Molecular Tools Developed for Nicotiana attenuata

A comprehensive toolbox for the N. attenuata ecological expression system has been developed in our Department. The most important compartment of this toolbox is a set of transgenic N. attenuata lines, transformed with our stable transformation system, posttranscriptionally silencing or ectopically overexpressing about 200 different genes with strong ecological relevance. This unique tool is being used to study the function of ecologically relevant genes in field experiments (Fig. 1) or in the greenhouse.

Alt-Text 1

For the identification of ecologically important genes in a forward genetics approach an AI-RIL (Advanced Intercross-Recombinant Inbred Lines) population from a cross of the two best studied N. attenuata ecotypes (“Utah” and “Arizona”) and a MAGIC (Multiparent Advanced Generation InterCross) population from 26 different ecotypes are being developed. To explore the ecological relevance of identified genes a VIGS based transformation system can be utilized.
The almost complete sequences of the N. attenuata genome and transcriptome are available. This sequence information was used to design complete transcriptomic microarrays based on AGILENT´s 4x44k and 8x60k platforms, which allow identification and transcriptional analyses of ecologically relevant genes. For the same purpose SAGE libraries and qPCR tools have been developed. Moreover, genomic and transcriptomic databases from different accessions of N. attenuata and different Nicotiana species and transcriptomic sequence data from insects feeding on N. attenuata are available. A microsatellite based method for the identification of different N. attenuata populations, ecotypes and individuals has also been developed. To study signaling between shoots and roots, a micrografting method can be applied for N. attenuata.

 

 

MAGIC
We established a MAGIC (Multiparent Advanced Generation InterCross) population from 26 different N. attenuata ecotypes.
N. attenuata accessions were collected over 20 years from more than 100 natural populations (Fig. 2).
 

For the selection of the parental plants used in the MAGIC cross, morphological and biochemical traits of 424 individuals from 75 accessions were characterized. To guarantee genetic diversity, the 26 individuals with the most extreme phenotypes, representing 26 different ecotypes including “Utah” and “Arizona” were selected as parents.
For the MAGIC cross first all 325 possible combinations of the 26 parents were performed in a half diallel cross without parents or reciprocal crosses, ♀ and ♂ were chosen randomly (Fig. 3a). The offspring with each 2 of the 26 chosen parents was used then in four subsequent rounds of crosses, designed in a way that each of the 325 combinations served as ♀ and most as ♂, that no reciprocal crosses occurred and that each offspring had 4, 8, 16 and all 26 different parents after 1, 2, 3, 4 rounds of crossing, respectively (Fig. 3b). To obtain the final MAGIC Recombinant Inbred Lines (RILs), the 325 obtained lines will be used in six generations of inbreeding (Fig. 3c).

Virus induced Gene Silencing (VIGS)
A VIGS system based on a tobacco rattle virus containing vector has been adapted for N. attenuata (Saedler and Baldwin, 2004) which allows the rapid silencing of endogenous genes (Fig. 4) in plants in a matter of weeks. Another application of this VIGS system is Plant mediated RNAi (PmRNAi) to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta [Kumar, P., Pandit, S. S., Baldwin, I. T. (2012). Tobacco Rattle Virus vector: A rapid and transient means of silencing Manduca sexta genes by plant mediated RNA interference. PLoS One, 7(2): e31347. doi:10.1371/journal.pone.0031347.]. For this, a 300 bp fragment of the target gene is cloned in the VIGS vector in antisense orientation. After infiltration of N. attenuata with this vector, in the course of virus replication, dsRNA of this fragment is formed. In M. sexta larvae feeding on an infiltrated plant reductions in the transcript levels of the targeted midgut gene can be observed.
At present 250 different constructs for VIGS are available in the department. Since the current VIGS procedure only works at relatively low temperatures of around 20°C we are now working on the development of a temperature resistant VIGS system that can be used under field conditions at temperatures of more than 30°C in the native habitat of N. attenuata.

