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1.  Molecular evolution and diversification of the Argonaute family of proteins in plants 
BMC Plant Biology  2015;15:23.
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.
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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0364-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4318128  PMID: 25626325
Argonaute; miRNA; Plants; Nicotiana attenuata; Herbivory; Evolution; Small-RNA
2.  Label-free nanoUPLC-MSE based quantification of antimicrobial peptides from the leaf apoplast of Nicotiana attenuata 
BMC Plant Biology  2015;15:18.
Overexpressing novel antimicrobial peptides (AMPs) in plants is a promising approach for crop disease resistance engineering. However, the in planta stability and subcellular localization of each AMP should be validated for the respective plant species, which can be challenging due to the small sizes and extreme pI ranges of AMPs which limits the utility of standard proteomic gel-based methods. Despite recent advances in quantitative shotgun proteomics, its potential for AMP analysis has not been utilized and high throughput methods are still lacking.
We created transgenic Nicotiana attenuata plants that independently express 10 different AMPs under a constitutive 35S promoter and compared the extracellular accumulation of each AMP using a universal and versatile protein quantification method. We coupled a rapid apoplastic peptide extraction with label-free protein quantification by nanoUPLC-MSE analysis using Hi3 method and identified/quantified 7 of 10 expressed AMPs in the transgenic plants ranging from 37 to 91 amino acids in length. The quantitative comparison among the transgenic plant lines showed that three particular peptides, belonging to the defensin, knottin and lipid-transfer protein families, attained the highest concentrations of 91 to 254 pmol per g leaf fresh mass, which identified them as best suited for ectopic expression in N. attenuata. The chosen mass spectrometric approach proved to be highly sensitive in the detection of different AMP types and exhibited the high level of analytical reproducibility required for label-free quantitative measurements along with a simple protocol required for the sample preparation.
Heterologous expression of AMPs in plants can result in highly variable and non-predictable peptide amounts and we present a universal quantitative method to confirm peptide stability and extracellular deposition. The method allows for the rapid quantification of apoplastic peptides without cumbersome and time-consuming purification or chromatographic steps and can be easily adapted to other plant species.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0398-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4318441  PMID: 25604123
Intercellular fluid; Cysteine-rich peptides; Heterologous expression; Transgenic plants; Vacuum infiltration; Data-independent acquisition; Defensin; Lipid-transfer protein; Knottin
3.  Herbivore associated elicitor-induced defences are highly specific among closely related Nicotiana species 
BMC Plant Biology  2015;15:2.
Herbivore-induced defence responses are often specific - different herbivores induce different defence responses in plants - and their specificity is largely mediated by chemical cues (herbivore-associated elicitors: HAEs) in insect oral or oviposition secretions. However, the specificity and the mechanisms of HAE-induced defence have not been investigated in the context of the evolutionary relationships among plant species. Here we compare the responses of six closely related Nicotiana species to a synthetic elicitor, N-linolenoyl-glutamic acid (C18:3-Glu) and HAE of two insect herbivores (the Solanaceae specialist Manduca sexta and generalist Spodoptera littoralis).
HAE-induced defences are highly specific among closely related Nicotiana species at three perspectives. 1) A single Nicotiana species can elicit distinct responses to different HAEs. N. pauciflora elicited increased levels of JA and trypsin proteinase inhibitors (TPI) in response to C18:3-Glu and the oral secretions of M. sexta (OSMs) but not to oral secretions of S. littoralis (OSSl). In contrast, N. miersii only responded to OSSl but not to the other two HAEs. The specific responses to different HAEs in Nicotiana species are likely due to the perception by the plant of each specific component of the HAE. 2) One HAE can induce different defence responses among closely related Nicotiana species. OSMs and C18:3-Glu induced JA and TPI accumulations in N. linearis, N. attenuata, N. acuminata and N. pauciflora, but not in N. miersii and N. obtusifolia. 3) The effect of HAE-induced defences differ for the Solanaceae specialist M. sexta and the generalist S. littoralis. Among the four tested Nicotiana species, while the growth rate of M. sexta was only reduced by the induced defences elicited by C18:3-Glu; the growth rate of S. littoralis can be reduced by the induced defences elicited by all three HAEs. This is likely due to differences in the susceptibility of the specialist M. sexta and generalist S. littoralis to induced defences of their host.
