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Summary: 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. [ABSTRACT FROM AUTHOR]

Laboratory studies have revealed that strigolatone (SL) and karrikin (KAR) signalling mediate responses to abiotic and biotic stresses, and reshape branching architecture that could increase reproductive performance and crop yields. To understand the ecological function of SL and KAR signalling, transgenic lines of wild tobacco Nicotiana attenuata, silenced in SL/KAR biosynthesis/signalling were grown in the glasshouse and in two field plots in the Great Basin Desert in Utah over four field seasons. Of the lines silenced in SL and KAR signalling components (irMAX2, irD14, irKAI2 and irD14 × irKAI2 plants), which exhibited the expected increases in shoot branching, only irMAX2 plants showed a strong leaf‐bleaching phenotype when grown in the field. In the field, irMAX2 plants had lower sugar and higher leaf amino acid contents, lower lifetime fitness and were more susceptible to herbivore attack compared to wild‐type plants. These irMAX2 phenotypes were not observed in glasshouse‐grown plants. Transcriptomic analysis revealed dramatic responses to high‐light intensity in irMAX2 leaves in the field: lutein contents decreased, and transcriptional responses to high‐intensity light, singlet oxygen and hydrogen peroxide increased. PAR and UV‐B manipulations in the field revealed that the irMAX2 bleaching phenotype is reversed by decreasing PAR, but not UV‐B fluence. We propose that NaMAX2 functions in high‐light adaptation and fitness optimisation by regulating high‐light responses independently of its roles in the SL and KAR signalling pathways. The work provides another example of the value of studying the function of genes in the complex environments in which plants evolved, namely nature. Summary statement: Strigolatone (SL) and karrikin (KAR) signalling are known reshape branching architecture. Transgenic lines of wild tobacco Nicotiana attenuata, silenced in SL/KAR biosynthesis/signalling were grown in the glasshouse and in field plots in the Great Basin Desert during four field seasons. Of the lines silenced in SL and KAR signalling components (irMAX2, irD14, irKAI2 and irD14 × irKAI2 plants), which exhibited increases in shoot branching, only irMAX2 plants showed a strong leaf‐bleaching phenotype when grown in the field, but not in the glasshouse. Experimentation revealed that NaMAX2 functions in high‐light adaptations independently of its roles in SL and KAR signalling. The work provides another example of the value of studying gene function in the environments in which plants evolved, namely nature. [ABSTRACT FROM AUTHOR]

Summary: Plants interact with arbuscular mycorrhizal fungi (AMF) and in doing so, change transcript levels of many miRNAs and their targets. However, the identity of an Argonaute (AGO) that modulates this interaction remains unknown, including in Nicotiana attenuata.We examined how the silencing of NaAGO1/2/4/7/and 10 by RNAi influenced plant‐competitive ability under low‐P conditions when they interact with AMF. Furthermore, the roles of seven miRNAs, predicted to regulate signaling and phosphate homeostasis, were evaluated by transient overexpression.Only NaAGO7 silencing by RNAi (irAGO7) significantly reduced the competitive ability under P‐limited conditions, without changes in leaf or root development, or juvenile‐to‐adult phase transitions. In plants growing competitively in the glasshouse, irAGO7 roots were over‐colonized with AMF, but they accumulated significantly less phosphate and the expression of their AMF‐specific transporters was deregulated. Furthermore, the AMF‐induced miRNA levels were inversely regulated with the abundance of their target transcripts. miRNA overexpression consistently decreased plant fitness, with four of seven‐tested miRNAs reducing mycorrhization rates, and two increasing mycorrhization rates. Overexpression of Na‐miR473 and Na‐miRNA‐PN59 downregulated targets in GA, ethylene, and fatty acid metabolism pathways.We infer that AGO7 optimizes competitive ability and colonization by regulating miRNA levels and signaling pathways during a plant's interaction with AMF. [ABSTRACT FROM AUTHOR]

