Transgenic cotton expressing Allium sativum leaf agglutinin exhibits resistance to whiteflies and aphids without negative effects on ladybugs.

Cotton (Gossypium hirsutum L.) is of great economic importance as a cultivated crop in many parts of the world. In addition to being a pillar of the textile industry, cotton and its byproducts are used for livestock feed, seed oil, and other products. Bacillus thuringiensis crystal toxin (Bt) expression in cotton provides effective protection against chewing insects but does not defend plants from piercing/sucking insect pests. With the aim to create transgenic plants with resistance against piercing/sucking pests, we used Agrobacterium-mediated genetic transformation of cotton cultivar Coker 312 to express the Allium sativum leaf agglutinin (ASLA) gene from the phloem-specific rolC promoter. The ASLA transgene was stably inherited and showed Mendelian segregation in the T1 generation. Transgenic lines, expressing the ASLA gene, showed explicit resistance against major sap-sucking pests. Green peach aphid (Myzus persicae Sulzer) choice assays showed that 75% of aphids preferred untransformed cotton plants relative to those expressing the ASLA gene. In detached leaf bioassays, plants expressing ASLA caused 82% aphid mortality and 44-53% reduction in fecundity. Clip cage bioassays with whiteflies (Bemisia tabaci Gennadius) showed 74-82% mortality and 44-60% decrease in fecundity due to ASLA gene expression. In whole plant bioassays, whiteflies showed 77% mortality and a 54% decrease in fecundity on ASLA transgenics. Importantly, we did not observe a negative effect of the ASLA gene on ladybugs (Coccinella septempunctata) that consumed these whiteflies. Together, our findings demonstrate the potential of ASLA-transgenic cotton for providing protection against two devastating insect pests, whiteflies and aphids. The ASLA-transgenic cotton appears promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Acylsugars, Nicotine and a Protease Inhibitor Provide Variable Protection for Nicotiana benthamiana in a Natural Setting.

Plants produce an immense diversity of defensive specialized metabolites. However, despite extensive functional characterization, the relative importance of different defensive compounds is rarely examined in natural settings. Here, we compare the efficacy of three Nicotiana benthamiana defensive compounds, nicotine, acylsugars and a serine protease inhibitor, by growing plants with combinations of knockout mutations in a natural setting, quantifying invertebrate interactions and comparing relative plant performance. Among the three tested compounds, acylsugars had the greatest defensive capacity, affecting aphids, leafhoppers, spiders and flies. Nicotine mutants displayed increased leafhopper feeding and aphid colonization. Plants lacking both nicotine and acylsugars were more susceptible to flea beetles and thrips. By contrast, knockout of the serine protease inhibitor did not affect insect herbivory in the field. Complementary experiments under controlled laboratory conditions with caterpillars, grasshoppers and aphids confirmed results obtained in a natural setting. We conclude that the three metabolite groups collectively provide broad-spectrum protection to N. benthamiana. However, there is a gradient in their effects on the interacting invertebrates present in the field. Furthermore, we demonstrate that, even if individual metabolites do not have a measurable defensive benefit on their own, they can have an additive effect when combined with other defensive compounds.

Catechol acetylglucose: a newly identified benzoxazinoid-regulated defensive metabolite in maize.

An enormous diversity of specialized metabolites is produced in the plant kingdom, with each individual plant synthesizing thousands of these compounds. Previous research showed that benzoxazinoids, the most abundant class of specialized metabolites in maize, also function as signaling molecules by regulating the production callose as a defense response. We searched for additional benzoxazinoid-regulated specialized metabolites, characterized them, examined whether they too function in herbivore protection, and determined how Spodoptera frugiperda (fall armyworm), a prominent maize pest, copes with these metabolites. We identified catechol acetylglucose (CAG) as a benzoxazinoid-regulated metabolite that is produced from salicylic acid via catechol and catechol glucoside. Genome-wide association studies of CAG abundance identified a gene encoding a predicted acetyltransferase. Knockout of this gene resulted in maize plants that lack CAG and over-accumulate catechol glucoside. Upon tissue disruption, maize plants accumulate catechol, which inhibits S. frugiperda growth. Analysis of caterpillar frass showed that S. frugiperda detoxifies catechol by glycosylation, and the efficiency of catechol glycosylation was correlated with S. frugiperda growth on a catechol-containing diet. Thus, the success of S. frugiperda as an agricultural pest may depend partly on its ability to detoxify catechol, which is produced as a defensive metabolite by maize.