Closely related Nicotiana species elicit highly specific defence responses to herbivore associated elicitors and provide an ideal framework for investigating the molecular mechanisms and evolutionary divergence of induced resistance in plants.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0406-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4304619  PMID: 25592329
Specificity of herbivore induced defence; Nicotiana; Jasmonic acid; Trypsin proteinase inhibitor; Induced resistance; Specialist and generalist
4.  The Nicotiana attenuata GLA1 lipase controls the accumulation of Phytophthora parasitica-induced oxylipins and defensive secondary metabolites 
Plant, cell & environment  2014;37(7):1703-1715.
Nicotiana attenuata plants silenced in the expression of GLYCEROLIPASE A1 (ir-gla1 plants) are compromised in the herbivore- and wound-induced accumulation of jasmonic acid (JA). However, these plants accumulate wild-type (WT) levels of JA and divinyl-ethers (DVE) during Phytophthora parasitica infection (Bonaventure et al., 2011). By profiling oxylipin-enriched fractions with targeted and untargeted LC-QTOF approaches, we demonstrate that the accumulation of 9-hydroxy-10E,12Z-octadecadienoic acid (9-OH-18:2) and additional C18 and C19 oxylipins is reduced by ca. 20-fold in P. parasitica infected ir-gla1 leaves compared to WT. This reduced accumulation of oxylipins was accompanied by a reduced accumulation of unsaturated free fatty acids and specific lysolipid species. Untargeted metabolic profiling of total leaf extracts showed that 87 metabolites accumulated differentially in leaves of P. parasitica-infected ir-gla1 plants with glycerolipids, hydroxylated-diterpene glycosides and phenylpropanoid derivatives accounting together for ca. 20% of these 87 metabolites. Thus, P. parasitica-induced oxylipins may participate in the regulation of metabolic changes during infection. Together, the results demonstrate that GLA1 plays a distinct role in the production of oxylipins during biotic stress responses, supplying substrates for 9-OH-18:2 and additional C18 and C19 oxylipin formation during P. parasitica infection whereas supplying substrates for the biogenesis of JA during herbivory and mechanical wounding.
PMCID: PMC4190502  PMID: 24450863
Phytophthora; lipase; oxylipin; divinyl-ethers; jasmonic acid; infection; resistance; defense; metabolism; lipid
5.  Phytochemical Investigation on Euphorbia macrostegia (Persian wood spurge) 
Euphorbia macrostegia or Persian wood spurge is one of the seventeen endemic plants of this genus in Iran. Three triterpenoids, 24-methylenecycloartan-3β-ol (1), butyrospermol (2) and cycloartenol (3) and three diglycerides, 1,2-di-O-α-linolenoyl-sn-glycerol (4), 1-O-linoleoyl-3-O-palmitoyl-sn-glycerol (5) and 1-O-α-linolenoyl-2-O-palmitoyl-sn-glycerol (6) were isolated from the hexane soluble part of methanol-dichloromethane extracts of the aerial parts of Euphorbia macrostegia Boiss. The structures of all compounds were elucidated using different spectroscopy methods including, 1H NMR, 13C NMR, HSQC, HMBC, EI-MS and IR. The triterpenes and the unsaturated fatty acids moieties of the diglycerides isolated from the plant were reported previously to have analgesic, anticancer, bactericidal and antifungal activity. Here, we show that E. macrostegia is a new source for the above mentioned biologically active compounds.
