Nicotiana Attenuata Research Articles

Roles of cytochrome P450 monooxygenases in triterpene biosynthesis and their potential impact on growth and development.

Pentacyclic triterpenoids, recognized for their natural bioactivity, display complex spatiotemporal accumulation patterns within the ecological model plant Nicotiana attenuata. Despite their ecological importance, the underlying biosynthetic enzymes and functional attributes of triterpenoid synthesis in N. attenuata remain unexplored. Here, we show that three cytochrome P450 monooxygenases (NaCYP716A419, NaCYP716C87, and NaCYP716E107) from N. attenuata oxidize the pentacyclic triterpene skeleton, as evidenced by heterologous expression in Nicotiana benthamiana. NaCYP716A419 catalyzed a consecutive three-step oxidation reaction at the C28 position of β-amyrin/lupeol/lupanediol, yielding the corresponding alcohol, aldehyde, and carboxylic acid. NaCYP716C87 hydroxylated the C2α position of β-amyrin/lupeol/lupanediol/erythrodiol/oleanolic acid/betulinic acid, while NaCYP716E107 hydroxylated the C6β position of β-amyrin/oleanolic acid. The genes encoding these three CYP716 enzymes are highly expressed in flowers and respond to induction by ABA, MeJA, SA, GA3, and abiotic stress treatments. Using VIGS technology, we revealed that silencing of NaCYP716A419 affects the growth and reproduction of N. attenuata, suggesting the ecological significance of these specialized metabolite biosynthetic steps.

Jasmonate/ethylene- and NaWRKY6/3-regulated Alternaria resistance depends on ethylene response factor 1B-like in Nicotiana attenuata.

Biosynthesis of the phytoalexins scopoletin and scopolin in Nicotiana species is regulated by upstream signals including jasmonate (JA), ethylene (ET), and NaWRKY3 in response to the necrotrophic fungus Alternaria alternata, which causes brown spot disease. However, how these signals are coordinated to regulate these phytoalexins remains unknown. By analyzing RNA sequencing data and RNAi, we identified NaERF1B-like (NaERF1B-L) as a key player in Nicotiana attenuata during A. alternata infection by regulating the transcripts of Feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), encoding a key enzyme for scopoletin biosynthesis, and NaVS1-like (NaVS1-L), a putative biosynthetic gene of the phytoalexin solavetivone. We further demonstrated that the synergistic induction of these two genes by JA and ET signaling is mediated by NaERF1B-L. Additionally, we found that the two closely related proteins, NaWRKY6 and NaWRKY3, physically interact to enhance NaERF1B-L expression by directly binding to and activating the NaERF1B-L promoter. Collectively, our current results demonstrate that NaERF1B-L plays a positive role in resistance to A. alternata by modulating phytoalexin biosynthesis through the integration of JA/ET and NaWRKY6/3 signaling. Our findings reveal a fine-tuned transcriptional regulatory hierarchy mediated by NaERF1B-L for brown spot disease resistance in wild tobacco.

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

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

A pooled-sample draft genome assembly provides insights into host plant-specific transcriptional responses of a Solanaceae-specializing pest, Tupiocoris notatus (Hemiptera: Miridae).

The assembly of genomes from pooled samples of genetically heterogenous samples of conspecifics remains challenging. In this study, we show that high-quality genome assemblies can be produced from samples of multiple wild-caught individuals. We sequenced DNA extracted from a pooled sample of conspecific herbivorous insects (Hemiptera: Miridae: Tupiocoris notatus) acquired from a greenhouse infestation in Tucson, Arizona (in the range of 30-100 individuals; 0.5 mL tissue by volume) using PacBio highly accurate long reads (HiFi). The initial assembly contained multiple haplotigs (>85% BUSCOs duplicated), but duplicate contigs could be easily purged to reveal a highly complete assembly (95.6% BUSCO, 4.4% duplicated) that is highly contiguous by short-read assembly standards (N 50 = 675 kb; Largest contig = 4.3 Mb). We then used our assembly as the basis for a genome-guided differential expression study of host plant-specific transcriptional responses. We found thousands of genes (N = 4982) to be differentially expressed between our new data from individuals feeding on Datura wrightii (Solanaceae) and existing RNA-seq data from Nicotiana attenuata (Solanaceae)-fed individuals. We identified many of these genes as previously documented detoxification genes such as glutathione-S-transferases, cytochrome P450s, and UDP-glucosyltransferases. Together our results show that long-read sequencing of pooled samples can provide a cost-effective genome assembly option for small insects and can provide insights into the genetic mechanisms underlying interactions between plants and herbivorous pests.