Summary: Hydroxy‐ and carboxyblumenol C‐glucosides specifically accumulate in roots and leaves of plants harboring arbuscular mycorrhizal fungi (AMF).To explore blumenol function in AMF relationships, we silenced an early key‐gene in blumenol biosynthesis, CCD1 (carotenoid cleavage dioxygenase 1), in the ecological model plant, Nicotiana attenuata, and analyzed whole‐plant performance in comparison with control and CCaMK‐silenced plants, unable to form AMF associations.Root blumenol accumulations reflected a plant's Darwinian fitness, as estimated by capsule production, and were positively correlated with AMF‐specific lipid accumulations in roots, with relationships that changed as plants matured when grown without competitors. When grown with wild‐type competitors, transformed plants with decreased photosynthetic capacity or increased carbon flux to roots had blumenol accumulations that predicted plant fitness and genotype trends in AMF‐specific lipids, but had similar levels of AMF‐specific lipids between competing plants, likely reflecting AMF‐networks.We propose that when grown in isolation, blumenol accumulations reflect AMF‐specific lipid allocations and plant fitness. When grown with competitors, blumenol accumulations predict fitness outcomes, but not the more complicated AMF‐specific lipid accumulations. RNA‐seq analysis provided candidates for the final biosynthetic steps of these AMF‐indicative blumenol C‐glucosides; abrogation of these steps will provide valuable tools for understanding blumenol function in this context‐dependent mutualism. [ABSTRACT FROM AUTHOR]

miR390 is a highly conserved miRNA in plant lineages known to function in growth and development processes, such as lateral root development, and in responses to salt and metal stress. In the ecological model species, Nicotiana attenuata, miR390's biological function remains unknown, which we explore here with a gain‐of‐function analysis with plants over‐expressing (OE‐) N. attenuata miR390 (Na‐miR390) in glasshouse and natural environments. OEmiR390 plants showed normal developmental processes, including lateral root formation or reproductive output, in plants grown under standard conditions in the glasshouse. OEmiR390 plants did not have dramatically altered interactions with arbuscular mycorrhizal fungi (AMF), Fusarium pathogens, or herbivores. However, Na‐miR390 regulated the plant's tolerance of herbivory. Caterpillar feeding elicits the accumulation of a suite of phytohormones, including auxin and jasmonates, which further regulate host‐tolerance. The increase in Na‐miR390 abundance reduces the accumulation of auxin but does not influence levels of other phytohormones including jasmonates (JA, JA‐Ile), salicylic acid (SA), and abscisic acid (ABA). Na‐miR390 overexpression reduces reproductive output, quantified as capsule production, when plants are attacked by herbivores. Exogenous auxin treatments of herbivore‐attacked plants restored capsule production to wild‐type levels. During herbivory, Na‐miR390 transcript abundances are increased; its overexpression reduces the abundances of auxin biosynthesizing YUCCA and ARF (mainly ARF4) transcripts during herbivory. Furthermore, the accumulation of auxin‐regulated phenolamide secondary metabolites (caffeoylputrescine, dicaffeoylspermidine) is also reduced. In N. attenuata, miR390 functions in modulating tolerance responses of herbivore‐attacked plants. [ABSTRACT FROM AUTHOR]

