 days when grown on maize cell walls or
xylan.

No detectable differences in the rate of lesion development, size, and
morphology were seen when the wild type and the xylanase mutant strains
were inoculated onto susceptible maize leaves (Figure 23).

DISCUSSION

The triple mutant was obtained by crossing a XYLI/XYL2 double

mutant with a XYL3 mutant and analyzing the progeny by Southern

analysis. From work by J oong-Hoon Ahn (personal communication) it is

55
Table 3. Crosses to obtain a triple xylanase mutant.

Cross #1: T448-4A (XYLI+/XYL2+/XYL3') X 447-3a (XYL1'IXYL2'IXYL3+)

Progeny genotypes Number of progeny obtained
XYLI'IXYL2'IXYL3+

XYLI+IXYL2+IXYL3'
XYL1 ’/XYL2+/XYL3+
XYL1“/XYL2'/XYL3+
Xl’LI'le’L2+/XYL3'
XYL 1 +/XYL2‘/XYL3'
XYL 1 +/XYL2+/XYL3+
XYLI'IXYLZ'IXYL3'

HNHCJIGCDQUI

Cross #2: T448-4A (XYL1+/XYL2*/XYL3') X 447-7 a (XYL1'IXYL2‘IXYL3+)

Progeny genotypes Number of progeny obtained
XYLI'IXYLZ‘IXYL3+
XYL1+/XYLZ+IXYL3'
XYL1'IXYL2VXYL3+
XYLI+IXYL27XYL3+
XYL1'IXYL2+/XYL3'
XYL1VXYLZ‘IXYL3’
XYL1+IXYLZ+IXYL3+
XYL1’IXYL2‘IXYL3‘

NOIHODQOODC

56

 

Probe —-» XYL1 XYL2 XYL3
XYLI+—++-—+— +-++--+- +-++——+—
GenotypeXYL2++—+—+-- ++-+-+—— ++—+—+—-

XYL3+++-+--- +++-+-—— +++-+---

14.1—

85-

6.4— ." .- - . C '-

4.8-

3.7-

—- fl 0 o

2.3—

1.9-

13- . . O.

0.7-
mmmh-mp Numb-mm mumh-ntp
B I I I I I
L'—.-____l I__-_.I l. — J
L,.__11£_.J I ll .1 I, 11. m 1
“Hubbub-W mount-mum monument-Inn
I I I I ' I I I
La.‘ -__.I q -_1 L,_- n_I
L'n—Et

156
' i m . "

 

Figure 20. DNA blot analysis of wild-type and disruption mutants.
Genomic DNAs from Cochliobolus carbonum wild type and disruption
mutants were digested with BamHI, fractionated, blotted, and hybridized
with either XYLI, XYL2, or XYL3. The predicted restriction map is shown
below the blots corresponding to each gene. The black shading indicates the
xylanase gene of interest while the light shading indicates the disruption
vector. B=BamHI endonuclease restriction sites.

57

 

 

 

 

 

 

 

 

 

Probe ' " __,
XYL3

0”” QLIIJJ! Q.

 

 

 

 

 

Figure 21. RNA blot of wild-type and disruption mutants.
Approximately 10ug of total RNA, isolated from fungal mats grown for 6 days
on maize cell walls, was fi'actionated on a 1.2% agarose gel containing
formaldehyde and blotted to a nylon membrane. The blot was hybridized
sequentially with the XYLI cDNA, XYL2 cDNA, the KpnI/Xbal fragment of
XYL3, and the glyceraldehyde-3-phosphate dehydrogenase gene (GPDI) from

C. heterostrophus.

58

A.Maize Cell Walls

 

 

 

 

 

 

 

 

E (m2
2’

m

w-t

o

a

a

H

a

B.Xylan
on

E my

3 om«

3' on»

0

3 om-

m

80.05..

am
XYL1 +—++—
XYL2 ++—+—
1013 +++——
3 Days 8 Days

Figure 22. Growth of wild-type and mutant strains. Dry weights of
fungal mats (in grams) at 3 and 8 days when grown on maize cell walls or
xylan. All treatments were done in duplicate. Error bars indicate the range.

