16th

IMPRS Symposium

March 7-8, 2017

Old Castle, Dornburg

Program

Tuesday, March 7th, 2017

08:00 Departure, central bus stop (opposite Jena Paradies train station)

08:45 Welcome and Announcements (Großer Kaisersaal)

Prof. Jonathan Gershenzon, spokesperson of the IMPRS

09:00 Plenary Lecture 1 (Großer Kaisersaal)

Glucosinolate structural evolution studied with reliable MS2 and NMR

identification

Prof. Niels Agerbirk, University of Copenhagen, Denmark, p.2

Chair: Anton Shekhov

10:00 Coffee break

1st talk session (Großer Kaisersaal)

Insect molecular and neuroethological adaptations to plants

Chair: Wiebke Häger

10:20 1 - Glutathione-dependent detoxification of p-hydroxybenzyl glucosinolate in

Spodoptera littoralis

Anton Shekhov (GER), p.5

10:40 2 - How do flea beetles overcome the ‘mustard-oil-bomb’?

Theresa Sporer (HEC), p.6

11:00 3 - Mannanases, but not always! Evolution and functional characterization of

glycoside hydrolase family 5 proteins in two species of phytophagous beetles

André Busch (HEC), p.7

11:20 4 - Detoxification of salicortin in the specialist herbivore Cerura vinula

Felix Feistel (NMR), p.8

11:40 5 - The ecological significance of trichoid sensilla in female silkmoths

Elisa Schuh (HAN), p.9

12:00 Lunch

13.00 Poster talks 1 (Großer Kaisersaal)

Odd numbers (1 slide and 1 min/poster)

Chair: Mohammed A. Khallaf

13:15 Poster session 1 – odd numbers (Kleiner Kaisersaal)

14:40 Coffee break & group photo

15:10 Introduction of new PhD representatives & IMPRS announcements (Großer

Kaisersaal)

PhD reps, Claudia Voelckel

2nd talk session (Großer Kaisersaal)

The chemical ecology of pollination

Chair: Sarah Körte

15:30 6 - Finding the best match: comparing preferences of Nicotiana attenuata and

its pollinators

Julia Bing (ITB), p.10

15:50 7 - In-vivo tracking of differential pollen tube growth in style confirms pre-

zygotic mate selection amongst wild paternal genotypes of Nicotiana attenuata

Xiang Li (ITB), p.11

16:10 8 - Where, when and how? The secrets of effective nudicaulin production in

Papaver nudicaule petals

Bettina Dudek (NMR), p.12

16:30 IMPRS Faculty Meeting (Großer Kaisersaal)

16:45 Bus returns to Jena (Students and guest speakers leave)

17:15 IMPRS faculty return to Jena (Carpooling)

18:15 Dinner with guest speakers – Zur Noll

19:00 Discussions with guest speakers and MPICE scientists about collaborative

projects

20:30 End of day 1

Wednesday, March 8th, 2017

08:00 Departure, central bus stop (opposite Jena Paradies train station)

09:00 Plenary Lecture 2 (Großer Kaisersaal)

How single parasite virulence effectors modulate plant development and

promote colonization of insect vectors

Prof. Saskia Hogenhout, University of East Anglia, Norwich, UK, p.4

Chair: Maria Paulmann

10:00 Coffee break

3rd talk session (Großer Kaisersaal)

Plant signaling and secondary metabolism in the context of defense and

symbiosis

Chair: Franziska Eberl

10:20 9 - About keeping the balance – How to antagonistic calmodulin-like proteins

mediate various plant stress responses

Monika Heyer (BOL), p.13

10:40 10 - Jasmonate signaling mediates the establishment of arbuscular

mycorrhizal symbiosis in Nicotiana attenuata by interfering with strigolactone

biosynthesis in a COI1-dependent manner

Ming Wang (ITB), p.14

11:00 11 - O-acylsugars function as generalized defense compounds of Nicotiana

attenuata against both pathogens and herbivores

Van Thi Luu (ITB), p.15

11:20 12 - Populus trichocarpa aromatic aminoacid decarboxylase enzymes and

their roles in plant direct and indirect defense

Jan Günther (GER), p.16

11:40 13 - How belowground herbivory influences aboveground defense

mechanisms of black poplar

Sandra Lackner (GER), p.17

12:00 Lunch

13:00 Poster talks 2 (Großer Kaisersaal)

Even numbers (1 slide and 1 min/poster)

Chair: Andrew J. O’Donnell

13:15 Poster session 2 – even numbers (Kleiner Kaisersaal)

14:40 Coffee break

4th talk session (Großer Kaisersaal)

Selected topics from ant olfaction and microbial evolution

Chair: Samir Giri

15:00 14 - Capacity and storage duration of an ant's olfactory memory

Roman Huber (HAN), p.18

15:20 15 - The meaning of fast and slow transcription

Peter Grossmann (FSU), p.19

15:40 16 - Circumstances favor the cooperative evolution of bacterial cross-feeding

Muhammad Atiqur Rahman (BOL), p.20

16:00 Closing remarks (Großer Kaisersaal)

Prof. Jonathan Gershenzon, spokesperson of the IMPRS

16:10 Talk and poster jury meets for discussions (Großer Kaisersaal)

16:30 Return to Jena

18:00 Café Rossini - Prof. Gershenzon announces winners of talk and poster prizes

18:20 Symposium feedback discussions and farewell of the guest speakers

20:00 End of day 2

Posters 1. DMNT-induced upregulation of defensive sporamin in sweet potato

Anja Meents (BOL), p.22

2. How to DREAM and what to learn from it Sarah Körte (HAN), p.23

3. Chlorophyll degradation in lepidopteran pest (Spodoptera littoralis) Vincensius S. P. Oetama (BOL), p.24

4. A male specific pheromone mediates the sexual and social behavior in D. mojavensis Mohammed A. Khallaf (HAN), p.25

5. Catabolism of flavonols in Arabidopsis thaliana by Sclerotinia sclerotiorum Jingyuan Chen (GER), p.26

6. Experience-dependent plasticity of olfactory circuits in Drosophila melanogaster Benjamin Fabian (HAN), p.27

7. Catechin and proanthocyanidins are effective antifungal defenses in poplar against biotrophic rust fungus (Melampsora spp.) Chhana Ullah (GER), p.28

8. Experimental evolution of metabolic cross-feeding among bacteria Samir Giri (BOL), p.29

9. Metabolic and evolutionary insight into regulation of diterpene resin acids in Norway

spruce

Andrew J. O’Donnell (GER), p.30

10. Public goods of Bacillus subtilis – how public are they really? Theresa Hölscher (FSU), p.31

11. Poplar-herbivore-pathogen interaction: herbivores take advantage of infected host plants

Franziska Eberl (GER), p.32

12. The evolutionary arms race between Psylliodes leaf beetles and their host plants Matilda Gikonyo (HEC), p.33

13. Cry-worthy cost of producing an ectopic defense protein

Erica McGale (ITB), p.34

14. Cytochrome P450s are involved in iridoid biosynthesis in leaf beetles

Nanxia Fu (BOL), p.35

15. Production of volatile and non-volatile isoprenoids during drought stress in P. glauca

young needles Erica Perreca (GER), p.36

16. Interaction of beetle polygalacturonases with plant cell wall proteins – heterologous expression of potential inhibitors Wiebke Häger (HEC), p.37

17. Evidence for GABA-induced systemic GABA accumulation upon wounding in Arabidopsis thaliana Jaiana Malabarba (Guest, BOL), p.38

Talks

2

Plenary 1

Glucosinolate structural evolution studied with reliable MS2 and NMR identification

Niels Agerbirk

University of Copenhagen, Denmark nia@plen.ku.dk

I was always fascinated by the structural diversity of secondary metabolites and couldn’t help

wondering how and why this diversity arose. As glucosinolates are so easy to work with and yet have

such a fascinating biochemistry because of the activation by myrosinase and multiple product types, it

was easy to choose them as my main focus. I also enjoyed the simple pleasure of elucidating

chemical structures. Hence my research subject was obvious: I wanted to determine glucosinolate

structures in a range of related plants, in order to find patterns that could show the course of evolution.

Furthermore, I wanted to be extra focused on finding small chemical differences and on understanding

chemical consequences of these small differences.

Fate gave me the chance to spend some years on the wild crucifer Barbarea vulgaris. While searching

for completely different metabolites, I accidentally discovered multiple novel glucosinolates. These

showed exactly the small differences I was so fascinated by, so I just had to go on with that.

I started to analyze Barbarea species systematically, and found small differences among them and

large differences from other species. As reliable phylogenetic information appeared in the literature, I

started to analyze more distantly related plants in parallel. A challenge here was to get reliable

identification of the many isomers possible within glucosinolates. The upcoming technique of HPLC-

MS proved to be a wonderful tool in this respect, allowing definite identifications when MS2

fragmentation patterns were backed up by NMR analysis of authentic standards. Na+ adducts were

particularly informative.