Genomic databases
In an ongoing project with the Max-Planck-Institute for Molecular Genetics the whole N. attenuata genome sized about 2.4 Gb is being sequenced. At present a draft version with an N50 contigs length of 27.6 kb comprising 2.2 Gb has been established and is available on our department´s internal BLAST server. A variety of tools for gene prediction, annotation and analysis of the genomic data has been established, among them the prediction tool AUGUSTUS, the annotation tool WebApollo and our GALAXY browser allowing access to tools for sequence analyses. Our current efforts are directed toward the improvement of the assembly. For this, we are using Pacific Biosciences (PacBio) sequencing and BioNano Genomics DNA mapping technologies. Whereas the PacBio method results in long reads up to 20 kp and allows sequencing of DNA regions not accessible to standard sequencing methods, the BioNano method will result in a complete graphic map of the genome, allowing the mapping and assigning of our current contigs and scaffolds to the exact positions in the N. attenuata genome. A combination of both methods with our current sequence data will lead to a considerable improvement of our N. attenuata draft genome. In addition, the genome of the related species Nicotiana obtusifolia has been sequenced with lower coverage. The availability of both sequences allows studies on the evolution of ecologically relevant traits in the genus Nicotiana.

Transcriptomic databases
The sequences of the nearly whole transcriptome of N. attenuata are available. mRNA from different tissues of N. attenuata collected at different time points after elicitations, was isolated, labeled and sequenced using Illumina and 454 technology. From these data the nearly complete transcriptome of N. attenuata was assembled de novo. To determine transcript abundance for each gene in specific tissues, N. attenuata tissues from different organs or growth stages (leaves, roots, stems, flower parts, seeds) were tagged individually prior to Illumina sequencing. Transcriptomic data of the two best-studied ecotypes “Utah” and “Arizona” were established. Sequence comparisons between both ecotypes will give insight in intraspecific genome variations and adaptation. The nearly complete transcriptome sequence of the related species N. obtusifolia is also available and was established in cooperation with Vertis Biotechnologie AG (www.vertis-biotech.com). mRNA was isolated, normalized and sequenced using the 454 technology. All next generations sequencing transcriptome assemblies can be analyzed on our group internal BLAST server.

In an ongoing project transcriptomic data from Nicotiana x obtusiata, the allotetraploid cross of N. attenuata and N. obtusifolia, and N. quadrivalvis and N. clevelandii were generated to investgate species formation.

To enable studies of plant-insect interactions the transcriptomes of the most common herbivores (Manduca sexta, M. quinquemaculata, Corimelaena extensa, Empoasca spec., Tupiocoris spec., Oecanthus fultoni and Trichobaris mucorea) feeding on N. attenuata and of the predator Geocoris spec., have been sequenced using Illumina technology.

Transgenic Plant Collection and Stable Transformation System
Since N. attenuata is a native species, the standard transformation approaches developed for cultivated tobacco could not be applied. A new Agrobacterium based transformation procedure with new transformation vectors optimized for it was developed (See transformation facility). We constructed around 150 binary plant transformation vectors each containing an inverted repeat sequence of a gene to be silenced constitutively and around 30 vectors for the constitutive ectopic expression of full length genes. Our latest cloning vectors for these types of constructs are the pRESC8/pRESC9 plasmids (Fig. 5) with a hygromycin plant selectable marker and the/nptII/ gene as bacterial selectable marker, which carry the /bla/ gene and the ColE1 origin of replication on their T-DNA. Using plasmid rescuing the presence of this bacterial replicon allows to recover the flanking sequence at the T-DNA insertion site and thus to localize mutual insertions in the plant genome.

Recently we were able to implement the highly sensitive LhGR/DEX – pOp6-promoter inducible system (Fig. 6a/6b) into our N. attenuata transformation system (Schäfer, M., Brütting, C., Gase, K., Reichelt, M., Baldwin, I. T., Meldau, S. (2013). “Real time” genetic manipulation: a new tool for ecological field studies. The Plant Journal, 76, 506-518.)

