Prezygotic mate selection is only partially correlated with the expression of NaS-like RNases and affects offspring phenotypes.

Nicotiana attenuata styles preferentially select pollen from among accessions with corresponding expression patterns of NaS-like-RNases (SLRs), and the postpollination ethylene burst (PPEB) is an accurate predictor of seed siring success. However, the ecological consequences of mate selection, its effect on the progeny, and the role of SLRs in the control of ethylene signaling remain unknown. We explored the link between the magnitude of the ethylene burst and expression of the SLRs in a set of recombinant inbred lines (RILs), dissected the genetic underpinnings of mate selection through genome-wide association study (GWAS), and examined its outcome for phenotypes in the next generation. We found that high levels of PPEB are associated with the absence of SLR2 in most of the tested RILs. We identified candidate genes potentially involved in the control of mate selection and showed that pollination of maternal genotypes with their favored pollen donors produces offspring with longer roots. When the maternal genotypes are only able to select against nonfavored pollen donors, the selection for such positive traits is abolished. We conclude that plants' ability of mate choice contributes to measurable changes in progeny phenotypes and is thus likely a target of selection.

A persistent major mutation in canonical jasmonate signaling is embedded in an herbivory-elicited gene network.

When insect herbivores attack plants, elicitors from oral secretions and regurgitants (OS) enter wounds during feeding, eliciting defense responses. These generally require plant jasmonate (JA) signaling, specifically, a jasmonoyl-L-isoleucine (JA-Ile) burst, for their activation and are well studied in the native tobacco Nicotiana attenuata. We used intraspecific diversity captured in a 26-parent MAGIC population planted in nature and an updated genome assembly to impute natural variation in the OS-elicited JA-Ile burst linked to a mutation in the JA-Ile biosynthetic gene NaJAR4. Experiments revealed that NaJAR4 variants were associated with higher fitness in the absence of herbivores but compromised foliar defenses, with two NaJAR homologues (4 and 6) complementing each other spatially and temporally. From decade-long seed collections of natural populations, we uncovered enzymatically inactive variants occurring at variable frequencies, consistent with a balancing selection regime maintaining variants. Integrative analyses of OS-induced transcriptomes and metabolomes of natural accessions revealed that NaJAR4 is embedded in a nonlinear complex gene coexpression network orchestrating responses to OS, which we tested by silencing four hub genes in two connected coexpressed networks and examining their OS-elicited metabolic responses. Lines silenced in two hub genes (NaGLR and NaFB67) co-occurring in the NaJAR4/6 module showed responses proportional to JA-Ile accumulations; two from an adjacent module (NaERF and NaFB61) had constitutively expressed defenses with high resistance. We infer that mutations with large fitness consequences can persist in natural populations due to compensatory responses from gene networks, which allow for diversification in conserved signaling pathways and are generally consistent with predictions of an omnigene model.

Single-cell multi-omics in the medicinal plant Catharanthus roseus.

Advances in omics technologies now permit the generation of highly contiguous genome assemblies, detection of transcripts and metabolites at the level of single cells and high-resolution determination of gene regulatory features. Here, using a complementary, multi-omics approach, we interrogated the monoterpene indole alkaloid (MIA) biosynthetic pathway in Catharanthus roseus, a source of leading anticancer drugs. We identified clusters of genes involved in MIA biosynthesis on the eight C. roseus chromosomes and extensive gene duplication of MIA pathway genes. Clustering was not limited to the linear genome, and through chromatin interaction data, MIA pathway genes were present within the same topologically associated domain, permitting the identification of a secologanin transporter. Single-cell RNA-sequencing revealed sequential cell-type-specific partitioning of the leaf MIA biosynthetic pathway that, when coupled with a single-cell metabolomics approach, permitted the identification of a reductase that yields the bis-indole alkaloid anhydrovinblastine. We also revealed cell-type-specific expression in the root MIA pathway.