As a result of these many coinciding developments, I now have a pretty good overview of

glucosinolate diversity in Barbarea and their allies (tribe Cardamineae, including horse radish,

watercress, Rorippa, Cardamine, etc). In my talk I’ll describe some of the technical progress in

glucosinolate identification, some of the diversity patterns observed and some chemical consequences

199

208

240

256

272

290

370

388

434

+MS2(452), 5.9min #746

0.0

0.2

0.4

0.6

0.8

1.0

6x10

Intens.

150 200 250 300 350 400 450 m/z

-H:

256 +H: 290 (c)

-H: 388 (g) -H:

199

-H:

240 +H: 274 (c)

-H: 372 (g)

A B

C

g

c

h

f g+f

i g+i

d69 d150

d150 MS2 of [M+Na]+

3

of specific small structural differences. I’ll finally propose biological roles of some of this diversity, and

speculate on further directions of fruitful research. Recently published genomes and transcriptomes of

Barbarea vulgaris may come in useful here.

4

Plenary 2

How single parasite virulence effectors modulate plant development and promote colonization of insect vectors

Saskia A. Hogenhout Department of Crop Genetics, John Innes Centre, Norwich Research Park, UK

saskia.hogenhout@jic.ac.uk

One of the most spectacular phenomena in biology is the complete hijacking of hosts by parasites such that these hosts become ‘zombies’ destined to advance survival of the parasites often to the detriment of the hosts. Phytoplasmas are obligate intracellular bacterial parasites of plants that induce dramatic changes in plant development, including proliferation of stems (witch’s brooms) and the reversion of flowers into leaf-like structures (phyllody). These bacterial parasites produce two virulence proteins (effectors), named SAP11 and SAP54, that promote the degradation of plant TCP and MADS-box transcription factors, respectively, thus altering leaf and flower development. Phytoplasmas are dependent on sap-feeding insects for transmission to plants. Interestingly, SAP11 and SAP54 also convert plants into more attractive hosts for egg laying and reproduction of the insect vectors. Thus, phytoplasma effectors interfere with key plant developmental processes. Moreover, these effectors increase the colonization of plants by insect vectors thereby promoting transmission of the obligate phytoplasmas to new plant hosts.

5

Talk 1

Glutathione-dependent detoxification of p-hydroxybenzyl glucosinolate in Spodoptera littoralis

Anton Shekhov

1, Christian Paetz

2, Michael Reichelt

1, Riya C. Menezes

3, Jonathan

Gershenzon1, Daniel G. Vassão

1

1Department of Biochemistry, Max Planck Institute for Chemical Ecology

2Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology

3 Research Group Mass Spectrometry, Max Planck Institute for Chemical Ecology

ashekhov@ice.mpg.de

Plants from the Brassicaceae family use a myrosinase-glucosinolate (gls) defense system to

chemically protect themselves against herbivory. Upon tissue rupture, myrosinases hydrolyse

glucosinolates, resulting in release of toxic compounds such as isothiocyanates (ITC), nitriles and

thiocyanates. Plants can accumulate up to 40 different glucosinolates with various side-chains

(classified broadly as aliphatic, benzenic and indolic) that undergo distinct post-hydrolysis breakdown

reactions. Consequently, the mixture of the products is equally chemically diverse. In order to be able

to feed on the plants, insects have to deal with these chemical plant defenses. As a result many

specialists have adapted to avoid and even to exploit the glucosinolate-myrosinase system for their

own benefit. Generalists, on the other hand, do not possess mechanisms to avoid the release of toxic

products and have to deal with their presence in the diet. We are investigating the metabolic fates of

distinct groups of glucosinolates in caterpillars of the generalist Spodoptera littoralis. While it is known

that these caterpillars metabolize aliphatic glucosinolates by conjugation of its electrophilic

isothiocyanates to glutathione (Schramm et al., 2012), the breakdown of several other glucosinolates

leads to less stable ITCs with highly contrasting reactivities, and their post-ingestive metabolism has

not been examined. We therefore performed feeding experiments with 14

C-labeled sinalbin (p-

hydroxybenzyl-GLS), whose derived ITC has several chemically reactive centers, and is unstable in

aqueous media. HPLC analysis of feces extracts revealed the major metabolite, which was identified

in non-radioactive samples as S-(p-hydroxybenzyl) cysteine. In addition, we detected other

metabolites from the corresponding mercapturic acid pathway (conjugates with glutathione,

cysteinylglycine and N-acetylcysteine), revealing glutathione’s involvement in the detoxification of

sinalbin in S. littoralis. However, the differences in reaction centers between aliphatic and benzenic

glucosinolate degradation products suggest that different underlying mechanisms are responsible for

this process. In order to shed light on the molecular aspects of this reaction, we are currently studying

the role of glutathione-S-transferases (GSTs) in the detoxification processes of all three groups of

glucosinolates.

6

Talk 2

How do flea beetles overcome the 'mustard-oil bomb'?

Theresa Sporer, Zhi-Ling Yang, Franziska Beran

Research Group Sequestration and Detoxification in Insects, Max Planck Institute for Chemical

Ecology

tsporer@ice.mpg.de

Plants of the order Brassicales defend themselves against herbivores with the so-called ‘mustard-oil

bomb’. In intact plant tissue, glucosinolates (GLS) are stored separately from the enzyme myrosinase,

but upon tissue damage, both components come together, which leads to the production of highly

reactive isothiocyanates. Phyllotreta flea beetles (Coleoptera: Galerucinae) are specialists on crucifers

and were previously shown to accumulate high amounts of GLS in their hemolymph. We want to

understand how Phyllotreta flea beetles prevent GLS hydrolysis by the plant myrosinase to sequester

intact GLS. One possible mechanism is that GLS sequestration is faster than hydrolysis by the plant

myrosinase. To determine how fast P. armoraciae beetles can sequester GLS, we allowed them to

feed for one minute on wild-type Arabidopsis thaliana (Col-0) leaves, dissected them subsequently into

gut and rest of body, and analyzed the plant derived GLS in these tissues via LC-MS/MS. We found

similar amounts of GLS in the gut and the rest of body, which shows a rapid but not complete uptake

of dietary GLS within one minute. Together with the observation of GLS being excreted with the feces,

these results indicate that P. armoraciae is able to control myrosinase activity during feeding and

digestion. Thus, we tested whether P. armoraciae gut content influences plant myrosinase activity in

enzyme assays by comparing myrosinase activity with and without P. armoraciae gut content extracts.

Indeed, myrosinase activity was reduced by 53% in assays incubated with beetle extracts. Our next

goal is to identify which compound(s) are responsible for this effect.

7

Talk 3

Mannanases, but not always! Evolution and functional characterization of glycoside hydrolase family 5 proteins in two species of phytophagous

beetles

André Busch, David G. Heckel, Yannick Pauchet Department of Entomology, Max Planck Institute for Chemical Ecology

abusch@ice.mpg.de

Hemicelluloses, such as xyloglucan, mannans and xylan, consist of a heterogeneous array of plant-

derived polysaccharides constituent of the plant cell wall. These polysaccharides are different in

structure and physiochemical properties but they share a β(1→4) linked sugar backbone.

Hemicellulose can be hydrolyzed by plant cell wall degrading enzymes (PCWDEs), which are widely

distributed in phytopathogenic microbes. However, in recent years it became apparent that also

phytophagous beetles produce their own PCWDEs.

Our previous work identified genes encoding putative mannanases belonging to the glycoside

hydrolase (GH) family 5 subfamily 10 (sf10) in the genomes of the leaf beetle Gastrophysa viridula

(Chrysomelidae, Chrysomelinae) and of the bean beetle Callosobruchus maculatus (Chrysomelidae,

Bruchinae), with one and four genes respectively. In contrast to other GH5 subfamilies, GH5sf10

proteins are scarcely distributed within the tree of life and have hardly been investigated so far. Hence,

we addressed the following questions: Are beetle-derived GH5sf10 active PCWDEs? If so, how did

they evolve and what is their physiological function?

Using heterologous protein expression and enzymatic assays, I could show that the G. viridula

GH5sf10 was a mannanase active on both galactomannan and glucomannan. I was also able to

demonstrate that only one out of four C. maculatus GH5sf10 was a mannanase with additional activity

on carboxymethyl cellulose (amorphous cellulose derivate). Unexpectedly, another C. maculatus

GH5sf10 evolved to use xylan instead of mannans as substrate, thus being a xylanase. RNAi

experiments in G. viridula indicated that (i) the sole GH5sf10 was entirely responsible for breaking

down mannans in the gut, and (ii) that this breakdown was rather accessory and may facilitate the

access to the nitrogen-rich plant cell content. Phylogenetic analyses indicated that coleopteran-

derived GH5sf10 cluster together with Chelicerata-derived proteins. Interestingly, other insect-derived

GH5sf10 cluster elsewhere, suggesting the possibility of several independent evolutionary origins

within the insects.