Abstract Background Induced defense responses to herbivores are generally believed to have evolved as cost-saving strategies that defer the fitness costs of defense metabolism until these defenses are needed. The fitness costs of jasmonate (JA)-mediated defenses have been well documented. Those of the early signaling units mediating induced resistance to herbivores have yet to be examined. Early signaling components that mediate herbivore-induced defense responses in Nicotiana attenuata, have been well characterized and here we examine their growth and fitness costs during competition with conspecifics. Two mitogen-activated protein kinases (MAPKs), salicylic acid (SA)-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) are rapidly activated after perception of herbivory and both kinases regulate herbivory-induced JA levels and JA-mediated defense metabolite accumulations. Since JA-induced defenses result in resource-based trade-offs that compromise plant productivity, we evaluated if silencing SIPK (irSIPK) and WIPK (irWIPK) benefits the growth and fitness of plants competiting with wild type (WT) plants, as has been shown for plants silenced in JA-signaling by the reduction of Lipoxygenase 3 (LOX3) levels. Results As expected, irWIPK and LOX3-silenced plants out-performed their competing WT plants. Surprisingly, irSIPK plants, which have the largest reductions in JA signaling, did not. Phytohormone profiling of leaves revealed that irSIPK plants accumulated higher levels of SA compared to WT. To test the hypothesis that these high levels of SA, and their presumed associated fitness costs of pathogen associated defenses in irSIPK plants had nullified the JA-deficiency-mediated growth benefits in these plants, we genetically reduced SA levels in irSIPK plants. Reducing SA levels partially recovered the biomass and fitness deficits of irSIPK plants. We also evaluated whether the increased fitness of plants with reduced SA or JA levels resulted from increased nitrogen or CO2 assimilation rates, and found no evidence that greater intake of these fitness-limiting resources were responsible. Conclusions Signaling mediated by WIPK, but not SIPK, is associated with large fitness costs in competing N. attenuata plants, demonstrating the contrasting roles that these two MAPKs play in regulating the plants’ growth-defense balance. We discuss the role of SIPK as an important regulator of plant fitness, possibly by modulating SA-JA crosstalk as mediated through ethylene signaling.

Abstract Background Plant microRNAs (miRNAs) play key roles in the transcriptional responses to environmental stresses. However, the role of miRNAs in responses to insect herbivory has not been thoroughly explored. To identify herbivory-responsive miRNAs, we identified conserved miRNAs in the ecological model plant Nicotiana attenuata whose interactions with herbivores have been well-characterized in both laboratory and field studies. Results We identified 59 miRNAs from 36 families, and two endogenous trans-acting small interfering RNAs (tasiRNA) targeted by miRNAs. We characterized the response of the precursor and mature miRNAs to simulated attack from the specialist herbivore Manduca sexta by quantitative PCR analysis and used ir-aoc RNAi transformants, deficient in jasmonate biosynthesis, to identify jasmonate-dependent and -independent miRNA regulation. Expression analysis revealed that groups of miRNAs and tasiRNAs were specifically regulated by either mechanical wounding or wounding plus oral secretions from M. sexta larvae, and these small RNAs were accumulated in jasmonate-dependent or -independent manners. Moreover, cDNA microarray analysis indicated that the expression patterns of the corresponding target genes were correlated with the accumulation of miRNAs and tasiRNAs. Conclusions We show that a group of miRNAs and tasiRNAs orchestrates the expression of target genes involved in N. attenuata’s responses to herbivore attack.

Abstract Background A plant’s endogenous clock (circadian clock) entrains physiological processes to light/dark and temperature cycles. Forward and reverse genetic approaches in Arabidopsis have revealed the mechanisms of the circadian clock and its components in the genome. Similar approaches have been used to characterize conserved clock elements in several plant species. A wild tobacco, Nicotiana attenuata has been studied extensively to understand responses to biotic or abiotic stress in the glasshouse and also in their native habitat. During two decades of field experiment, we observed several diurnal rhythmic traits of N. attenuata in nature. To expand our knowledge of circadian clock function into the entrainment of traits important for ecological processes, we here report three core clock components in N. attenuata. Results Protein similarity and transcript accumulation allowed us to isolate orthologous genes of the core circadian clock components, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1), and ZEITLUPE (ZTL). Transcript accumulation of NaLHY peaked at dawn and NaTOC1 peaked at dusk in plants grown under long day conditions. Ectopic expression of NaLHY and NaZTL in Arabidopsis resulted in elongated hypocotyl and late-flowering phenotypes. Protein interactions between NaTOC1 and NaZTL were confirmed by yeast two-hybrid assays. Finally, when NaTOC1 was silenced in N. attenuata, late-flowering phenotypes under long day conditions were clearly observed. Conclusions We identified three core circadian clock genes in N. attenuata and demonstrated the functional and biochemical conservation of NaLHY, NaTOC1, and NaZTL.

