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PP69CH23_Douglas ARI 10 April 2018 7:17

Annual Review of Plant Biology

Strategies for Enhanced CropResistance to Insect PestsAngela E. DouglasDepartment of Entomology and Department of Molecular Biology and Genetics,Cornell University, Ithaca, New York 14850, USA; email: [email protected]

Annu. Rev. Plant Biol. 2018. 69:637–60

First published as a Review in Advance onNovember 16, 2017

The Annual Review of Plant Biology is online atplant.annualreviews.org

https://doi.org/10.1146/annurev-arplant-042817-040248

Copyright c© 2018 by Annual Reviews.All rights reserved

Keywords

insect pest control, microbiome, phytophagous insects, plant immunity,RNAi, secondary metabolites

Abstract

Insect pests are responsible for substantial crop losses worldwide throughdirect damage and transmission of plant diseases, and novel approaches thatcomplement or replace broad-spectrum chemical insecticides will facilitatethe sustainable intensification of food production in the coming decades.Multiple strategies for improved crop resistance to insect pests, especiallystrategies relating to plant secondary metabolism and immunity and micro-biome science, are becoming available. Recent advances in metabolic engi-neering of plant secondary chemistry offer the promise of specific toxicityor deterrence to insect pests; improved understanding of plant immunityagainst insects provides routes to optimize plant defenses against insects;and the microbiomes of insect pests can be exploited, either as a target oras a vehicle for delivery of insecticidal agents. Implementation of these ad-vances will be facilitated by ongoing advances in plant breeding and genetictechnologies.

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ANNUAL REVIEWS Further

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https://www.annualreviews.org/doi/full/10.1146/annurev-arplant-042817-040248


Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638THE INSECT PESTS OF CROPS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639TECHNOLOGIES FOR CROP RESISTANCE AGAINST INSECT PESTS. . . . . . 641

The Complementary Roles of Conventional Breedingand Genetic Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641

Bt and Other Protein Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641RNA Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642Genome Editing by CRISPR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642

FROM PLANT SECONDARY METABOLISM TO CROPRESISTANCE TRAITS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643The Diversity of Plant Secondary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643Engineering Crop Secondary Metabolism for Resistance to Insects . . . . . . . . . . . . . . . . 644

PLANT IMMUNITY AND CROP RESISTANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645The Molecular Structure of Plant Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645Insect Elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646Insect Effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647

MANAGING THE INSECT MICROBIOME FOR IMPROVEDCROP RESISTANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649The Ubiquity of Microbiomes in Plant-Insect Interactions . . . . . . . . . . . . . . . . . . . . . . . . 649Suppression of Microbial Partners Required by Insect Pests . . . . . . . . . . . . . . . . . . . . . . . 650Microbial Delivery Vehicle for Novel Insecticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651

OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652

INTRODUCTION

Globally, an estimated 20–40% of crop yield is lost to pests and disease (50), and insect pestsrepresent a significant portion of this loss, both by direct damage and indirectly through thetransmission of plant disease (93). Reducing the burden of insect crop pests is a key priority,particularly since an estimated 70–100% increase in global food production will be required by2050 to feed the burgeoning human population (50, 106, 137). There is a growing consensusthat this target should be met by sustainable intensification, i.e., increased productivity withoutecological degradation (33), and that these changes in agricultural production will involve increasedautomation and biotechnological innovation (70). The demand for novel strategies to controlinsect crop pests is particularly acute because the use of broad-spectrum chemical insecticides,which have been the mainstay of crop protection against insects over the last 50 years, is setto decline. Increasing insect resistance, the greater regulatory burden linked to environmentaltoxicity and concerns for human health, and the high cost of bringing new products to market arealready having impacts on traditional insecticide R&D (14).

How is crop loss to insect pests to be reduced? Part of the solution lies in the problem:Agricultural crops provide attractive and nutritious food for many phytophagous insects. Mostcrop plants, especially improved varieties that have been bred in the last century and are grownwith high fertilizer inputs, have a high nutrient content and reduced physical and chemical defensesrelative to their wild relatives (25, 94). Thus, the challenge is to increase crop resistance to insectpests without compromising crop yield and quality. Recent advances in our understanding of the

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molecular basis of insect-plant interactions and in plant biotechnology are providing the meansto meet this challenge.

This review addresses recent research advances that are underpinning strategies to enhancecrop resistance to insect pests or that have the potential to do so. The pace of discovery, both ofessential insect traits and of reagents to perturb these traits, has increased dramatically throughmolecular biology and genomics, and opportunities for interventions are continuing to expandas technologies (e.g., sustained expression of multiple stacked transgenes in plants) are optimizedand as new technologies (e.g., editing of plant genomes by CRISPR) are introduced. It is nowroutine to address crop resistance to insect pests in molecular terms. Specifically, the insect trait isdefined in terms of one or multiple gene targets, and plant resistance is defined as a gene or suiteof genes with a product or products that inactivate or otherwise disable the product or productsof the target insect gene or genes. A second consequence of the genomic revolution has beenthe development of molecular methods to investigate the composition and function of microbialcommunities, including unculturable forms, leading to the recognition that the sustained vigorand fitness of both plants and insects are dependent on interactions with resident microorganisms,collectively known as the microbiome (18, 42). It is becoming increasingly evident that membersof the microbiome can influence insect-plant interactions and can contribute to strategies forenhanced crop resistance to insect pests.

The main purpose of this article is to explore three broad routes for the discovery of novelsources of resistance to insects: to customize plant secondary chemistry for specific toxicity ordeterrence to insect pests, to harness plant immunity against insect pests, and to target the mi-crobiome of insect pests. Some aspects of these topics are supported by well-developed researchdisciplines, and others are more speculative. To provide context for assessing the opportunitiesand limitations of these novel approaches, the article starts with two complementary overviewsof, first, the diversity of phytophagous crop pests (i.e., insects that feed on living plant tissues)and, second, strategies for engineering resistance traits into crops. The article concludes with anoutlook that addresses the principal opportunities and barriers for enhanced crop resistance toinsect pests.

THE INSECT PESTS OF CROPS

A key factor contributing to the importance of insects (the class Insecta) as crop pests is theirdiversity. The insects are the most species-rich group of animals, and two-thirds of known species(>600,000 species) are phytophagous; i.e., they feed on living plant material (excluding pollenand nectar). Virtually every plant species is utilized by at least one phytophagous insect species,and often by multiple species. Most phytophagous insects have a very restricted plant range—i.e.,they use just one or a few related plant species—but a minority of phytophagous insects, includingsome of the most important crop pests, can utilize many plant species.

Phytophagous insects display a variety of feeding modes, most of which can be categorized intotwo broad groups: (a) chewing and (b) piercing and sucking. These two feeding styles are broadlypredictive of the type of crop damage caused by an insect pest.

The chewing insects have mouthparts that break off and ingest portions of whole plant tissuefrom the root, shoot, or specialized organs such as flowers and fruit. This feeding behavior cancause substantial mechanical damage to the plant and can impair critical functions, such as waterand mineral acquisition by the roots, photosynthetic carbon capture by shoot tissues, and seedproduction. Many chewing insects feed from the surface of the plant, often dispersing amongdifferent parts of a single plant and to different plants. For some chewing insect species, one ormultiple life stages live within the plant tissues (the adult females deposit eggs deep within the

www.annualreviews.org • Strategies for Enhanced Crop Resistance 639

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Insect orders

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Figure 1The incidence of phytophagous insect species across the 31 orders of the class Insecta. The 9 orders withphytophagous members are indicated with an asterisk, and the percentage of phytophagous species in eachorder is shown above each bar. Figure drawn from data in supplemental table 4 of Reference 144.

plant, or larval insects may bore their way into the plant, varying with species); these groups includethe leaf miners, stem borers, and root borers.