8

Talk 4

Detoxification of salicortin in the specialist herbivore Cerura vinula

Felix Feistel1, Christian Paetz

1, Bernd Schneider

1, Daniel Veit

2, Regina Seibt

3, Riya C. Menezes

4

1Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology

2Technical Service, Max Planck Institute for Chemical Ecology

3Department of Entomology, Max Planck Institute for Chemical Ecology

4Research Group Mass Spectrometry, Max Planck Institute for Chemical Ecology

ffeistel@ice.mpg.de

Salicortin is a plant phenolic glucoside produced as chemical defense against herbivory. Specialist

herbivores like the lepidopteran caterpillar of Cerura vinula are able to surpass this defense. We

examined the main frass constituents of C. vinula, resulting in identification of eleven quinate esters

containing benzoate and/or salicylate. Whether the quinates are a result of salicinoid breakdown or

de-novo biosynthetic products was examined in feeding experiments with highly [13

C]-enriched

salicortin. Using HRMS and NMR analyses we could confirm that salicortin detoxification proceeds

likely by deglucosylation and ester hydrolysis followed by oxidative transformation of saligenin into

salicylic acid, and further to formation of quinate esters.

9

Talk 5

The ecological significance of trichoid sensilla in female silkmoths

Elisa Schuh1, Bill S. Hansson

2, Silke Sachse

1, Sonja Bisch-Knaden

2

1Research Group Olfactory Coding, Max Planck Institute for Chemical Ecology

2Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology

ebadeke@ice.mpg.de

Sensory hairs on the antenna of male silkmoths (Bombyx mori) house two neurons that are involved in long-range attraction to conspecific females via female-produced sex pheromones. Female silkmoths also possess these so-called trichoid sensilla, which are described to house neurons exclusively tuned to linalool and benzoic acid, respectively. The specific ecological significance of those volatiles remained unknown. We therefore asked which ecological role the trichoid sensilla on the antenna of female silkmoths might play. In order to address this question, we collected volatiles of mulberry leaves, the only host plant of B.

mori larvae. Furthermore, we collected the headspace of female and male moths to identify potential

pheromonal compounds in addition to the female-produced sex pheromone. We analyzed these odour

collections using gas chromatography, and tested the physiological activity of the most prominent

compounds by single sensillum recordings from female trichoid sensilla. In total we tested a panel of

73 monomolecular odorants with a potential ecological significance for female silkmoths.

In mulberry extracts we found several aromatic compounds, terpenes and a few green leaf volatiles.

The analysis of volatile collections of female and male moths revealed predominantly terpenes. Our

single sensillum recordings revealed a broader response profile of trichoid sensilla than reported in

earlier studies. While the ‘linalool cell’ is also highly tuned to α-terpineol, cis-jasmone, and some

aromatic compounds, the ‘benzoic acid cell’ showed equally high spike frequencies to the acids 3-

methyl butanoic acid and pentanoic acid as well as to benzaldehyde. Interestingly, there was one

mulberry compound, indole, that elicited an inhibititory response, lasting for 4-5 seconds. Beside this

sensillum type, we found a second type of trichoid sensilla with a different response profile. The two

neurons housed in this sensillum type responded to none of the acids, but mostly to methyl salicylate,

2-phenylethanol and cis-jasmone.

Our data show that neurons housing in trichoid sensilla of female silkmoths are broadly tuned and

detect mainly host plant volatiles. However, the significance of the neuron responding to acids is still

unknown.

10

Talk 6

Finding the best match: Comparing preferences of Nicotiana attenuata and its pollinators

Julia Bing

1, Danny Kessler

1, Ian T. Baldwin

1

1Department of Molecular Ecology, Max Planck Institute for Chemical Ecology

jbing@ice.mpg.de

The self-compatible wild tobacco plant Nicotiana attenuata grows in genetically diverse populations

and is visited by a variety of pollinators, such as hummingbirds, hawkmoths and bees. These floral

visitors show differences in their response to floral traits, such as scent or nectar, which the plant uses

to allure potential floral visitors. Pollinators choose based on their adaptations to different floral traits.

But not only pollinators make a choice, tobacco does as well. If pollen of different paternal genotypes

has been transferred to the stigma of N. attenuata, the plant is able to select its mates from this

diverse pool of pollen.

For this, two questions have to be considered:

1. What floral traits are important for the different pollinators?

2. Which pollinators match the plant’s preferences?

To answer this, we conduct natural and semi-natural experiments with transgenic lines and native

accessions that show differences in floral traits. Since ethylene plays an important role in enabling the

mate selection process we used transformed plants with silenced ethylene production (ACO) in

comparison to empty vector control plants (EV). Pairs of EV and ACO plants were planted in a

population of different native accessions that vary in floral traits important for pollinator attraction.

Seeds produced after pollinator visitation will be used for genotyping. The comparison of the seed set

from both transformed lines will shed light on which pollen was brought by the pollinators (ACO plant)

and what pollen was selected by the plant to set seeds (EV plant). That allows us to uncouple the

events of pre-pollination sexual selection (pollinator choice) and post-pollination sexual selection (mate

choice of plant) to get an idea if there are pollinators that transfer exactly the pollen genotypes to the

flowers that also the plant would choose based on its mate selection pattern.

11

Talk 7

In-vivo tracking of differential pollen tube growth in style confirms pre-zygotic mate selection amongst wild paternal genotypes of Nicotiana

attenuata

Xiang Li1, Ian T. Baldwin

1, Samik Bhattacharya

1

1Department of Molecular Ecology, Max Planck Institute for Chemical Ecology

xli@ice.mpg.de

Predominantly selfing Nicotiana attenuata allows non-self-pollen to sire offspring by opportunistic

outcrossing in nature. While self-pollen consistently produced more offspring in mixed pollinations than

the non-self-pollen genotypes, a consistent pattern of pre-zygotic mate selection amongst the non-self-

pollen genotypes was observed. However, the demonstration of pre-zygotic in vivo pollen tube (PT)

competition between natural genotypes was challenging due to the unavailability of naturally occurring

diagnostic visual parameters. We identified two parallel PT transmitting zones in N. attenuata styles,

which enabled tracking the growth of competing pollen genotypes carefully placed on either half of the

stigma, orthogonal to the stigma lobes. In this study, we wanted to visualize the pre-zygotic, stylar

mate selection in wild N. attenuata where the wild accession (UtWT) consistently produced more

seeds from the pollen of a native genotype (G2, favored) over the other (G10, non-favored) in equal

mixed pollination with the self-pollen. Pollinations on the two transmitting zones of an UtWT stigma

with UtWT and G10 pollen and subsequent tracking of PTs growth in the same style, 4h and 8h after

pollinations, provided first visual evidence of pre-zygotic pistil-mediated PT competition and

significantly slower PT growth for G10 was observed in vivo. Similar in vivo tracking of competitive

UtWT and G10 PTs’ growth kinetics in irACO and ETR1 styles (impaired in ethylene production or

perception, respectively) showed random bias for both pollen genotypes, highlighting the loss of mate

selection in the ethylene-deficient styles. In this research, we visually demonstrated PT competition

within style between two wild pollen genotypes as evidence for the ethylene-mediated pre-zygotic

mate selection in N. attenuata, that subsequently led to the non-random seed sets from outcrossing.

12

Talk 8

Where, when and how? The secrets of effective nudicaulin production in P. nudicaule petals

Bettina Dudek

1, Anne-Christin Warskulat

1, Jaime Martinez-Harms

2, Sybille Lorenz

3, Bernd Schneider

1

1Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology

2Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology

3Research Group of Mass Spectrometry/Proteomics,

Max Planck Institute for Chemical Ecology

bdudek@mpg.ice.de Poppy flowers from Papaver nudicaule owe their bright yellow colour a unique class of alkaloids, known as nudicaulins. For 80 years, details about the chemical structure and the biosynthesis of nudicaulins remained obscure until their indole-phenylpropanoid hybrid structure was elucidated in 2013. Since then, the interest turned to the study of their remarkable biosynthesis. This talk will provide an insight into the location, time course and mechanisms of nudicaulin production as well as pigment storage in the petals. The role of pelargonidin and indole as the final precursors and their reaction will be highlighted: Several pelargonidin glucosides are formed early during the petal development and stored inside the vacuoles, colouring the maturing petals red. Later on, the nudicaulins gradually accumulate due to the reaction of pelargonidin with indole, finally changing the colour of the petals to yellow. In contrast to known conditions in plant cells, this specific reaction requires a very acidic environment. We were able to induce nudicaulin formation in vivo and in vitro indicating a spontaneous biosynthetic step. Additionally, we observed indole in the flower bouquet of yellow Papaver nudicaule in high amounts. The combination of odour and colour enables honeybees to recognise the bright yellow flowers and might therefore be crucial for reproductive success. Proteomic and transcriptomic investigations into the concerted formation of indole as an odour component and nudicaulins as flower pigments as well as the regulation of the linked biosynthetic pathways are ongoing.