PMCID: PMC4277637  PMID: 25561930
Euphorbiaceae; Euphorbia macrostegia; Cycloartane triterpenoids; Diglycerides of fatty acids
6.  A robust, simple, high-throughput technique for time-resolved plant volatile analysis in field experiments 
Plant volatiles (PVs) mediate interactions between plants and arthropods, microbes, and other plants, and are involved in responses to abiotic stress. PV emissions are therefore influenced by many environmental factors, including herbivore damage, microbial invasion, and cues from neighboring plants, but also light regime, temperature, humidity, and nutrient availability. Thus an understanding of the physiological and ecological functions of PVs must be grounded in measurements reflecting PV emissions under natural conditions. However, PVs are usually sampled in the artificial environments of laboratories or climate chambers. Sampling of PVs in natural environments is difficult, limited by the need to transport, maintain, and power instruments, or use expensive sorbent devices in replicate. Ideally, PVs should be measured in natural settings with high replication, spatiotemporal resolution, and sensitivity, and at modest costs. Polydimethysiloxane (PDMS), a sorbent commonly used for PV sampling, is available as silicone tubing (ST) for as little as 0.60 €/m (versus 100-550 € apiece for standard PDMS sorbent devices). Small (mm-cm) ST pieces (STs) can be placed in any environment and used for headspace sampling with little manipulation of the organism or headspace. STs have sufficiently fast absorption kinetics and large capacity to sample plant headspaces on a timescale of minutes to hours, and thus can produce biologically meaningful “snapshots” of PV blends. When combined with thermal desorption (TD)-GC-MS analysis – a 40-year-old and widely available technology – STs yield reproducible, sensitive, spatiotemporally resolved, quantitative data from headspace samples taken in natural environments.
PMCID: PMC4190661  PMID: 24684685
Polydimethylsiloxane; PDMS; ST; headspace analysis; plant volatiles; HIPVs; indirect defense; Nicotiana attenuata; Manduca sexta; field research
7.  Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata 
BMC Plant Biology  2014;14(1):326.
Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown.
Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses.
Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0326-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4263023  PMID: 25430398
Defense; Fatty acid-amino acid conjugates; Herbivore; Jasmonic acid; Mitogen-activated protein kinase (MAPK); Nicotiana attenuata; Systemic response
8.  Improving the accuracy of expression data analysis in time course experiments using resampling 
BMC Bioinformatics  2014;15(1):352.
As time series experiments in higher eukaryotes usually obtain data from different individuals collected at the different time points, a time series sample itself is not equivalent to a true biological replicate but is, rather, a combination of several biological replicates. The analysis of expression data derived from a time series sample is therefore often performed with a low number of replicates due to budget limitations or limitations in sample availability. In addition, most algorithms developed to identify specific patterns in time series dataset do not consider biological variation in samples collected at the same conditions.
Using artificial time course datasets, we show that resampling considerably improves the accuracy of transcripts identified as rhythmic. In particular, the number of false positives can be greatly reduced while at the same time the number of true positives can be maintained in the range of other methods currently used to determine rhythmically expressed genes.
The resampling approach described here therefore increases the accuracy of time series expression data analysis and furthermore emphasizes the importance of biological replicates in identifying oscillating genes. Resampling can be used for any time series expression dataset as long as the samples are acquired from independent individuals at each time point.
Electronic supplementary material
The online version of this article (doi:10.1186/s12859-014-0352-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4220062  PMID: 25344112
Resampling; Gene expression data; ARSER; Biological replicates; Circadian rhythms; HAYSTACK
9.  “Real time” genetic manipulation: a new tool for ecological field studies 
Field experiments with transgenic plants often reveal the functional significance of genetic traits important for plant performance 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 to study ecological interactions in real-time, genetic traits playing essential roles in development, or 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 RNAi 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 CK’s in both the glasshouse and field to understand resistance to the native herbivore Tupiocoris notatus, which attack plants at small spatial scales. By spatially restricting ipt expression and elevating CK levels in single leaves, T. notatus damage increased, demonstrating CK’s role 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.