Insects with piercing-and-sucking feeding habits have sharp, elongated mouthparts that canpenetrate individual cells or the vascular tissue of the plant. For insects that feed on plant cellcontents, the mouthparts perform repeated cycles of penetration into a plant cell, ingestion, with-drawal, and transfer to a different plant cell. Insects that tap into the phloem or xylem vessels mayfeed for many hours or days via a single penetration. The direct damage caused by these lesionsand removal of plant cell contents can be substantial, especially when a plant is infested by manyinsects. Additionally, piercing-and-sucking insects are important vectors of plant disease agents,including viruses and bacteria.

Despite the very large number of phytophagous insect species, phytophagy has a restricteddistribution across the insect phylogeny, with representatives in just 9 (29%) of the 31 orders(Figure 1). Many of the most important crop pests are members of just two insect orders thatinclude >100,000 phytophagous species and that display the chewing feeding habit: the Lepi-doptera (butterflies and moths), whose caterpillar larvae are both ancestrally and near-universallyphytophagous, and the Coleoptera (beetles), in which phytophagy by both larvae and adults hasevolved multiple times. Four insect orders have tens of thousands of phytophagous species: theHemiptera, which feed exclusively on fluids (plant cell contents, phloem, and xylem) via thepierce-and-suck method; the Diptera, in which phytophagy has evolved multiple times (e.g., tip-ulid crane flies, cecidomyiid midges, and tephritid flies) and with a diversity of feeding styles; andtwo groups with the chewing feeding habit, the predominantly phytophagous order of Orthoptera(grasshoppers and locusts) and the sawflies and wood wasps within the Hymenoptera (ants, bees,

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and wasps). The remaining insect orders with phytophagous representatives are the Thysanoptera(thrips), which include important pests, and the Phasmatoda (stick insects) and Collembola, eachcomprising <10,000 species.

TECHNOLOGIES FOR CROP RESISTANCE AGAINST INSECT PESTS

The Complementary Roles of Conventional Breedingand Genetic Technologies

Once plant traits conferring resistance to insect pests are identified, the priority is to introducethem to crop genotypes that already have multiple desirable traits (e.g., growth form, yield, andflavor). Conventional breeding is possible where the trait of interest is present in the same species(for example, in a land race) or sexually compatible plants of related species, and the timescales forbreeding projects can be reduced by marker-assisted selection (31). However, genetic technologies,including genetic transformation and genome editing, can dramatically accelerate the productionof cultivars with insect resistance and are essential for crops with limited genetic diversity in thedesired traits.

Bt and Other Protein Toxins

The success of crop genetic technologies is epitomized by the insecticidal Bt toxins produced bythe soil bacterium Bacillus thuringiensis (97). More than 200 Bt genes are known, each active againsta restricted range of insects and thus conferring exquisite specificity with minimal direct impact onnontarget organisms, including natural enemies of the insect pest. The first crops engineered toexpress Bt toxins and grown commercially were Bt corn and Bt cotton, which were approved in theUnited States in 1995, and by 2016, Bt varieties accounted for 79% and 84% of the US acreage forcorn and cotton, respectively (138). Internationally, the use of Bt crops is increasing, although withmuch regional variation (66). Diverse Bt crops are under development. For example, Bt eggplant(Solanum melongena) with resistance against the lepidopteran Leucinodes orbonalis was approved forcommercial release in Bangladesh in 2013, and field trials of insect-resistant chickpea and pigeonpea were approved in India in 2016 (66). Also, crops are increasingly being engineered to expressmultiple traits. The US market in Bt corn and Bt cotton is dominated by crop varieties withtwo or more Bt toxin genes stacked with herbicide resistance, and field trials of insect-resistantand drought-tolerant Bt corn are under way in Portugal and Mozambique (66). Although fieldresistance to Bt crops has been reported for several insect pests, the scale of the problem is reducedby proactive resistance management, especially refuges for non-Bt host plants and pyramiding ofmultiple Bt genes against a single pest (20, 127).

Genetic technologies are enhancing the efficacy of Bt toxins. Domains from different Bt toxinshave been recombined to enhance efficacy; e.g., Cry2AX1 (a fusion product of Cry2Aa and Cry2Ac)protects against multiple lepidopterans (23). Bt toxins have also been engineered to confer activityagainst hemipteran pests, which are not susceptible to most naturally occurring Bt toxins (97).For example, the Bt toxin Cyt2Aa has been modified to include a 12-amino-acid sequence thatbinds to the aphid alanyl aminopeptidase N protein, which is expressed abundantly on the apicalmembrane of the gut epithelium, resulting in high aphid mortality (28), and genetic modification ofCry51Aa2 increases the activity >200-fold against Lygus species, conferring significantly improvedprotection of cotton (61). These innovations pave the way for future development of engineeredcrop resistance against hemipteran pests. Hemipterans are increasingly important pests in manyBt crop systems, probably because the Bt-mediated control of lepidopteran and coleopteran pestshas reduced chemical insecticide applications that previously controlled hemipterans and, in someagrosystems, has also eliminated the principal competitors of hemipterans (81, 148).


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The arsenal of naturally occurring and engineered Bt toxins can be complemented by multi-ple other proteins with insecticidal activity. Protein toxins of various soil bacteria, plant lectins,plant inhibitors of insect digestive enzymes, and components of the venoms of insect predators(e.g., spiders, scorpions) have potential. For example, the soil bacterium Pseudomonas chlororaphisproduces a small protein that, when expressed in plants, is toxic to coleopteran pests, includingthe western corn rootworm, but not lepidopteran or hemipteran insects (115); an α-amylase in-hibitor engineered into pea (Pisum sativum) confers resistance to the pea weevil Bruchus pisorum(86); mannose-binding lectins of various monocot plants, notably the snowdrop lectin, Galanthusnivalis agglutinin (GNA), are toxic to various insects, but not mammals (27); and spider venomsinclude thousands of insecticidal peptides (117). In principle, the insect resistance profile of plantscan be designed by appropriate stacking of genes, including genes engineered for optimal efficacyand specificity. For example, fusion of a scorpion neurotoxin domain (As1T) active against lepi-dopteran pests and the GNA lectin domain active against hemipterans confers resistance againstmultiple lepidopteran and hemipteran pests in Arabidopsis, tobacco, and rice (80). In another ex-ample, the toxicity of an orally delivered spider toxin Hv1a is enhanced, for aphid pests, by fusionwith a luteovirus coat protein (13) and, for lepidopteran pests, by fusion with the GNA lectin (51).

RNA Interference

RNA interference (RNAi) offers the opportunity to design insecticides that have even greaterflexibility than protein toxins with regard to both mode of action and specificity. Double-strandedRNA (dsRNA) specific to an essential gene of an insect pest is internalized into cells, where it isprocessed by Dicer enzymes to small interfering RNA (siRNA) molecules that guide the Argonauteprotein of the RNA-induced silencing complex (RISC) to degrade complementary mRNAs and,in some instances, to interfere with translation of the target mRNA (119). The specificity ofRNAi can, in principle, be controlled very precisely by selecting sequences that evolve at differentrates (a wider range of insect taxa can be targeted by more conserved sequences). dsRNA is mostcommonly delivered by genetic modification of the plant (102), but topical application of dsRNAby sprays or drenches has also been reported to control lepidopteran and hemipteran pests (77).