59

556-2

 

Figure 23. Pathogenicity test of xylanase mutants. Maize leaves were
inoculated with 104 spores per milliliter of the indicated strain. Photograph
was taken 5 days after inoculation.

60
known that XYLI and XYL2 are definitely on different chromosomes.

However, it is not clear if XYL1 and XYL3 are on the same or different
chromosomes because they hybridize to a location on CHEF gels that
contains more than one chromosome. Even if they are on the same
chromosome, it is clear that XYL1 and XYL3 are not tightly linked (Table 3).

Northern analysis (Figure 2 1) suggests that XYLI may be upregulated
when XYL2 and/or XYL3 is disrupted. The expression of these genes needs
to be studied further to confirm and extend this observation.

The disruption of all three xylanase genes in the same strain did not
affect the growth of the fungus on xylan or maize cell walls. However, the
oat spelt xylan used in our growth studies contains only about 60% xylose,
with the remainder being mainly arabinose and glucose (W eimer, 1985).
Therefore, metabolism of the non-xylan components of oat spelt "xylan"
might account for the growth of the triple mutant on xylan. Alternatively,
growth may be due to Xyl4 (xylanase III; Holden and Walton, 1992; Apel et
al., 1993) or another unknown xylan degrading activity.

Pathogenicity tests indicate that XYL1, XYL2 and XYL3 are
dispensable for pathogenicity of C. carbonum on maize and do not contribute
to virulence. From these experiments, it cannot be concluded that xylanases
are unimportant for pathogenicity since C. carbonum produces other xylan

degrading enzymes such as Xyl4.

CHAPTER 6

CONCLUSIONS

Three endo-xylanase genes were cloned from the plant pathogenic
fungus Cochliobolus carbonum. XYLI, XYL2, and XYL3 are all predicted to
encode family G, 22-kDa xylanases. Xy12 and Xy13 are 60% and 42% identical
to Xyll at the amino acid level. Xy12 and Xy13 are 39% identical.
Transformation-mediated gene disruption was used to create mutants in all
three genes alone and in combination. A triple XYLI/XYLZ/XYL3 mutant
was created by crossing. The mutants were tested for enzyme production in
vitro, growth in culture on xylan and maize cell walls, and pathogenicity on
maize.

XYL1 has one intron. It encodes two xylanase isoforms that together
are responsible for 85-94% of the total extracellular endo-xylanase activity of
C. carbonum when‘grown on maize cell walls. Growth of the XYL1 mutant
was indistinguishable from the growth of the wild type in culture. The
residual xylanase activity is apparently sufficient to support growth. XYL1
is also expressed at the mRNA level in planta. The XYL1 mutant is still fully

pathogenic on maize.

61

62
XYL2 has two introns, one of which is at the same position as the

intron in XYL1. It is expressed at the mRNA level in culture but in a XYL2
mutant the residual xylanase activity seen in the XYL1 mutant is still
present. N o enzyme activity can be associated with XYL2. However,
immunoblot analysis indicates that the protein product of XYLZ is present in
culture filtrates but at a low and enzymatically undetectable level. XYL2
mRNA is not detected in planta and disruption of XYL2 has no effect on
pathogenicity.

XYL3 has one predicted intron at the same position as the intron in
XYLI. A cDNA of XYL3 was not obtained because it is not expressed in
culture. XYL3 is expressed in planta but a XYL3 mutant grows on xylan and
maize cell walls and is still pathogenic.

A triple xylanase mutant grows as well as the wild type on xylan and
maize cell walls. The triple mutant is still pathogenic.

The question of whether xylanase activity is necessary for growth on
xylan and for pathogenicity is still an open question due to the residual
xylanase activity remaining in the triple mutant. This xylanase activity has
been purified and is due to a 33—kDa family F endo-xylanase (J. Scott-Craig,
personal communication). The gene, XYL4, encoding this xylanase is being
cloned. Furthermore, C. carbonum makes at least one B-xylosidase, which is

also in the process of being cloned (R. Ransom, personal communication).

63
At this time it is important to clone and disrupt XYL4 alone and in

combination with XYL1, XYL2, and XYL3. If this quadruple mutant cannot
grow on xylan or is non-pathogenic, then by studying strains with different
combinations of the four xylanase genes mutated, it can be determined which
enzymes are important for each process.

