studies of ergot alkaloid biosynthesis genes in clavicipitaceous fungi

105 Pages·2016·2.03 MB·English

by  Caroline Machado

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University of Kentucky Doctoral Dissertations Graduate School
2004
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Caroline Machado
University of Kentucky, [email protected]
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Machado, Caroline, "STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN CLAVICIPITACEOUS
FUNGI" (2004). University of Kentucky Doctoral Dissertations. 433.
https://uknowledge.uky.edu/gradschool_diss/433
This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been
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ABSTRACT OF DISSERTATION
Caroline Machado
The Graduate School
University of Kentucky
2004
STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN
CLAVICIPITACEOUS FUNGI
ABSTRACT OF DISSERTATION
A dissertation submitted in partial fulfillment of the requirements for
the degree of Doctor of Philosophy in the College of Agriculture
at the University of Kentucky
By
Caroline Machado
Lexington, Kentucky
Director: Dr. Christopher L. Schardl, Professor of Plant Pathology
Lexington, Kentucky
2004
Copyright by Caroline Machado 2004
ABSTRACT OF DISSERTATION
STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN
CLAVICIPITACEOUS FUNGI
Neotyphodium species, endophytic fungi associated with cool-season grasses,
enhance host fitness and stress tolerance, but also produce biologically active alkaloids
including ergot alkaloids associated with fescue toxicosis in grazing animals. One
approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway.
The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis,
was cloned previously from Claviceps spp. and its function was demonstrated by
expression in yeast. Putative homologs have been cloned from Neotyphodium
coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass).
In order to confirm the function of dmaW in ergot alkaloid production, dmaW in
Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW
knockout mutant produced no detectable ergovaline or simpler ergot alkaloids.
Complementation with Claviceps fusiformis dmaW restored ergovaline production. These
results confirmed that the cloned endophyte gene was dmaW, and represented the first
genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis.
Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum)
has two homologs of dmaW. Considering the possible field applications in future, the
Cre/lox site-specific recombination system was chosen because of the potential to
sequentially knock out both homologs and obtain marker-free dmaW mutants of N.
coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the
marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N.
coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate
ergovaline production, indicating that the dmaW-1 was probably also active in N.
coenophialum.
A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps
purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of
ergot alkaloids. Identification of nine common genes between them suggests the possible
role of these genes in the early part of the ergot alkaloid biosynthetic pathway.
KEYWORDS: Neotyphodium endophytes, Ergot alkaloid biosynthesis, Cre/lox, dmaW,
marker exchange
Caroline Machado
Date: 5/06/04
STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN
CLAVICIPITACEOUS FUNGI
By
Caroline Machado
Christopher L. Schardl
_____________________________
Director of Dissertation
Lisa J. Vaillancourt
______________________________
Director of Graduate Studies
5/06/04
______________________________
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Unpublished dissertations submitted for the Doctors degree and deposited in the
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Extensive copying or publication of the dissertation in whole or in part also requires the
consent of the Dean of the Graduate School of the University of Kentucky.
DISSERTATION
Caroline Machado
The Graduate School
University of Kentucky
2004
STUDIES OF ERGOT ALKALOID BIOSYNTHESIS GENES IN
CLAVICIPITACEOUS FUNGI
DISSERTATION
A dissertation submitted in partial fulfillment of the requirements for
the degree of Doctor of Philosophy in the College of Agriculture
at the University of Kentucky
By
Caroline Machado
Lexington, Kentucky
Director: Dr. Christopher L Schardl, Professor of Plant Pathology
Lexington, Kentucky
2004
Copyright by Caroline Machado 2004
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my advisor Dr. Christopher L. Schardl for
his constant encouragement, motivation and patient guidance throughout the dissertation period.
I would like to thank my committee members Dr. Lisa Vaillancourt, Dr. Tom Pirone, Dr. Lowell
Bush, Dr. Deane Falcone and Dr. Tim Phillips for their great help and valuable suggestions. I
also would like to thank Dr. Daniel Panaccione from West Virginia University for guiding me in
ergot alkaloid analysis at his facility. I am thankful to Dr. Pete Mirabito from the Department of
Biology, University of Kentucky for providing me the plasmid containing cre gene.
I would like to thank Dr. Jinghong Wang who helped me to get started with the project,
Dr. Christina Moon for tips on GPS system and Jimmy Blankenship for teaching me the
computer skills and for the friendship and moral support throughout the graduate student career.
I appreciate all the help provided by all the Schardl lab members; for creating a very friendly
working atmosphere in the laboratory, you are the best. I also would like to thank Walter Hollin
and Al Byrd for their great help in the laboratory. Thanks to Jenny Wiseman for helping me with
sequencing work.
I am indebted to my husband Arun, whose constant support and motivation helped me to
reach the goal, thanks Arun for taking over the kitchen, feeding me and for the inspiration during
the most difficult time of writing dissertation. I would like to express my deep gratitude to my
mummy and papa for all the sacrifices, hard work and love, which made me reach this stage. I
also would like to thank my brother and sister for all the support and encouragement. I like to
thank my mother-in-law for her support and blessings.
I would like to thank all the faculty and staff in the Department of Plant Pathology for the
support and encouragement during the course of study.
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