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Fungi in the genus Metarhizium (Hypocreales: Clavicipitaceae) are insect-pathogens and endophytes that can benefit their host plant through growth promotion and protection against stresses. Cochliobolus heterostrophus (Drechsler) Drechsler (Pleosporales: Pleosporaceae) is an economically-significant phytopathogenic fungus that causes Southern Corn Leaf Blight (SCLB) in maize. We conducted greenhouse and lab-based experiments to determine the effects of endophytic M. robertsii J.F. Bisch., Rehner & Humber on growth and defense in maize (Zea mays L.) infected with C. heterostrophus. We inoculated maize seeds with spores of M. robertsii and, at the 3 to 4-leaf stage, the youngest true leaf of M. robertsii-treated and untreated control plants with spores of C. heterostrophus. After 96 h, we measured maize height, above-ground biomass, endophytic colonization by M. robertsii, severity of SCLB, and expression of plant defense genes and phytohormone content. We recovered M. robertsii from 74% of plants grown from treated seed. The severity of SCLB in M. robertsii-treated maize plants was lower than in plants inoculated only with C. heterostrophus. M. robertsii-treated maize inoculated or not inoculated with C. heterostrophus showed greater height and above-ground biomass compared with untreated control plants. Height and above-ground biomass of maize co-inoculated with M. robertsii and C. heterostrophus were not different from M. robertsii-treated maize. M. robertsii modulated the expression of defense genes and the phytohormone content in maize inoculated with C. heterostrophus compared with plants not inoculated with C. heterostrophus and control plants. These results suggest that endophytic M. robertsii can promote maize growth and reduce development of SCLB, possibly by induced systemic resistance mediated by modulation of phytohormones and expression of defense and growth-related genes in maize., Competing Interests: The authors have declared that no competing interests exist.

Background: Maize (Zea mays L.) is a major cereal crop, with the United States accounting for over 40% of the worldwide production. Corn leaf aphid [CLA; Rhopalosiphum maidis (Fitch)] is an economically important pest of maize and several other monocot crops. In addition to feeding damage, CLA acts as a vector for viruses that cause devastating diseases in maize. We have shown previously that the maize inbred line Mp708, which was developed by classical plant breeding, provides heightened resistance to CLA. However, the transcriptomic variation conferring CLA resistance to Mp708 has not been investigated., Results: In this study, we contrasted the defense responses of the resistant Mp708 genotype to those of the susceptible Tx601 genotype at the transcriptomic (mRNA-seq) and volatile blend levels. Our results suggest that there was a greater transcriptomic remodeling in Mp708 plants in response to CLA infestation compared to the Tx601 plants. These transcriptomic signatures indicated an activation of hormonal pathways, and regulation of sesquiterpenes and terpenoid synthases in a constitutive and inducible manner. Transcriptomic analysis also revealed that the resistant Mp708 genotype possessed distinct regulation of ethylene and jasmonic acid pathways before and after aphid infestation. Finally, our results also highlight the significance of constitutive production of volatile organic compounds (VOCs) in Mp708 and Tx601 plants that may contribute to maize direct and/or indirect defense responses., Conclusions: This study provided further insights to understand the role of defense signaling networks in Mp708's resistance to CLA.