Orally delivered RNAi is particularly effective against many coleopteran insects, routinely me-diating >80% reduction in expression of target genes and conferring significant crop protection,e.g., in corn against the western corn rootworm (6), and in potato against the Colorado potatobeetle Leptinotarsa decemlineata (149). Insecticidal RNAi against the western corn rootworm isprogressing through the regulatory process in the United States. Specifically, SmartStax R© PRO(Monsanto) includes a dsRNA sequence against the Snf7 gene of the western corn rootworm,stacked with Cry3Bb1 (also with activity against the western corn rootworm) and an herbicideresistance gene (85). The Snf7 protein is a class E vacuolar sorting protein, and its downregulationresults in perturbation to protein deubiquitination and autophagy in the insect midgut and fat body(104). RNAi against lepidopteran and hemipteran pests is used widely in research but can be less re-liable than in the Coleoptera (119, 132). Strategies to enhance the efficacy of in planta RNAi againstinsect pests include expression of long hairpin RNA (hpRNA) in the chloroplast to minimize pro-cessing by the plant RNAi machinery (5, 149) and stacking the hpRNA against the gene of interestwith hpRNA against nonspecific nucleases expressed in the gut of the target insect (121, 122).

Genome Editing by CRISPR

Genome editing technology dramatically expands the opportunities for introducing resistancetraits into crops (57). The most widely used CRISPR (clustered regularly interspersed short

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palindromic repeats) system in eukaryotes, including plants, has been designed from the CRISPR/Cas9 bacterial defense against viruses. Briefly, the Cas9 protein is a DNA-specific nuclease thatmakes a double-stranded break in DNA at a site guided by the binding of a synthetic guideRNA. Multiple CRISPR protocols are available, including those with the capacity to generatesite-specific indels (often yielding frameshift mutations), to replace or insert specific sequences,and (by using a deactivated Cas9) to suppress gene expression. In relation to insect pests, the firstapplications of CRISPR in crops confer resistance to insect-vectored viruses, especially the gemi-niviruses, which have DNA genomes (3, 53). CRISPR is also the technology of choice to producenew crop varieties in response to insect pest genotypes that break plant resistance mechanisms.This is because resistant and susceptible alleles of plant resistance loci generally differ by just oneor a few nucleotides. Specifically, CRISPR can be used to edit the susceptible allele to the resis-tance allele, thereby eliminating the need for extensive crosses and back crosses by conventionalmethods. The relative ease with which CRISPR can be applied to edit all copies of a gene makesCRISPR the technology of choice for polyploid crops.

In summary, plant genetic technologies have a proven track record for crop protection againstinsect pests, and ongoing innovations in plant biotechnology offer the prospect of more sophis-ticated and effective routes for plant protection against the major groups of phytophagous insectpests over the coming years. In this context, the remainder of this review addresses three majoropportunities for the development of novel insect pest strategies: plant secondary metabolites,plant immunity, and microbiome science.

FROM PLANT SECONDARY METABOLISM TO CROPRESISTANCE TRAITS

The Diversity of Plant Secondary Metabolism

Plants synthesize a rich and diverse array of secondary metabolites, currently estimated at >200,000molecules in multiple biosynthetic classes, including terpenes, alkaloids, phenylpropanoids andaromatic polyketides (e.g., flavonoids, stilbenoids), cyanogenic glycosides, and glucosinolates (60,65, 84). Many of these compounds are toxic or deterrent to insects, and some function as indirectdefenses by attracting parasitic wasps or other natural enemies of phytophagous insects (116).However, various insects can tolerate or detoxify certain plant secondary metabolites, and somespecies utilize plant secondary compounds as cues required for feeding or oviposition (116). Fur-thermore, there is a wealth of evidence that many phytophagous insects have coevolved with thesecondary metabolite profile of their host plants (54, 103, 133), and this is an important consider-ation in the design and management of crop resistance based on secondary metabolite chemistry.

Many crops display much-reduced synthesis of secondary metabolites and toxicity to animalsrelative to related wild plant species (94). For example, domestication of the tomato (Lycopersiconesculentum) was accompanied by the loss of two genes (zFPP and ShZIS) coding enzymes thatsynthesize the sesquiterpene 7-epizingiberene. When these genes were transferred from a wildtomato species to the cultivated tomato with expression in trichomes, the plant was better protectedagainst multiple insects. Specifically, oviposition by the whitefly Bemisia tabaci (Hemiptera) on thetrichome-rich adaxial leaf surface was suppressed, development of the tobacco hornworm Manducasexta (Lepidoptera) was delayed, and L. decemlineata (Coleoptera) was deterred from feeding (11).Some plant taxa have, however, retained toxicity through domestication. For example, the potatotuber is rich in glycoalkaloids, the cassava tuber contains cyanogenic glycosides, and many legumeseeds harbor alkaloids. It has been argued that domestication of some crops included selectionfor toxicity, namely that toxins confer protection against predators, including insects, but areinactivated by cooking and other processing prior to human consumption (83).


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Engineering Crop Secondary Metabolism for Resistance to Insects

Recent advances in plant genetics and molecular biology create the opportunity to address thegenetically impoverished and variable secondary metabolite profile of crops for improved resis-tance to insect pests. The goal is to manage the toxin content of crops more precisely, so thatsustained defense against insect pests is combined with reduced risk of chronic or acute toxicity tothe consumer. However, the relationship between an engineered gene activity and the secondarymetabolite profile of the plant is considerably more complex than for the engineering of proteintoxins, such as Bt, because the gene products are not the bioactive compounds but the enzymes thatsynthesize these compounds. The problems are twofold. First, many of the enzymes in secondarymetabolism have low substrate specificity, with the consequence that the composition of reactionproducts can be driven by the availability of multiple alternative substrates and potentially influ-enced by the metabolic flux distribution across the entire metabolic network. In other words, theprofile of secondary metabolites can be variable, and the outputs from manipulations are often notfully predictable (92). For example, the insertion of genes for farnesyl diphosphate synthase andsqualene synthase into tobacco, with the goal to increase squalene production, resulted in multi-ple unexpected metabolic changes, including dramatically elevated levels of the alkaloid nicotine(98). These unforeseen metabolic phenotypes are not peculiar to engineered plants. They are alsoreported in conventional breeding programs; for example, somatic hybrids of the potato Solanumtuberosum and the wild relative Solanum brevidens contained toxic levels of a steroidal alkaloid,demissidine, that was undetectable in either parent species (74).

The second difficulty in engineering plant metabolism is that inserting single or several genesdesigned to increase flux through specific reactions or to add in novel reactions can often have min-imal effects because metabolic pathways are tightly regulated with flux controls distributed acrossmultiple reactions. Various strategies can ameliorate these constraints. For example, metabolicmodeling can inform the choice of a reaction or reactions to achieve the desired change in sec-ondary metabolism. It is also becoming possible to silence or overexpress specific transcriptionfactors and miRNAs that regulate the spatiotemporal expression of secondary metabolism geneexpression, to identify candidate routes by which secondary metabolite profiles can be managed(147). Additionally, targeting of expression to the plastid, where the metabolic network is generallymore tolerant of flux variance than in the cytoplasm, can deliver the desired metabolic changes.Multiple studies on plant secondary metabolite profiles have used these approaches, often in com-bination (2, 16, 87, 145, 146).

