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, post-transcriptionally silencing or ectopically overexpressing more than 200 different genes (table below) that are important for N. attenuata’s ecological performance. A large proportion of the targeted genes originate from “ask the plant” experiments in which the transcriptomes of plants involved in an ecological interaction (herbivory, pollination, competition, mycorrhization, root microbial colonization etc.) were queried by DDRT-PCR, SuperSage, various cDNA and oligo microarrays and RNAseq to identify differentially regulated genes. Many of the differentially regulated genes were first silenced with a virus induced gene silencing system based on the Tobacco Rattle Virus optimized for rapid gene silencing in N. attenuata (VIGS below), before being stably transformed.

The collection of transformed lines represents a unique tool that is being used to study the function of ecologically relevant genes in field experiments (Fig. 1a), tents, mesocosms, glasshouse (Fig. 1b) and even the wind tunnels of our institute (Ecological toolbox). All of these lines are available in T2 or T3 generations as fully characterized homozygous lines harboring single insertions of the silencing/overexpression constructs and hence can be crossed to make all binary combinations of transformants in hemizygous lines. In most cases, the silencing phenotype is comparable between homozygous single construct- and hemizygous binary construct-harboring lines.

To complement the above-mentioned reverse-genetics approach using RNAi for the identification of ecologically important genes, a forward genetics approach has been adopted using an AI-RIL (Advanced Intercross-Recombinant Inbred Lines) population created from a cross of the two best studied N. attenuata ecotypes (“Utah” and “Arizona”) and a MAGIC (Multiparent Advanced Generation InterCross) population created from 26 different ecotypes that expressed unique defense, metabolomics, and morphological phenotypes (MAGIC below). Both of these RILs are in the final stages of breeding and phenotyping and we are now planning field experiments both at our field station in Utah as well as at field sites in Arizona.

The assembly of the almost completed sequences of the N. attenuata and N. obtusifolia genomes and transcriptomes are available (Genomic databases below). This sequence information was used to design complete transcriptomic microarrays based on AGILENT´s 4x44k and 8x60k platforms, which allow for the 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 RNAseq data from different plant tissues and plants attacked by different insect species (Ecological tool box) 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.

Fig. 2: Collection sites of seeds from different natural N. attenuata populations in the past 20 years. Individuals from these collections were phenotypically characterized to choose the ecotypes with the most extreme phenotypes.

For the selection of the parental plants used in the MAGIC cross, morphological and biochemical traits of 424 individuals from 230 accessions were characterized. To guarantee genetic diversity, the 26 individuals with the most extreme morphological, chemical, and defensive 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 in four subsequent rounds of crossings, designed so that each of the 325 combinations served as ♀ and most as ♂, so 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 inbred for 6 consecutive generations (Fig. 3c).

A VIGS system based on a tobacco rattle virus containing vector has been adapted for N. attenuata (Saedler & Baldwin, 2004) which allows for 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 insect genes of the plant’s specialist herbivores, including Manduca sexta (Kumar et al., 2012). In this example, a 300 bp fragment of the target gene is cloned in the VIGS vector in antisense orientation. After Agro-infiltration of N. attenuata with this vector, dsRNA of this fragment is formed as a result of TRV replication. Native M. sexta larvae feeding on plants stabily transformed with this construct in the field were silenced in the expression the targeted midgut gene.

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.

In an ongoing project with the Max-Planck-Institute for Molecular Genetics, the genomes of N. attenuata (2.4 Gb) and N. obtusifolia (1.5 Gb) are being sequenced. At present a draft N. attenuata 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 focused at improving the assembly. For this, we are using Pacific Biosciences (PacBio) sequencing and BioNano Genomics DNA optical mapping technologies. PacBio method results in long reads up to 60 kb and allows assembling of DNA regions, particularly the repeat regions which yields suboptimal results using Illumina short reads sequencing. This assembly would be further improved by 10x Genomics linked read technology, which links short Illumina sequence reads using DNA barcoding, and results in large contiguous assemblies. Additionally, BioNano optical maps generated from high molecular weight DNA provides a complete graphic map of the genome, allowing chromosome length scaffolding of assembled contigs to the near exact positions in the N. attenuata chromosomes. We anticipate that a combination of all these methods with our current sequence data will lead to a near complete N. attenuata genome, which will soon be made publicly available. In addition, the genome of the related species Nicotiana obtusifolia has been sequenced with lower coverage. The availability of both sequences allows for studies of the evolution of ecologically relevant traits in the genus Nicotiana. Link to the NaDH database.

High throughput pyrosequencing of microbial communities

Roots and leaves of field-grown N. attenuata plants can be colonized by diverse microbial communities living as endophytes (within the plant) or as epiphytes (on the plant surface). We isolated DNA from field grown plants to amplify bacterial 16S rDNA using universal bacterial primers. For the comparison of microbial communities among different genotypes we used high throughput 454 pyrosequencing offered by an external company (Santhanam, et al., 2014). We also investigated whether the composition of fungal communities is influenced by the calcium calmodulin-dependent protein kinase gene (CCaMK) using a transgenic irCCaMK knock down line and primers for the fungal internal transcribed spacer (ITS) region.