Abstract To adjust their development to the environment, plants rely on specific signals that travel from shoot to root and vice versa. Here we describe an efficient micrografting protocol for Nicotiana attenuata, a useful tool for identifying these signals and understanding their functions. Additionally we analyzed transcript accumulation profiles of scions and rootstocks of grafts performed with wild-type and stably transformed N. attenuata. Our results are consistent with the source-to-sink movement of an sRNA silencing signal.

Summary: The ubiquitous volatile linalool is metabolized in plants to nonvolatile derivatives. We studied Nicotiana attenuata plants which naturally vary in (S)‐(+)‐linalool contents, and lines engineered to produce either (R)‐(‐)‐ or (S)‐(+)‐linalool. Only (S)‐(+)‐linalool production was associated with slower growth of a generalist herbivore, and a large fraction was present as nonvolatile derivatives. We found that variation in volatile linalool and its nonvolatile glycosides mapped to the same genetic locus which harbored the biosynthetic gene, NaLIS, but that free linalool varied more in environmental responses. This study reveals how (S)‐(+)‐linalool and conjugates differ in their regulation and possible functions in resistance. [ABSTRACT FROM AUTHOR]

Long non‐coding RNA (lncRNA) are important regulators of many biological processes in plants, including defence against pathogens; whether lncRNAs mediate defence against herbivore attack is yet to be explored. With wild tobacco, Nicotiana attenuata, and its well‐characterized interactions with herbivores, we identified a total of 1,290 significantly up‐ or down‐regulated lncRNAs in response to a precise herbivore elicitation treatment. Of these, long intergenic non‐coding RNAs (lincRNAs) were the most abundant. Based on their expression patterns, these up‐regulated lincRNAs were classified as early (<1 hr) or late (>3 hr) responders. The early responding lincRNAs had accumulation patterns that tracked the herbivore‐elicited burst of bioactive jasmonates (JAs) and the expression of regulator genes in JA signalling which regulate plant defences against herbivores. Silencing two of these early responders in N. attenuata (JAL1 and JAL3) significantly attenuated the accumulation of JAs, JA‐mediated defensives and the plant's resistance to M. sexta attack, suggesting roles in regulating JA‐mediated plant defence. By lincRNA sequencing of JA‐deficient lines, many late responder lincRNAs were found to be transcriptionally regulated by JA signalling. This study uncovers a new role of lncRNAs in JA‐mediated herbivore resistance. The role of long non‐coding RNAs (lncRNAs) in plant defence against herbivores has not yet been explored. Here we show that the transcripts of more than 500 lncRNAs are up‐regulated after herbivore‐elicitation, some of them are associated with jasmonate‐mediated herbivore resistance. [ABSTRACT FROM AUTHOR]

Abstract 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.

Summary: The circadian clock contextualizes plant responses to environmental signals. Plants use temporal information to respond to herbivory, but many of the functional roles of circadian clock components in these responses, and their contribution to fitness, remain unknown.We investigate the role of the central clock regulator TIMING OF CAB EXPRESSION 1 (TOC1) in Nicotiana attenuata's defense responses to the specialist herbivore Manduca sexta under both field and glasshouse conditions. We utilize 15N pulse‐labeling to quantify nitrogen incorporation into pools of three defense compounds: caffeoylputrescine (CP), dicaffeoyl spermidine (DCS) and nicotine.Nitrogen incorporation was decreased in CP and DCS and increased in nicotine pools in irTOC1 plants compared to empty vector (EV) under control conditions, but these differences were abolished after simulated herbivory. Differences between EV and irTOC1 plants in nicotine, but not phenolamide production, were abolished by treatment with the ethylene agonist 1‐methylcyclopropene. Using micrografting, TOC1's effect on nicotine was isolated to the root and did not affect the fitness of heterografts under field conditions.These results suggest that the circadian clock contributes to plant fitness by balancing production of metabolically expensive nitrogen‐rich defense compounds and mediating the allocation of resources between vegetative biomass and reproduction. [ABSTRACT FROM AUTHOR]