The xylanases of C. carbonum are redundant. C. carbonum has
multiple xylanase genes and makes multiple xylanases. However, the
relationship between the enzymes and the genes is not simple: XYL1 encodes
two of the three enzymes seen in culture, and XYL4 probably encodes the
fourth. Although C. carbonum has multiple xylanase genes, the genes are
not equivalent in their pattern of expression: XYL1 and XYL3 are expressed
in planta, and XYLI and XYL2 are expressed in culture. Whether Xyll,
Xy12, and Xy13 have similar enzymatic activities could not be determined
since only Xyll can be assayed.

Redundancy is a common theme in cell wall degrading enzymes from
plant pathogenic bacteria and fungi. Magnaporthe grisea and Alternaria
brassicicola each make at least two cutinases (Sweigard et al., 1992; Yao and
Koller, 1995). Glomerella cingulata has a large pectin lyase gene family
(Bowen et al., 1995) and Erwinia chrysanthemi makes at least eight pectin
lyases (Kelemu and Collmer, 1993). The reasons for this redundancy are not
known. One explanation is that the enzymes are so important that the

organism protects itself against mutation by having more than one gene.

64
Another explanation is that facultative pathogens have specialized enzymes

for saprophytic versus pathogenic growth. Yao and Koller (1995) proposed
that A. brassicicola makes one cutinase specialized for saprophytic growth
and another for pathogenic growth. A third explanation is that different
isozymes are specialized for hydrolyzing polymers that are slightly but
significantly different. Perhaps, Xyll, Xy12, and Xy13 are adapted to
hydrolyze slightly different xylans. Takenishi and Tsujisaka (197 5) found
that Aspergillus niger secretes three xylanases. Xylanase I most effectively
hydrolyses cotton xylan while xylanase II contributes to the hydrolysis of rice
straw arabinoxylan by the removal of arabinosyl substituents (Takenishi and
Tsujisaka, 1975).

More studies need to be completed before we will know the true
purpose of the multiple xylanases in C. carbonum. They may be important
for pathogenicity, for saprophytic growth, or for another unknown purpose.
In order to elucidate the role of xylanases or other cell wall degrading
enzymes, all isozymes need to be characterized and all related genes must be

disrupted in a single strain.

APPENDIX

APPENDIX

EXPRESSION OF A FUNGAL ENDO-XYLANASE
GENE IN TOBACCO

ABSTRACT

There are several potential applications for xylanases but the high cost
of xylanases produced by fermentation is a significant commercial barrier.
As an approach to producing large quantities of xylanase less expensively, we
have expressed the XYLI gene of the fungus Cochliobolus carbonum in
Nicotiana tabacum. Several XYL1 gene constructs were tested. All the XYL1
gene constructs were driven by the double CaMV 35S promoter from p1079
(DuPont Chemical Company, Wilmington, DE). These include a XYLI cDNA
with and without the native signal peptide as well as XYL1 containing an
engineered endoplasmic reticulum-retention signal (KDEL) at the carboxyl
end. Agrobacterium-mediated transformation was used to obtain transgenic
tobacco. Transgenic plants were screened for XYL1 mRNA by tissue printing
(Reid and Pont-Lezica, 1992). RNA was isolated from plants that screened
positively for XYLI message by tissue printing. The RNA was blotted and

hybridized with the XYL1 cDNA. Some plants from each construct group

65

66
showed high levels of mRNA expression of XYL1. However, not all plants

that were positive by tissue printing produced XYLI mRN A. Additionally,
none of the transgenic plants produced any Xyll protein as determined by
protein blots reacted with antibody to a Trichoderma viride 22-kDa xylanase
which cross-reacts with Xyll. None of the transgenic plants had xylan-
degrading activity.
INTRODUCTION

Enzymes that can depolymerize proteins and polysaccharides of plant
cell walls are produced by many pathogenic fungi and bacteria. These
enzymes have potential usefulness for commercial purposes. Xylanases can
be used in food manufacturing to improve baking with high fiber material, to
clarify fruit juices and wine, to improve nutritional and product properties of
cereal fibers, and to produce food thickeners (Zeikus et al., 1991).
Additionally, xylanases can be used in the paper and fiber manufacturing
industry for biobleaching of kraft pulps and the biopulping of wood (Senior
and Hamilton, 1992; Zeikus et al., 1991).