This inducible promoter is very valuable for experiments under native field conditions, because in contrast to constitutive promoters it allows sufficient control over gene expression for the study of ecological interactions in real time, of genetic traits that play essential roles in development, or of dose dependent effects (Fig. 7). This allows us to ask completely new research questions. More than 230 different transgenic plant lines have been generated, among them a set of about 30 different vectors for the pOp6-promoter driven induced silencing or overexpression of genes. We still continue to produce new lines for newly identified genes with high ecological relevance. An overview of the most important transgenic N. attenuata lines available in our group can be found in the table at the end of the chapter.

Microarrays
The availability of nearly complete transcriptomic and genomic sequence data for N. attenuata enabled the development of 8x60k microarrays for the Agilent Technologies microarray platform (
http://www.genomics.agilent.com/) covering the complete N. attenuata transcriptome. An also available 4x44k microarray for the AGILENT platform [GEO Accession GPL13527] bases on a 454 sequencing approach [BioProject PRJNA223344]. For this microarray the 43,533 N. attenuata contigs with the best coverage were used to design 60mer oligonucleotides. Statistical tools for the evaluation of the microarray data have been developed. A database with the results from hundreds of hybridizations from our ecological experiments is available.

SAGE (Serial Analysis of Gene Expression)
We have generated in collaboration with GenXPro GmbH (
http://www.genxpro.info/) two SAGE libraries with more than 350,000 total tags from N. attenuata wounded and FAC (Fatty Acid Conjugates) elicited leaves (Gilardoni, P., Schuck, S., Jüngling, R., Rotter, B., Baldwin, I. T., Bonaventure, G. (2010). SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): Identification of early mediators of insect responses. BMC Plant Biology, 10, 66. doi:10.1186/1471-2229-10-66.). These tags represent ca. 23,000 unique transcripts and approx. 500 of these tags are statistically > 2-fold up-regulated after FAC treatment compared to wounding whereas 250 are > 2-fold down-regulated. Some of these tags have been selected for gene function characterization in N. attenuata using VIGS and RNAi gene-specific silencing techniques. Consistent with the high sensitivity of the SAGE technique to detect very low abundant transcripts (potentially encoding for regulatory factors), a large number of differentially expressed tags in FAC treated samples correspond to low copy number tags, and preliminary sequence alignment analysis has classified them as encoding for potential regulatory components (e.g., transcription factors, protein kinases, protein phosphatases, calcium binding proteins).

qPCR
Quantitative PCR can be performed on two MX3005P cyclers For transcriptional studies, primers and probes for many ecologically important genes are available.

Genotyping
Primer pairs amplifying N. tabacum microsatellite markers (Bindler et al., Theor Appl Genet., 2007) were screened with N. attenuata gDNA from different populations for amplification of polymorphic loci. By using fluorescent-labeled primers, size determination of the identified PCR fragments (Fig. 8) on an ABI 3100 sequencer (Fig. 9) confirmed that 6 primer pairs are suitable for genotyping. These will allow us to genotype individual N. attenuata plants that we work with in natural habitats. The initial plant material grinding for the HTP gDNA isolation procedure is performed in a SPEX SamplePrep 2000 Geno/Grinder® (Fig. 10).

Micrografting
A micrografting method for the connection of shoots and roots from different transgenic N. attenuata plants has been developed and established (Fragoso, V., Goddard, H., Baldwin, I. T., Kim, S.-G. (2011). A simple and efficient micrografting method for stably transformed Nicotiana attenuata plants to examine shoot-root signaling. Plant Methods, 7: 34.). This simple procedure (Fig. 11) allows us to study signal transduction between the above- and below-ground organs of the plant and is also extensively used for experiments in the field [link to Ecological Platform]