COI1 F-box proteins regulate DELLA protein levels, growth, and photosynthetic efficiency in maize.

The F-box protein Coronatine Insensitive (COI) is a receptor for the jasmonic acid signaling pathway in plants. To investigate the functions of the 6 maize (Zea mays) COI proteins (COI1a, COI1b, COI1c, COI1d, COI2a, and COI2b), we generated single, double, and quadruple loss-of-function mutants. The pollen of the coi2a coi2b double mutant was inviable. The coi1 quadruple mutant (coi1-4x) exhibited shorter internodes, decreased photosynthesis, leaf discoloration, microelement deficiencies, and accumulation of DWARF8 and/or DWARF9, 2 DELLA family proteins that repress the gibberellic acid (GA) signaling pathway. Coexpression of COI and DELLA in Nicotiana benthamiana showed that the COI proteins trigger proteasome-dependent DELLA degradation. Many genes that are downregulated in the coi1-4x mutant are GA-inducible. In addition, most of the proteins encoded by the downregulated genes are predicted to be bundle sheath- or mesophyll-enriched, including those encoding C4-specific photosynthetic enzymes. Heterologous expression of maize Coi genes in N. benthamiana showed that COI2a is nucleus-localized and interacts with maize jasmonate zinc-finger inflorescence meristem domain (JAZ) proteins, the canonical COI repressor partners. However, maize COI1a and COI1c showed only partial nuclear localization and reduced binding efficiency to the tested JAZ proteins. Together, these results show the divergent functions of the 6 COI proteins in regulating maize growth and defense pathways.

Four enzymes control natural variation in the steroid core of Erysimum cardenolides.

Plants commonly produce families of structurally related metabolites with similar defensive functions. This apparent redundancy raises the question of underlying molecular mechanisms and adaptive benefits of such chemical variation. Cardenolides, a class defensive compounds found in the wallflower genus Erysimum (L., Brassicaceae) and scattered across other plant families, show substantial structural variation, with glycosylation and hydroxylation being common modifications of a steroid core, which itself may vary in terms of stereochemistry and saturation. Through a combination of chemical mutagenesis and analysis of gene coexpression networks, we identified four enzymes involved in cardenolide biosynthesis in Erysimum that work together to determine stereochemistry at carbon 5 of the steroid core: Ec3ßHSD, a 3ß-hydroxysteroid dehydrogenase, Ec3KSI, a ketosteroid isomerase, EcP5ßR2, a progesterone 5ß-reductase, and EcDET2, a steroid 5α-reductase. We biochemically characterized the activity of these enzymes in vitro and generated CRISPR/Cas9 knockout lines to confirm activity in vivo. Cardenolide biosynthesis was not eliminated in any of the knockouts. Instead, mutant plants accumulated cardenolides with altered saturation and stereochemistry of the steroid core. Furthermore, we found variation in carbon 5 configuration among the cardenolides of 44 species of Erysimum, where the occurrence of some 5ß-cardenolides is associated with the expression and sequence of P5ßR2. This may have allowed Erysimum species to fine-tune their defensive profiles to target specific herbivore populations over the course of evolution. SIGNIFICANCE STATEMENT: Plants use an array of toxic compounds to defend themselves from attack against insects and other herbivores. One mechanism through which plants may evolve more toxic compounds is through modifications to the structure of compounds they already produce. In this study, we show how plants in the wallflower genus Erysimum use four enzymes to fine-tune the structure of toxic metabolites called cardenolides. Natural variation in the sequence and expression of a single enzyme called progesterone 5ß-reductase 2 partly explains the variation in cardenolides observed across the Erysimum genus. These alterations to cardenolide structure over the course of evolution suggests that there may be context-dependent benefits to Erysimum to invest in one cardenolide variant over another.