13

Talk 9

About keeping the balance - How two antagonistic calmodulin-like proteins mediate various plant stress responses

Monika Heyer

1, Sandra Scholz

2, Michael Reichelt

3, Jyothilakshmi Vadassery

4, Ralf Oelmüller

2,

Wilhelm Boland1, Axel Mithöfer

1

1Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology

2Department of Plant Physiology, Friedrich Schiller University Jena

3Department of Biochemistry, Max Planck Institute for Chemical Ecology

4National Institute of Plant Genome Research, New Delhi, India

mheyer@ice.mpg.de

Plants are challenged by various environmental stimuli throughout their lifetime. In response to abiotic

as well as to biotic stresses, calcium (Ca2+

) is released into the plant cell cytoplasm early after the

recognition of the stimulus. A specific decoding of such Ca2+

signals is essential for the plant to react

appropriately to each stimulus. Binding of Ca2+

ions by calcium sensor proteins is an important first

step of decoding, leading to downstream responses like the specific elevation of phytohormones or the

release of defensive compounds. In Arabidopsis, 250 calcium binding proteins are known. Among

them, the family of calmodulin-like proteins (CMLs) is of great importance since they are unique for

plants. This study is focusing on two of them: CML37 and CML42.

Recently, it has been shown that both CMLs regulate the response to the insect herbivore Spodoptera

littoralis and to drought stress. Besides, they seem to mediate the plant’s reaction to the necrotrophic

fungus, Alternaria brassicicola. In all cases, CML37 turns out as a positive regulator of the stress

response whereas CML42 is a negative one. Thus, CML37 and CML42 might act as antagonistic

regulators of stress responses in Arabidopsis plants. To further investigate this hypothesis, double

knock out mutants lines were constructed and analyzed.

14

Talk 10

Jasmonates signaling mediates the establishment of arbuscular mycorrhizal symbiosis in Nicotiana attenuata by interfering with

strigolactone biosynthesis in a COI1-dependent manner

Ming Wang, Yang Wang, Karin Groten, Ran Li, Ian T. Baldwin Department of Molecular Ecology, Max Planck Institute for Chemical Ecology

mwang@ice.mpg.de

Full establishment of arbuscular mycorrhizal (AM) symbioses between more than 80% land plants and fungi of the Glomeromycota is thought to be largely controlled by phytohormones. Evidence for a role for jasmonates (JA) in AM colonization with Medicago truncatula, tomato and rice, has been confounded by the use of various mutants in JA biosynthesis and signaling which has produced inconsistent results. Clearly a need for a systematic examination of JA biosynthesis or signaling on AM colonization in a single native host plant that does not have a long history of artificial selection is required for a clearer understanding of this important symbiosis. Here, we examined AM colonization in Nicotiana attenuata in JA biosynthetic and signaling deficient lines (iraoc, ircoi1, respectively) to evaluate if JA production or perception is required. Silencing JA perception (ircoi1) significantly increased AM colonization, while silencing JA biosynthesis (iraoc) did not. Furthermore, in the JA signaling pathway, downstream of COI1, two AM responsive JAZ proteins (JAZd and JAZl) were functionally characterized by Virus-induced Gene Silencing (VIGS), and they were found to significantly inhibit AM colonization when their expression was silenced. Together these results reveal that JA production is not required for AM colonization but that JA signaling strongly represses AM development through the function of specific suppressor of JAZs in a COI1-dependant manner. Moreover, in the ircoi1 line (but not the irAOC line), the expression of strigolactone (SL) biosynthetic genes were found to be upregulated. These results suggest that SL production might be increased in these plants, which would be expected to enhance AM spores germination in the early stage of symbiosis. These results highlight the impact of JA signaling on SL production, which could profoundly influence the establishment of AM symbiosis.

15

Talk 11

O-acyl sugars function as generalized defense compounds of Nicotiana attenuata against both pathogens and herbivores

Van Thi Luu

1, Alexander Weinhold

2, Chhana Ullah

3, Stefanie Dressel

1, Matthias Schoettner

1, Klaus

Gase1, Emmanuel Gaquerel

4, Shuqing Xu

1, Ian T. Baldwin

1

1Department of Molecular Ecology, Max Planck Institute for Chemical Ecology

2Department of Molecular Interaction Ecology, German Centre for Integrative Biodiversity (iDiv),

Leipzig, Germany 3Department of Biochemistry, Max Planck Institute for Chemical Ecology

4Centre for Organismal Studies, University of Heidelberg, Germany

tluu@ice.mpg.de Plants are simultaneously threatened by herbivores and pathogens, and it has been hypothesized that many plant defenses evolved as dual functions. In many plants, trichomes are the first layer of protection directly against attacks from herbivores and pathogens. They act not only as physical barriers, but they are also known as the place to produce and store various defensive metabolites (Van Dam and Hare, 1998; Fahn, 2000; Roda et al., 2003; Schilmiller et al., 2008; Last et al., 2012). Nicotiana attenuata, a wild tobacco species, native in Great Basin Desert, USA, is fully covered with glandular trichomes (Roda et al., 2003; Weinhold and Baldwin, 2011). Among many secondary metabolites produced in the trichomes of N. attenuata, O-acyl sugars (O-AS) is the most abundant group, which can be produced up to 1.5mg/gFW. Studies have shown that this group of O-AS can act as both direct and indirect defensive against aphids, spider mites and other soft-body insects (Rodriguez et al., 1993; Chortyk et al., 1996; McKenzie and Puterka, 2004, Weinhold et al., 2011). However, whether this group of O-AS can directly protect plants from attacks from native pathogens and specialist herbivores remains unknown. In this study, we characterized natural variation in O-AS among 26 accessions and examined their influence on two native fungal pathogens, Fusarium brachygibbosum U4 and Alternaria sp. U10, and the specialist herbivore, Manduca sexta. At least 15 different O-AS structures belonging to three classes were found in N. attenuata leaves. A three-fold quantitative variation in total leaf O-AS was found among the natural accessions. Experiments with natural accessions and crosses between high- and low-O-AS accessions revealed that total O-AS levels were associated with resistance against herbivores and pathogens. Removing O-AS from the leaf surface increased M. sexta growth rate and plant fungal susceptibility. O-AS supplementation in artificial diets and germination medium reduced M. sexta growth and fungal spore germination respectively. Finally, silencing the expression of a putative branched-chain alpha-ketoacid dehydrogenase E1 beta subunit encoding gene (NaBCKDE1B) in the trichomes reduced total leaf O-AS by 20-30% and increased susceptibility to Fusarium pathogens. I conclude that O-AS function as direct defenses to protect plants from attack from both native pathogenic fungi and a specialist herbivore, and infer that their diversification is likely shaped by the functional interactions among these biotic stresses.

16

Talk 12

Populus trichocarpa aromatic aminoacid decarboxylase enzymes and its roles in plant direct and indirect defense

Jan Günther, Jonathan Gershenzon, Tobias Köllner

Department of Biochemistry, Max Planck Institute for Chemical Ecology jguenther@ice.mpg.de

Plants are under a continuous influence of biotic and abiotic stresses. As a defense against herbivores, many plants like the tree model organism Populus trichocarpa emit a complex blend of defensive volatile compounds upon attack of herbivores like the gypsy moth caterpillar, Lymantria dispar. These volatile compounds comprise terpenes, green leaf volatiles (GLV´s), nitrogenous compounds and aromatic compounds like 2-phenylethanol, a dominant flower and fruit volatile. A plants emission of volatile alcohols reduce the fitness of the herbivore as they might attract herbivore enemies (indirect defense) or by directly interfering with the insect metabolism (direct defense). We identified aromatic aminoacid decarboxylase enzymes (AADC´s) that might contribute to the biosynthesis of volatile alcohols upon herbivory via RNA-Seq. Biochemical characterisation revealed the AADC´s that are responsible for the biosynthesis of 2-phenylacetaldehyde from L-phenylalanine. 2-phenylacetaldeyhde can be then further oxidized to 2-phenylethanol by aldehyde reductases (PtPAR1, 2, 4, 5). Surprisingly, RNAi-meidated knock-down of the responsible AADC enzymes in Populus canescens resulted not only in a reduced emission of 2-phenylethanol but also to the decreased accumulation of 2-phenylethanol-β-D-glucoside that seems to be biosynthetically connected. This migth lead to the conclusion that volatile 2-phenylethanol and water soluble 2-phenylethanol-β-D-glucoside have a different function or target in the plants defense system tough the precursor might be biosynthesized by the same plant enzymes. We propose the reason for this signal diversifiaction of volatile alcohols.

17

Talk 13

How belowground herbivory influences aboveground defense mechanisms of black poplar

Sandra Lackner, Nathalie Lackus, Thomas Fabisch, Jonathan Gershenzon, Tobias Köllner, Sybille B.