PMCID: PMC4190501  PMID: 23906159
pOp6; LhGR; dexamethasone; fieldwork; Nicotiana attenuata; Tupiocoris notatus; cytokinin; Manduca sexta; herbivory; pds
10.  The roots of plant defenses: Integrative multivariate analyses uncover dynamic behaviors of roots’ gene and metabolic networks elicited by leaf herbivory 
High-throughput analyses have frequently been used to characterize herbivory-induced reconfigurations in plant primary and secondary metabolism in above and below-ground tissues but the conclusions drawn from these analyses are often limited by the univariate methods used to analyze the data. Here we use our previously described multivariate time series data analysis to evaluate leaf herbivory-elicited transcriptional and metabolic dynamics in the roots of Nicotiana attenuata. We observed large, but transient, systemic responses in the roots that contrasted with the pattern of co-linearity observed in the up- and down-regulation of genes and metabolites across the entire time series in treated and systemic leaves. Using this newly developed approach for the analysis of whole-plant molecular responses in a time course multivariate data-set, we simultaneously analyzed stress responses in leaves and roots in response to the elicitation of a leaf. We found that transient systemic responses in roots resolved into two principal trends characterized by: (a) an inversion of root-specific semidiurnal (12h) transcript oscillations and (b) transcriptional changes with major amplitude effects that translated into a distinct suite of root-specific secondary metabolites (e.g. alkaloids synthesized in the roots of N. attenuata). These findings underscore the importance of understanding tissue-specific stress responses in the correct day-night phase context and provide a holistic framework for the important role played by roots in aboveground stress responses.
PMCID: PMC4190575  PMID: 24456376
Systems biology; Transcriptomics; Metabolomics; Plant stress responses; Multivariate analysis; Time course experiments; Nicotiana attenuata
11.  The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses 
BMC Plant Biology  2014;14(1):268.
Verticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years.
Here we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host.
We conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0268-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4198706  PMID: 25297988
Calcium; Defense; Ethylene; Jasmonic acid; Piriformospora indica; Salicylic acid; Verticillium dahliae
12.  Ecological Observations of Native Geocoris pallens and G. punctipes Populations in the Great Basin Desert of Southwestern Utah 
Big-eyed bugs (Geocoris spp. Fallén, Hemiptera: Lygaeidae) are ubiquitous, omnivorous insect predators whose plant feeding behavior raises the question of whether they benefit or harm plants. However, several studies have investigated both the potential of Geocoris spp. to serve as biological control agents in agriculture and their importance as agents of plant indirect defense in nature. These studies have demonstrated that Geocoris spp. effectively reduce herbivore populations and increase plant yield. Previous work has also indicated that Geocoris spp. respond to visual and olfactory cues when foraging and choosing their prey and that associative learning of prey and plant cues informs their foraging strategies. For these reasons, Geocoris spp. have become models for the study of tritrophic plant-herbivore-predator interactions. Here, we present detailed images and ecological observations of G. pallens Stål and G. punctipes (Say) native to the Great Basin Desert of southwestern Utah, including observations of their life histories and color morphs, dynamics of their predatory feeding behavior and prey choice over space and time, and novel aspects of Geocoris spp.’s relationships to their host plants. These observations open up new areas to be explored regarding the behavior of Geocoris spp. and their interactions with plant and herbivore populations.
PMCID: PMC4185340  PMID: 25298571
13.  Isolating Fungal Pathogens from a Dynamic Disease Outbreak in a Native Plant Population to Establish Plant-Pathogen Bioassays for the Ecological Model Plant Nicotiana attenuata 
PLoS ONE  2014;9(7):e102915.