Serine proteinase inhibitors from Nicotiana benthamiana, a nonpreferred host plant, inhibit the growth of Myzus persicae (green peach aphid).

BACKGROUND: The green peach aphid (Myzus persicae) is a severe agricultural crop pest that has developed resistance to most current control methods, requiring the urgent development of novel strategies. Plant proteinase inhibitors (PINs) are small proteins that protect plants against pathogens and/or herbivores, likely by preventing efficient protein digestion. RESULTS: We identified 67 protease genes in the transcriptomes of three M. persicae lineages (USDA-Red, G002 and G006). Comparison of gene expression levels in aphid guts and whole aphids showed that several proteases, including a highly expressed serine protease, are significantly overexpressed in the guts. Furthermore, we identified three genes encoding serine protease inhibitors (SerPIN-II1, 2 and 3) in Nicotiana benthamiana, which is a nonpreferred host for M. persicae. Using virus-induced gene silencing (VIGS) with a tobacco rattle virus (TRV) vector and overexpression with a turnip mosaic virus (TuMV) vector, we demonstrated that N. benthamiana SerPIN-II1 and SerPIN-II2 cause reduced survival and growth, but do not affect aphid protein content. Likewise, SerPIN-II3 overexpression reduced survival and growth, and serpin-II3 knockout mutations, which we generated using CRISPR/Cas9, increased survival and growth. Protein content was significantly increased in aphids fed on SerPIN-II3 overexpressing plants, yet it was decreased in aphids fed on serpin-II3 mutants. CONCLUSION: Our results show that three PIN-IIs from N. benthamiana, a nonpreferred host plant, effectively inhibit M. persicae survival and growth, thereby representing a new resource for the development of aphid-resistant crop plants. © 2024 Society of Chemical Industry.

Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids.

The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.

Aphid Resistance Segregates Independently of Cardenolide and Glucosinolate Content in an Erysimum cheiranthoides (Wormseed Wallflower) F2 Population.

Plants in the genus Erysimum produce both glucosinolates and cardenolides as a defense mechanism against herbivory. Two natural isolates of Erysimum cheiranthoides (wormseed wallflower) differed in their glucosinolate content, cardenolide content, and their resistance to Myzus persicae (green peach aphid), a broad generalist herbivore. Both classes of defensive metabolites were produced constitutively and were not further induced by aphid feeding. To investigate the relative importance of glucosinolates and cardenolides in E. cheiranthoides defense, we generated an improved genome assembly, genetic map, and segregating F2 population. The genotypic and phenotypic analysis of the F2 plants identified quantitative trait loci, which affected glucosinolates and cardenolides, but not the aphid resistance. The abundance of most glucosinolates and cardenolides was positively correlated in the F2 population, indicating that similar processes regulate their biosynthesis and accumulation. Aphid reproduction was positively correlated with glucosinolate content. Although the overall cardenolide content had little effect on aphid growth and survival, there was a negative correlation between aphid reproduction and helveticoside abundance. However, this variation in defensive metabolites could not explain the differences in aphid growth on the two parental lines, suggesting that processes other than the abundance of glucosinolates and cardenolides have a predominant effect on aphid resistance in E. cheiranthoides.

Punicalagin, a pomegranate polyphenol sensitizes the activity of antibiotics against three MDR pathogens of the Enterobacteriaceae.