NaWRKY70 is a key regulator of Nicotiana attenuata resistance to Alternaria alternata through regulation of phytohormones and phytoalexins biosynthesis.

Jasmonate (JA) and abscisic acid (ABA) are two major phytohormones involved in pathogen resistance. However, how their biosynthesis is regulated is not well understood. We silenced NaWRKY70 in wild tobacco Nicotiana attenuata and determined its role in regulating genes involved in the production of JA, ABA and the phytoalexin capsidiol in response to the fungal pathogen Alternaria alternata using techniques including electrophoretic mobility shift, chromatin immunoprecipitation, transient overexpression and virus-induced gene silencing. Silencing NaWRKY70 dramatically reduced both basal and A. alternata-induced jasmonoyl-isoleucine (JA-Ile) and ABA. Further evidence showed that NaWRKY70 directly binds to the W-boxes of the promoters of NaAOS and NaJAR4 (JA biosynthesis), NaNCED1 and NaXD1-like (ABA biosynthesis), and NaMPK4 (ABA signaling) to activate their expression, while binding but repressing the expression of NaCYP707A4-like3 (ABA degradation). Additionally, NaWRKY70 regulates capsidiol production through its key enzyme genes NaEASs and NaEAHs, and interacts with its regulator NaERF2-like to enhance their expression, whereas ABA negatively regulates capsidiol biosynthesis. Our results highlight the key role of NaWRKY70 in controlling both JA-Ile and ABA production, as well as capsidiol production, thus providing new insight into the defense mechanism of plant resistance to A. alternata.

Genome-Wide Identification and Expression Profiling of Potato (Solanum tuberosum L.) Universal Stress Proteins Reveal Essential Roles in Mechanical Damage and Deoxynivalenol Stress.

Universal stress proteins (USPs) play an important regulatory role in responses to abiotic stress. Most of the research related to USPs so far has been conducted on plant models such as Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa L.), and cotton (Gossypium hirsutum L.). The potato (Solanum tuberosum L.) is one of the four major food crops in the world. The potato is susceptible to mechanical damage and infection by pathogenic fungi during transport and storage. Deoxynivalenol (DON) released by Fusarium can seriously degrade the quality of potatoes. As a result, it is of great significance to study the expression pattern of the potato StUSP gene family under abiotic stress conditions. In this study, a total of 108 USP genes were identified from the genome of the Atlantic potato, divided into four subgroups. Based on their genetic structure, the physical and chemical properties of their proteins and other aspects of their biological characteristics are comprehensively analyzed. Collinear analysis showed that the homologous genes of StUSPs and four other representative species (Solanum lycopersicum, Arabidopsis, Oryza sativa L., and Nicotiana attenuata) were highly conserved. The cis-regulatory elements of the StUSPs promoter are involved in plant hormones, environmental stress, mechanical damage, and light response. RNA-seq analysis showed that there are differences in the expression patterns of members of each subgroup under different abiotic stresses. A Weighted Gene Coexpression Network Analysis (WGCNA) of the central gene showed that the differential coexpression gene is mainly involved in the plant-pathogen response process, plant hormone signal transduction, and the biosynthesis process of secondary metabolites. Through qRT-PCR analysis, it was confirmed that StUSP13, StUSP14, StUSP15, and StUSP41 may be important candidate genes involved in the response to adversity stress in potatoes. The results of this study provide a basis for further research on the functional analysis of StUSPs in the response of potatoes to adversity stress.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the β-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

Radiocarbon Flux Measurements Reveal Mechanistic Insight into Heat-Stress Induction of Nicotine Biosynthesis in Nicotiana attenuata.