The wild tobacco species Nicotiana attenuata has been intensively used as a model plant to study its interaction with insect herbivores and pollinators in nature, however very little is known about its native pathogen community. We describe a fungal disease outbreak in a native N. attenuata population comprising 873 plants growing in an area of about 1500 m2. The population was divided into 14 subpopulations and disease symptom development in the subpopulations was monitored for 16 days, revealing a waxing and waning of visible disease symptoms with some diseased plants recovering fully. Native fungal N. attenuata pathogens were isolated from diseased plants, characterized genetically, chemotaxonomically and morphologically, revealing several isolates of the ascomycete genera Fusarium and Alternaria, that differed in the type and strength of the disease symptoms they caused in bioassays on either detached leaves or intact soil-grown plants. These isolates and the bioassays will empower the study of N. attenuata-pathogen interactions in a realistic ecological context.
PMCID: PMC4103856  PMID: 25036191
14.  Silencing ribulose-1,5-bisphosphate carboxylase/oxygenase expression does not disrupt nitrogen allocation to defense after simulated herbivory in Nicotiana attenuata 
Plant Signaling & Behavior  2013;8(12):e27570.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant protein on the planet and in addition to its central role in photosynthesis it is thought to function as a nitrogen (N)-storage protein and a potential source of N for defense biosynthesis in plants. In a recent study in the wild tobacco Nicotiana attenuata, we showed that the decrease in absolute N invested in soluble proteins and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis; 15N flux studies revealed that N for defensive phenolamide synthesis originates from recently assimilated N rather than from RuBisCO turnover. Here we show that a transgenic line of N. attenuata silenced in the expression of RuBisCO (asRUB) invests similar or even larger amounts of N into phenolamide biosynthesis compared with wild type plants, consistent with our previous conclusion that recently assimilated N is channeled into phenolamide synthesis after elicitation. We suggest that the decrease in leaf proteins after simulated herbivory is a tolerance mechanism, rather than a consequence of N-demand for defense biosynthesis.
PMCID: PMC4091567  PMID: 24390158
growth-defense trade-off; caffeoyl-putrescine; dicaffeoyl-spermidine; ribulose-1,5-bisphosphate carboxylase/oxygenase; total soluble protein
15.  Alternative splicing and nonsense-mediated decay of circadian clock genes under environmental stress conditions in Arabidopsis 
BMC Plant Biology  2014;14:136.
The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level.
We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5′ splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD.
Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.
PMCID: PMC4035800  PMID: 24885185
Arabidopsis thaliana; Circadian clock; Transcription factor; Alternative splicing; Nonsense-mediated decay (NMD); Environmental stress
16.  A comparison of performance of plant miRNA target prediction tools and the characterization of features for genome-wide target prediction 
BMC Genomics  2014;15(1):348.
Deep-sequencing has enabled the identification of large numbers of miRNAs and siRNAs, making the high-throughput target identification a main limiting factor in defining their function. In plants, several tools have been developed to predict targets, majority of them being trained on Arabidopsis datasets. An extensive and systematic evaluation has not been made for their suitability for predicting targets in species other than Arabidopsis. Nor, these have not been evaluated for their suitability for high-throughput target prediction at genome level.
We evaluated the performance of 11 computational tools in identifying genome-wide targets in Arabidopsis and other plants with procedures that optimized score-cutoffs for estimating targets. Targetfinder was most efficient [89% ‘precision’ (accuracy of prediction), 97% ‘recall’ (sensitivity)] in predicting ‘true-positive’ targets in Arabidopsis miRNA-mRNA interactions. In contrast, only 46% of true positive interactions from non-Arabidopsis species were detected, indicating low ‘recall’ values. Score optimizations increased the ‘recall’ to only 70% (corresponding ‘precision’: 65%) for datasets of true miRNA-mRNA interactions in species other than Arabidopsis. Combining the results of Targetfinder and psRNATarget delivers high true positive coverage, whereas the intersection of psRNATarget and Tapirhybrid outputs deliver highly ‘precise’ predictions. The large number of ‘false negative’ predictions delivered from non-Arabidopsis datasets by all the available tools indicate the diversity in miRNAs-mRNA interaction features between Arabidopsis and other species. A subset of miRNA-mRNA interactions differed significantly for features in seed regions as well as the total number of matches/mismatches.
