Table Of ContentUUnniivveerrssiittyy ooff KKeennttuucckkyy
UUKKnnoowwlleeddggee
University of Kentucky Doctoral Dissertations Graduate School
2010
PPHHYYLLLLOOPPLLAANNIINNSS:: NNOOVVEELL AANNTTIIFFUUNNGGAALL PPRROOTTEEIINNSS OONN PPLLAANNTT
LLEEAAFF SSUURRFFAACCEESS
Ryan William Shepherd
University of Kentucky, [email protected]
RRiigghhtt cclliicckk ttoo ooppeenn aa ffeeeeddbbaacckk ffoorrmm iinn aa nneeww ttaabb ttoo lleett uuss kknnooww hhooww tthhiiss ddooccuummeenntt bbeenneefifittss yyoouu..
RReeccoommmmeennddeedd CCiittaattiioonn
Shepherd, Ryan William, "PHYLLOPLANINS: NOVEL ANTIFUNGAL PROTEINS ON PLANT LEAF SURFACES"
(2010). University of Kentucky Doctoral Dissertations. 763.
https://uknowledge.uky.edu/gradschool_diss/763
This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been
accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of
UKnowledge. For more information, please contact [email protected].
ABSTRACT OF DISSERTATION
Ryan William Shepherd
The Graduate School
University of Kentucky
2004
PHYLLOPLANINS: NOVEL ANTIFUNGAL PROTEINS
ON PLANT LEAF SURFACES
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
Ryan William Shepherd
Lexington, Kentucky
Director: Dr. George J. Wagner, Professor of Agronomy
Lexington, Kentucky
2004
Copyright © Ryan William Shepherd 2004
ABSTRACT OF DISSERTATION
PHYLLOPLANINS: NOVEL ANTIFUNGAL PROTEINS
ON PLANT AERIAL LEAF SURFACES
Secreted surface proteins are an innate immune defense component employed by animals
to inhibit invading microbes. Surface proteins have not been documented in plants, even though
the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered
novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum,
and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-
expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete
pathogen Peronospora tabacina.
We investigated the site of phylloplanin biosynthesis using biochemical techniques.
These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal
peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation
fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude
that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not
by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose
elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly
provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest
that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface.
Phylloplanins constitute a first-point-of-contact, rapid response, innate immune
deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied
representatives of a novel protein class in the plant kingdom. Further study of leaf surface
proteins is justified to understand further their roles in plant defense, and to investigate their
potential in agricultural biotechnology.
Additionally, we describe miscellaneous observations we have made during the course
of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf
surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known
antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its
function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be
antibacterial. Further study of this protein is warranted. We note that proteins can also be
recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further
characterized these proteins.
In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene
encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is
reported, and evidence was found for a unique mechanism of surface deposition.
KEYWORDS: Phylloplane, Phyllosphere, Peronospora tabacina, Innate Immunity, Hydathode
Ryan W. Shepherd
PHYLLOPLANINS: SECRETED ANTIFUNGAL PROTEINS
ON AERIAL PLANT SURFACES
By
Ryan William Shepherd
George J. Wagner
Director of Dissertation
Arthur G. Hunt
Director of Graduate Studies
RULES FOR THE USE OF DISSERTATIONS
Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of
Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the
rights of the authors. Bibliographical references may be noted, but quotations or summaries of
parts may be published only with the permission of the author, and with the usual scholarly
acknowledgements.
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
Ryan William Shepherd
The Graduate School
University of Kentucky
2004
PHYLLOPLANINS: NOVEL ANTIFUNGAL PROTEINS
ON PLANT LEAF SURFACES
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
Ryan William Shepherd
Lexington, Kentucky
Director: Dr. George J. Wagner, Professor of Agronomy
Lexington, Kentucky
2004
Copyright © Ryan William Shepherd 2004
ACKNOWLEDGEMENTS
I am indebted to many people for their help over the past years. First and foremost, I
want to thank Dr. George Wagner, my advisor, for giving me a job in his lab and introducing me
to the uncharted realms of plant physiology and the leaf surface. He’s the smartest man I’ve ever
met, and I’ve been very lucky to work with him, learn from him, and conduct research with him.
It is a true honor to know him and call him a mentor and a friend. I also wish to thank Dr.
Robert L. Houtz, not only for being on my committee, but also for insisting that I give pepsin
digestion a try to recover the unknown phylloplanin gene. Without his help, I’d still today be
scratching my head as a graduate student and wondering if these strange proteins were real. Mr.
W. Troy Bass also has taught me a great deal about blue mold. He has been very helpful and
energetic about the preparation and conduct of the antifungal assays, and he is a great guy to
work with. I also want to thank the other members of my committee, Dr. Deane L. Falcone and
Dr. Arthur G. Hunt, both of whom have provided valuable insights in times of need. Thanks also
to Dr. Said Ghabrial, my outside examiner.
My parents, Gene and Cheryl Shepherd, have emphasized education from the start, so
any success that I’ve had as a student is directly attributable to them. In graduate school, I’ve
always been encouraged by my parents, my grandparents, my brothers Owen and Colin and my
sister Megan to keep doing the experiments. Dr. Antoaneta Kroumova, Dr. Victor Koren’kov,
and other members of Dr. Wagner’s lab have given me much encouragement and technical
assistance. Additionally, many others from KTRDC and the UK Agronomy Department are
deserving of thanks, ranging from other graduate students to post-docs to technicians and
professors. They are a terrific group of people and it has been a pleasure to work with them for
the past years. Finally, I want to thank Ralitza Kroumova, my present fiancé and future wife,
for her enduring support and endless inspiration. I find myself very fortunate and I dedicate this
work to her.
iii
Description:example, trichome density is reported to increase on developing willow leaves if older portions of the plant are .. utility of the phylloplanin promoter region as a novel leaf surface protein delivery system. phylloplanins, and showed that peptides of less than 11 kDa (e.g. plant defensins) were no