The application of metabolic engineering for improved crop resistance builds on the evidencethat genetic modification of secondary metabolite profiles in the model plant Arabidopsis andselected crops can promote plant defenses against insects. Two examples are illustrative. WhenArabidopsis was transformed to express a terpene synthase (specifically a dual S-linalool/(3S)-E-nerolidol synthase, FaNES1, isolated from strawberry Fragaria × ananassa) in the plastid, theleaves contained 40- to 60-fold-higher concentrations of linalool, together with glycosylated andhydroxylated derivatives (2). This metabolite profile was strongly repellent to the one insect tested,the aphid Myzus persicae. Similarly, Arabidopsis has been engineered to produce the cyanogenicglycoside dhurrin, and the flea beetle Phyllotreta nemorum (a major pest of crucifer crops) displayeddramatically reduced adult feeding and very high larval mortality on the transformed plants relativeto the wild-type and multiple transformed controls (131). Although these studies provide excellentproof of principle, application to crop production requires resolution of the twin problems of(a) the negative effects on plant growth and yield and (b) toxicity to the end consumer.

Secondary metabolite chemistry has also been manipulated to engineer indirect defenses ofplants. An illustration of the opportunities and frustrations of this approach is provided by the aphidalarm pheromone, the sesquiterpene (E)-β-farnesene (Eβf ). When Eβf is released from an aphid,

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it triggers rapid dispersal of nearby aphids and also attracts aphid predators and parasitic wasps. Eβfis also synthesized by many plants (34), and its secretion from the trichomes of the potato speciesSolanum berthaultii has been demonstrated to contribute to resistance against aphids (59). WhenArabidopsis was transformed with the Eβf synthase gene of peppermint, Mentha piperita, it releasedEβf constitutively, repelling aphids and attracting Diaeretiella rapae, a parasitoid of crucifer-feedingaphids (7). Similar results were obtained in the laboratory for wheat, including repellency againstthree pest aphid species, and increased foraging by the parasitoid Aphidius ervi (16). However, infield trials, the abundance of neither aphids nor their parasitoids differed significantly betweenthe Eβf-emitting wheat and control plants (16). This disappointing result is likely attributable tolong-term habituation of the insects to the constitutively produced volatile. Inducible productionof Eβf in response to aphid feeding is anticipated to support more reliable crop protection. Furtherdevelopment of this modification depends on advances in our understanding of the molecular basisof the plant response to aphid feeding.

The mixed results obtained for protection of Eβf-producing wheat against aphid pests (16)illustrate the general point that management of plant secondary chemistry in agricultural systemscan be complex. The impact of secondary metabolites on the target insect can vary with the spa-tiotemporal pattern and concentration of the product, and the overall impact of plant secondarymetabolites on crop production can be shaped by multiple, interlocking interactions. For example,Eβf is a potent oviposition stimulant for the European corn borer Ostrinia nubilalis (9), precludingits use as an aphid deterrent in corn, and manipulation of plant terpene chemistry for protectionagainst phytophagous insects can perturb essential chemical cues used by pollinators (45).

PLANT IMMUNITY AND CROP RESISTANCE

The Molecular Structure of Plant Immunity

Plant immunity refers to the capacity of plants to detect and respond to the molecular patternsthat are characteristic of foreign organisms and cellular perturbations caused by mechanical orchemical damage. It is argued that plant cells can detect insect products as foreign by processes thatmatch their response to microbial pathogens (see the sidebar titled Plant Immunity to Pathogensand Figure 2). Specifically, conserved insect elicitors are proposed to activate basal immune

PLANT IMMUNITY TO PATHOGENS

The current paradigm for interactions between the plant immune system and microbial pathogens, illustrated inFigure 2, focuses on two complementary elements of the cell-autonomous defense system, known as pathogen-triggered immunity (PTI) and effector-triggered immunity (ETI). Conserved molecular motifs of pathogens (elic-itors) are detected by transmembrane receptors (pattern recognition receptors) at the cell surface, inducing PTI,which is defined as a conserved basal defensive response. Various pathogens produce protein effectors that suppressPTI, and this process results in selection for ETI, which is a more robust and often longer-lasting plant immuneresponse than PTI. Intracellular NLRs (nucleotide-binding leucine-rich repeat receptors), which inactivate thepathogen effector, play a central role in ETI. In many plant-pathogen systems, pathogen effector genes and plantNLR genes engage in a gene-for-gene coevolutionary arms race, yielding multiple alleles of plant NLR genes thatconfer resistance against pathogen genotypes with certain effector gene alleles. In the nomenclature of classical ge-netics (as first elucidated by Harold Flor for flax resistance to the rust fungus Melampsora lini ), the plant NLR genesconferring resistance are known as R genes, and the pathogen effector genes are known as avr (avirulence) genes.


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Pattern recognitionreceptor

Elicitor released fromnatural enemy

Basal immunity(PTI)

Virulencemediated by effectors

of natural enemy

ETI

Suppression ofbasal defense

Positive interaction

Negative interaction

Selection pressures

Figure 2The molecular structure of plant immunity. This paradigm was developed for plant-pathogen interactions(see the sidebar titled Plant Immunity to Pathogens) but is relevant to interactions with many naturalenemies of plants, including insects. The plant is envisaged to possess two complementary cell-autonomoussystems: the basal defense, often known as pathogen-triggered immunity (PTI), against conserved molecularfeatures of elicitor molecules released by the natural enemy, and effector-triggered immunity (ETI), raisedagainst molecules (generically known as effectors) of the natural enemy that suppress PTI. Some interactionsbetween the plant immune system and natural enemies do not conform perfectly to the PTI/elicitor versusETI/effector dichotomy. For example, some elicitors are not highly conserved for both insects (see text) andbacterial pathogens (32), and ETI and PTI are often not independent (e.g., ETI can induce elevated PTI, asshown in the figure). Interactions between the plant immune system and natural enemies are indicated withsolid lines (arrow, positive interaction; bar, negative interaction), and selection pressures leading to evolutionand diversification of effectors and ETI are denoted by dashed lines.

responses, equivalent to the conserved pathogen-triggered immunity (PTI), and insect effectorsare predicted to coevolve with plant effector-triggered immunity (ETI), yielding dominant allelesof single genes that confer resistance to the insect pest (64). Here, the evidence for insect elicitorsand effectors is considered in turn.

Insect Elicitors

Insects inadvertently reveal their presence to plants by constituents in their oral secretions (saliva,gut regurgitant) and egesta (frass, honeydew) and by the oviposition fluids released by femaleinsects depositing eggs into plant tissue (109). These candidate insect elicitors of plant immuneresponses include fatty acid–amino acid conjugates (commonly an unsaturated C18-fatty acidcoupled to glutamine or glutamate, e.g., volicitin), caeliferins (sulfooxy fatty acids), and variousproteins (e.g., glucose oxidase). Generally, these elicitors activate the jasmonic acid ( JA) signal-ing pathway, leading to direct defenses, e.g., polyphenol oxidase activity, and indirect defensescomprising volatiles that attract specific natural enemies of the phytophagous insect.

The paradigm of plant immunity that developed from research on plant pathogens (Figure 2)predicts that the insect elicitors are conserved molecules, with the expectation that single moleculesderived from insects can be used to manage the immune defenses of multiple crops against multipleeconomically important pests. The data do not fully support this expectation. For example, theelicitor glucose oxidase released in the saliva of the tomato fruitworm Helicoverpa zea induces

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JA-mediated plant defenses in tomato (136) but suppresses production of the defensive compoundnicotine in tobacco (89), and caterpillar frass can either induce or suppress plant defense responsesthat suppress caterpillar growth, varying with the insect species, crop, and location of the insectson the plant (108). This variability suggests that the evolutionary diversification of elicitors maybe less constrained in insects than in pathogens [although examples of evolutionary diversificationof pathogen elicitors are known (32)].

