Antimicrobial peptide expression in a wild tobacco plant reveals the limits of host-microbe-manipulations in the field

Plant-microbe associations are thought to be beneficial for plant growth and resistance against biotic or abiotic stresses, but for natural ecosystems, the ecological analysis of microbiome function remains in its infancy. We used transformed wild tobacco plants (Nicotiana attenuata) which constitutively express an antimicrobial peptide (Mc-AMP1) of the common ice plant, to establish an ecological tool for plant-microbe studies in the field. Transgenic plants showed in planta activity against plant-beneficial bacteria and were phenotyped within the plants´ natural habitat regarding growth, fitness and the resistance against herbivores. Multiple field experiments, conducted over 3 years, indicated no differences compared to isogenic controls. Pyrosequencing analysis of the root-associated microbial communities showed no major alterations but marginal effects at the genus level. Experimental infiltrations revealed a high heterogeneity in peptide tolerance among native isolates and suggests that the diversity of natural microbial communities can be a major obstacle for microbiome manipulations in nature

Host-plant-mediated effects of Nadefensin on herbivore and pathogen resistance in Nicotiana attenuata

<p>Abstract</p> <p>Background</p> <p>The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for <it>Nicotiana attenuata</it>, which is commonly attacked by both pathogens (<it>Pseudomonas spp</it>.) and herbivores (<it>Manduca sexta</it>) in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Na<it>defensin </it>is an effective induced defense gene against the bacterial pathogen <it>Pseudomonas syringae </it>pv <it>tomato </it>(PST DC3000), which is also elicited by attack from <it>M. sexta </it>larvae, but whether this defense protein influences <it>M. sexta's </it>growth and whether <it>M. sexta</it>-induced Na<it>defensin </it>directly or indirectly influences PST DC3000 resistance are unknown.</p> <p>Results</p> <p><it>M. sexta </it>larvae consumed less on WT and on Na<it>defensin</it>-silenced <it>N. attenuata </it>plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by <it>M. sexta </it>larvae, but larval mass gain was unaffected. PST DC3000-infected Na<it>defensin</it>-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, <it>M. sexta </it>larvae consumed less and gained less mass. WT and Na<it>defensin</it>-silenced plants previously damaged by <it>M. sexta </it>larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants.</p> <p>Conclusion</p> <p>These results demonstrate that Na-defensin directly mediates defense against PST DC3000 and indirectly against <it>M. sexta </it>in <it>N. attenuata</it>. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Na<it>defensin</it>-silenced plants differentially reduced <it>M. sexta's </it>leaf consumption and mass gain. In plants that were previously damaged by <it>M. sexta</it>, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more effective than Na<it>defensin </it>alone in resisting PST DC3000.</p

Opportunistic out-crossing in Nicotiana attenuata (Solanaceae), a predominantly self-fertilizing native tobacco

BACKGROUND: Although Nicotiana attenuata is entirely self-compatible, chemical and other floral traits suggest selection for the maintenance of advertisement for moth pollinators. RESULTS: Experimental exclusions of pollinators from plants with emasculated flowers in natural populations in southern Utah during an outbreak of the hawkmoth Hyles lineata revealed that 24% of the seed set could be attributed to insect pollination, and eliminated wind pollination and apomixis as contributing to seed set. Hence these moths can mediate gene flow when self-pollen is unavailable. To quantify gene flow when self-pollen is available, plants were transformed with two marker genes: hygromycin-B resistance and β-glucuronidase. The utility of these genetic markers to measure gene flow between plants was examined by mixing pollen from plants homozygous for both genes with self-pollen in different ratios and hand-pollinating emasculated flowers of plants growing in a natural population. The proportion of transformed seeds was positively correlated with the amount of transformed pollen applied to stigmas. In glasshouse experiments with the hawkmoth Manduca sexta and experimental arrays of transformed and wild-type plants, pollination mediated by moths accounted for 2.5% of the seed set. CONCLUSIONS: Even though moth pollination is rare and highly variable for this largely selfing plant, N. attenuata opportunistically employs a mixed-mating system

Fitness benefits of trypsin proteinase inhibitor expression in Nicotiana attenuata are greater than their costs when plants are attacked.

BACKGROUND: The commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs) expression in Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores. RESULTS: In order to determine whether the putative benefits of TPI production outweigh its cost, we transformed N. attenuata to silence endogenous TPI production or restore it in a natural mutant that was unable to produce TPIs. We compared the lifetime seed production of N. attenuata genotypes of the same genetic background with low or no TPI to that of genotypes with high TPI levels on which M. sexta larvae were allowed to feed freely. Unattacked low TPI-producing genotypes produced more seed capsules than did plants with high TPI levels. Caterpillar attack reduced seed capsule production in all genotypes and reversed the pattern of seed capsule production among genotypes. M. sexta larvae attacking genotypes with high TPI activity consumed more TPI, less protein, and move later to the young leaves. Larval masses were negatively correlated (R(2 )= 0.56) with seed capsule production per plant. CONCLUSIONS: Our results demonstrate that the fitness benefits of TPI production outweigh their costs in greenhouse conditions, when plants are attacked and that despite the ongoing evolutionary interactions between plant and herbivore, TPI-mediated decreases in M. sexta performance translates into a fitness benefit for the plant