BACKGROUND: Multidrug resistance (MDR) in the family Enterobacteriaceae is a perniciously increasing threat to global health security. The discovery of new antimicrobials having the reversing drug resistance potential may contribute to augment and revive the antibiotic arsenal in hand. This study aimed to explore the anti-Enterobacteriaceae capability of bioactive polyphenols from Punica granatum (P. granatum) and their co-action with antibiotics against clinical isolates of Enterobacteriaceae predominantly prevalent in South Asian countries. METHODS: The Kandhari P. granatum (Pakistani origin) extracts were tested for anti-Enterobacteriaceae activity by agar well diffusion assay against MDR Salmonella enterica serovar Typhi, serovar Typhimurium and Escherichia coli. Predominant compounds of active extract were determined by mass spectrometry and screened for bioactivity by agar well diffusion and minimum inhibitory concentration (MIC) assay. The active punicalagin was further evaluated at sub-inhibitory concentrations (SICs) for coactivity with nine conventional antimicrobials using a disc diffusion assay followed by time-kill experiments that proceeded with SICs of punicalagin and antimicrobials. RESULTS: Among all P. granatum crude extracts, pomegranate peel methanol extract showed the largest inhibition zones of 25, 22 and 19 mm, and the MICs as 3.9, 7.8 and 7.8 mg/mL for S. typhi, S. typhimurium and E. coli, respectively. Punicalagin and ellagic acid were determined as predominant compounds by mass spectrometry. In plate assay, punicalagin (10 mg/mL) was active with hazy inhibition zones of 17, 14, and 13 mm against S. typhi, S. typhimurium and E. coli, respectively. However, in broth dilution assay punicalagin showed no MIC up to 10 mg/mL. The SICs 30 µg, 100 µg, and 500 µg of punicalagin combined with antimicrobials i.e., aminoglycoside, ß-lactam, and fluoroquinolone act in synergy against MDR strains with % increase in inhibition zone values varying from 3.4 ± 2.7% to 73.8 ± 8.4%. In time-kill curves, a significant decrease in cell density was observed with the SICs of antimicrobials/punicalagin (0.03-60 µg/mL/30, 100, 500 µg/mL of punicalagin) combinations. CONCLUSIONS: The P. granatum peel methanol extract exhibited antimicrobial activity against MDR Enterobacteriaceae pathogens. Punicalagin, the bacteriostatic flavonoid act as a concentration-dependent sensitizing agent for antimicrobials against Enterobacteriaceae. Our findings for the therapeutic punicalagin-antimicrobial combination prompt further evaluation of punicalagin as a potent activator for drugs, which otherwise remain less or inactive against MDR strains.

Aphid resistance segregates independently of cardiac glycoside and glucosinolate content in an Erysimum cheiranthoides (wormseed wallflower) F2 population.

Plants in the genus Erysimum produce both glucosinolates and cardiac glycosides as defense against herbivory. Two natural isolates of Erysimum cheiranthoides (wormseed wallflower) differed in their glucosinolate content, cardiac glycoside content, and resistance to Myzus persicae (green peach aphid), a broad generalist herbivore. Both classes of defensive metabolites were produced constitutively and were not induced further by aphid feeding. To investigate the relative importance of glucosinolates and cardiac glycosides in E. cheiranthoides defense, we generated an improved genome assembly, genetic map, and segregating F2 population. Genotypic and phenotypic analysis of the F2 plants identified quantitative trait loci affecting glucosinolates and cardiac glycosides, but not aphid resistance. The abundance of most glucosinolates and cardiac glycosides was positively correlated in the F2 population, indicating that similar processes regulate their biosynthesis and accumulation. Aphid reproduction was positively correlated with glucosinolate content. Although overall cardiac glycoside content had little effect on aphid growth and survival, there was a negative correlation between aphid reproduction and helveticoside abundance. However, this variation in defensive metabolites could not explain the differences in aphid growth on the two parental lines, suggesting that processes other than the abundance of glucosinolates and cardiac glycosides have a predominant effect on aphid resistance in E. cheiranthoides.

Cardiac glycosides protect wormseed wallflower (Erysimum cheiranthoides) against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. Erysimum cheiranthoides (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model. We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in E. cheiranthoides and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using E. cheiranthoides cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field. EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side-chain cleavage, and EcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores, but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores. These results begin elucidation of the E. cheiranthoides cardiac glycoside biosynthetic pathway and demonstrate in vivo that cardiac glycoside production allows Erysimum to escape from some, but not all, specialist herbivores.