Available transgenic N. attenuata lines

Gene

Experimental trait

Type

Circadian clock

ADO3

adagio-protein 2

ir

LHY

late elongated hypocotyl

ir/ov

TOC1

timing of CAB expression

ir

ZTL1

zeitlupe

ir

PRR5

pseudo-response regulator

ir/ov

Cytokinin pathway

CKX2

cytokinin oxidase

senescence

ov

CRE1

cytokinin receptor

senescence

ir

SGT1

suppressor of G-two allele of SKP1

senescence

ir

Ethylene pathway

ACO1

1-aminocyclopropane 1-carboxylic acid oxidase

ethylene biosynthesis

ir

LOX3/ETR1

ir silencing LOX2 and ov ETR1-1

ethylene signalling

ir/ov

ETR1-1

ethylene receptor mutant

ethylene reception

ov

Floral traits

CHAL

chalcone synthase

benzyl acetone synthesis

ir

NON1

non-opening

ir

SWEET9

sucrose transporter

nectar production

i

Growth

CWII

cell wall incertase inhibitor

ir

PME

pectin methyl esterase

ir

RALF

rapid alkalization factor

root development

ir

CAD

cinnamyl alcohol dehydrogenase

lignin biosynthesis

ir

IPT1 to 5

isopentenyl transferase

senescence

ov

HK2HK3

histidine kinase

senescence

ir

Indirect/direct defense

AT2

acetyltransferase

ir

HQT

hydroxycinnamoyl CoA quinate transferase

chlorogenic acid biosynthesis

ir

FPPS

farnesyl pyrophosphate synthase

terpene biosynthesis

ir

GGPPS

geranylgeranyl pyrophosphate synthase

terpene biosynthesis

ir

RT

UDP-rhamnosyltransferase

DTG glycosylation

ir

TPS10

terpene synthase

Sesquiterpene biosynthesis

ov

HPL1

hydroperoxide lyase

GLV biosynthesis

as

PMT

putrescine n-methyl transferase

nicotine biosynthesis

ir

TPI

trypsin protease inhibitor

protease inhibition

ir/ov

Kinase signaling

SIPK

salicylic acid-activated MAP kinase

herbivore related signaling

ir

CCAMK

Calcium/calmodulin-dependent protein kinase

Mycorrhiza interaction

ir

CDPK 2 to 4

calcium dependent protein kinase

JA biosynthesis

ir

lecRK1

lectin-domain receptor-like kinase

herbivore related signaling

ir

MEK 1 and 2

mitogen activated protein kinase kinase

herbivore related signaling

ir

MPK4

mitogen-activated protein kinase

herbivore related signaling

ir

SIPKK

MAP kinase kinase

herbivore related signaling

ir

WIPK

wound inducible protein kinase

herbivore related signaling

ir

Light response

PHOT1

phototropin

light reception

ir

CRY1 and 2

cryptochrome 1a

light reception

ir

PHY A, B1/2

phytochrome

Far red response

ir

UVR8

UVB receptor

UV response

ir

Oxylipin pathway

alphaDOX

alpha-dioxygenase

2-HOT biosynthesis

ir

COI1

coronatine insensitive 1

Jasmonate reception

ir

COI2

coronatine insensitive 2

Jasmonate reception

ir

JMT

jasmonic acid carboxyl methyltransferase

Jasmonate metabolism

ov

JAR4 and 6

jasmonic acid resistance protein

JA pathway

ir

JAZ A to H

jasmonate ZIM domain

Jasmonate metabolism

ir

JIH1

jasmonoyl-L-isoleucine hydrolase

JA pathway

ir

MJE

methy jasmonate esterase

JA pathway

ir

MJE/JMT

ir silencing MJE and ovAtJMT

JA pathway

ir/ov

OPR3

transforms 12-oxo-PDA to OPC-8:3

JA pathway

ir

LOX 1-3, 6

lipoxygenase

fatty acid peroxidation

ir

ACX

acyl-CoA oxidase

JA biosynthesis

ir

AOC

allene oxide cyclase

JA biosynthesis

ir

AOS1

allene oxide synthase

JA biosynthesis

as

Pathogen related

DEF1 and 2

defensin

antimicrobial peptide

ov

NPR1

non responding pathogen resistance protein