SUMMARY: Circadian organ movements are ubiquitous in plants. These rhythmic outputs are thought to be regulated by the circadian clock and auxin signalling, but the underlying mechanisms have not been clarified. Flowers of Nicotiana attenuata change their orientation during the daytime through a 140° arc to balance the need for pollinators and the protection of their reproductive organs. This rhythmic trait is under the control of the circadian clock and results from bending and re‐straightening movements of the pedicel, stems that connect flowers to the inflorescence. Using an explant system that allowed pedicel growth and curvature responses to be characterized with high spatial and temporal resolution, we demonstrated that this movement is organ autonomous and mediated by auxin. Changes in the growth curvature of the pedicel are accompanied by an auxin gradient and dorsiventral asymmetry in auxin‐dependent transcriptional responses; application of auxin transport inhibitors influenced the normal movements of this organ. Silencing the expression of the circadian clock component ZEITLUPE (ZTL) arrested changes in the growth curvature of the pedicel and altered auxin signalling and responses. IAA19‐like, an Aux/IAA transcriptional repressor that is circadian regulated and differentially expressed between opposite tissues of the pedicel, and therefore possibly involved in the regulation of changes in organ curvature, physically interacted with ZTL. Together, these results are consistent with a direct link between the circadian clock and the auxin signalling pathway in the regulation of this rhythmic floral movement. Significance Statement: Circadian organ movements are widespread among plants. Yet, the molecular components that operate in these movements have remained unknown. Here, we demonstrate that the growth movements of the pedicel, the organ responsible for the circadian changes in orientation of Nicotiana attenuata flowers, are under the control of the circadian clock and auxin signalling; a result that opens potential avenues to investigate the relationship among different types of movements in plants. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi (AMF) establish symbiotic associations with a majority of terrestrial plants to form underground common mycorrhizal networks (CMNs) that connect neighbouring plants. Because Nicotiana attenuata plants do not respond to herbivory‐elicited volatiles from neighbours, we used this ecological model system to evaluate if CMNs function in interplant transmission of herbivory‐elicited responses. A mesocosm system was designed to establish and remove CMNs linking N. attenuata plants to examine the herbivory‐elicited metabolic and hormone responses in CMNs‐connected "receiver" plants after the elicitation of "donor" plants by wounding (W) treated with Manduca sexta larval oral secretions (OS). AMF colonization increased constitutive jasmonate (JA and JA‐Ile) levels in N. attenuata roots but did not affect well‐characterized JAs‐regulated defensive metabolites in systemic leaves. Interestingly, larger JAs bursts, and higher levels of several amino acids and particular sectors of hydroxygeranyllinalool diterpene glycoside metabolism were elevated in the leaves of W + OS‐elicited "receivers" with CMN connections with "donors" that had been W + OS‐elicited 6 hr previously. Our results demonstrate that AMF colonization alone does not enhance systemic defence responses but that sectors of systemic responses in leaves can be primed by CMNs, suggesting that CMNs can transmit and even filter defence signalling among connected plants. Mycorrhizal colonization alone does not enhance systemic defence responses in Nicotiana attenuata plants but that sectors of systemic responses in leaves can be primed by mycorrhizal networks, suggesting that mycorrhizal networks can transmit and filter defence signalling among connected plants. [ABSTRACT FROM AUTHOR]

