Mechanisms of jasmonate induced activation of defense responses in Nicotiana attenuata [Elektronische Ressource] / von Rayko Halitschke

Rayko Halitschke

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2004
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Mechanisms of jasmonate-induced activation of defense
responses in Nicotiana attenuata


Dissertation
zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.)


vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller Universität Jena






von Diplomchemiker
Rayko Halitschke

geboren am 16. November 1974 in Weimar





















Gutachter




1.
2.
3.

Tag der Doktorprüfung:

Tag der öffentlichen Verteidigung:

Table of Contents I
Table of Contents

I
Manuscript Overview II

1. Introduction 1

2. Manuscripts
2.1. Manuscript I
Active components in larval oral secretions and regurgitant 9

2.2. Manuscript II
FACs regulate herbivore-induced transcriptional changes 27

2.3. Manuscript III
Nicotiana attenuata transformation 50

2.4. Manuscript IV
LOX-dependent herbivore resistance in a native tobacco 68

2.5. Manuscript V
Oxylipin-mediated defense signaling in wild tobacco 101

2.6. Manuscript VI
Influence of induced plant defenses on herbivore community composition 127

3. Discussion 152

4. Conclusion 160

5. Zusammenfassung 161

6. Literature Cited 164

7. Appendix 168
7.1. Supplementary Data 168
7.2. Publication List 169
7.3. Lebenslauf 171
7.4 Danksagung 172
7.5. Selbständigkeitserklärung 173
Manuscript Overview II
Manuscript I

Molecular Interactions Between the Specialist Herbivore Manduca sexta
(Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. III. Fatty
Acid-Amino Acid Conjugates in Herbivore Oral Secretions Are Necessary and
Sufficient for Herbivore-Specific Plant Responses

Rayko Halitschke, Ursula Schittko, Georg Pohnert, Wilhelm Boland and Ian T.
Baldwin

Plant Physiology (2001) 125: 711-717

This manuscript describes the isolation and characterization of fatty acid-amino acid
conjugates (FACs) as elicitors in the oral secretions and regurgitant of Manduca sexta
and Manduca quinquemaculata larvae. These compounds are responsible for the
recognition of the feeding herbivore by Nicotiana attenuata and elicit several
herbivore-specific defense responses including altered transcript accumulation, a
jasmonic acid burst, and the release of volatile organic compounds.
I collected, analyzed, and manipulated (by ion-exchange treatment) the caterpillar oral
secretions and regurgitant and planned the jasmonic acid accumulation experiment,
which I performed together with Ursula Schittko. Furthermore, I designed and
performed the experiments to characterize the induced volatile emissions. The
characterization of transcript accumulation was planned and realized by U. Schittko
and the fatty acid-amino acid conjugates were synthesized by Georg Pohnert in the
department of Professor Wilhelm Boland.

Manuscript Overview III
Manuscript II

Molecular Interactions Between the Specialist Herbivore Manduca sexta
(Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata. VI.
Microarray Analysis Reveals That Most Herbivore-Specific Transcriptional
Changes Are Mediated by Fatty Acid-Amino Acid Conjugates

Rayko Halitschke, Klaus Gase, Dequan Hui, Dominik D. Schmidt, and Ian T.
Baldwin

Plant Physiology (2003) 131: 1894-1902

The experiments presented in this manuscript quantitatively evaluate the contribution
of the elicitors identified and characterized in Manuscript I to the biological activity
of the oral secretions and regurgitant. A cDNA microarray was designed for the
expression analysis of herbivore-responsive genes and used to compare the expression
patterns in mechanically wounded plants, which received either water or Manduca
ssp. oral secretions or different solutions of herbivorous elicitors (FACs and glucose
oxidase; GOX). A majority of the genes (56%) induced by application of Manduca
ssp. oral secretions were similarly induced by FAC application. These patterns
involved the upregulation of genes involved in oxylipin signaling and defense
responses and the downregulation of photosynthetic genes. In contrast, the
transcriptional response to a second elicitor, GOX, showed the opposite regulation
pattern compared with the response to the larval oral secretions.
I designed the experiments analyzing the transcriptional response to the oral
secretions and regurgitants of the two Manduca species and FACs and performed the
plant treatments, harvest, RNA isolation, and analysis of microarray data. The glucose
oxidase experiment was performed by Dominik Schmidt and the cDNA AFLP
analysis was performed by Dequan Hui. Klaus Gase designed the microarray and was
responsible for the technical development and realization of the microarray analysis.

Manuscript Overview IV
Manuscript III

Agrobacterium-mediated transformation of Nicotiana attenuata, a model
ecological expression system

Tamara Krügel, Michelle Lim, Klaus Gase, Rayko Halitschke, and Ian T. Baldwin

Chemoecology (2002) 12: 177-183

Manuscript III describes the development of a transformation procedure for N.
attenuata. The standard transformation protocols for cultivated tobacco (N. tabacum)
had to be significantly modified for the transformation of N. attenuata. The developed
method was used to manipulate the expression of genes involved in oxylipin signaling
by antisense expression-mediated gene silencing. By silencing the expression of a
lipoxygenase (LOX) and allene oxide synthase (AOS) – two enzymes of the
octadecanoid pathway – we dramatically reduced the accumulation of wound-induced
jasmonic acid (JA) accumulation. Furthermore, we manipulated the release of green
leaf volatiles (GLVs) by silencing expression of hydroperoxide lyase. The transgenic
plants generated by this procedure represent the tools for the following experiments to
elucidate the role of individual oxylipin signals in the defensive response of N.
attenuata.
Klaus Gase designed and developed the transformation vectors and the DNA
extraction procedure described in the manuscript. Tamara Krügel und Michelle Lim
developed, optimized, and performed the transformation procedure and generated the
first generation of transgenic plants (T ). I was responsible for the genetic and 0
biochemical characterization of the transformed plants. This involved the
confirmation of transformation, segregation analysis, propagation and phenotype
characterization. I developed the high-throughput screening procedure for JA and
GLV analysis and analyzed these metabolites in the transformed plants.

Manuscript Overview V
Manuscript IV

Antisense LOX expression increases herbivore performance by decreasing
defense responses and inhibiting growth-related transcriptional reorganization
in Nicotiana attenuata

Rayko Halitschke and Ian T. Baldwin

Plant Journal (2003) 36: 794-807

Manuscript IV describes the isolation and characterization of three distinct classes of
LOX genes in N. attenuata. I Identified the isoform (NaLOX3) showing expression
patterns that strongly correlate with induced JA accumulation for further
investigations. I investigated the consequences of reduced JA signaling capacity in
transgenic plants (as-lox), which were generated by the procedure described in
manuscript III. The impaired JA biosynthesis suppressed direct and indirect defense
responses and resulted in reduced resistance against herbivory by M. sexta larvae.
I was responsible for the experimental planning and realization including bioassays,
chemical and statistical analysis. I isolated and characterized the LOX genes and
characterized the antisense transformed plants.

Manuscript Overview VI
Manuskript V

Silencing of hydroperoxide lyase and allene oxide synthase reveals substrate and
defense signaling cross-talk in Nicotiana attenuata

Rayko Halitschke, Jörg Ziegler, Markku Keinänen, and Ian T. Baldwin

In review: Plant Journal

This manuscript describes the isolation and characterization of a hydroperoxide lyase
(HPL) gene of N. attenuata and the co