In parallel with research on the identity and diversity of insect elicitors, there has been con-siderable research on the interaction between these elicitors and plant immunity. Intriguingly,plant responses to some insect elicitors are not protective for the plant (contrary to the predic-tions of the plant-pathogen paradigm; see Figure 2) but appear to be advantageous to the insect.Specifically, some insect elicitors activate defenses against biotrophic pathogens orchestrated bythe phytohormone salicylic acid (SA). This plant response is advantageous to the insect undermany circumstances because antagonistic cross talk between the SA-dependent pathway and theJA-dependent pathway results in suppression of JA-mediated defense against the insect. In effect,these insect pests are exploiting a weakness in the regulation of the complex signaling networkscontrolling plant immune defenses. A number of insect elicitors of the SA-dependent pathwayhave been characterized. For example, SA-dependent defense in maize is induced by two maizechitinases (Endochitinase A and Pr4) that are consumed by caterpillars of the fall armywormSpodoptera frugiperda feeding on the plant tissue and that are then released back onto the plant sur-face in caterpillar frass. The resultant SA-mediated suppression of JA-mediated defense improvesinsect performance (107). Similarly, the honeydew released by the pea aphid Acyrthosiphon pisumcontains SA and probably other molecules that activate SA-mediated defense in the fava bean Viciafaba, suppressing JA signaling to the benefit of the aphid (118). Other elicitors produced by insectsare, however, microbial. The gut regurgitant of L. decemlineata contains bacteria that trigger SAsignaling in tomato plants, with consequent reductions in JA-regulated plant defenses, includingreductions in a cysteine protease inhibitor and polyphenol oxidase (29). This response is inducedby the bacterial flagellin protein, which is a well-known bacterial elicitor of plant immunity. Theimportance of this protein for insect performance is demonstrated by the reduced growth rates ofthe beetle larvae on plants in which SA signaling is ablated genetically or in which the bacteria aresuppressed by antibiotic treatment (29).

Taken together, these fundamental studies on the interaction between insect-derived elicitorsand the plant immune system reveal that some phytophagous insect pests can manipulate thebasal immune responses of plants. This manipulation is indicative of coevolutionary interactionsbetween insects and plant immunity, raising the possibility of natural variation in plant immuneresponses to specific insect pests, especially in land races and related wild plant species. Such vari-ation can potentially be exploited by conventional breeding or genetic engineering for improvedcrop resistance.

Insect Effectors

Various lines of evidence suggest that phytophagous insects produce specific effectors that sup-press, in a fashion comparable to effector molecules of pathogens, the basal plant defenses inducedby some elicitors (15). The plant receptors for many pathogen effectors are intracellular NLRscoded by plant resistance genes (R genes). The R genes offer superb opportunities for breeding forenhanced crop resistance because dominant alleles of R genes can confer resistance against multiplegenotypes of specific pathogens (see the sidebar titled Plant Immunity to Pathogens and Figure 2).Because the NLRs coded by R genes against pathogens are intracellular, the ETI paradigm is ex-pected to have greatest relevance to insects that interact directly with the contents of living plant


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Table 1 Examples of candidate plant R genes conferring resistance to insect pests

Crop Insect pest Candidate R gene Evidence Reference(s)

Melon (Cucumismelo)

Aphid (Aphis gossypii ) Vat (Virus-Aphid-Transmission) gene

Gene fully characterized at themolecular level

39

Medicago truncatula Blue alfalfa aphid(Acyrthosiphon kondoi )

AKR (Acyrthosiphonkondoi resistance) gene

Gene fully characterized at themolecular level

72

Tomato (Lycopersiconesculentum)

Potato aphid (Macrosiphumeuphorbiae), tomato psyllid(Bactericerca cockerelli ),whitefly (Bemisia tabaci )

Mi-1.2 gene Gene fully characterized at themolecular level

22, 91, 113

Soybean (Glycinemax)

Soybean aphid (Aphisglycines)

Rag (Resistance to Aphisglycines) genes

At least two of the five Raggenes map to chromosomalregions coding NLR(nucleotide-binding leucine-richrepeat receptor) genes

63

Cultivated rice(Oryza sativa) andwild Oryza species

Brown planthopper(Nilaparvata lugens)

Various Several of ∼30 genesdemonstrated to code NLRproteins

44, 68, 130, 141

Cultivated rice(O. sativa)

Rice gall midge (Orseoliaoryzae)

Eleven rice genes(Gm1–Gm11)

Mapped to chromosomalregions that include NLRgenes

8

Cultivated wheat(Triticum aestivum)

Hessian fly (Mayetioladestructor)

Various Mapped to clusters ofNLR-coding genes

125

cells. Consistent with this reasoning, some of the best genetic evidence for gene-for-gene coevo-lutionary interactions involving R genes relates to (a) insects with mouthparts that penetrate to thesieve elements and imbibe phloem sap (e.g., aphids, whiteflies) (41) and to (b) cecidomyiid midgesthat pierce plant cells close to the meristem, inducing them to produce nutrient-rich fluids (62).

Where identified, the plant R genes against phloem-feeding hemipterans and cecidomyiidmidges code for intracellular NLR proteins, just as in the case of the R genes against pathogens(Table 1). However, the physiological mechanisms by which R genes mediate resistance againstinsects are not well understood and appear to be diverse. For example, Vat (Virus-Aphid-Transmission)-mediated resistance in melon against Aphis gossypii involves the hypersensitive re-sponse, including localized cell death in the immediate vicinity of the feeding site, as well asperoxidase activity consisting of a micro-oxidative burst and callose deposition; these responsesare very rapid, evident within 10–20 min of aphid infestation (140). By contrast, resistance intomato conferred by Mi-1.2 is independent of the hypersensitive response (82) and involves SAand MAPK signaling cascades (78), whereas JA signaling has been implicated in the R gene–mediated resistance of Medicago truncatula to Acyrthosiphon kondoi (56).

A key limitation of R gene–mediated resistance in crop protection is that it selects for evolu-tionary diversification of the insect effector proteins, thereby breaking the resistance. This coevo-lutionary arms race (Figure 2) has been demonstrated, for example, for Aphis glycines on soybean(139), Nilaparvata lugens on rice (26), and Hessian fly on wheat (125). Pyramiding of multipleR genes can enhance the durability of crop resistance (see, e.g., References 69, 120, and 139),although this strategy can depress crop yield. As recent research on rice resistance to the riceblast fungus Magnaporthe oryzae illustrates (37), improved understanding of the molecular mecha-nisms underlying the interactions between plant immunity (especially R genes and ETI) and insect

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effectors can assist in the choice of R genes conferring resistance with minimal reduction in cropyield. In other words, identification of R genes and their mode of action is a prerequisite for usingthese genes to design crops that combine sustained yield with durable resistance against insect pests.

Most progress in research on crop R genes has been achieved with the Hessian fly and aphids.The genome of the Hessian fly bears multiple candidate effector genes (150). For example, thecandidate effector gene vH13 is genetically dominant in interactions with wheat bearing the H13gene (i.e., the R gene that confers resistance against Hessian flies with the vH13 gene), as predictedby the gene-for-gene paradigm (52). Specifically, Hessian fly larvae with the vH13 gene suffer veryhigh mortality on wheat with the H13 resistance gene, but larvae that either have null mutationsin vH13 or are treated with dsRNA that suppresses vH13 expression can survive and develop onthe H13 wheat (1). The molecular basis of this interaction is, however, unknown: vH13 codes fora small taxon-specific protein with no homology to other publicly available sequences (1).