Fatty alcohols, a minor component of the tree tobacco surface wax, are associated with defence against caterpillar herbivory.

Despite decades of research resulting in a comprehensive understanding of epicuticular wax metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. In this study, we examined the effects of CRISPR-induced knockout mutations in four Nicotiana glauca (tree tobacco) wax metabolism genes. These mutations cause a wide range of changes in epicuticular wax composition, leading to altered interactions with insects and snails. Three interaction classes were examined: chewing herbivory by seven caterpillars and one snail species, phloem feeding by Myzus persicae (green peach aphid) and oviposition by Bemisia tabaci (whitefly). Although total wax load and alkane abundance did not affect caterpillar growth, a correlation across species, showed that fatty alcohols, a minor component of N. glauca surface waxes, negatively affected the growth of both a generalist caterpillar (Spodoptera littoralis) and a tobacco-feeding specialist (Manduca sexta). This negative correlation was overshadowed by the stronger effect of anabasine, a nicotine isomer, and was apparent when fatty alcohols were added to an artificial lepidopteran diet. By contrast, snails fed more on waxy leaves. Aphid reproduction and feeding activity were unaffected by wax composition but were potentially affected by altered cutin composition. Wax crystal morphology could explain the preference of B. tabaci to lay eggs on waxy wild-type plants relative to both alkane and fatty alcohol-deficient mutants. Together, our results suggest that the varied responses among herbivore classes and species are likely to be a consequence of the co-evolution that shaped the specific effects of different surface wax components in plant-herbivore interactions.

Cardiac glycosides protect wormseed wallflower (Erysimum cheiranthoides) against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. Erysimum cheiranthoides (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model.We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in E. cheiranthoides and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using E. cheiranthoides cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field.EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side chain cleavage, and EcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores.These results begin elucidation of the E. cheiranthoides cardiac glycoside biosynthetic pathway and demonstrate in vivo that cardiac glycoside production allows Erysimum to escape from some, but not all, specialist herbivores.

Temporal matches between monarch butterfly and milkweed population changes over the past 25,000 years.

In intimate ecological interactions, the interdependency of species may result in correlated demographic histories. For species of conservation concern, understanding the long-term dynamics of such interactions may shed light on the drivers of population decline. Here, we address the demographic history of the monarch butterfly, Danaus plexippus, and its dominant host plant, the common milkweed Asclepias syriaca (A. syriaca), using broad-scale sampling and genomic inference. Because genetic resources for milkweed have lagged behind those for monarchs, we first release a chromosome-level genome assembly and annotation for common milkweed. Next, we show that despite its enormous geographic range across eastern North America, A. syriaca is best characterized as a single, roughly panmictic population. Using approximate Bayesian computation with random forests (ABC-RF), a machine learning method for reconstructing demographic histories, we show that both monarchs and milkweed experienced population expansion during the most recent recession of North American glaciers 10,000-20,000 years ago. Our data also identify concurrent population expansions in both species during the large-scale clearing of eastern forests (∼200 years ago). Finally, we find no evidence that either species experienced a reduction in effective population size over the past 75 years. Thus, the well-documented decline of monarch abundance over the past 40 years is not visible in our genomic dataset, reflecting a possible mismatch of the overwintering census population to effective population size in this species.

Cytokinin Promotes Jasmonic Acid Accumulation in the Control of Maize Leaf Growth.

Plant organ growth results from the combined activity of cell division and cell expansion. The co-ordination of these two processes depends on the interplay between multiple hormones that determine the final organ size. Using the semidominant Hairy Sheath Frayed1 (Hsf1) maize mutant that hypersignals the perception of cytokinin (CK), we show that CK can reduce leaf size and growth rate by decreasing cell division. Linked to CK hypersignaling, the Hsf1 mutant has an increased jasmonic acid (JA) content, a hormone that can inhibit cell division. The treatment of wild-type seedlings with exogenous JA reduces maize leaf size and growth rate, while JA-deficient maize mutants have increased leaf size and growth rate. Expression analysis revealed the increased transcript accumulation of several JA pathway genes in the Hsf1 leaf growth zone. A transient treatment of growing wild-type maize shoots with exogenous CK also induced the expression of JA biosynthetic genes, although this effect was blocked by the co-treatment with cycloheximide. Together, our results suggest that CK can promote JA accumulation, possibly through the increased expression of specific JA pathway genes.

