Klaus Gase

Nicotine's Defensive Function in Nature

Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D4-nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.

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

Summary. Research into the genetic basis of the ecological sophistication of plants is hampered by the availability of transformable systems with a wealth of well-described ecological interactions. We present an Agrobacterium-mediated transformation system for the model ecological expression system, Nicotiana attenuata, a native tobacco that occupies the post-fire niche in the Great Basin Desert of North America. We describe a transformation vector and a transformation procedure that differs from the standard cultivated tobacco transformation protocols in its use of selectable markers, explants, media and cultivation conditions. We illustrate its utility in the transformations with genes coding for key enzymes in the oxylipin cascade (lipoxygenase, allene oxide synthase, hydroperoxide lyase) in antisense orientations and present high-throughput screens useful for the detection of altered phenotypes for the oxylipin cascade (green leaf volatiles and jasmonic acid after wounding).

Field experiments with transformed plants reveal the sense of floral scents

Plants use many means to attract pollinators, including visual cues and odor. We investigated how nonpigment floral chemistry influences nectar removal, floral visitation, florivory, rates of outcrossing, and fitness through both male and female functions. We blocked expression of biosynthetic genes of the dominant floral attractant [benzyl acetone (Nachal1)] and nectar repellent [nicotine (Napmt1/2)] in all combinations in the native tobacco Nicotiana attenuata and measured their effects on plants in their native habitat. Both repellent and attractant were required to maximize capsule production and seed siring in emasculated flowers and flower visitation by native pollinators, whereas nicotine reduced florivory and nectar robbing.

Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses

Evidence for the in planta defensive function of trypsin protease inhibitors (TPIs) comes from observations of enhanced herbivore resistance after heterologous TPI expression or the manipulation of signal cascades that activate numerous defense responses, including TPI production; no studies have altered the expression of an endogenous pi gene to examine defensive function. We isolated two genes with seven- and six-repeat TPI domains from Nicotiana attenuata from the potato (Solanum tuberosum) PI-II family. To determine whether endogenous TPIs in N. attenuata function defensively against the native herbivores, hornworm (Manduca sexta) and mirids (Tupiocoris notatus), we expressed 175 bp of the seven-domain pi from N. attenuata in an antisense orientation in a TPI-producing genotype to reduce TPI expression and expressed the full-length seven-domain pi in a sense orientation under control of a constitutive promoter to restore TPI activity in a natural genotype from Arizona unable to produce TPIs. Constitutive and inducible TPI production in two antisense lines were diminished by 80% to 90% and 33% to 52%, respectively, and sense expression restored 67% of the activity found in the TPI-producing genotype after caterpillar attack in the TPI-deficient A genotype. Hornworm larvae fed on genotypes with low or no TPI activity grew faster, had higher survivorship, and produced heavier pupae than those that fed on genotypes with high TPI activity. T. notatus showed higher preference for genotypes with low or no TPI activity than for genotypes with high TPI levels. We conclude that endogenous TPIs are an effective defense against these native herbivores.

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

Evidence is accumulating that insect-specific plant responses are mediated by constituents in the oral secretions and regurgitants (R) of herbivores, however the relative importance of the different potentially active constituents remains unclear. Fatty acid-amino acid conjugates (FACs) are found in the R of many insect herbivores and have been shown to be necessary and sufficient to elicit a set of herbivore-specific responses when the native tobacco plantNicotiana attenuata is attacked by the tobacco hornworm,Manduca sexta. Attack by this specialist herbivore results in a large transcriptional reorganization in N. attenuata, and 161 genes have been cloned from previous cDNA differential display-polymerase chain reaction and subtractive hybridization with magnetic beads analysis. cDNAs of these genes, in addition to those of 73 new R-responsive genes identified by cDNA-amplified fragment-length polymorphism display of R-elicited plants, were spotted on polyepoxide coated glass slides to create microarrays highly enriched in Manduca spp.- and R-induced genes. With these microarrays, we compare transcriptional responses in N. attenuata treated with R from the two most damaging lepidopteran herbivores of this plant in nature, M. sexta and Manduca quinquemaculata, which have very similar FAC compositions in their R, and with the two most abundant FACs in Manduca spp. R. More than 68% of the genes up- and down-regulated by M. sexta R were similarly regulated byM. quinquemaculata R. A majority of genes up-regulated (64%) and down-regulated (49%) by M. sexta R were similarly regulated by treatment with the two FACs. In contrast, few genes showed similar transcriptional changes after H2O2- and R-treatment. These results demonstrate that the two most abundant FACs in Manduca spp. R can account for the majority ofManduca spp.-induced alterations of the wound response of N. attenuata.

Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphingidae) and Its Natural Host Nicotiana attenuata: V. Microarray Analysis and Further Characterization of Large-Scale Changes in Herbivore-Induced mRNAs

We extend our analysis of the transcriptional reorganization that occurs when the native tobacco, Nicotiana attenuata, is attacked by Manduca sexta larvae by cloning 115 transcripts by mRNA differential display reverse transcription-polymerase chain reaction and subtractive hybridization using magnetic beads (SHMB) from the M. sexta-responsive transcriptome. These transcripts were spotted as cDNA with eight others, previously confirmed to be differentially regulated by northern analysis on glass slide microarrays, and hybridized with Cy3- and Cy5-labeled probes derived from plants after 2, 6, 12, and 24 h of continuous attack. Microarray analysis proved to be a powerful means of verifying differential expression; 73 of the cloned genes (63%) were differentially regulated (in equal proportions from differential display reverse transcription-polymerase chain reaction and SHMB procedures), and of these, 24 (32%) had similarity to known genes or putative proteins (more from SHMB). The analysis provided insights into the signaling and transcriptional basis of direct and indirect defenses used against herbivores, suggesting simultaneous activation of salicylic acid-, ethylene-, cytokinin-, WRKY-, MYB-, and oxylipin-signaling pathways and implicating terpenoid-, pathogen-, and cell wall-related transcripts in defense responses. These defense responses require resources that could be made available by decreases in four photosynthetic-related transcripts, increases in transcripts associated with protein and nucleotide turnover, and increases in transcripts associated with carbohydrate metabolism. This putative up-regulation of defense-associated and down-regulation of growth-associated transcripts occur against a backdrop of altered transcripts for RNA-binding proteins, putative ATP/ADP translocators, chaperonins, histones, and water channel proteins, responses consistent with a major metabolic reconfiguration that underscores the complexity of response to herbivore attack.

Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR

BackgroundAfter transformation, plants that are homozygous and contain one copy of the transgene are typically selected for further study. If real-time PCR is to be used to determine copy number and zygosity, it must be able to distinguish hemizygous from homozygous and one-copy from two-copy plants. That is, it must be able to detect two-fold differences.ResultsWhen transgenic Nicotiana attenuata plants which had been previously determined by Southern analysis to contain one or two copies of the transgene, were analyzed by real-time PCR (2-ΔΔCt method), the method failed to confirm the results from the Southern analysis. In a second data set we analyzed offspring of a hemizygous one-copy plant, which were expected to segregate into three groups of offspring in a 1:2:1 ratio: no transgene, hemizygous, homozygous. Because it was not possible to distinguish homozygous from hemizygous plants with real-time PCR, we could not verify this segregation ratio.ConclusionsDetection of two-fold differences by real-time PCR is essential if this procedure is to be used for the characterization of transgenic plants. However, given the high variability between replicates, a detection of two-fold differences is in many cases not possible; in such cases Southern analysis is the more reliable procedure.

Herbivory-induced changes in the small-RNA transcriptome and phytohormone signaling in Nicotiana attenuata

Phytohormones mediate the perception of insect-specific signals and the elicitation of defenses during insect attack. Large-scale changes in a plants transcriptome ensue, but how these changes are regulated remains unknown. Silencing of RNA-directed RNA polymerase 1 (RdR1) makes Nicotiana attenuata highly susceptible to insect herbivores, suggesting that defense elicitation is under the direct control of small-RNAs (smRNAs). Using 454-sequencing, we characterized N. attenuatas smRNA transcriptome before and after insect-specific elicitation in wild-type (WT) and RdR1-silenced (irRdR1) plants. We predicted the targets of N. attenuata smRNAs in the genes related to phytohormone signaling (jasmonic acid, JA-Ile, and ethylene) known to mediate resistance responses, and we measured the elicited dynamics of phytohormone biosynthetic transcripts and phytohormone levels in time-course experiments with field- and glasshouse-grown plants. RdR1 silencing severely altered the induced transcript accumulation of 8 of the 10 genes, reduced JA, and enhanced ethylene levels after elicitation. Adding JA completely restored the insect resistance of irRdR1 plants. irRdR1 plants had photosynthetic rates, growth, and reproductive output indistinguishable from that of WT plants, suggesting unaltered primary metabolism. We conclude that the susceptibility of irRdR1 plants to herbivores is due to altered phytohormone signaling and that smRNAs play a central role in coordinating the large-scale transcriptional changes that occur after herbivore attack. Given the diversity of smRNAs that are elicited after insect attack and the recent demonstration of the ability of ingested smRNAs to silence transcript accumulation in lepidopteran larvae midguts, the smRNA responses of plants may also function as direct defenses.

Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9

Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.

Silencing Geranylgeranyl Diphosphate Synthase in Nicotiana attenuata Dramatically Impairs Resistance to Tobacco Hornworm

In bioassays with artificial diets, the 17-hydroxygeranyllinalool diterpenoid glycosides (HGL-DTGs) of Nicotiana attenuata function as antifeedants for the plants adapted herbivore, tobacco hornworm (Manduca sexta). To determine whether HGL-DTGs have a defensive function in planta, we suppressed HGL-DTG production by silencing the source of the geranylgeranyl diphosphates (GGPPs) required for geranyllinalool biosynthesis, a key intermediate. We used virus-induced gene silencing to suppress transcript levels of GGPP synthase gene (Naggpps) and farnesyl diphosphate (FPP) synthase gene (Nafpps), northern blotting and real-time polymerase chain reaction to quantify transcript accumulations, and radio gas chromatography to analyze prenyltransferase specificity. Silencing Nafpps had no effect on the accumulation of HGL-DTGs but decreased leaf steroid content, demonstrating that DTG-synthesizing enzymes do not use GGPP derived from FPP and confirming FPPs role as a steroid precursor. Unlike plants silenced in the phytoene desaturase gene (Napds), which rapidly bleached, Naggpps-silenced plants had reduced HGL-DTG but not carotenoids or chlorophyll contents, demonstrating that Naggpps supplies substrates for GGPP biosynthesis for HGL-DTGs, but not for phytoene or phytol. Expression of Naggpps in Escherichia coli revealed that the recombinant protein catalyzes the GGPP synthesis from isopentenyl diphosphate and dimethylallyl diphosphate. When fed on silenced plants, hornworm larvae gained up to 3 times more mass than those that fed on empty vector control plants or plants silenced in Nafpps, the trypsin protease inhibitor gene, or the putrescine N-methyltransferase gene. We conclude that HGL-DTGs or other minor undetected diterpenoids derived from GGPP function as direct defenses for N. attenuata and are more potent than nicotine or trypsin protease inhibitors against attack by hornworm larvae.