Recent research on aphids has been dominated by the recognition that the insect effectors arelikely salivary proteins, which have been identified by transcriptomic and proteomic studies (19,105, 134), and that the function of these effectors can be investigated by quantifying the per-formance of insects on plants expressing individual insect salivary proteins (15). Multiple studieshave revealed that some salivary proteins expressed in plants significantly affect aphid survival orfecundity (67). For example, the enhanced performance of the green peach aphid M. persicae onArabidopsis expressing the salivary protein Mp55 is associated with reduced plant defenses, includ-ing lower levels of callose deposition, hydrogen peroxide, and the glucosinolate 4-methoxyindol-3-ylmethyl glucosinolate (47). Moreover, the reduced performance of M. persicae on Nicotianabenthamiana expressing a different salivary protein, Mp10, has been linked to increased expressionof defense-related plant genes associated with JA and SA signaling (112). Candidate effectors ofinsects expressed in plants can, however, generate artefactual (positive or negative) results becausethe concentration and localization of the effector protein may differ between plants bearing sali-vating insects and plants expressing the transgene, resulting in different plant immune responses(12, 47, 67, 112). These concerns can be addressed, at least in part, by complementary experi-mental approaches. For example, the reduced performance of M. persicae following RNAi againstMp55 validates the improved performance of this aphid on plants expressing Mp55 (47). Similarly,the interpretation that the aphid salivary protein C002 promotes plant utilization is supported bymultiple lines of evidence: The fecundity of M. persicae is elevated on plants expressing C002 butreduced on plants expressing dsRNA against C002 (15, 100), and survivorship of A. pisum admin-istered ds-C002 by injection is sharply curtailed when insects are reared on plants but is unaffectedwhen the insects are reared on artificial diets (90).

Further progress in harnessing plant immune responses for crop resistance to insects will befacilitated by advances in molecular understanding of the interactions. Although various elicitorsand candidate effectors of phytophagous insects have been identified, the putative plant receptorsfor these molecules remain to be characterized in most systems other than wheat/Hessian fly.

MANAGING THE INSECT MICROBIOME FOR IMPROVEDCROP RESISTANCE

The Ubiquity of Microbiomes in Plant-Insect Interactions

There is now overwhelming evidence that the nutrition, the immune function, and the generalwell-being of plants and animals are dependent on the activities of the microbial communitiesborne on their surfaces and in their tissues. Interactions with microorganisms are of particularimportance to plant-insect interactions, with evidence that plant microbial communities, including


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Table 2 Strategies to target microbial partners of insect crop pests

Insect feeding habit Insect order Examples Reference(s)

(a) Closed systems: targeting interactions between insects and obligate microbial symbioses1

Piercing-sucking Hemiptera Most phloem/xylem-feeding species, including aphids,whiteflies, planthoppers, and sharpshooters; a minority ofphytophagous heteropteran bugs

43, 126

Chewing Coleoptera Widespread but poorly studied in weevils (Curculionidae)and in bruchid beetles (Bruchidae)

40

(b) Open systems: microbial delivery vehicles for agents that target insect or microbial functions2

Piercing-sucking HemipteraThysanoptera

Gut bacteria in many stinkbugs and seed bugs(phytophagous heteropteran bugs); gut bacteria allied toErwinia in western flower thrips (Frankliniella occidentalis)

36, 49, 126

Chewing LepidopteraColeopteraOrthopteraDiptera

Gut bacteria and yeasts, often including transient taxa 24, 38, 123

1Closed systems feature intracellular microbial symbionts in specialized somatic cells (bacteriocytes) with obligate transmission to the ovary and, usually,insertion into the cytoplasm of the unfertilized egg (transovarial transmission). If the maternally derived microbial cells are eliminated, the insect hostcannot acquire a replacement microbiota from the environment or other insects.2Open systems feature gut microorganisms that are acquired by feeding and persist (often proliferating to generate sustaining populations) for varyinglengths of time and that are shed in feces. Generally, the gut microbiota is reduced or lost when insects molt, and insects are routinely colonized byfecal-oral cycling (with specialized behavior in some species) or from the environment.

fungal endophytes and mycorrhizal fungi, can influence herbivory (30, 55, 73, 99) and that theplant range of some insects is shaped by their microbiome (21). Although most research to date hasfocused almost exclusively on the fundamentals of microbial impacts on plant-insect interactions,there is a growing appreciation of opportunities for crop protection. Two approaches are especiallypromising: plant delivery of microbicides that suppress microorganisms associated with the insect,including microbial partners required by the insect pest and plant pathogens vectored by theinsect, and the use of plant-associated microorganisms for delivery of novel insecticides. Insectpests suitable for these approaches are summarized in Table 2.

Suppression of Microbial Partners Required by Insect Pests

Plants with microbicidal capability against insect-associated microorganisms have been engineeredprincipally to combat bacterial pathogens that are vectored by hemipteran insects, particularlyXylella fastidiosa, the agent of Pierce’s disease vectored by xylem-feeding sharpshooters, and Can-didatus Liberibacter species (including Ca. L. asiaticus, the agent of huanglongbing/citrus greeningdisease) vectored by phloem-feeding psyllids. The preferred class of microbicides is antimicrobialpeptides (AMPs), which contribute to the immune function of all plants and animals. AMPs displayfar greater toxicity against microorganisms than against animal or plant cells, and, because they aregenetically coded, they can be engineered readily to enhance potency against the target microbeand to combine multiple functions within a single fusion protein. Proof of concept comes fromtwo independent studies that transformed grape with constructs coding the insect AMP cecropinB either with a segment of a second AMP, melittin (79), or with elastase (35). Under glasshouseconditions, both constructs conferred excellent protection against Pierce’s disease, as indicated byabrogation of disease symptoms and Xylella titers in the xylem vessels of the plant (35, 79). However,

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the construct failed to protect against Xylella in field trials (79), probably because AMP expressionwas depressed in field plants exposed to abiotic stress that is not experienced in the glasshouseplants. The efficacy of various AMPs for the control of Ca. L. asiaticus has also been tested (124),and two spinach defensins (SoD2 and SoD7) engineered into citrus are currently undergoingfield trials for protection against huanglongbing (https://geneticexperts.org/epa-exemption-for-transgenic-orange-trees-with-spinach-genes-to-combat-citrus-greening/).

Although the primary focus of microbicidal function of AMPs engineered into plants has beentheir impact on specific bacterial pathogens, these peptides are active against a diversity of mi-croorganisms. In particular, AMPs may alter the composition and activity of beneficial endophytesin the plant, resulting in increased susceptibility to opportunistic pathogens, reduced tolerance toabiotic stress, or other deleterious consequences. This potentially important drawback to the useof AMPs or other antimicrobials is analogous to the negative effects of antibiotics used in humanmedicine. Although this issue has not been investigated systematically in relation to plants, it isnoteworthy that “measurable broad spectrum antibacterial activity” was identified in grapevinestransformed with the cecropin B–melittin construct (79).

A further implication of plants with augmented antimicrobial function relates to the insectsthat vector the bacterial pathogens. The xylem-feeding sharpshooters and phloem-feeding psyllidsthat vector Xylella and Ca. Liberibacter, respectively, possess intracellular bacterial symbionts thatthey absolutely require for growth and reproduction (17, 42). These are closed associations; i.e.,once the insect is deprived of its intracellular symbionts, for example, by plant-derived antimi-crobials, it cannot recover the bacteria from the environment or other insects (Table 2a). Thiscreates the opportunity to construct AMPs that combine activity against the bacterial pathogensvectored by the insect and the intracellular bacterial symbionts required by the insect. It is poten-tially relevant that the genomes of plant sap–feeding insects that have been sequenced to date aremuch depleted in immune genes, including recognizable AMPs, and this evolutionary simplifica-tion of immune system function has been related to the selection pressure to avoid inadvertentdestruction of the obligate microbial symbionts (58). This reasoning suggests that the microbialsymbionts of plant sap–feeding insects may have very limited resistance and tolerance to AMPsand so may be exquisitely susceptible to a wide range of antimicrobials. This trait should facilitatethe design of antimicrobials that combine efficacy against the symbiont and plant pathogen ininsect vectors with minimization of the negative effects on the plant microbiome. Furthermore,closed associations with intracellular microorganisms are a general trait of the phytophagoushemipteran insects feeding through the life cycle on xylem or phloem sap (Table 2a), facilitatingthe future development of highly specific antimicrobials against the microbial symbionts in theseinsects.