The effect of high-temperature (HT) stress on nicotine biosynthesis in Nicotiana attenuata was examined. Nicotine content was measured in mature leaves, young sink leaves, and in roots from well-watered plants grown at 25 °C as controls and from plants exposed to 38 °C and 43 °C temperatures applied for 24, 48, 72, and 96 h duration. At 38 °C, all leaf nicotine levels were significantly less than control plants for up to 72 h exposure but rose sharply thereafter to levels significantly greater than controls with 96 h exposure. In contrast, plants exposed to 43 °C never exhibited a reduction in leaf nicotine content and showed an increase in content with just 48 h exposure. Using radioactive 11CO2 and 13NO3-, we found that HT stress reduced both CO2 fixation and nitrate uptake. Furthermore, radiocarbon flux analysis revealed that 'new' carbon partitioning (as 11C) into the 11C-radiolabeled amino acid (AA) pool was significantly reduced with HT stress as were yields of [11C]-aspartic acid, an important AA in nicotine biosynthesis, and its beta-amido counterpart [11C]-asparagine. In contrast, [12C]-aspartic acid levels appeared unaffected at 38 °C but were elevated at 43 °C relative to controls. [12C]-Asparagine levels were noted to be elevated at both stress temperatures. Since HT reductions in carbon input and nitrogen uptake were noted to impede de novo AA biosynthesis, protein degradation at HT was examined as a source of AAs. Here, leaf total soluble protein (TSP) content was reduced 39% with long exposures to both stress temperatures. However, Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which was 41% TSP appeared unaffected. Altogether, these results support the theory that plant proteins other than Rubisco degrade at elevated temperatures freeing up essential AAs in support of nicotine biosynthesis.

Synergistic induction of phytoalexins in Nicotiana attenuata by jasmonate and ethylene signaling mediated by NaWRKY70.

Production of the phytoalexins scopoletin and scopolin is regulated by jasmonate (JA) and ethylene signaling in Nicotiana species in response to Alternaria alternata, the necrotrophic fungal pathogen that causes brown spot disease. However, how these two signaling pathways are coordinated to control this process remains unclear. In this study, we found that the levels of these two phytoalexins and transcripts of their key enzyme gene, feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), were synergistically induced in Nicotiana attenuata by co-treatment with methyl jasmonate (MeJA) and ethephon. By combination of RNA sequencing and virus-induced gene silencing, we identified a WRKY transcription factor, NaWRKY70, which had a similar expression pattern to NaF6'H1 and was responsible for A. alternata-induced NaF6'H1 expression. Further evidence from stable transformed plants with RNA interference, knock out and overexpression of NaWRKY70 demonstrated that it is a key player in the synergistic induction of phytoalexins and plant resistance to A. alternata. Electrophoretic mobility shift, chromatin immunoprecipitation-quantitative PCR, and dual-luciferase assays revealed that NaWRKY70 can bind directly to the NaF6'H1 promoter and activate its expression. Furthermore, the key regulator of the ethylene pathway, NaEIN3-like1, can directly bind to the NaWRKY70 promoter and activate its expression. Meanwhile, NaMYC2s, important JA pathway transcription factors, also indirectly regulate the expression of NaWRKY70 and NaF6'H1 to control scopoletin and scopolin production. Our data reveal that these phytoalexins are synergistically induced by JA and ethylene signaling during A. alternata infection, which is largely mediated by NaWRKY70, thus providing new insights into the defense responses against A. alternata in Nicotiana species.

Evaluating potential of leaf reflectance spectra to monitor plant genetic variation

Remote sensing of vegetation by spectroscopy is increasingly used to characterize trait distributions in plant communities. How leaves interact with electromagnetic radiation is determined by their structure and contents of pigments, water, and abundant dry matter constituents like lignins, phenolics, and proteins. High-resolution (“hyperspectral”) spectroscopy can characterize trait variation at finer scales, and may help to reveal underlying genetic variation—information important for assessing the potential of populations to adapt to global change. Here, we use a set of 360 inbred genotypes of the wild coyote tobacco Nicotiana attenuata: wild accessions, recombinant inbred lines (RILs), and transgenic lines (TLs) with targeted changes to gene expression, to dissect genetic versus non-genetic influences on variation in leaf spectra across three experiments. We calculated leaf reflectance from hand-held field spectroradiometer measurements covering visible to short-wave infrared wavelengths of electromagnetic radiation (400–2500 nm) using a standard radiation source and backgrounds, resulting in a small and quantifiable measurement uncertainty. Plants were grown in more controlled (glasshouse) or more natural (field) environments, and leaves were measured both on- and off-plant with the measurement set-up thus also in more to less controlled environmental conditions. Entire spectra varied across genotypes and environments. We found that the greatest variance in leaf reflectance was explained by between-experiment and non-genetic between-sample differences, with subtler and more specific variation distinguishing groups of genotypes. The visible spectral region was most variable, distinguishing experimental settings as well as groups of genotypes within experiments, whereas parts of the short-wave infrared may vary more specifically with genotype. Overall, more genetically variable plant populations also showed more varied leaf spectra. We highlight key considerations for the application of field spectroscopy to assess genetic variation in plant populations.