Exploring the metabolic basis of growth/defense trade-offs in complex environments with Nicotiana attenuata plants cosilenced in NaMYC2a/b expression.

In response to challenges from herbivores and competitors, plants use fitness-limiting resources to produce (auto)toxic defenses. Jasmonate signaling, mediated by MYC2 transcription factors (TF), is thought to reconfigure metabolism to minimize these formal costs of defense and optimize fitness in complex environments. To study the context-dependence of this metabolic reconfiguration, we cosilenced NaMYC2a/b by RNAi in Nicotiana attenuata and phenotyped plants in the field and increasingly realistic glasshouse setups with competitors and mobile herbivores. NaMYC2a/b had normal phytohormonal responses, and higher growth and fitness in herbivore-reduced environments, but were devastated in high herbivore-load environments in the field due to diminished accumulations of specialized metabolites. In setups with competitors and mobile herbivores, irMYC2a/b plants had lower fitness than empty vector (EV) in single-genotype setups but increased fitness in mixed-genotype setups. Correlational analyses of metabolic, resistance, and growth traits revealed the expected defense/growth associations for most sectors of primary and specialized metabolism. Notable exceptions were some HGL-DTGs and phenolamides that differed between single-genotype and mixed-genotype setups, consistent with expectations of a blurred functional trichotomy of metabolites. MYC2 TFs mediate the reconfiguration of primary and specialized metabolic sectors to allow plants to optimize their fitness in complex environments.

California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting.

BACKGROUND: Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant's native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research. RESULTS: Here, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance. CONCLUSIONS: The new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense in Nicotiana attenuata.

The native diploid tobacco Nicotiana attenuata produces abundant, potent anti-herbivore defense metabolites known as 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 family-1 UGT members. The transcript levels of three UGTs were highly correlated with the transcript levels two key HGL-DTG biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLS's role in HGL-DTG biosynthesis was confirmed by virus-induced gene silencing. Silencing the Uridine diphosphate (UDP)-rhamnosyltransferase gene UGT91T1 demonstrated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. Plants with stable silencing of UGT74P3 and UGT74P5 were severely developmentally deformed, pointing to a phytotoxic effect of the aglycone. The application of synthetic 17-HGL and silencing of the UGTs in HGL-DTG-free plants confirmed this phytotoxic effect. Feeding assays with tobacco hornworm (Manduca sexta) larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation of 17-HGL is therefore a critical step that contributes to the resulting metabolites' defensive function and solves the autotoxicity problem of this potent chemical defense.

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco.

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(-)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.

Mate Selection in Self-Compatible Wild Tobacco Results from Coordinated Variation in Homologous Self-Incompatibility Genes.

In flowering plants, intraspecific mate preference is frequently related to mating systems: the rejection of self pollen in self-incompatible (SI) plants that prevents inbreeding is one of the best described examples. However, in other mating systems, more nuanced patterns of pollen rejection occur. In the self-compatible (SC) Nicotiana attenuata, in which SI is not found and all crosses are compatible, certain pollen genotypes are consistently selected in mixed pollinations. However, the molecular mechanisms of this polyandrous mate selection remain unknown. Style-expressed NaS-like-RNases and pollen-expressed NaSLF-like genes, homologous to SI factors in Solanaceae, were identified and examined for a role in N. attenuata's mate selection. A comparison of two NaS-like-RNases and six NaSLF-like genes among 26 natural accessions revealed specific combinations of co-expression and direct protein-protein interactions. To evaluate their role in mate selection, we silenced the expression of specific NaS-like-RNases and NaSLF-like proteins and conducted diagnostic binary mixed pollinations and mixed pollinations with 14 different non-self pollen donors. Styles expressing particular combinations of NaS-like-RNases selected mates from plants with corresponding NaS-like-RNase expression patterns, while styles lacking NaS-like-RNase expression were non-selective in their fertilizations, which reflected the genotype ratios of pollen mixtures deposited on the stigmas. DNA methylation could account for some of the observed variation in stylar NaS-like-RNase patterns. We conclude that the S-RNase-SLF recognition mechanism plays a central role in polyandrous mate selection in this self-compatible species. These results suggest that after the SI-SC transition, natural variation of SI homologous genes was repurposed to mediate intraspecific mate selection.