Microbial Delivery Vehicle for Novel Insecticides

Microorganisms have great potential for the delivery of insecticides to insects with open microbialassociations; i.e., the insects can be colonized by externally applied microorganisms (Table 2b).For example, the great majority of insects possess a gut microbiome, including plant-associatedmicroorganisms for phytophagous insects, that is acquired by the oral route (42, 48). Of particularinterest is the use of microorganisms as a vehicle for RNAi delivery. A microorganism is modifiedto express dsRNA specific to an essential gene of the insect pest and, following ingestion andcolonization of the insect gut, deliver a sustained supply of dsRNA molecules. Recent studies havefocused on microorganisms isolated from the insect pest (as opposed to heterologous bacteria,such as Escherichia coli ), to facilitate colonization without activation of the insect immune system.Two studies illustrate the efficacy of the approach. The first concerns a gut bacterium, allied with


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https://geneticexperts.org/epa-exemption-for-transgenic-orange-trees-with-spinach-genes-to-combat-citrus-greening/

https://geneticexperts.org/epa-exemption-for-transgenic-orange-trees-with-spinach-genes-to-combat-citrus-greening/


Pantoea ananatis and known informally as BFo2α (49), that has been isolated from the westernflower thrip Frankliniella occidentalis and transformed with an expression plasmid bearing eitherthe eGFP gene or dsRNA targeting the insect α-tubulin gene. When fed to the insects, the BFo2α

bacterium transformed with eGFP colonized the proximal hindgut and persisted over multipledays, demonstrating that it was competitive with the indigenous microbiota. Feeding the flowerthrips with the ds-α-tubulin gene resulted in a 60% knockdown in expression over 2 days andsignificantly elevated mortality over 4 days (143). The second study concerns the spotted-wingfruit fly Drosophila suzukii, an invasive pest of small fruits that feeds on fruit-associated yeasts andbacteria. D. suzukii fed on Saccharomyces cerevisiae yeasts expressing the ds-γ-tubulin gene constructand incurred high larval mortality and reduced adult fecundity (88).

Under what circumstances is microbial delivery of RNAi preferable to in planta RNAi againstinsect pests (see section titled RNA Interference)? The key advantage of microbial deliveryis that genetic manipulation of microorganisms, especially bacteria, is cheap and straightfor-ward, as opposed to the time-consuming and often difficult procedures for the stable transfor-mation of crops (142). This difference is particularly acute both for generalist insects, such asF. occidentalis and D. suzukii, for which protection of multiple different plant species is required,and for crops (e.g., many vegetables and ornamentals) with market demand for multiple vari-eties or cultivars. There are, however, environmental concerns relating to the containment ofthe manipulated microorganisms. Uncontrolled dispersal of the microorganisms and horizontaltransfer of the transgenes to other bacteria could suppress the insect pest and possibly relatednonpest species in nonagricultural environments, with substantial ecological consequences. So-lutions are potentially available but will require development. For example, control over deliverymay be facilitated by encapsulation methods (4) that permit microbial release only under condi-tions of an insect gut (e.g., extreme pH, high protease activity). Additionally, the capacity of thetransformed microorganisms to persist and proliferate in the environment may be constrained bygenetic strategies for synthetic auxotrophy, i.e., the genetic requirement for a synthetic compoundthat becomes limiting after a set number of cell divisions of the released microbial cells (114). Ofcourse, such environmental safeguards would, by definition, limit one perceived advantage of mi-crobial delivery of RNAi: that microbial-encoded controls can persist in the insect indefinitelyand spread to other individuals via fecal-oral transmission (142). These difficulties are sharedwith the related strategy of paratransgenesis, i.e., the use of genetically manipulated microorgan-isms to control pathogen transmission, which has been applied to suppress vector competence inhematophagous insects (46, 111). Resolution of these generic challenges for managing the mi-crobiome of insect pests will create new opportunities for both human health and agriculturalproduction.

OUTLOOK

Strategies to control insect crop pests need to take into account that many aspects of crop produc-tion, including large-scale monoculture, intensive crop production schedules, and elimination ofnatural enemies by broad-spectrum chemical pesticides, promote colonization, feeding, and pop-ulation increase of insect pests. Furthermore, the highly simplified agricultural ecosystems canfavor generalist insects (i.e., species that can utilize multiple crops) and can facilitate the establish-ment of invasive species. Some of the most important agricultural pests are generalists or invasives(or both), forming superabundant populations that are often resistant to multiple insecticides andthat can devastate crop production. These challenges are set to intensify as the growing humanpopulation increases the demand for agricultural production, creating an ever-greater need foreffective crop protection against insect pests.

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The research community is well positioned to take up this mounting challenge by combin-ing the wealth of knowledge of plant-insect interactions and plant biotechnology. As this reviewdiscusses, multiple strategies for enhanced crop protection are at various stages of development.This multiplicity is a strength. Different approaches will be required for the following: differentenvironmental circumstances, especially the increased variability in rainfall and temperature asso-ciated with climate change (75); different crops, including new crop varieties designed for changingconditions (71) and new crop species generated by targeted domestication of plant species withdesirable traits (95); and different insect pests that arise through ecological release by changesin agricultural practice or climate and by human-mediated transport to different geographicalregions (10, 96, 135).

The repeated experience of insect pest control in the twentieth century has taught us that therecan be no permanent solution for insect crop pests because insects are under intense selectionpressure to break control measures, including plant resistance mechanisms (135). The durabilityof resistance traits in plants can be enhanced by adherence to well-designed resistance managementstrategies, as used with some success for Bt crops (20, 127). These approaches will increasingly becomplemented by new technologies that offer the potential to design crops that can be patchedin real time against evolving insect pests. For example, CRISPR can be used to edit a susceptibleallele of a plant immunity gene to a resistant allele; recent advances in metabolic engineeringare achieving in vivo changes in specific secondary metabolites by the swapping in/out of genescoding enzymes with different kinetics or substrate affinity (92); and microbes used for insectparatransgenesis, including microbial delivery of RNAi, can be reformulated for swift response toevolutionary change in insect pests (142).

The application of the wealth of knowledge and developing technologies to crop resistancestrategies is dependent not only on the science, but also on the regulatory framework for thecommercial release of novel crops. Regulation is necessarily conservative for the protection of theenvironment and human health (for different perspectives on this topic, see References 128 and129), but the cost and regional variation in regulatory procedures are major constraints on thetranslation of innovation to product (57, 101, 110). As CRISPR and other genetic technologiescome to the fore and metabolic engineering and microbiome science yield more reliable benefitsfor crop production, there is a new opportunity for regulation policy to achieve the optimalbalance between protection against the hazards of new technologies and the need for sustainableintensification of food production (33, 57, 76).