Deciphering mitogen activated protein kinase pathway activated during insect attack in Nicotiana attenuata

Plant yields are compromised due to abiotic and biotic stresses. A crucial biotic stress instigated by insect attack, is a major concern that limits crop production. To overcome the deleterious effect of herbivory, pesticides are used but long-term usage of pesticides can be harmful to the environment and human health. Understanding the plants’ inherent defense mechanism by interpreting the interaction pattern of defense-related proteins and signalling components and manipulating them to strengthen defense status, is one of the alternative approaches of green biotechnology. During insect attack, host plants initiate innumerable signalling pathways to activate defense response; Mitogen Activated Protein Kinase (MAPK) Pathway is a crucial component of signalling pathway that regulate the expression of downstream defense-related genes. MAPK pathway has three components: MAPKKK, MAPKK and MAPK. Earlier studies have shown participation of SIPK and WIPK (MAPKs) as well as MEK2 (MAPKK) during insect infestation and its association with plant defense. However, information on the third component and elucidation of the complete MAPK pathway are still elusive. Therefore, this study aims to identify the unknown component and decipher MAPK pathway in Nicotiana attenuata involved in plant defense against herbivory by identifying herbivory-inducible MAPKKKs and and their interaction with known partners of the MAPK pathway by docking and MD simulation. The possible pathway was predicted to be MAPKKK Na12134/Na04522-MEK2-SIPK/WIPK. Further, validation of the above interaction by in vitro and in vivo methods is highly recommended. Communicated by Ramaswamy H. Sarma

Field-work reveals a novel function for MAX2 in a native tobacco's high-light adaptions.

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.

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

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

Arbuscular mycorrhizal fungi influence the intraspecific competitive ability of plants under field and glasshouse conditions

Main conclusionNicotiana attenuata’s capacity to interact with arbuscular mycorrhizal fungi influences its intraspecific competitive ability under field and glasshouse conditions, but not its overall community productivity.Arbuscular mycorrhizal (AM) fungi can alter the nutrient status and growth of plants, and they can also affect plant–plant, plant–herbivore, and plant–pathogen interactions. These AM effects are rarely studied in populations under natural conditions due to the limitation of non-mycorrhizal controls. Here we used a genetic approach, establishing field and glasshouse communities of AM-harboring Nicotiana attenuata empty vector (EV) plants and isogenic plants silenced in calcium- and calmodulin-dependent protein kinase expression (irCCaMK), and unable to establish AM symbioses. Performance and growth were quantified in communities of the same (monocultures) or different genotypes (mixed cultures) and both field and glasshouse experiments returned similar responses. In mixed cultures, AM-harboring EV plants attained greater stalk lengths, shoot and root biomasses, clearly out-competing the AM fungal-deficient irCCaMK plants, while in monocultures, both genotypes grew similarly. Competitive ability was also reflected in reproductive traits: EV plants in mixed cultures outperformed irCCaMK plants. When grown in monocultures, the two genotypes did not differ in reproductive performance, though total leaf N and P contents were significantly lower independent of the community type. Plant productivity in terms of growth and seed production at the community level did not differ, while leaf nutrient content of phosphorus and nitrogen depended on the community type. We infer that AM symbioses drastically increase N. attenuata’s competitive ability in mixed communities resulting in increased fitness for the individuals harboring AM without a net gain for the community.

Argonaute7 (AGO7) optimizes arbuscular mycorrhizal fungal associations and enhances competitive growth in Nicotiana attenuata.

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.

Expression Analysis and Interaction Protein Screening of CoZTL in Camellia oleifera Abel

Camellia oleifera Abel., which produces fruits of high comprehensive utilization value, is an important woody oil tree in China. ZEITLUPE (ZTL) is a blue light receptor and clock component protein that is involved in various physiological and biochemical processes. However, the expression pattern and function of C. oleifera ZTL (CoZTL) remain unclear. In this study, the coding sequence of the CoZTL gene was isolated and the protein function was explored using bioinformatics and expression analyses and heterologous expression techniques. The results showed that the CoZTL protein was highly conserved during evolution and was on the same branch of the evolutionary tree as the ZTL proteins from Ipomoea nil and Nicotiana attenuata. CoZTL was mainly expressed in the fruit shells and stems of C. oleifera, and its expression level fluctuated greatly during flower bud development. Transgenic CoZTL-overexpressing Arabidopsis plants showed delayed flowering under long-day conditions as well as light-dependent promotion of hypocotyl elongation. Furthermore, yeast two-hybrid library screening revealed that seven C. oleifera proteins (CoAAT, Coβ-GAL, CoLAT52-like, CoCAR4-like, CoAO, CoUQCC1, and CoADF 2) interacted with CoZTL. Our results indicate that CoZTL plays an important role in C. oleifera flowering and hypocotyl growth.