SA receptor

ir

EDS1

enhanced desease susceptibility

pathogen defesnse

ir

ICS1

isochorismate synthase

SA biosynthesis

ir

NAHG1

salycilate hydroxylase

SA metabolism

ov

Photosynthesis

COP

constitutively photomorphogenic

ir

PDS

phytoene desaturase

ir

RBOH

NAPDH oxidase

ir

RCA

RuBPCase activase

ir

RUB

RuBPCase

as

Primary metabolism

TD

threonine deaminase

isoleucine biosynthesis

ir

GAL83

sucrose non fermenting protein

ir

SBP

sedoheptulose-1,7-bisphosphatase

ov

Small RNAs

AGO1 to 10

argonaute

ir

DCL 2 to 4

dicer like

ir

RDR 1 to 3

RNA dependent RNA polymerase

ir

Transcription factors

MYB 5/8

MYB transcripion factor

herbivore related signaling

ir

WRKY 3/6

WRKY transcription factor

herbivore related signaling

ir/ov

Others

AOX

alternative oxidase

herbivore related signaling

ir

UPF

up frameshift protein

ir

GER

germin

ir

GT

UDP-glycosyltransferase

ir

HMGR

3-Hydroxy-3-methylglutaryl-CoA-reductase

ir

HER

herbivore elicitor regulated

ir

LHGR

regulator for POP6 promoter

POP6 promoter induction

ov

LEC

lectin

ov

DR5

DR5 element

root specific activation

ir

HSPRO

ortholog of sugar beet Hs1pro-1

root-fungi-interaction

ir

BIP

luminal binding protein

secretion

as

ir: inverted repeat silencing; as: antisense silencing; ov: overexpression

 

 

Selected literature using stably transformed lines

Circadian clock:
Yon, F., Seo, P. J., Ryu, J. Y., Park, C.-M., Baldwin, I. T., Kim, S.-G. (2012). Identification and characterization of circadian clock genes in a native tobacco, Nicotiana attenuata. BMC Plant Biology, 12: 172. doi:10.1186/1471-2229-12-172.

Ethylene pathway
von Dahl, C. C., Winz, R., Halitschke, R., Kühnemann, F., Gase, K., Baldwin, I. T. (2007).
Tuning the herbivore-induced ethylene burst: the role of transcript accumulation and ethylene perception in Nicotiana attenuata. The Plant Journal, 51(2), 293-307. doi:10.1111/j.1365-313X.2007.03142.x.

Floral traits
Kessler, D., Gase, K., Baldwin, I. T. (2008). Field experiments with transformed plants reveal the sense of floral scents. Science, 321(5893), 1200-1202. doi:10.1126/science.1160072.

Kinase signaling
Schäfer, M., Meza Canales, I. D., Navarro-Quezada, A., Brütting, C., Radomira, V., Baldwin, I. T., Meldau, S. (2014). Cytokinin levels and signaling respond to wounding and the perception of herbivore elicitors in Nicotiana attenuata. Journal of Integrative Plant Biology. doi:10.1111/jipb.12227.

Oxylipin pathway
Oh, Y., Baldwin, I. T., Galis, I. (2013). A jasmonate ZIM-domain protein NaJAZd regulates floral jasmonic acid levels and counteracts flower abscission in Nicotiana attenuata plants. PLoS One, 8(2): e57868. doi:10.1371/journal.pone.0057868.

Photosynthesis
Mitra, S., Baldwin, I. T. (2014). RuBPCase activase mediates growth-defense tradeoffs: silencing RCA redirects jasmonic acid (JA) flux from JA-Ile to MeJA to attenuate induced defense responses in Nicotiana attenuata. New Phytologist, 201, 1385-1395. doi:10.1111/nph.12591.

Small RNAs
Bozorov, T. A., Pandey, S. P., Dinh, T. S., Kim, S.-G., Heinrich, M., Gase, K., Baldwin, I. T. (2012). Dicer-like proteins and their role in plant-herbivore interactions in Nicotiana attenuata. Journal of Integrative Plant Biology, 54(3), 189-206. doi:10.1111/j.1744-7909.2012.01104.x.