Inoculation of Maize with Sugarcane Mosaic Virus Constructs and Application for RNA Interference in Fall Armyworms.

Virus-mediated transient gene overexpression and gene expression silencing can be used to screen gene functions in plants. Sugarcane mosaic virus (SCMV) is a positive strand RNA virus in the Potyviridae family that has been modified to be used as vector to infect monocots, including maize (Zea mays), for transient gene overexpression and gene expression silencing. Relative to stable transformation, SCMV-mediated transient expression in maize has the advantages of being faster and less expensive. Here, we describe a protocol for cloning constructs into the plasmid vector pSCMV-CS3. After maize seedlings are transformed with pSCMV-CS3 constructs by particle bombardment, the virus replicates and spreads systemically in the plants. Subsequent infections of maize seedlings can be accomplished by rub inoculation with sap from SCMV-infested plants. As an example of a practical application of the method, we also describe virus-induced gene silencing (VIGS) of fall armyworm (Spodoptera frugiperda) gene expression. Transgenic viruses are created by cloning a segment of the fall armyworm target gene into pSCMV-CS3 prior to maize transformation. Caterpillars are fed on the virus-infected maize plants, which make dsRNA to silence the expression of the fall armyworm target gene after ingestion. This use of SCMV for plant-mediated VIGS in insects allows rapid screening of gene functions when caterpillars are feeding on their host plants. Graphical overview.

Convergent and divergent evolution of plant chemical defenses.

The majority of the several hundred thousand specialized metabolites produced by plants function in defense against insects and other herbivores. Despite this diversity, identical metabolites or structurally distinct metabolites hitting the same targets in herbivorous animals have evolved repeatedly. This convergent evolution may reflect the constraints of plant primary metabolism in providing metabolic precursors, as well as the limited number of readily accessible targets in animals. These restrictions may make it uncommon for plants to develop completely novel toxic and deterrent metabolites, despite the ongoing evolution of resistance mechanisms in insect herbivores. Defensive compounds that are unique to individual genera or species often have long biosynthetic pathways that may complicate the repeated evolution of these metabolites in different plant species.

Non-volatile metabolites mediate plant interactions with insect herbivores.

Non-volatile metabolites constitute the bulk of plant biomass. From the perspective of plant-insect interactions, these structurally diverse compounds include nutritious core metabolites and defensive specialized metabolites. In this review, we synthesize the current literature on multiple scales of plant-insect interactions mediated by non-volatile metabolites. At the molecular level, functional genetics studies have revealed a large collection of receptors targeting plant non-volatile metabolites in model insect species and agricultural pests. By contrast, examples of plant receptors of insect-derived molecules remain sparse. For insect herbivores, plant non-volatile metabolites function beyond the dichotomy of core metabolites, classed as nutrients, and specialized metabolites, classed as defensive compounds. Insect feeding tends to elicit evolutionarily conserved changes in plant specialized metabolism, whereas its effect on plant core metabolism varies widely based the interacting species. Finally, several recent studies have demonstrated that non-volatile metabolites can mediate tripartite communication on the community scale, facilitated by physical connections established through direct root-to-root communication, parasitic plants, arbuscular mycorrhizae and the rhizosphere microbiome. Recent advances in both plant and insect molecular biology will facilitate further research on the role of non-volatile metabolites in mediating plant-insect interactions.

Quantification of plant cardenolides by HPLC, measurement of Na+/K+-ATPase inhibition activity, and characterization of target enzymes.

The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na+/K+-ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na+/K+-ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na+/K+-ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na+/K+-ATPase activity by plant extracts, and (3) extraction of insect Na+/K+-ATPases for inhibition assays.