Argonaute 8 (AGO8) Mediates the Elicitation of Direct Defenses against Herbivory.

In Nicotiana attenuata, specific RNA-directed RNA polymerase (RdR1) and the Dicer-like (DCL3 and DCL4) proteins are recruited during herbivore attack to mediate the regulation of defense responses. However, the identity and role(s) of Argonautes (AGOs) involved in herbivory remain unknown. Of the 11 AGOs in the N. attenuata genome, we silenced the expression of 10. Plants silenced in NaAGO8 expression grew normally but were highly susceptible to herbivore attack. Larvae of Manduca sexta grew faster when consuming inverted-repeat stable transformants (irAGO8) plants but did not differ from the wild type when consuming plants silenced in AGO1 (a, b, and c), AGO2, AGO4 (a and b), AGO7, or AGO10 expression. irAGO8 plants were significantly compromised in herbivore-induced levels of defense metabolites such as nicotine, phenolamides, and diterpenoid glycosides. Time-course analyses revealed extensively altered microRNA profiles and the reduced accumulation of MYB8 transcripts and of the associated genes of the phenolamide and phenylpropanoid pathways as well as the nicotine biosynthetic pathway. A possible AGO8-modulated microRNA-messenger RNA target network was inferred. Furthermore, comparative analysis of domains revealed the diversity of AGO conformations, particularly in the small RNA-binding pocket, which may influence substrate recognition/binding and functional specificity. We infer that AGO8 plays a central role in the induction of direct defenses by modulating several regulatory nodes in the defense signaling network during herbivore response. Thus, our study identifies the effector AGO of the herbivore-induced small RNA machinery, which in N. attenuata now comprises RdR1, DCL3/4, and AGO8.

Wild tobacco genomes reveal the evolution of nicotine biosynthesis.

Nicotine, the signature alkaloid of Nicotiana species responsible for the addictive properties of human tobacco smoking, functions as a defensive neurotoxin against attacking herbivores. However, the evolution of the genetic features that contributed to the assembly of the nicotine biosynthetic pathway remains unknown. We sequenced and assembled genomes of two wild tobaccos, Nicotiana attenuata (2.5 Gb) and Nicotiana obtusifolia (1.5 Gb), two ecological models for investigating adaptive traits in nature. We show that after the Solanaceae whole-genome triplication event, a repertoire of rapidly expanding transposable elements (TEs) bloated these Nicotiana genomes, promoted expression divergences among duplicated genes, and contributed to the evolution of herbivory-induced signaling and defenses, including nicotine biosynthesis. The biosynthetic machinery that allows for nicotine synthesis in the roots evolved from the stepwise duplications of two ancient primary metabolic pathways: the polyamine and nicotinamide adenine dinucleotide (NAD) pathways. In contrast to the duplication of the polyamine pathway that is shared among several solanaceous genera producing polyamine-derived tropane alkaloids, we found that lineage-specific duplications within the NAD pathway and the evolution of root-specific expression of the duplicated Solanaceae-specific ethylene response factor that activates the expression of all nicotine biosynthetic genes resulted in the innovative and efficient production of nicotine in the genus Nicotiana Transcription factor binding motifs derived from TEs may have contributed to the coexpression of nicotine biosynthetic pathway genes and coordinated the metabolic flux. Together, these results provide evidence that TEs and gene duplications facilitated the emergence of a key metabolic innovation relevant to plant fitness.

O-Acyl Sugars Protect a Wild Tobacco from Both Native Fungal Pathogens and a Specialist Herbivore.