These uses are advantageous for the consumer and the environment.
For example, limited hydrolysis of kraft pulps with xylanases enhances the
extractability of lignins by conventional bleaching chemicals, resulting in
reduction of chemical bleaching requirements and improvement in viscosity
(Senior et al., 1992; Yang et al, 1992). The use of xylanases in the treatment

of bread dough decreases the dough strength, yielding loaves with an open

67
structure (Kulp, 1968). Adding xylanases to ensiled herbage improves the

rate of lactic acid production and produces a better quality silage (Gilbert
and Hazlewood, 1991). Xylanases improve the rate of plant-fiber degradation
resulting in increased plant cell wall utilization in ruminant animals. This
increases the amount of available nutrients to the animal, which results in
increased milk production in dairy cattle (Gilbert and Hazlewood, 1991;
Choung and Chamberlain, 1992).

While there are several potential commercial applications for
xylanases, the high cost of xylanases produced by fermentation is a
significant barrier to large volume production of xylanases (Zeikus et al.,
1991). Expression in plants might be a way to produce xylanases and other
enzymes less expensively. This was our purpose in expressing XYL1, the
gene encoding the major endo-xylanase from Cochliobolus carbonum, in
tobacco.

MATERIALS AND METHODS
Gene constructions

Specific PCR primers were used to amplify the XYL1 cDNA with and
without the signal peptide, and with and without a KDEL endoplasmic
reticulum retention signal. Primers used were:
JDW194=AATGG'I‘TTCTT'1‘CACCTCCATCATC,
JDW195=CATGCAGAACACCCCCAACGG,

JDW196=GTTATGGGCAGTTGACGGTGATCT, and

68
JDW l97=G'I'I‘AGAG'I'1‘CATC'I"I'I"I‘GGGCAG'1"I‘GACGG. The product used

in pPA5 was from primers JDW194 and JDW196; pPA6 was from primers
JDW194 and JDW197; pPA7 was from primers JDW195 and JDW196; and
pPA8 was from primers JDW195 and JDW197. The PCR products were
cloned into the EcoRV site of pBluescript. After the clones were confirmed by
sequencing, the 0.7-kb SmaI/Apal fragment containing each PCR product
was subcloned into the PstI site of p1079 (DuPont Chemical Company,
Wilmington, DE) in the proper orientation behind a double CaMV enhancer
region. These clones were then digested with Xbal to release a 1.8-kb
fragment containing the subcloned PCR fragment of 0.7 kb, with the 358
promoter double enhancer region (0.8 kb) and the nopaline synthase 3’
terminator region (0.3 kb). The fragment was blunted with T4 DNA
polymerase and subcloned into a blunted HindIII site in the plasmid pB112 l
(Clonetech) for Agrobacterium transformation. Agrobacterium strain
LBA4404 was transformed by electroporation under the conditions
recommended by the manufacturer (Bio-Rad; 25uF, 2.5kV, 2000).
Plant materials, growth and transformation

Growth of Nicotiana tabacum SR1 plants and the transformation of
the plants was done as described by Newman et al. (1993).
Nucleic acid manipulations

Tissue prints were done by squashing a leaf from each tobacco plant

onto a nylon membrane using a pestle covered with plastic wrap (Reid and

69
Pont-Lezica, 1992). Fresh plastic wrap was used for each leaf to avoid cross-

contamination. The blot was wetted in 2x SSPE and the RNA was fixed to
the membrane using a UV Stratalinker 1800 at 1200 mjoules according to
the manufacturer’s instructions (Stratagene). The filters were washed for
5 hr in a solution containing 50 mM Tris-H01, pH 8.0, l M NaCl, 1 mM
EDTA, and 0.1% SDS (Reid and Pont-Lezica, 1992).

RNA was isolated from leaves by the method of Yoder ( 1988) except
cresol and glass beads were omitted. For the RNA blots 10 pg of total RNA
was loaded per lane of a 1.2% agarose gel (Sambrook et al., 1989). RNA
electrophoresis was done in the presence of formaldehyde at 70V for 4 hr
(Selden, 1987). GIBCO-BRL RNA standards (0.24 - 9.5 kb) were used for
transcript size estimation.