Unsicker

Department of Biochemistry, Max Planck Institute for Chemical Ecology slackner@ice.mpg.de

Upon insect herbivore attack, plants induce a series of indirect and direct defenses, e.g. phenolic glycosides, volatile organic compounds or protease inhibitors. When simultaneously challenged by a root feeding insect, these well-known patterns become more complex. Most of the work trying to explain the dynamics of combined below- and aboveground attack was done in herbaceous plants. So far, only little is known about how a woody plant species reacts to the combined attack of different antagonists. The aim of this study was to investigate the effect of belowground herbivory by cockchafer (Melolontha melolontha) grub, on aboveground defense mechanism of black poplar (Populus nigra) trees and the consequences for its natural aboveground herbivore, the gypsy moth caterpillar (Lymnatria dispar). After 4 days of belowground herbivory by M. melolontha grubs, young black poplar trees were infested with gypsy moth caterpillars for 40 hours. Volatiles were collected and after that, leaf harvest defense hormones, phenolic compounds and protease inhibitors were measured. Furthermore, gene expression of volatile biosynthesizing enzymes was investigated. Salicinoids in the leaves were significantly induced by aboveground and combined Ja der herbivory. Protease inhibitor activity significantly decreased upon belowground attack and terpene emission from the leaves also decreased in trees with belowground grub herbivory. Other groups of volatiles, such as nitrogenous compounds and aromatics, were not affected by belowground herbivory and this was true for both the constitutive and the caterpillar induced blend. The synthase transcript patterns of the volatile biosynthesizing enzymes correlated with the emission patterns of the corresponding products. ABA levels were strongly induced after grub herbivory but SA and JA levels were not affected by belowground herbivory. In a choice essay, L. dispar caterpillars preferred feeding on leaves from trees that experienced belowground herbivory.The results of our study show that belowground herbivory heavily influences the way trees react to aboveground attack. In future experiments, the consequences of belowground herbivory on aboveground interactions of trees with their antagonist and mutualist communities will be studied.

18

Talk 14

Capacity and storage duration of an ant's olfactory memory

Roman Huber, Bill S. Hansson, Markus Knaden Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology

rhuber@ice.mpg.de The desert ant Cataglyphis fortis, a model organism for navigational studies, survives in the hostile salt pans of Tunisia using many navigational strategies. One of these strategies is to employ olfactory cues for finding food and also for pinpointing the nest entrance when returning home afterwards. It has also been shown that ants learn to associate odors with a specific target. However, it remains unknown how many odors an ant can learn and how long it can remember these odors. We here investigated the capacity and the storage duration of the olfactory memory of the ants. We presented food spiked with different individual odors, respectively, and tested the ants’ olfactory memory afterwards. We found that Cataglyphis fortis learnt to associate the individual odors with a food reward already after one training trial. Furthermore, when we trained the ants with food that was alternately spiked with one of 14 individual odors, they were able to learn each of them. Finally the storage duration of a learned odor lasted at least for 26 days. These results indicate that ants, which during their life time might experience different food sources, are able to learn the odors of all of them to later be able to locate similar food sources efficiently.

19

Talk 15

The meaning of fast and slow transcription

Peter Großmann1, Christoph Kaleta

1, Christian Kost

2, Tobias Goris

1

1Research GroupTheoretical Systems Biology, Friedrich Schiller University Jena

2Department of Ecology, University of Osnabrück, Germany

pgrossmann@ice.mpg.de

Two individuals with identical genomes can have different phenotypes thanks to epigenetic factors. One of these factors is DNA supercoiling, a change in the DNA secondary structure, in response to transcription. The speed of trancription determines the strength of the supercoiling and is therefore of interest to shed light on this experimentally hardly accessible topic. But there is also an ecological perspective on the transcription speed in that it should determine reaction times to environmentally induced regulatory inputs at the promoter. Fast transcription would cause short reaction times, but would also cost the cell more energy to remove accumulating supercoils. The transcription speed could thus be seen as a measure of importance of a gene to the cell. From RNAP ChIP-chip, transcriptome and RNA turn-over data, we have calculated the transcription speed genome-wide in E. Coli. We were able to demonstrate the link between gene importance and transcription speed on the transcription factor gene set and found a significant enrichment of ontological groups in the low and high speed range. Surprisingly, stress response genes do not belong to the group of fast transcribed genes. Our data qualitatively explains the sequential down-regulation of flagellum-associated genes in cold-shock response, highlighting the advantage of considering the transcription speed in time series measurements.

20

Talk 16

Circumstances favor the cooperative evolution of bacterial cross-feeding

Muhammad Atiqur Rahman

1, 3, Sebastian Germerodt

3, Christian Kost

2

1Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology

2Department of Ecology, University of Osnabrück, Germany

3Department of Bioinformatics, Friedrich Schiller University Jena

mrahman@ice.mpg.de

Cooperative cross-feeding is the reciprocal trade of metabolites among species of microbes. The

circumstances favoring the evolution of cooperative cross-feeding are still to be researched. We

speculated that under certain environment, cross-feeding evolves as a unidirectional process and then

shifts to reciprocal exchange among cooperative species. To explore this, we tested a set of

experiments by initially introducing (in 1:100 ratio) wildtype-donor, over-producer and auxotroph(s)

independently and one after another in wildtype-loner community and finally the cross-feeder(s). In a

parallel, we also checked the same aspect through a simulation model. We observed that the feeding

interactions among species impaired gradually due to supplementation of growth limiting metabolites

in external environment. Under limiting conditions, only the existence of wild-donor greatly benefited

auxotroph(s) either in high donor density-low metabolite diffusion or low donor density-high metabolite

diffusion dynamics. Following this, the introduction of cross-feeders in the extant community, the

highest and simultaneous invasion of cross-feeding partners noticed only if they received equal or

higher benefits from the interacting partner in return to offset the cost. These findings suggest that the

evolution of cooperative cross-feeding in nature may be more challenging than other types of microbial

interactions. However, the determinants identified here may enable to explain the ecological

consequences behind these observations.

Posters

22

Poster 1

DMNT-induced upregulation of defensive Sporamin in sweet potato

Anja Meents1,4

, Shi-Peng Chen2, Stefan Bartram

1, Michael Reichelt

3, Kai-Wun Yeh

2, Wilhelm Boland

1,

Axel Mithöfer1

1Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology

2National Taiwan University, Institute of Plant Biology, Taiwan

3Department of Biochemistry, Max Planck Institute for Chemical Ecology

4Institute of General Botany and Plant Physiology, Friedrich Schiller University Jena

ameents@ice.mpg.de

Plants are constantly subjected to a plethora of environmental challenges. In order to cope with

increasing biotic as well as abiotic stress factors, distinct signaling pathways are activated within the

plant resulting in the mobilization of powerful physico-chemical weapons. Especially indirect defense

mechanisms, comprising emission of volatile organic compounds (VOCs), have gained attention due

to their function as signaling compounds for inter-plant communication and attractants for predators

during insect attack. Severe agricultural yield losses caused by pests reveal the necessity for inducible

strategies to retaliate against herbivore infestation. Sweet potato (Ipomoea batatas) as the fifth most

important tuber crop worldwide plays a key role in global agriculture, serving as an easily cultivatable,

nutrient-rich staple food source. Different insect-resistant cultivars of sweet potato are available.

Especially I. batatas cv. Tainong 57 showed the wound-inducible systemic expression of Sporamin, a

tuber storage protein that also acts as trypsin inhibitor. The homoterpene 4,8-dimethyl-1,3,7-

nonatriene (DMNT) was identified to be the most prominent compound emitted by I. batatas after

infestation by the insect herbivore Spodoptera littoralis. Interestingly, DMNT turned out to be a potent

elicitor on defense upregulation in untreated sweet potato plants. The coherences between volatile

emission after damage and its putative impact on defense upregulation through Sporamin and

jasmonate production will be addressed.

23

Poster 2

How to DREAM and what to learn from it

Sarah Koerte, Ewald Groβe-Wilde, Markus Knaden, Bill S. Hansson

Department of Neuroethology, Max Planck Institute for Chemical Ecology skoerte@ice.mpg.de

Insects gain essential information for their survival and reproduction from detecting and processing chemosensory signals in their environment. In the course of insect evolution, dedicated receptor types emerged, allowing the perception of a plethora of different chemical signals. In the vinegar fly Drosophila melanogaster (D. mel), the perception of volatile compounds (olfaction) is mediated by predominantly odorant receptors (ORs), but also by gustatory receptors (GRs) and ionotropic receptors (IRs). The majority of D. mel ORs and IRs have been functionally characterized (deorphanized) in vivo, which assigned the receptors chemosignals leading to their activation. Nevertheless, most D. mel GRs as well as some ORs and IRs remain orphans despite many different experimental approaches. However, substantial progress in understanding the rules governing chemoperception is not possible without ascertaining all receptors gated by a given chemosignal. Recently, it was discovered that exposure to high concentrations of odorants, above those levels found naturally, leads to decreases in the expression of certain odorant receptor genes (von der Weid et al. 2015). These alterations in expression can be observed after only a few hours of exposure and are reversible if the odor source is removed. This phenomenon holds not only the potential to uncover new receptor-ligand combinations in D. mel, but also the possibility to have a fast screening method to find receptor-ligand pairs in non-model Drosophila clade species. Moreover, this would enable a huge step forward towards deciphering the fundamentals of chemoperception, especially in organisms that are not as extensively studied as D. mel.