Although, many plant miRNA target prediction tools may be optimized to predict targets with high specificity in Arabidopsis, such optimized thresholds may not be suitable for many targets in non-Arabidopsis species. More importantly, non-conventional features of miRNA-mRNA interaction may exist in plants indicating alternate mode of miRNA target recognition. Incorporation of these divergent features would enable next-generation of algorithms to better identify target interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-348) contains supplementary material, which is available to authorized users.
PMCID: PMC4035075  PMID: 24885295
miRNA; Target prediction; Plants; Deep-sequencing; Non-model plants
17.  Differences in Nicotine Metabolism of Two Nicotiana attenuata Herbivores Render Them Differentially Susceptible to a Common Native Predator 
PLoS ONE  2014;9(4):e95982.
Nicotiana attenuata is attacked by larvae of both specialist (Manduca sexta) and generalist (Spodoptera exigua) lepidopteran herbivores in its native habitat. Nicotine is one of N. attenuata's important defenses. M. sexta is highly nicotine tolerant; whether cytochrome P450 (CYP)-mediated oxidative detoxification and/or rapid excretion is responsible for its exceptional tolerance remains unknown despite five decades of study. Recently, we demonstrated that M. sexta uses its nicotine-induced CYP6B46 to efflux midgut-nicotine into the hemolymph, facilitating nicotine exhalation that deters predatory wolf spiders (Camptocosa parallela). S. exigua's nicotine metabolism is uninvestigated.
Methodology/Principal Findings
We compared the ability of these two herbivores to metabolize, tolerate and co-opt ingested nicotine for defense against the wolf spider. In addition, we analyzed the spider's excretion to gain insights into its nicotine metabolism. Contrary to previous reports, we found that M. sexta larvae neither accumulate the common nicotine oxides (cotinine, cotinine N-oxide and nicotine N-oxide) nor excrete them faster than nicotine. In M. sexta larvae, ingestion of nicotine as well as its oxides increases the accumulation of CYP6B46 transcripts. In contrast, S. exigua accumulates nicotine oxides and exhales less (66%) nicotine than does M. sexta. Spiders prefer nicotine-fed S. exigua over M. sexta, a preference reversed by topical or headspace nicotine supplementation, but not ingested or topically-coated nicotine oxides, suggesting that externalized nicotine but not the nicotine detoxification products deter spider predation. The spiders also do not accumulate nicotine oxides.
Nicotine oxidation reduces S. exigua's headspace-nicotine and renders it more susceptible to predation by spiders than M. sexta, which exhales unmetabolized nicotine. These results are consistent with the hypothesis that generalist herbivores incur costs of detoxification, which include the ecological costs of greater predation risks, in addition to the previously demonstrated energetic, physiological and metabolic costs.
PMCID: PMC3995989  PMID: 24755743
18.  Analysis of Plant-Bacteria Interactions in Their Native Habitat: Bacterial Communities Associated with Wild Tobacco Are Independent of Endogenous Jasmonic Acid Levels and Developmental Stages 
PLoS ONE  2014;9(4):e94710.