Routine hybridization of the tissue prints was done with the sub-
cloned XYLI cDNA labeled with 3‘é’P-dCTP by random primer labeling
(Sambrook et al., 1989) in 50% formamide, 5X SSPE, 2X Denhardt's solution,
0.1% SDS, and 0.1 mg of denatured salmon sperm DNA per ml at 42°C
overnight (Pitkin et al., 1996). The blots were washed in 2X SSPE and 0. 1%
SDS two times for 15 min at room temperature and in 0.1X SSPE and 0.1%
SDS two times for 30 min each at 42°C.

The Northern blot was pre-hybridized for 1 hour at 65°C in 4X SET,
(20X SET = 3 M NaCl, 0.6 M Tris-H01, and 0.04 M EDTA, pH 7.4), 0.1%

tetrasodium pyrophosphate, 0.2% SDS, and 100 ug/ml heparin before being

70
hybridized with the XYL1 cDNA labeled with 32P-dCTP by random primer

labeling (Sambrook et al., 1989) overnight at 65°C in 4X SET, 0. 1%
tetrasodium pyrophosphate, 0.2% SDS, 10% (w/v) dextran sulfate, and
625 ug/ml heparin. The RNA blot was washed in 2X SSPE and 0.1% SDS
two times for 15 min at room temperature and in 0.1X SSPE and 0.1% SDS
two times for 20 min each at 65°C (Singh and Jones, 1984).
Xylanase activity assays

Protein extracts were obtained from the leaves of the transgenic plants
and were assayed for xylan degrading activity using the Remazol Brilliant
Blue Assay as described by Herbers et al. (1995), except that the assay was
done at 37°C rather than at 60°C.

Leaves from the transgenic plants were harvested and ground in 4 ml
50 mM sodium acetate, pH 5. The solution was centrifuged for 10 min at
3200 rpm (1513 x g) and the supernatant was decanted. The samples were
desalted using a BioRad Econo-Pak 10DG desalting column according to the
manufacturer's instructions. The samples were then concentrated to 100 pl
using an Amicon Centricon 3 microconcentrator and xylanase activity was
assayed by a reducing sugar assay (Lever, 1972). Each sample was assayed
in a 300 Ill reaction volume containing a solution of 1 % oat spelt xylan
(Fluka), 50 mM sodium acetate, pH 5.0, at 37°C for 30 min. Twenty five pl
aliquots from the reactions were taken at 0 min and 30 min and added to 1.5

ml of p-hydroxybenzoic acid hydrazide working solution which was heated to

71
100 °C for 10 min prior to measuring the absorption at 410 nm. The p-

hydroxybenzoic acid hydrazide working solution was prepared from a 5%
(w/v) stock solution in 0.5 N HCl by diluting 1:4 with 0.5 N N aOH.
Absorbance of the 0 min sample was subtracted fi'om the 30 min absorbance
and xylose was used as a standard.
Immunoblot analysis

The protein from leaf extracts was fractionated on a 17% acrylamide
SDS-PAGE gel. The gel was blotted to nitrocellulose and reacted with anti-
serum raised in rabbit against the 22-kDa xylanase of Trichoderma viride
(Dean et al., 1989) at a 1:1000 dilution. The antibody was a gift from Dr.
James Anderson (USDA Beltsville, MD). The protein was visualized with a
goat anti-rabbit antibody conjugated with alkaline phosphatase (Cooper
Biomedical, Inc., Malvern, PA). Markers correspond to the prestained low
molecular weight standards from Gibco-BRL.
RESULTS

Nicotiana tabacum plants were transformed with each of four
constructs (Table 4). The constructs are various combinations with or
without the fungal signal peptide or an endoplasmic reticulum retention
signal (KDEL). The transgenic plants were initially screened by tissue
printing. The plants which showed XYLI mRNA by tissue printing were

analyzed by RNA blot analysis. As seen in Figure 24, the wild type control

72
Table 4. XYL1 constructs used and transformation results.

 

 

Clone Signal KDEL Transformants # Transformants
NJme Peptide Recovered with XYL1 mRNA
pPA5 Yes No 17 9

pPA6 Yes Yes 20 7

pPA7 No No 16 4

pPA8 No Yes 12 2

plants do not have any XYL1 mRNA, as expected, while plants 5-6, 5-13, 6-
17, 7-5, and 8-2 do have XYLI mRNA. Plants 6-6, 7-15, and 8-10 do not have
detectable XYLI mRNA even though they show positive expression by tissue
printing.