In regard to the specifics of this study, we will use RealTime quantitative Poly-Chain-Reaction (RT

qPCR) to analyze olfactory tissue from control and high odor concentration exposed D. mel

individuals, confirming odorant receptor-ligand combinations found in behavioral and

electrophysiological studies. Furthermore, we will find out if the DREAM (Deorphanization of

receptors based on expression alterations in mRNA levels; von der Weid et al. 2015)

technique can also be expanded to other chemosensory receptor classes beyond ORs, thus

characterizing currently orphan IRs and GRs. Ultimately, we want to use the DREAM

method to identify receptor-ligand pairs in non-model Drosophilidae species.

24

Poster 3

Chlorophyll degradation in lepidopteran pest (Spodoptera littoralis)

Vincensius S.P. Oetama1, Stefan Pentzold

1, Amarsanaa Badgaa

2, Wilhelm Boland

1

1Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology

2Institute of Chemistry and Chemical Technology, MAS, Mongolia

voetama@ice.mpg.de

Chlorophyll is a natural pigment that is synthesized and conjointly degraded in an enormous number

every year. It is well studied in plants whereas there are limited findings on other organisms. As

herbivores are organisms that often consume leaves – the chlorophyll main producers –, it is a good

start to learn the degradation mechanism. Previous studies using the notorious lepidopteran pest

species, the Egyptian cotton leaf worm (Spodoptera littoralis) have shown that i.) regurgitate has the

catalytic activity to breakdown chlorophyll and ii.) catabolites were detected inside the gut and frass

(Chlorophllide, Pheophorbide, and Pyropheophorbide).

In this study, the suspected liable protein – Chlorophyllide binding protein (CHBP) – has been

identified within the transcriptome and proteome analysis from regurgitate. The similar gene was also

found in other lepidopteran, such as Bombyx mori and Helicoverpa amiverga. First experiments on the

gene expression level in different developmental stages and tissues indicate that CHBP is mainly

expressed in the digestive tract in the 5th instar. Furthermore, transcript silencing via RNAinterference

was applied to larvae, and first results showed the metabolite changing as it is detected with LC/MS.

We also found a lower survival rate in larvae injected with gene specific dsRNA. Additionally, gene

expression decreased about 70 to 80% after RNAi treatment. This study may have an impact in

herbivore-plant interactions considering CHBP’s role in survival rate, prospectively it would be of

importance in agriculture to fight against lepidopteran pest.

25

Poster 4

A male specific pheromone mediate the sexual and social behavior in D. mojavensis

Mohammed A. Khallaf

1, Jerrit Weissflog

2, Aleš Svatoš

2, Bill Hansson

1, Hany Dweck

1*, Markus

Knaden1*

1Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology

2Research Group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology

*These authors contributed equally to this work mkhallaf@ice.mpg.de

Courtship is the behavior that fruit flies do best. Once they land on a food patch colonized by

conspecifics, males almost immediately court females. Pheromones play an important role as

chemical signals to stimulate male courtship and female receptivity. Males of the melanogaster-

species clade generically produce male-produced cis-vaccenyl acetate (cVA), which induces female

receptivity and functions as an aggregation pheromone. The males transfer it to females during mating

as an anti-aphrodisiac signal for further males. However, little is known what regulates the social and

sexual behaviors in the non cVA-producing species, like Drosophila mojavensis mojavensis. In this

study, we identify previously anonymous male-specific compounds (two monoenes, (R/S)10Z-

Heptadecen-2-yl acetate (R10HDA) and Heptadecan-2-yl acetate, and one diene, 19Z,22Z-

Octacosadienyl acetate), which D. mojavensis males transfer to females during copulation. The former

compound activates AT4-like OSNs of this species. We found that perfuming with R10HDA increases

the copulation success of males, but reduces the attractiveness of virgin females. In addition, R10HDA

in combination with food triggers aggregation behavior in D.moj moj females. Our chemical analysis

for the different D. mojavensis subspecies revealed that R10HDA production is not conserved among

the sub-species. Interestingly, R10HDA works as a sexual isolation barrier in the four D mojavensis

sub-species. Taken together, we identified a novel male-specific pheromone detected by AT4 OSNs

which induces female receptivity, reduces the attractiveness of the newly mated females, and induces

aggregation in combination with food odors. This male-specific acetate is produced exclusively in the

ejaculatory bulb, and works as an isolation barrier between the D. mojavensis sub-species. These

cVA-like functions of the newly discovered pheromone increase our understanding of the evolution of

Drosophila pheromones and how reproductive isolation barriers between species are created mainly

by species-specific signals.

26

Poster 5

Catabolism of flavonols in Arabidopsis thaliana by Sclerotinia sclerotiorum

Jingyuan Chen

1, Jonathan Gershenzon

1, Almuth Hammerbacher

1

1Department of Biochemistry, Max Planck Institute for Chemical Ecology

jchen@ice.mpg.de

Flavonoids are widely distributed in nature, and many studies have revealed that some flavonoids have antibiotic activity against fungal plant infection. Fungi can however also produce enzymes that metabolize and detoxify these plant chemical defenses. The phytopathogenic fungus Sclerotinia sclerotiorum is a devastating necrotrophic pathogen that can cause stem rot disease in a vast range of plant species and results in large losses of crop yields worldwide. In order to determine how A.thaliana interacts with S.sclerotiorum, changes in the levels of defense compounds (glucosinolates, flavonoids, camalexin and coumaroyl agmatine) in A. thaliana were investigated. HPLC analyses showed that the concentration of total flavonoids in A. thaliana inoculated with the aggressive strains UF-70 decreased 3 days post inoculation when compared with mock inoculated A. thaliana. The result suggested that S. sclerotiorum may have a strategy to block accumulation of flavonoids in the host plant.

An in vitro growth experiment with artificial medium which contained different phenolic compounds showed that S. sclerotiorum can degrade quercetin and kaempferol (which are precursors of flavonoids biosynthesis) more quickly than others. Therefore, the candidate gene encoding an enzyme involved in degradation of quercetin and kaempferol of S. sclerotiorum was identified and named SsQDO. In order to investigate the role of this flavonoid degradation gene in fungal virluence, a SsQDO knock-out mutant ΔQDO was generated based on wild type UF-70. The ability to degrade quercetin and kaempferol in the mutant was deleted but the growth rate of mycelium was not affected. Inoculation with A. thaliana showed that QDO knock-out mutants decreased pathogenicity.

Our results suggested that polyphenol degradation may play an important role in virulence and survival of S. sclerotiorum during interation with the host plant.

27

Poster 6

Experience-dependent plasticity of olfactory circuits in Drosophila melanogaster

Benjamin Fabian

1, 2, Veit Grabe

1, Rolf G. Beutel

2, Bill S. Hansson

1, Silke Sachse

1

1Department of Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology

2Institute of Systematic Zoology and Evolutionary Biology with Phyletic Museum, Friedrich Schiller

University Jena

benjaminfabian.bf@gmail.com

The sense of olfaction is very crucial for insects in order to navigate in a complex environment of volatile odorants. Olfactory cues play a major role in locating suitable substrates for feeding and oviposition, and are necessary for finding potential mating partners or for the avoidance of predators and parasitoids. The structure and function of the olfactory system of Drosophila melanogaster is well understood and documented. However, little is known about whether and to which extent individual experience is able to transform certain parts of this system. Odor molecules bind to specific olfactory receptors on the antennal and maxillary palp surface. From these receptors, information about the constitution of the chemical environment is passed to higher neuronal centers, namely the antennal lobe, the mushroom body and the lateral horn. The first modulation of this information takes place in the glomeruli of the antennal lobe. Each of them consists of odor-specific olfactory sensory neurons (OSNs), local interneurons (LNs) and projection neurons (PNs). Studies have shown that glomeruli increase in size when they are stimulated by the presence of their associated odor molecules for several days, and decrease in size again after a few days without the stimulus. We try to find the cause that underlies this plasticity. For this, flies are cyclically exposed to geosmin, a strongly aversive odor molecule. The geosmin molecules bind to the Or56a receptor, which is expressed in OSNs that converge to the DA2 glomerulus in the antennal lobe. The glomerulus of treated flies is then analyzed to document the volume increase and number of OSNs and PNs. For the visualization of neurons, we express photoactivatable GFP in certain glomeruli in transgenic flies. To allow general conclusions for the experience-dependent plasticity of olfactory circuits, we aim for additional experiments with other odors to stimulate different sets of glomeruli.

28

Poster 7

Catechin and proanthocyanidins are effective anti-fungal defenses in poplar against biotrophic rust fungus (Melampsora spp.)