Jasmonic acid (JA) mediates defense responses against herbivores and necrotrophic pathogens but does it influence the recruitment of bacterial communities in the field? We conducted field and laboratory experiments with transformed Nicotiana attenuata plants deficient in jasmonate biosynthesis (irAOC) and empty vector controls (EV) to answer this question. Using both culture-dependent and independent techniques, we characterized root and leaf-associated bacterial communities over five developmental stages, from rosette through flowering of plants grown in their natural habitat. Based on the pyrosequencing results, alpha and beta diversity did not differ among EV and irAOC plants or over ontogeny, but some genera were more abundant in one of the genotypes. Furthermore, bacterial communities were significantly different among leaves and roots. Taxa isolated only from one or both plant genotypes and hence classified as ‘specialists’ and ‘generalists’ were used in laboratory tests to further evaluate the patterns observed from the field. The putative specialist taxa did not preferentially colonize the jasmonate-deficient genotype, or alter the plant's elicited phytohormone signaling. We conclude that in N. attenuata, JA signaling does not have a major effect on structuring the bacterial communities and infer that colonization of plant tissues is mainly shaped by the local soil community in which the plant grows.
PMCID: PMC3984252  PMID: 24728407
19.  Salicylic acid-dependent gene expression is activated by locomotion mucus of different molluscan herbivores 
Slugs and snails specifically secrete mucus to aid their locomotion. This mucus is the contact material between molluscan herbivores and plants. We have recently shown that the locomotion mucus of the slug Deroceras reticulatum contains salicylic acid (SA).1 When applied to wounded leaves of Arabidopsis thaliana this mucus induces the activity of the SA-responsive pathogenesis related 1 (PR1) promotor1. Here we analyzed PR1 promotor activity in response to treatments with locomotion mucus of eight slugs and snails. Although none of the mucus contained SA, their application still elicited PR1 promotor activity. These data provide further insights into the complex interactions between molluscan herbivores and plants.
PMCID: PMC4203486  PMID: 25346792
plant; mollusk; slug; snail; PR1; salicylic acid
20.  Multiple interactions of NaHER1 protein with abscisic acid signaling in Nicotiana attenuata plants 
Plant Signaling & Behavior  2013;8(11):e26365.
Previously, we identified a novel herbivore elicitor-regulated protein in Nicotiana attenuata (NaHER1) that is required to suppress abscisic acid (ABA) catabolism during herbivore attack and activate a full defense response against herbivores. ABA, in addition to its newly defined role in defense activation, mainly controls seed germination and stomatal function of land plants. Here we show that N. attenuata seeds silenced in the expression of NaHER1 by RNA interference (irHER1) accumulated less ABA during germination, and germinated faster on ABA-containing media compared to WT. Curiously, epidermal cells of irHER1 plants were wrinkled, possibly due to the previously demonstrated increase in transpiration of irHER1 plants that may affect turgor and cause wrinkling of the cells. We conclude that NaHER1 is a highly pleiotropic regulator of ABA responses in N. attenuata plants.
PMCID: PMC4091387  PMID: 24022276
Nicotiana attenuata; abscisic acid (ABA); ABA metabolism; germination
21.  Ecology in the genomics era of a degraded planet 
eLife  2014;3:e02394.
Modern research in ecology draws upon a wide range of different techniques and can offer fresh insights on how to tackle a range of challenges facing the natural world.
PMCID: PMC3917232  PMID: 24520168
eLife; natural habitats; diversity; population growth
22.  Salicylic Acid, a Plant Defense Hormone, Is Specifically Secreted by a Molluscan Herbivore 
PLoS ONE  2014;9(1):e86500.
Slugs and snails are important herbivores in many ecosystems. They differ from other herbivores by their characteristic mucus trail. As the mucus is secreted at the interface between the plants and the herbivores, its chemical composition may play an essential role in plant responses to slug and snail attack. Based on our current knowledge about host-manipulation strategies employed by pathogens and insects, we hypothesized that mollusks may excrete phytohormone-like substances into their mucus. We therefore screened locomotion mucus from thirteen molluscan herbivores for the presence of the plant defense hormones jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). We found that the locomotion mucus of one slug, Deroceras reticulatum, contained significant amounts of SA, a plant hormone that is known to induce resistance to pathogens and to suppress plant immunity against herbivores. None of the other slugs and snails contained SA or any other hormone in their locomotion mucus. When the mucus of D. reticulatum was applied to wounded leaves of A. thaliana, the promotor of the SA-responsive gene pathogenesis related 1 (PR1) was activated, demonstrating the potential of the mucus to regulate plant defenses. We discuss the potential ecological, agricultural and medical implications of this finding.