All of the transgenic plants were tested for xylan degrading activity by
the Remazol Brilliant Blue Assay (Herbers et al., 1995). None of the plants
showed any xylan degrading activity above wild type controls (data not
shown). Plants WT10, WTll, 5-6, 5-13, 6-6, 6-17, 7-5, 7-15, 8-2, and 8-10
were assayed for xylan degrading activity by the reducing sugar assay which
is six times more sensitive than the Remazol Brilliant Blue assay (Biely et
al., 1985). Even with this assay no xylan degrading activity was found in
any of the plants. Additionally, an immunoblot of protein extracts from these
plants showed no evidence of xylanase protein expression in the transgenic
plants (data not shown). All proteins that reacted with the anti-xylanase

antibody were present in untransformed tobacco controls.

73

5
WT10
WT11
5-6
5-13
6-6
6-17
7-5

9:33
4.40-

2.40-

1.40-

0.24-

7-15

8—2
8-10

 

Figure 24. RNA blot of X YLI transgenic plants. WT 10 and WT 11 are
two untransformed control plants. 5-6 and 5-13 are plants transformed with
plasmid pPA5. 6-6 and 6-17 are plants transformed with plasmid pPA6. 7 -5
and 7-15 are plants transformed with plasmid pPA7. 8-2 and 8-10 are plants

transformed with pPA8.

Total RNA was isolated from a selection of plants that showed XYL1
expression by the leaf print method. The RNA (30 pg per lane) was run on a
1.2% agarose/formaldehyde gel, blotted to Nytran and probed with a XYL1

cDNA

74
It appears that the transgenic plants are producing XYL1 transcript

but are not producing any Xyll protein. One possible reason for this is that
the codon bias of Cochliobolus carbonum is not compatible with expression in
tobacco. As seen in Table 5, however, there appears to be no significant
difl'erence in codon usage between XYL1, 56 tobacco genes or 20 genes
encoding the highly-expressed small subunit of ribulose 1,5 bisphosphate
carboxylase.

DISCUSSION

XYL1, the gene encoding the major endo-xylanase from Cochliobolus
carbonum, was expressed at the mRNA level in tobacco both with and
without its native signal peptide and with and without an endoplasmic
reticulum retention signal (KDEL). While plants transformed with each
construct produce large amounts of transcript none of the plants produce any
xylanase protein.

This project was started before it was realized that another group was
doing a similar set of experiments with a xylanase from Clostridium
thermocellum (Herbers et al., 1995). The xylanase used in these studies was
originally a 90-kDa protein but only a coding region yielding a 37-kDa
protein was expressed in transgenic plants. They also fused their sequence
to the signal peptide of the proteinase inhibitor II protein from potato.
Herbers et al. (1995) did obtain plants that had active xylan degrading

protein activity.

75
Table 5 Codon usage comparison

 

a Wada, 1992
b Murray, 1989
RuBP SSU = Ribulose 1,5 bisphosphate carboxylase small subunit.

76
It is not known why Xyll is not present in the transgenic plants.

Perhaps the signal peptide or some other portion of the protein is unstable in
the plant cell environment and the protein is degraded. Inaccurate
polyadenylation may be occurring if there are cryptic polyadenylation signals
within the coding sequence of XYL1. Also, perhaps the transcript is not
translated since we do not see any protein accumulation or even partial
breakdown products with an anti-xylanase antibody. The first step toward
solving this problem would be to see if the transcript can be translated. To
test this, XYL1 transcript could be in vitro translated with wheat germ
extracts (Struhl, 1993). However, it may be better to start over by
constructing a xylanase with a signal peptide proven to work in tobacco and
perhaps, as a control, putting a different cell wall degrading enzyme in
tobacco to see if it is produced and is functional.

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