Chhana Ullah

1, Sybille B. Unsicker

1, C. Peter Constabel

2, Axel Schmidt

1, Jonathan Gershenzon

1,

Almuth Hammerbacher1

1Department of Biochemistry, Max Planck Institute for Chemical Ecology

2Centre for Forest Biology and Department of Biology, University of Victoria, Canada

cullah@ice.mpg.de

Phenolic secondary metabolites are often thought to protect plants against attacks by microbes. However, the role of phenolic compounds in defense against pathogen infection in woody plants has not often been comprehensively investigated. We studied the biosynthesis, occurrence and anti-fungal activity of flavan-3-ols in black poplar (Populus nigra L.), which include both monomers, such as catechin, and oligomers known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases (LAR) and two anthocyanidin reductases (ANR) from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) synthesized higher amounts of catechin and PAs than uninfected trees, which accumulated more at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth in vitro. Poplar genotypes with constitutively higher levels of catechin and PAs were more resistant to rust infection. Overexpression of a transcription factor MYB134 in hybrid poplar increased flavan-3-ol contents and reduced susceptibility to rust fungus M. medusae. Furthermore, silencing the PnMYB134 transcription factor in P. nigra decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective anti-fungal defenses in poplar.

29

Poster 8

Experimental evolution of metabolic cross-feeding among bacteria

Samir Giri

1,2, Christian Kost

1,2

1Experimental Ecology and Evolution Research Group, Max Planck Institute for Chemical Ecology

2Department of Ecology, University of Osnabrück, Germany

sgiri@ice.mpg.de

Metabolic cross-feeding interactions in which two or more bacterial species exchange costly

metabolites are very common in bacterial communities. Such cross-feeding interactions promote

growth and enable behaviors that neither party can perform alone. Evolutionary theory predicts

cooperating organisms should favour genealogically related individuals over unrelated ones. Whether

or not the relatedness among two bacterial lineages to engage in cooperative cross-feeding

interactions remains elusive.

Previous work of our laboratory has shown that cooperative cross-feeding interactions rapidly evolved

between two Escherichia coli genotypes that were auxotrophic for two different amino acids. Evolved

auxotrophic consortia displayed significantly increased consortium-level fitness relative to derived

populations of metabolically autonomous, prototrophic cells. Moreover, derived auxotrophic consortia

showed an extensive degree of phenotypic diversity, formed multicellular clusters and a significantly

improved growth relative to the ancestor.

The main objective is to unravel how the relatedness among coevolving auxotrophic genotypes affects

the evolution of cross-feeding interactions. To address this issue, pairs of auxotrophic genotypes will

be set up within and between different bacterial species and experimentally coevolved to compare

their propensity to engage in cooperative cross-feeding interactions. Consequently, I plan to

investigate the influence of positive assortment, local adaptation and how coevolving genotypes affect

the evolution of phenotypic diversity during cooperative cross-feeding interactions. The expected

results will provide unprecedented insights into the role relatedness plays for the evolution of cross-

feeding among different bacterial genotypes and may thus help to explain the widespread distribution

of such interactions in nature.

30

Poster 9

Metabolic and evolutionary insight into regulation of diterpene resin acids in Norway Spruce

Andrew J. O’Donnell, Bianca Fiedler, Sascia Hilck, Jonathan Gershenzon, Axel Schmidt

Department of Biochemistry, Max Planck Institute for Chemical Ecology aodonnell@ice.mpg.de

Plant diterpenoids are a large family of diverse bioactive compounds that are ubiquitous across the plant phylogeny. Excluding those committed to plant primary metabolism, many diterpenoids in secondary metabolism appear to be confined to single plant lineages. One example of lineage-specific diterpenoids is the diterpene resin acids (DRAs) in conifers such as Picea abies (Norway Spruce), which are implicated in defense against herbivores and pathogenic fungi in spruce and other conifers (Keeling and Bohlmann 2006). Although significant advancements in our understanding of DRA biosynthesis have been made in the past (Ro and Bohlmann 2006; Hall et al. 2011; Geisler et al. 2016), a comprehensive picture of regulation, resistance, and evolution of these ecologically-relevant compounds remains out of reach. Here we show through bark chemical and transcriptome analyses, coupled with molecular phylogenetics of DRA biosynthetic genes, that 1) DRAs are differentially regulated in pathogen-susceptible and resistant spruce lines, and 2) cytochrome P450 monooxygenases in conifers were likely recruited into DRA metabolism from ancient brassinosteroid pathways.

31

Poster 10

Public goods of Bacillus subtilis – how public are they really?

Theresa Hölscher, Ákos T. Kovács

Terrestrial Biofilms Group, Institute of Microbiology, Friedrich Schiller University Jena thoelscher@ice.mpg.de

Several studies show that cooperation can determine the structure of microbial communities and can

increase the overall fitness of the community although being costly to the individual cells. One aspect

of cooperation is the production of public goods i.e. products that are secreted into the environment

that benefit the producing cell as well as non-producers. The maintenance of cooperation can be

explained for example by the microbial colonization of spatially heterogeneous environments where

the spatial structure stabilizes cooperation. This might particularly be important for the colonization of

surfaces e.g. the formation of colony biofilms or sliding communities on agar (Grau et al., 2015) where

the production of public goods is crucial. Bacillus subtilis is a Gram-positive bacterium that was

previously used for studies on the stability of cooperation in colony biofilms (van Gestel et al., 2014).

However, there is less information about the influence of public goods in expanding populations such

as sliding colonies.

Therefore, this study aimed to analyze the importance of different public goods produced during sliding

of B. subtilis and to expose how well they are shared in the expanding population. Competition

experiments were performed under sliding promoting conditions with different combinations of the

wild-type and mutants unable to produce a certain good. Further, the metabolic cost of production was

determined for each public good. Being important for sliding, we analyzed the impact of surfactin, the

amphiphilic protein BslA and extracellular polysaccharides as public goods.

Competition experiments revealed that while certain public goods can be exploited by non-producers,

others are kept private benefiting only the producer linage. Our experiments demonstrate that the

tested public goods are not shared equally and thus have varying influence on the population structure

during sliding.

32

Poster 11

Poplar-herbivore-pathogen interaction: Herbivores take advantage of infected host plants

Franziska Eberl

1, Almuth Hammerbacher

1,2, Jonathan Gershenzon

1, Sybille B. Unsicker

1

1Department of Biochemistry, Max Planck Institute for Chemical Ecology

2Institute for Forestry and Agricultural Biotechnology, University of Pretoria, South Africa

feberl@ice.mpg.de In a natural forest ecosystem, trees are under constant attack by numerous herbivores and pathogens. However, most studies on plant-herbivore and plant-pathogen interactions focus on single-attacker scenarios in annual herbaceous plants instead of long-lived trees. Here, we investigated the effect of combined attacks on the defense chemistry of a tree species, as well as the influence that the attackers have on each other. We infested black poplar (Populus nigra) trees with a biotrophic rust fungus (Melampsora larici-populina) and herbivorous caterpillars (Lymantria dispar) alone and in combination, and measured volatile emission as well as phytohormone levels. In general, both the pathogen and the herbivores induced volatile emission, specifically terpenes. In the combined treatment, however, the herbivore-induced emission was reduced. Phytohormone measurements suggest an antagonistic crosstalk caused by the simultaneous presence of herbivores and pathogen. Furthermore, the generalist herbivore, Lymantria dispar, was attracted to the odor of rust-infected leaves and preferred to feed on them. Caterpillars reared on pathogen-infected leaves also gained significantly more weight than caterpillars reared on non-infected leaves. Whether specific changes in primary or secondary metabolites in response to rust fungus infection account for the differences in caterpillar performance await further elucidation. We observed a negative effect of pathogen infection on the herbivore-induced volatile emission from black poplar, and showed that a generalist herbivore can benefit from the pathogen infection of its host tree. Our results underline the importance of studying complex systems with more than one plant attacker in order to understand the dynamics and mechanisms of insect, plant and pathogen interactions in natural ecosystems.

33

Poster 12

The evolutionary arms race between Psylliodes leaf beetles and their host plants

Matilda Gikonyo

1, Heiko Vogel

2, Franziska Beran

1

1Research Group Sequestration and Detoxification in Insects, Max Planck Institute for Chemical

Ecology 2Department of Entomology, Max Planck Institute for Chemical Ecology

mgikonyo@ice.mpg.de The flea beetle genus Psylliodes (Chrysomelidae: Galerucinae) comprises more than 140 species, which are associated with plants belonging to 27 different plant families. The goal of this project is to trace host plant shifts and subsequent species diversifications in this genus by determining the phylogenetic relationships of Psylliodes species. Previous work showed that common molecular markers such as cytochrome c oxidase I are not appropriate in resolving the Psylliodes species phylogeny. Therefore, we searched for suitable single copy nuclear genes in the available in-house transcriptomes of eight different Psylliodes species. A phylogenetic analysis of these eight beetle species based on 24 identified marker genes (e.g. carbamoyl phosphate synthase, RNA polymerase II 215kD and cannonball) resulted in a phylogenetic tree with strong node supports. The preliminary result also suggested that within this genus, a host plant shift from Solanaceae to Brassicaceae occurred. Since it is not possible to sequence the transcriptome of each Psylliodes species, we designed primers which can be used to amplify these new marker genes from genomic DNA. Since there is no reference genome available of any Psylliodes species, we mapped the intron-exon structure of the corresponding orthologous genes from Tribolium castaneum and Dendroctonus ponderosae. We then designed primers targeting the longest conserved exon of each marker gene, and the primers are currently being tested and optimized using genomic DNA of different Psylliodes species. Our next goal is to include additional Psylliodes species in this phylogenetic analysis. In the future, this species phylogeny will provide the framework to identify key evolutionary adaptations of Psylliodes to chemical plant defenses.