Bisulfite genomic sequencing

We used bisulfite genomic sequencing for promoter methylation analyses in N. attenuata plants (Weinhold et al., 2013). Primers were designed to be universally suitable for the analysis of the NOS promoter of most transgenic plant lines in our department. Transgenic N. attenuata plants can show a very dynamic increase in promoter methylation during vegetative development which is inherited to the following generation and leads to epigenetic gene silencing.

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 technologies. 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. Additionally, de novo transcriptome assembly is performed on all the 26 MAGIC parents and a comparitive transcriptomic analysis between these ecotypes will give insight in intraspecific genome variations and adaptation. We are now in the process of sequencing the transcriptome of one set of MAGIC population post elicitation in the field, which would then enable us to perform eQTL and identify varition in gene expression associated with herbivory. The nearly complete transcriptome sequence of the related species N. obtusifolia is also available and was established in cooperation with Vertis Biotechnologie AG. mRNA was isolated, normalized and sequenced using the 454 technology. All next generations sequencing transcriptome assemblies can be currently 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 investigate allopolyploid species formation. These are being used to study how the genetic architecture of complex ecological adaptions are altered during allopolyploidization to better understand why this common form of speciation is so strongly associated with adaptive radiations [(Pearse et al., (2006); Anssour et al., (2008) Anssour & Baldwin (2010); Bezzi et al., (2010)].

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. We anticipate using these sequences in numerous future PmRNAi experiments in the field and laboratory to unravel the mysteries of how these insects manage to feed on N. attenuata or the herbivores of N. attenuata. Link to the NaDH database.

Tools to study smRNA mediated interactions

In close collaboration with Shree Pandey’s group at IISER Kolkata we are investigating the world of regulatory small-RNAs. RNA-directed RNA polymerases (RdRs), DICER-Like proteins (DCLs) and the Argonautes (AGOs) from the three most important components of the small-RNA pathway. We have generated knock-down-lines for all the three functional RdRs (irRdR1, 2 and 3; Pandey & Baldwin, 2007; Pandey & Baldwin, 2008; Pandey et al., 2008), the four DCLs (Bozorov et al., 2011) and eight AGOs. Using NGS approach, we have developed an expression atlas of N. attenuata smRNAs (Pandey et al., 2008). The Pandey group is in parallel developing databases and computational resources (such as Srivastava et al., 2014) for N. attenuata smRNA genomics that is proving helpful to understand their function and evolution (Singh et al., 2015).

Transgenic Plant Collection and Stable Transformation System

The standard transformation procedures developed for cultivated tobacco did not work for N. attenuata and hence a completely new Agrobacterium based transformation procedure (Krügel et al., 2002) with new transformation vectors optimized for the transformation of the elongated hypocotyl and the regeneration of plants from the resulting callus was developed. 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 overexpression 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 with the nptII gene as bacterial selectable marker, which allows flanking sequence at the insertion site to be recovered. Other vectors with nourseothricin as a selectable marker are also available, which allow plants to be transformed twice.

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 et al., 2013).

Fig. 7: Dexamethasone (DEX) induced gene silencing of the phytoene desaturase (pds) gene in N. attenuata under field conditions approximately 2 weeks after induction. (a) Bleached plant after treatment with 100µM DEX. (b) Treatment of different branches with different local DEX concentrations results in different degrees of bleaching. Pictures: Martin Schäfer.

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 covering the complete N. attenuata transcriptome. A 4x44k microarray for the AGILENT platform [GEO Accession GPL13527] based on a 454 sequencing approach [BioProject PRJNA223344] is also available. 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 two SAGE libraries with more than 350,000 total tags from N. attenuata wounded and FAC (Fatty Acid Conjugates) elicited leaves (Gilardoni et al., 2010). 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

Genotyping Primer pairs amplifying N. tabacum microsatellite markers (Bindler et al., A microsatellite marker based linkage map of tobacco. Theor Appl Genet. 2007; 114:341–349) 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)

Molecular Biology Standard Lab equipment

Our lab is well equipped with standard molecular biology instrumentation. Here is an overview of the equipment:

Modification and Preparation of DNA and RNA

• 6 PCR-Machines
• 8 Incubators
• 1 Concentrator
• 6 Micro-Centrifuges
• 3 Table-Centrifuges (refrigerated)
• 1 Gel Documentation System

Cultivation of Bacteria

• 3 Table Shaking Incubators
• 1 Horizontal Shaking Incubator

Analysis of the DNA and RNA

• 1 Agilent 2100 Bioanalyzer (Fig. 9)
• 1 Tecan Infinite 200
• 1 Tecan Microplate Reader
• 2 Photometers
• 1 ABI Genetic Analyzer 3100 (Fig. 12)
• 1 Isotope Imaging System (Phosphoimager)
• 2 qPCR Machines Mx3005P

A selection of available transgenic N. attenuata lines

Selected literature using stably transformed lines (for a full list see: departmental publications):

• 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 signaling
  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.