O-Acyl sugars (O-AS) are abundant trichome-specific metabolites that function as indirect defenses against herbivores of the wild tobacco Nicotiana attenuata; whether they also function as generalized direct defenses against herbivores and pathogens remains unknown. 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 3-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 α-ketoacid dehydrogenase E1 ß-subunit-encoding gene (NaBCKDE1B) in the trichomes reduced total leaf O-AS by 20% to 30% and increased susceptibility to Fusarium pathogens. We conclude that O-AS function as direct defenses to protect plants from attack by both native pathogenic fungi and a specialist herbivore and infer that their diversification is likely shaped by the functional interactions among these biotic stresses.

Functional specialization of Nicotiana attenuata phytochromes in leaf development and flowering time.

Phytochromes mainly function in photoautotrophic organisms to adjust growth in response to fluctuating light signals. The different isoforms of plant phytochromes often display both conserved and divergent roles, presumably to fine-tune plant responses to environmental signals and optimize fitness. Here we describe the distinct, yet partially redundant, roles of phytochromes NaPHYA, NaPHYB1 and NaPHYB2 in a wild tobacco species, Nicotiana attenuata using RNAi-silenced phytochrome lines. Consistent with results reported from other species, silencing the expression of NaPHYA or NaPHYB2 in N. attenuata had mild or no influence on plant development as long as NaPHYB1 was functional; whereas silencing the expression of NaPHYB1 alone strongly altered flowering time and leaf morphology. The contribution of NaPHYB2 became significant only in the absence of NaPHYB1; plants silenced for both NaPHYB1 and NaPHYB2 largely skipped the rosette-stage of growth to rapidly produce long, slender stalks that bore flowers early: hallmarks of the shade-avoidance responses. The phenotyping of phytochrome-silenced lines, combined with sequence and transcript accumulation analysis, suggest the independent functional diversification of the phytochromes, and a dominant role of NaPHYB1 and NaPHYB2 in N. attenuata's vegetative and reproductive development.

Changes in cytokinins are sufficient to alter developmental patterns of defense metabolites in Nicotiana attenuata.

Plant defense metabolites are well known to be regulated developmentally. The optimal defense (OD) theory posits that a tssue's fitness values and probability of attack should determine defense metabolite allocations. Young leaves are expected to provide a larger fitness value to the plant, and therefore their defense allocations should be higher when compared with older leaves. The mechanisms that coordinate development with defense remain unknown and frequently confound tests of the OD theory predictions. Here we demonstrate that cytokinins (CKs) modulate ontogeny-dependent defenses in Nicotiana attenuata. We found that leaf CK levels highly correlate with inducible defense expressions with high levels in young and low levels in older leaves. We genetically manipulated the developmental patterns of two different CK classes by using senescence- and chemically inducible expression of CK biosynthesis genes. Genetically modifying the levels of different CKs in leaves was sufficient to alter ontogenic patterns of defense metabolites. We conclude that the developmental regulation of growth hormones that include CKs plays central roles in connecting development with defense and therefore in establishing optimal patterns of defense allocation in plants.

Molecular evolution and diversification of the Argonaute family of proteins in plants.

BACKGROUND: Argonaute (AGO) proteins form the core of the RNA-induced silencing complex, a central component of the smRNA machinery. Although reported from several plant species, little is known about their evolution. Moreover, these genes have not yet been cloned from the ecological model plant, Nicotiana attenuata, in which the smRNA machinery is known to mediate important ecological traits. RESULTS: Here, we not only identify 11 AGOs in N. attenuata, we further annotate 133 genes in 17 plant species, previously not annotated in the Phytozome database, to increase the number of plant AGOs to 263 genes from 37 plant species. We report the phylogenetic classification, expansion, and diversification of AGOs in the plant kingdom, which resulted in the following hypothesis about their evolutionary history: an ancestral AGO underwent duplication events after the divergence of unicellular green algae, giving rise to four major classes with subsequent gains/losses during the radiation of higher plants, resulting in the large number of extant AGOs. Class-specific signatures in the RNA-binding and catalytic domains, which may contribute to the functional diversity of plant AGOs, as well as context-dependent changes in sequence and domain architecture that may have consequences for gene function were found. CONCLUSIONS: Together, the results demonstrate that the evolution of AGOs has been a dynamic process producing the signatures of functional diversification in the smRNA pathways of higher plants.