34

Poster 13

“Cry-worthy” cost of producing an ectopic defense protein

Erica McGale1, Celia Diezel

1, Meredith Schuman

1,2, Samik Bhattacharya

1,3, Ian T. Baldwin

1

1Department of Molecular Ecology, Max Planck Institute for Chemical Ecology

2German Center for Integrative Biodiversity (iDiv), Leipzig, Germany

3Department of Botany, University of Osnabrück, Germany

emcgale@ice.mpg.de Plants are the primary producers in most terrestrial ecosystems and utilize sophisticated strategies to defend their produce against consumers. These defenses are costly when not needed. Thus, wild plants have developed signaling systems to regulate endogenous defenses, largely dependent on the jasmonate (JA) hormones. Even with inducibility these systems remain costly, as seen in previously reported Nicotiana attenuata lines silenced in allene oxide cyclase by inverted repeat constructs

(irAOC), which had significantly larger plant biomasses.

Breeding of crop plants for yield and aggressive artificial pest management have resulted in losses of endogenous defenses in many agricultural crops. Advances in genetic engineering have brought reinforcements to these plants through novel traits: most commonly, the production of Cry toxins from Bacillus thuringiensis (Bt) targeting specific insect clades. The overall benefit of these plants in terms

of yield is still debated in scientific and news outlets.

We engineered the wild tobacco Nicotiana attenuata to ectopically produce Cry1Ac (ovCRY) and report that under field conditions, costs of producing Cry1Ac were mostly greater than costs of endogenous JA-mediated defenses in growth and reproduction. Although these costs have been recognized in current debates and previous works, our study now details the initial Cry expression costs in a natural setting through important comparisons to plants with wild-type defense (empty vector controls, EV) and JA-deficient plants (irAOC).

35

Poster 14

Cytochrome P450s are involved in iridoid biosynthesis in leaf beetles

Nanxia Fu, Antje Burse, Wilhelm Boland Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology

nfu@ice.mpg.de

Leaf beetles are specialist herbivores which spend almost their entire life cycles on the same host

plants. To protect themselves against potential predators or microbial invaders, the larvae of the

mustard leaf beetle Phaedon cochleariae (Chrysomelidae, Chrysomelina) de novo synthesize the

defensive compound chrysomelidial, a five-membered cyclic monoterpenoid (iridoid). The juvenile

beetles store chrysomelidial in nine pairwise dorsal glandular reservoirs and release the defensive

secretion upon disturbance. Once the alarm has ceased, the defensive droplets are sucked back for

recycle usage. Although the de novo iridoid pathway has been well established, the enzyme that

catalyzes the synthesis of the early precursor 8-hydroxygeraniol from geraniol has remained elusive.

Previous studies showed that both geraniol and glucosidically bound 8-hydroxygeraniol are

accumulated at first in fat body tissue and then transferred via the hemolymph into the glandular

reservoirs for further conversion into the biologically active iridoid. In the iridoid-producing plant

Catharanthus roseus, a cytochrome P450 monooxgenase CYP76B6 catalyzes the hydroxylation of

geraniol. In P. cochleariae, we identified 32 cytochrome P450s from the fat body membrane proteins

by using a proteomics approach. The subsequent combination of the proteomics data with tissue

expression pattern analysis and RNA interference screening indicated that a candidate P450

(PcC12641) enzyme is involved in the early steps of the iridoid metabolism. Hence, like their plant

counterpart, P. cochleariae may have also recruited a P450 enzyme from the insect CYP4G family to

oxidatively produce the early iridoid precursor 8-hydroxygeraniol.

36

Poster 15

Production of volatile and non volatile isoprenoids during drought stress in P. glauca young needles

Erica Perreca

1, Jonathan Gershenzon

1, Louwrance Peter Wright

2

1Department of Biochemistry, Max Planck Institute for Chemical Ecology

2Zeiselhof Research Farm, Menlo Park, South Africa

eperreca@ice.mpg.de

The plastidial methylerythritol phosphate (MEP) pathway synthesizes volatile and non volatile

isoprenoids from common five-carbon (C5) isoprene building blocks, isopentenyl diphosphate (IPP)

and its isomer dimethylallyl diphosphate (DMAPP). Volatile isoprenoids are involved in ecological

interactions and in defense against biotic and abiotic stresses. Non volatile isoprenoids are involved in

primary metabolism. In our experiment we subjected a conifer producing isoprene Picea glauca, to

drought stress for 20 days, during the flushing period of the shoots. Afterwards we evaluated the

production of volatile (isoprene and monoterpenes) and non volatile isoprenoids (carotenoids and

chlorophylls) in the young needles. Isoprene, the first and simplest product of the pathway, seems to

be affected only during severe stress, probably because of its defense role against abiotic stress like

drought. We are interested in investigating how drought stress could affect the MEP pathway, altering

the capacity of defense and the ecological interactions between the plant and its environment during

the growing stage.

37

Poster 16

Interaction of beetle polygalacturonases with plant cell wall proteins – Heterologous expression of potential inhibitors

Wiebke Häger, Roy Kirsch, Yannick Pauchet, David Heckel

Department of Entomology, Max Planck Institute for Chemical Ecology wihaeger@ice.mpg.de

The herbivorous mustard leaf beetle Phaedon cochleariae feeds on brassicaceous plants and

possesses various carbohydrases for the digestion of plant cell wall polysaccharides. Amongst those,

polygalacturonases (PGs) facilitate the breakdown of the cell wall polysaccharide pectin. Several

plant-derived, cell wall-associated polygalacturonase-inhibiting proteins (PGIPs) are known to inhibit

microbial PGs and thus contribute to the plant´s defence against phytopathogenic fungi and bacteria.

However, direct interactions between beetle PGs and plant inhibitory proteins have not yet been

investigated.

We performed an interaction study of P. cochleariae PGs with crude cell wall protein extracts from the

beetle’s food plant Chinese cabbage (Brassica rapa ssp. pekinensis) and identified plant PGIPs and

other leucine-rich repeat (LRR) proteins as candidates for beetle PG inhibition. To investigate whether

beetle PGs, like their microbial counterparts, could be inhibited by plant PGIPs, candidates should be

heterologously expressed to test their effect on beetle PGs in vitro. Since challenges were

encountered during the heterologous expression of potential PG inhibitors – concerning success,

sufficient yield, protein stability in follow-up experiments and reproducibility – alternative strategies to

overcome these difficulties are presented.

38

Poster 17

Evidence for GABA-induced systemic GABA accumulation upon wounding in Arabidopsis thaliana

Jaiana Malabarba

1, Sandra S. Scholz

2, Michael Reichelt

1, Monika Heyer

1, Frank Ludewig

3, Axel

Mithöfer1

1Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Germany

2Institute of General Botany and Plant Physiology, Friedrich Schiller University Jena

3Department of Biology, University of Erlangen-Nuremberg, Germany

jmalabarba@ice.mpg.de

The non‐proteinogenic amino acid γ‐aminobutyric acid (GABA) is present in all plant species analyzed so far. Its synthesis is stimulated by either acidic conditions occurring after tissue disruption or higher cytosolic calcium level. In mammals, GABA acts as inhibitory neurotransmitter but its function in plants is still not well understood. Besides its involvement in abiotic stress resistance, GABA has a role in the jasmonate-independent defense against invertebrate pests. While the biochemical basis for GABA accumulation in wounded leaves is obvious, the underlying mechanisms for wounding-induced GABA accumulation in systemic leaves remained unclear. Here, the Arabidopsis thaliana knock-out mutant lines pop2-5, unable to degrade GABA, and tpc1-2, lacking a wounding-induced systemic cytosolic calcium elevation, were employed for a comprehensive investigation of systemic GABA accumulation. A wounding-induced systemic GABA accumulation was detected in tpc1-2 plants demonstrating that an increased calcium level was not involved. Similar, after both mechanical wounding and Spodoptera littoralis feeding, GABA accumulation in pop2-5 plants was significantly increased in local and systemic leaves. Moreover, compared to wild type plants, GABA content was significantly higher in the pop2-5 line and, consequently, larvae feeding on these GABA-enriched mutant plants showed significantly reduced growth. Upon exogenous application of a D2-labeled GABA to wounded leaves of pop2-5 plants, its uptake but no translocation to unwounded leaves was detected. In contrast, an accumulation of endogenous GABA was observed in vascular connected systemic leaves. These results indicate that the systemic accumulation of GABA upon wounding is neither depending on a translocation of GABA nor on an increase of systemic cytosolic calcium.