PMCID: PMC3899270  PMID: 24466122
23.  An integrative statistical method to explore herbivory-specific responses in plants 
Plant Signaling & Behavior  2013;8(10):e25638.
Spatial-temporal coordination between multiple processes/pathways is a key determinant of whole-organism transcriptome and metabolome reconfigurations in plant’s response to biotic stresses. To explore tissue-based interdependencies in Nicotiana attenuata’s resistance to insect attack, we performed time course analyses of the plant’s transcriptome and metabolome in herbivory-elicited source leaves and unelicited sink leaves and roots. To dissect the multidimensionality of these responses, we have recently designed a novel approach of constructing interactive motifs by combining an extended self-organizing maps (SOM) based dimensionality reduction method with bootstrap-based non-parametric ANOVA models. In this previous study, we used this method to study nonlinearities in gene-metabolite associations involved in the acyclic diterpene glucoside pathway. Here, we extend the application of this method to the extraction of genes showing herbivory-elicitation specifically in systemic (distal from the treatment sites) tissues using motif analysis for different combinations of treatment applied to Nicotiana attenuata.
PMCID: PMC4091209  PMID: 23857359
metabolomics; network analysis; plant stress responses; systems biology; transcriptomics
24.  Defence on demand: mechanisms behind optimal defence patterns 
Annals of Botany  2012;110(8):1503-1514.
The optimal defence hypothesis (ODH) predicts that tissues that contribute most to a plant's fitness and have the highest probability of being attacked will be the parts best defended against biotic threats, including herbivores. In general, young sink tissues and reproductive structures show stronger induced defence responses after attack from pathogens and herbivores and contain higher basal levels of specialized defensive metabolites than other plant parts. However, the underlying physiological mechanisms responsible for these developmentally regulated defence patterns remain unknown.
This review summarizes current knowledge about optimal defence patterns in above- and below-ground plant tissues, including information on basal and induced defence metabolite accumulation, defensive structures and their regulation by jasmonic acid (JA). Physiological regulations underlying developmental differences of tissues with contrasting defence patterns are highlighted, with a special focus on the role of classical plant growth hormones, including auxins, cytokinins, gibberellins and brassinosteroids, and their interactions with the JA pathway. By synthesizing recent findings about the dual roles of these growth hormones in plant development and defence responses, this review aims to provide a framework for new discoveries on the molecular basis of patterns predicted by the ODH.
Almost four decades after its formulation, we are just beginning to understand the underlying molecular mechanisms responsible for the patterns of defence allocation predicted by the ODH. A requirement for future advances will be to understand how developmental and defence processes are integrated.
PMCID: PMC3503495  PMID: 23022676
Optimal defence hypothesis; growth; development; defence; herbivores; pathogens; jasmonic acid; auxin; gibberellins; cytokinins; brassinosteroids; plant–herbivore interactions
25.  Just in time 
Plant Signaling & Behavior  2013;8(6):e24410.
The optimal defense hypothesis (ODH) provides a functional explanation for the inhomogeneous distribution of defensive structures and defense metabolites throughout a plant’s body: tissues that are most valuable in terms of fitness and have the highest probability of attack are generally the best defended. In a previous review,1 we argue that ontogenically-controlled accumulations of defense metabolites are likely regulated through an integration of developmental and defense signaling pathways. In this addendum, we extend the discussion of ODH patterns by including the recent discoveries of circadian clock-controlled defenses in plants.
PMCID: PMC3909060  PMID: 23603968
optimal defense hypothesis; circadian clock; jasmonic acid; herbivory; Nicotiana attenuata

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