NaWRKY3 is a master transcriptional regulator of the defense network against brown spot disease in wild tobacco.

WRKY transcription factors are involved in plant defense against pathogens. No WRKYs have been reported to be involved in resistance to tobacco brown spot disease caused by Alternaria alternata. Here, we found that NaWRKY3 plays a critical role in Nicotiana attenuata defense against A. alternata. NaWRKY3 bound and regulated many defense genes, including: lipoxygenase 3, ACC synthase 1, and ACC oxidase 1, three jasmonate- and ethylene-biosynthetic genes; feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), the biosynthetic gene for the phytoalexins scopoletin and scopolin; and three A. alternata resistance genes, the long non-coding RNA L2, NADPH oxidase (NaRboh D), and berberine bridge-like (NaBBL28). Silencing L2 reduced jasmonate concentrations and NaF6'H1 expression. NaRboh D-silenced plants were severely impaired in reactive oxygen species production and stomatal closure responses. NaBBL28 was the first A. alternata resistance BBL identified and was involved in the hydroxylation of 17-hydroxygeranyllinalool diterpene glycosides. NaWRKY3 bound to its own promoter but repressed its expression. Thus, we demonstrated that NaWRKY3 is a fine-tuned master regulator of the defense network against A. alternata in N. attenuata by regulating several signaling pathways and defense metabolites. This is the first time such an important WRKY has been identified in Nicotiana species, providing new insights into defense against A. alternata.

Arbuscular mycorrhizal fungi-indicative blumenol-C-glucosides predict lipid accumulations and fitness in plants grown without competitors.

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.

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

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

NaMLP, a new identified Kunitz trypsin inhibitor regulated synergistically by JA and ethylene, confers Spodoptera litura resistance in Nicotiana attenuata.

We identified a miraculin-like protein (NaMLP) who is a new Kunitz trypsin inhibitor regulated synergistically by JA and ethylene signals and confers Spodoptera litura resistance in wild tobacco Nicotiana attenuata. The findings revealed a new source of trypsin inhibitor activities after herbivory, and provide new insights into the complexity of the regulation of trypsin inhibitor-based defense after insect herbivore attack. Upon insect herbivore attack, wild tobacco Nicotiana attenuata accumulates trypsin protease inhibitor (TPI) activities as a defense response from different protease inhibitor (PI) coding genes, including WRKY3-regulated NaKTI2, and JA-dependent NaPI. However, whether any other TPI gene exists in N. attenuata is still unclear. A miraculin-like protein gene (NaMLP) was highly up-regulated in N. attenuata after Alternaria alternata infection. However, silencing or overexpression of NaMLP had no effect on the lesion diameter developed on N. attenuata leaves after A. alternata inoculation. Meanwhile, the transcripts of NaMLP could be induced by wounding and amplified by Spodoptera litura oral secretions (OS). S. litura larvae gained significantly more biomass on NaMLP-silenced plants but less on NaMLP overexpressed plants. Although NaMLP showed low sequence similarity to NaKTI2, it had conserved reaction sites of Kunitz trypsin inhibitors, and exhibited TPI activities when its coding gene was overexpressed transiently or stably in N. attenuata. This was consistent with the worst performance of S. litura larvae on NaMLP overexpressed lines. Furthermore, NaMLP-silenced plants had reduced TPI activities and better S. litura performance. Finally, OS-elicited NaMLP was dramatically reduced in JA-deficient AOC silencing and ethylene-reduced ACO-silencing plants, and the expression of NaMLP could be significantly induced by methyl jasmonate or ethephon alone, but dramatically amplified by co-treatment of both methyl jasmonate and ethephon. Thus, our results demonstrate that in addition to JA-regulated NaPI, and WRKY3/6-dependent NaKTI2, N. attenuata plants also up-regulates TPI activities via NaMLP, which confers S. litura resistance through JA and ethylene signaling pathways in a synergistic way.