Capsidiol is a sesquiterpenoid phytoalexin produced in Nicotiana and Capsicum species in response to pathogen attack. Whether capsidiol plays a defensive role and how its biosynthesis is regulated in the wild tobacco Nicotiana attenuata when the plant is attacked by Alternaria alternata (tobacco pathotype), a notorious necrotrophic fungus causing brown spot disease, are unknown. Transcriptome analysis indicated that a metabolic switch to sesquiterpene biosynthesis occurred in young leaves of N. attenuata after A. alternata inoculation: many genes leading to sesquiterpene production were strongly up-regulated, including the capsidiol biosynthetic genes 5- epi - aristolochene synthase (EAS) and 5- epi - aristolochene hydroxylase (EAH). Consistently, the level of capsidiol was increased dramatically in young leaves after fungal inoculation, from not detectable in mock control to 50.68±3.10 µg g−1 fresh leaf at 3 d post-inoculation. Capsidiol-reduced or capsidiol-depleted plants, which were generated by silencing EAH s or EAS s by virus-induced gene silencing, were more susceptible to the fungus. In addition, this sesquiterpene when purified from infected plants exhibited strong anti-fungal activities against A. alternata in vitro. Furthermore, an ERF2-like transcription factor was found to positively regulate capsidiol production and plant resistance through the direct transactivation of a capsidiol biosynthetic gene, EAS12. Taken together, our results demonstrate that capsidiol, a phytoalexin highly accumulated in N. attenuata plants in response to A. alternata infection, plays an important role in pathogen resistance independent of jasmonate and ethylene signaling pathways, and its biosynthesis is transcriptionally regulated by an ERF2-like transcription factor. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Benzyl acetone (4‐phenylbutan‐2‐one; BA), the dominant floral fragrance of the wild tobacco Nicotiana attenuata, is known to attract night‐time pollinators, such as Manduca sexta hawkmoths. For this pollinator, BA is not only essential for the pollen transport between conspecific plants, but also for the moth's short‐distance handling of flowers at night. The emission of BA from the corolla limb, however, starts up to 4 hr before dusk, peaks at dusk and continues throughout the first half of the night. While the function of the nocturnal part of the emission is well studied, the function of the early part of the peak emission remained unknown.By using plants silenced in the expression of the biosynthetic gene of BA (Nachal1), we investigated the function of this early peak emission of BA in the plants´ native habitat in Southwestern Utah.By emitting BA before dusk, N. attenuata is able to prevent the establishment and resulting floral damage by a florivore, the cucumber beetle (Diabrotica undecimpunctata). Plants lacking BA emissions (CHAL) were not only more often colonized by florivores, but also suffered significantly more damage, than control empty vector plants (EV). Choice assays revealed that D. undecimpunctata feeding required the absence of BA. While feeding damage to CHAL flowers was higher than to EV flowers in the early night hours, the effect disappeared during the second half of the night, when flowers stop emitting BA. The close proximity of BA‐emitting flowers to non‐emitting CHAL flowers prevented D. undecimpunctata feeding. In the field, the emission of BA at dusk was sufficient to protect plants for the entire night, as beetles search for new hosts during the early evening hours and remain for the duration of the night, once a host is chosen.Floral BA emission before dusk may have evolved as a consequence of antagonistic interactions with florivores. A single floral volatile can thus simultaneously function as an important floral attractant for pollinators and as effective feeding deterrent against florivores in the same plant species. A plain language summary is available for this article. [ABSTRACT FROM AUTHOR]

The beneficial effects of plant‐–bacterial interactions in controlling plant pests have been extensively studied with single bacterial isolates. However, in nature, bacteria interact with plants in multitaxa consortia, systems which remain poorly understood. Previously, we demonstrated that a consortium of five native bacterial isolates protected their host plant Nicotiana attenuata from a sudden wilt disease. Here we explore the mechanisms behind the protection effect against the native pathosystem. Three members of the consortium, Pseudomonas azotoformans A70, P. frederiksbergensis A176 and Arthrobacter nitroguajacolicus E46, form biofilms when grown individually in vitro, and the amount of biofilm increased synergistically in the five‐membered consortium, including two Bacillus species, B. megaterium and B. mojavensis. Fluorescence in situ hybridization and scanning electron microscopy in planta imaging techniques confirmed biofilm formation and revealed locally distinct distributions of the five bacterial strains colonizing different areas on the plant‐root surface. One of the five isolates, K1 B. mojavensis produces the antifungal compound surfactin, under in vitro and in vivo conditions, clearly inhibiting fungal growth. Furthermore, isolates A70 and A176 produce siderophores under in vitro conditions. Based on these results we infer that the consortium of five bacterial isolates protects its host against fungal phytopathogens via complementary traits. The study should encourage researchers to create synthetic communities from native strains of different genera to improve bioprotection against wilting diseases. [ABSTRACT FROM AUTHOR]