A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

<p>Abstract</p> <p>Background</p> <p>Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices.</p> <p>Results</p> <p>A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA)) and small polar molecules (e.g., jasmonic acid (JA), salicylic acid (SA)) containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of <it>Nicotiana attenuata </it>leaf material with previously described analytical methods.</p> <p>Conclusion</p> <p>The method presented was used to detect 16 compounds in leaf extracts of <it>N. attenuata </it>plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the molecules must contain at least one free carboxyl group.</p

The two α-dox genes of Nicotiana attenuata: overlapping but distinct functions in development and stress responses

<p>Abstract</p> <p>Background</p> <p>Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In <it>Nicotiana attenuata</it>, herbivory strongly induces the expression of an <it>α-dox1 </it>gene. To determine its role, we silenced its expression using <it>Agrobacterium</it>-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second <it>α-dox </it>isoform. This led us to identify the corresponding <it>α-dox2 </it>gene in <it>N. attenuata </it>and examine the regulation of both <it>α-dox </it>genes as well as the consequences of their silencing in plant development and anti-herbivore defense.</p> <p>Results</p> <p>The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both <it>α-dox </it>genes resulting in a nearly complete suppression of α-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for <it>α-dox1</it>, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product <it>in planta </it>(2-HOT) and showed no signs of oxidative stress. In contrast to <it>α-dox1</it>, the expression of <it>α-dox2 </it>gene is not induced by wounding or elicitors in the oral secretions of <it>Manduca sexta</it>. Instead, <it>α-dox2 </it>is expressed in roots and flowers which lack <it>α-dox1 </it>expression, but both genes are equally regulated during leaf maturation. We transiently silenced <it>α-dox </it>gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of <it>α-dox1 </it>or <it>α-dox2 </it>had no effects on plant growth, the co-suppression of both <it>α-dox </it>genes decreased plant growth. Plants transiently silenced for both <it>α-dox </it>genes had increased constitutive levels of JA and ABA but silencing <it>α-dox1 </it>alone resulted in lower <it>M. sexta</it>-induced levels of JA, 2-HOT and ABA.</p> <p>Conclusions</p> <p>Thus, both <it>α-dox </it>isoforms function in the development of <it>N. attenuata</it>. In leaf maturation, the two <it>α-dox </it>genes have overlapping functions, but only <it>α-dox2 </it>is involved in root and flower development and only <it>α-dox1 </it>functions in anti-herbivore defense.</p

Two mitogen-activated protein kinase kinases, MKK1 and MEK2, are involved in wounding- and specialist lepidopteran herbivore Manduca sexta-induced responses in Nicotiana attenuata

In a wild tobacco plant, Nicotiana attenuata, two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), play central roles in modulating herbivory-induced phytohormone and anti-herbivore secondary metabolites. However, the identities of their upstream MAPK kinases (MAPKKs) were elusive. Ectopic overexpression studies in N. benthamiana and N. tabacum suggested that two MAPKKs, MKK1 and MEK2, may activate SIPK and WIPK. The homologues of MKK1 and MEK2 were cloned in N. attenuata (NaMKK1 and NaMEK2) and a virus-induced gene silencing approach was used to knock-down the transcript levels of these MAPKK genes. Plants silenced in NaMKK1 and NaMEK2 were treated with wounding or simulated herbivory by applying the oral secretions of the specialist herbivore Manduca sexta to wounds. MAPK activity assay indicated that after wounding or simulated herbivory NaMKK1 is not required for the phosphorylation of NaSIPK and NaWIPK; in contrast, NaMEK2 and other unknown MAPKKs are important for simulated herbivory-elicited activation of NaSIPK and NaWIPK, and after wounding NaMEK2 probably does not activate NaWIPK but plays a minor role in activating NaSIPK. Consistently, NaMEK2 and certain other MAPKKs, but not NaMKK1, are needed for wounding- and simulated herbivory-elicited accumulation of jasmonic acid (JA), JA–isoleucine, and ethylene. Furthermore, both NaMEK2 and NaMKK1 regulate the levels of trypsin proteinase inhibitors. The findings underscore the complexity of MAPK signalling pathways and highlight the importance of MAPKKs in regulating wounding- and herbivory-induced responses

Microarrays in ecological research: A case study of a cDNA microarray for plant-herbivore interactions

BACKGROUND: Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs) of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER) data produced from microarrays. RESULTS: We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes (4 of known function, 2 of unknown) with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV) were used to calculate array-based variances (array CV), which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA) were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA revealed the contribution of individual gene ERs to the associations among arrays. CONCLUSIONS: While the costs of 'boutique' array fabrication are rapidly declining, familiar methods for the analysis of the data they create are still missing. The case history illustrated here demonstrates the ease with which this powerful technology can be adapted to ecological research

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

BACKGROUND: After 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. RESULTS: When 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. CONCLUSIONS: Detection 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