SUMMARY POINTS

1. Phytophagous insects, i.e., insects that feed on living plant tissues, damage crops eitherby chewing (breaking off and ingesting plant tissue) or by piercing and sucking (imbibingthe liquid contents of plant cells or phloem/xylem sap). Phytophagous insects, espe-cially lepidopteran caterpillars, beetles (Coleoptera), and hemipterans (including aphids,whiteflies, planthoppers, and stinkbugs), depress crop yield by feeding damage and trans-mission of plant disease agents.

2. The implementation of novel plant resistance traits against insect pests is facilitated byadvances in plant biotechnology, including genetic technologies for expression of proteintoxins (e.g., Bt crops), RNAi, and genome editing by CRISPR.


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3. Many crops have an impoverished secondary metabolite profile relative to related wildplant species, rendering the former susceptible to insect pests. Genetic modification ofsecondary metabolite pathways can repel insect pests and attract their natural enemies,although the magnitude and detail of the metabolic changes in plant tissues induced bygenetic manipulations can vary, sometimes in unpredicted ways.

4. Chewing insects interact with plant immunity via their secretions (e.g., saliva, regurgitant,frass) and wounding. These interactions often induce plant defensive traits against theinsect, but some insect products manipulate plant signaling networks to suppress insect-specific defensive responses.

5. Insects with the pierce-and-suck feeding habit release effector molecules into plant cells,and the resultant coevolutionary interactions with the plant immune system have selectedfor alleles of plant genes (R genes) conferring resistance against specific genotypes of theinsect pest.

6. Some crop pests, notably phloem-feeding hemipterans, are dependent on specific verti-cally transmitted microorganisms. Targeting of these microbial taxa, for example, by inplanta expression of antimicrobial peptides, has the potential to suppress populations ofthese crop pests.

7. Many phytophagous insects possess gut microorganisms ingested with their plant food.Members of these microbial communities can be engineered to deliver insecticidal agents,for example, by RNAi.

DISCLOSURE STATEMENT

The author is in receipt of funds from Bayer Crop Science for research that is not related directly tothe contents of this review. Apart from that, the author has no affiliations, memberships, funding,or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

This work was supported by foundational grants from the National Institute of Food and Agri-culture (2015-67013-23421 and GRANT12216941).

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https://doi.org/10.1002/bies.201600247

PP69_FrontMatter ARI 5 April 2018 9:11

Annual Review ofPlant Biology

Volume 69, 2018

Contents

My Secret LifeMary-Dell Chilton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Diversity of Chlorophototrophic Bacteria Revealed in the Omics EraVera Thiel, Marcus Tank, and Donald A. Bryant � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Genomics-Informed Insights into Endosymbiotic Organelle Evolutionin Photosynthetic EukaryotesEva C.M. Nowack and Andreas P.M. Weber � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �51

Nitrate Transport, Signaling, and Use EfficiencyYa-Yun Wang, Yu-Hsuan Cheng, Kuo-En Chen, and Yi-Fang Tsay � � � � � � � � � � � � � � � � � � � � �85

Plant VacuolesTomoo Shimada, Junpei Takagi, Takuji Ichino, Makoto Shirakawa,

and Ikuko Hara-Nishimura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 123

Protein Quality Control in the Endoplasmic Reticulum of PlantsRichard Strasser � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 147

Autophagy: The Master of Bulk and Selective RecyclingRichard S. Marshall and Richard D. Vierstra � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 173

Reactive Oxygen Species in Plant SignalingCezary Waszczak, Melanie Carmody, and Jaakko Kangasjarvi � � � � � � � � � � � � � � � � � � � � � � � � � � 209

Cell and Developmental Biology of Plant Mitogen-Activated ProteinKinasesGeorge Komis, Olga Samajova, Miroslav Ovecka, and Jozef Samaj � � � � � � � � � � � � � � � � � � � � � 237

Receptor-Like Cytoplasmic Kinases: Central Players in Plant ReceptorKinase–Mediated SignalingXiangxiu Liang and Jian-Min Zhou � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

Plant Malectin-Like Receptor Kinases: From Cell Wall Integrity toImmunity and BeyondChristina Maria Franck, Jens Westermann, and Aurelien Boisson-Dernier � � � � � � � � � � � � 301

Kinesins and Myosins: Molecular Motors that Coordinate CellularFunctions in PlantsAndreas Nebenfuhr and Ram Dixit � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

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The Oxylipin Pathways: Biochemistry and FunctionClaus Wasternack and Ivo Feussner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 363

Modularity in Jasmonate Signaling for Multistress ResilienceGregg A. Howe, Ian T. Major, and Abraham J. Koo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

Essential Roles of Local Auxin Biosynthesis in Plant Developmentand in Adaptation to Environmental ChangesYunde Zhao � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 417

Genetic Regulation of Shoot ArchitectureBing Wang, Steven M. Smith, and Jiayang Li � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 437

Heterogeneity and Robustness in Plant Morphogenesis: From Cellsto OrgansLilan Hong, Mathilde Dumond, Mingyuan Zhu, Satoru Tsugawa,

Chun-Biu Li, Arezki Boudaoud, Olivier Hamant, and Adrienne H.K. Roeder � � � � � � 469

Genetically Encoded Biosensors in Plants: Pathways to DiscoveryAnkit Walia, Rainer Waadt, and Alexander M. Jones � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 497

Exploring the Spatiotemporal Organization of Membrane Proteins inLiving Plant CellsLi Wang, Yiqun Xue, Jingjing Xing, Kai Song, and Jinxing Lin � � � � � � � � � � � � � � � � � � � � � � � 525

One Hundred Ways to Invent the Sexes: Theoretical and ObservedPaths to Dioecy in PlantsIsabelle M. Henry, Takashi Akagi, Ryutaro Tao, and Luca Comai � � � � � � � � � � � � � � � � � � � � � � 553

Meiotic Recombination: Mixing It Up in PlantsYingxiang Wang and Gregory P. Copenhaver � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 577

Population Genomics of Herbicide Resistance: Adaptation viaEvolutionary RescueJulia M. Kreiner, John R. Stinchcombe, and Stephen I. Wright � � � � � � � � � � � � � � � � � � � � � � � � � 611

Strategies for Enhanced Crop Resistance to Insect PestsAngela E. Douglas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 637

Preadaptation and Naturalization of Nonnative Species: Darwin’s TwoFundamental Insights into Species InvasionMarc W. Cadotte, Sara E. Campbell, Shao-peng Li, Darwin S. Sodhi,

and Nicholas E. Mandrak � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 661

Macroevolutionary Patterns of Flowering Plant Speciationand ExtinctionJana C. Vamosi, Susana Magallon, Itay Mayrose, Sarah P. Otto,

and Herve Sauquet � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 685

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When Two Rights Make a Wrong: The Evolutionary Genetics ofPlant Hybrid IncompatibilitiesLila Fishman and Andrea L. Sweigart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 707

The Physiological Basis of Drought Tolerance in Crop Plants:A Scenario-Dependent Probabilistic ApproachFrancois Tardieu, Thierry Simonneau, and Bertrand Muller � � � � � � � � � � � � � � � � � � � � � � � � � � � � 733

Paleobotany and Global Change: Important Lessons for Species toBiomes from Vegetation Responses to Past Global ChangeJennifer C. McElwain � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 761

Trends in Global Agricultural Land Use: Implications forEnvironmental Health and Food SecurityNavin Ramankutty, Zia Mehrabi, Katharina Waha, Larissa Jarvis,

Claire Kremen, Mario Herrero, and Loren H. Rieseberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 789

Errata

An online log of corrections to Annual Review of Plant Biology articles may be found athttp://www.annualreviews.org/errata/arplant

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strategies for enhanced crop resistance to insect pestsbiologia.ucr.ac.cr › profesores › garcia...

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