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.

'Real time' genetic manipulation: a new tool for ecological field studies.

Field experiments with transgenic plants often reveal the functional significance of genetic traits that are important for the performance of the plants in their natural environments. Until now, only constitutive overexpression, ectopic expression and gene silencing methods have been used to analyze gene-related phenotypes in natural habitats. These methods do not allow 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. We applied the sensitive dexamethasone (DEX)-inducible pOp6/LhGR expression system to the ecological model plant Nicotiana attenuata and established a lanolin-based DEX application method to facilitate ectopic gene expression and RNA interference-mediated gene silencing in the field and under challenging conditions (e.g. high temperature, wind and UV radiation). Fully established field-grown plants were used to silence phytoene desaturase and thereby cause photobleaching only in specific plant sectors, and to activate expression of the cytokinin (CK) biosynthesis gene isopentenyl transferase (ipt). We used ipt expression to analyze the role of CKs in both the glasshouse and the field to understand resistance to the native herbivore Tupiocoris notatus, which attacks plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, damage by T. notatus increased, demonstrating the role of CKs in this plant-herbivore interaction at a small scale. As the arena of most ecological interactions is highly constrained in time and space, these tools will advance the genetic analysis of dynamic traits that matter for plant performance in nature.

The use of VIGS technology to study plant-herbivore interactions.

Plants employ a large variety of defense strategies to resist herbivores, which require transcriptional reprogramming of cells and profound changes in plant metabolism. Due to the large number of genes involved in defense processes, rapid screening strategies are essential for elucidating the contributions of individual genes in the responses of plants to herbivory. However, databases and seed banks of mutant plants which allow rapid retrieval of mutant genotypes are limited to a few model plant species, namely, Arabidopsis thaliana and Oryza sativa (rice). In other plants, virus-induced gene silencing (VIGS) offers an efficient alternative for screening the functions of individual genes in order to prioritize the allocations of the large time investments required to establish stably transformed RNAi-silenced lines. With VIGS, it is usually possible to achieve strong, specific silencing of target genes in the ecological models Nicotiana attenuata and Solanum nigrum, allowing the rapid assessment of gene silencing effects on phytohormone accumulation, signal transduction and accumulation of defense metabolites. VIGS plants are also useful in bioassays with specialist and generalist herbivores, allowing direct verification of gene function in plant resistance to herbivores.

Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata.

Many plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotiana attenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds, increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show that these important pollinators learn to tolerate high-nicotine nectar but prefer low-nicotine nectar, and show no signs of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, are unpredictably variable among flowers, not only among populations, but also within populations, and even among flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrations increase outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. attenuata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic gene for nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized and transplanted into native populations. When only exposed to hummingbird pollinators, seeds produced by flowers with nicotine in their nectar had a greater number of genetically different sires, compared to seeds from nicotine-free flowers. As the variation in nectar nicotine levels among flowers in an inflorescence decreased in N. attenuata plants silenced in various combinations of three Dicer-like (DCL) proteins, small RNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.

DICER-like proteins and their role in plant-herbivore interactions in Nicotiana attenuata.

DICER-like (DCL) proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant-herbivore interactions remains unstudied. We identified and stably silenced four distinct DCL genes by RNAi in Nicotiana attenuata (Torrey ex. Watson), a model for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal. Manduca sexta larvae performed significantly better on ir-dcl3 and ir-dcl4 plants, but not on ir-dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir-dcl4 and double ir-dcl2 ir-dcl3 plants were impaired in JA accumulation, while JA-Ile was increased in ir-dcl3 plants. Ir-dcl3 and ir-dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir-dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti-herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions.

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression.

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.