Fungi in the genus Metarhizium (Hypocreales: Clavicipitaceae) are insect pathogens that can establish as endophytes and can benefit their host plant. In field experiments, we observed a positive correlation between the prevalence of M. robertsii and legume cover crops, and a negative relationship with brassicaceous cover crops and with increasing proportion of cereal rye in mixtures. Here, we report the effects of endophytic M. robertsii on three cover crop species under greenhouse conditions. We inoculated seeds of Austrian winter pea ( Pisum sativum L., AWP), cereal rye ( Secale cereale L.), and winter canola ( Brassica napus L.) with conidia of M. robertsii to assess the effects of endophytic colonization on cover crop growth. We recovered M. robertsii from 59%, 46%, and 39% of seed-inoculated AWP, cereal rye, and canola plants, respectively. Endophytic M. robertsii significantly increased height and above-ground biomass of AWP and cereal rye but did not affect chlorophyll content of any of the cover crop species. Among inoculated plants from which we recovered M. robertsii , above-ground biomass of AWP was positively correlated with the proportion of colonized root but not leaf tissue sections. Our results suggest that winter cover crops may help to conserve Metarhizium spp. in annual cropping systems., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Plants produce suites of defenses that can collectively deter and reduce herbivory. Many defenses target the insect digestive system, with some altering the protective peritrophic matrix (PM) and causing increased permeability. The PM is responsible for multiple digestive functions, including reducing infections from potential pathogenic microbes. In our study, we developed axenic and gnotobiotic methods for fall armyworm ( Spodoptera frugiperda ) and tested how particular members present in the gut community influence interactions with plant defenses that can alter PM permeability. We observed interactions between gut bacteria with plant resistance. Axenic insects grew more but displayed lower immune-based responses compared with those possessing Enterococcus , Klebsiella , and Enterobacter isolates from field-collected larvae. While gut bacteria reduced performance of larvae fed on plants, none of the isolates produced mortality when injected directly into the hemocoel. Our results strongly suggest that plant physical and chemical defenses not only act directly upon the insect, but also have some interplay with the herbivore's microbiome. Combined direct and indirect, microbe-mediated assaults by maize defenses on the fall armyworm on the insect digestive and immune system reduced growth and elevated mortality in these insects. These results imply that plant-insect interactions should be considered in the context of potential mediation by the insect gut microbiome., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The corn leaf aphid (CLA; Rhopalosiphum maidis ) is a phloem sap-sucking insect that attacks many cereal crops, including maize ( Zea mays ). We previously showed that the maize inbred line Mp708, which was developed by classical plant breeding, provides enhanced resistance to CLA. Here, using electrophysiological monitoring of aphid feeding behavior, we demonstrate that Mp708 provides phloem-mediated resistance to CLA. Furthermore, feeding by CLA on Mp708 plants enhanced callose deposition, a potential defense mechanism utilized by plants to limit aphid feeding and subsequent colonization. In maize, benzoxazinoids (BX) or BX-derived metabolites contribute to enhanced callose deposition by providing heightened resistance to CLA. However, BX and BX-derived metabolites were not significantly altered in CLA-infested Mp708 plants, indicating BX-independent defense against CLA. Evidence presented here suggests that the constitutively higher levels of 12-oxo-phytodienoic acid (OPDA) in Mp708 plants contributed to enhanced callose accumulation and heightened CLA resistance. OPDA enhanced the expression of ethylene biosynthesis and receptor genes, and the synergistic interactions of OPDA and CLA feeding significantly induced the expression of the transcripts encoding Maize insect resistance1-Cysteine Protease, a key defensive protein against insect pests, in Mp708 plants. Furthermore, exogenous application of OPDA on maize jasmonic acid-deficient plants caused enhanced callose accumulation and heightened resistance to CLA, suggesting that the OPDA-mediated resistance to CLA is independent of the jasmonic acid pathway. We further demonstrate that the signaling function of OPDA, rather than a direct toxic effect, contributes to enhanced CLA resistance in Mp708., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

In this study we examined global changes in protein expression in both roots and leaves of maize plants attacked by the root herbivore, Western corn rootworm (WCR, Diabrotica virgifera virgifera). The changes in protein expression Are indicative of metabolic changes during WCR feeding that enable the plant to defend itself. This is one of the first studies to look above- and below-ground at global protein expression patterns of maize plants grown in soil and infested with a root herbivore. We used advanced proteomic and network analyses to identify metabolic pathways that contribute to global defenses deployed by the insect resistant maize genotype, Mp708, infested with WCR. Using proteomic analysis, 4878 proteins in roots and leaves were detected and of these 863 showed significant changes of abundance during WCR infestation. Protein abundance patterns were analyzed using hierarchical clustering, protein correlation and protein-protein interaction networks. All three data analysis pipelines showed that proteins such as jasmonic acid biosynthetic enzymes, serine proteases, protease inhibitors, proteins involved in biosynthesis and signaling of ethylene, and enzymes producing reactive oxygen species and isopentenyl pyrophosphate, a precursor for volatile production, were upregulated in roots during WCR infestation. In leaves, highly abundant proteins were involved in signal perception suggesting activation of systemic signaling. We conclude that these protein networks contribute to the overall herbivore defense mechanisms in Mp708. Because the plants were grown in potting mix and not sterilized sand, we found that both microbial and insect defense-related proteins were present in the roots. The presence of the high constitutive levels of reduced ascorbate in roots and benzothiazole in the root volatile profiles suggest a tight tri-trophic interaction among the plant, soil microbiomes and WCR-infested roots suggesting that defenses against insects coexist with defenses against bacteria and fungi due to the interaction between roots and soil microbiota. In this study, which is one of the most complete descriptions of plant responses to root-feeding herbivore, we established an analysis pipeline for proteomics data that includes network biology that can be used with different types of "omics" data from a variety of organisms.

The underlying adaptive mechanisms by which insect strains are associated with specific plants are largely unknown. In this study, we investigated the role of herbivore-induced defenses in the host plant association of fall armyworm (Spodoptera frugiperda) strains. We tested the expression of herbivore-induced defense-related genes and the activity of plant-defensive proteins in maize and Bermuda grass upon feeding by fall armyworm strains. The rice strain caterpillars induced greater accumulation of proteinase inhibitors in maize than the corn strain caterpillars. In Bermuda grass, feeding by the corn strain suppressed induction of trypsin inhibitor activity whereas the rice strain induced greater activity levels. Differences in elicitation of these plant defenses by the two strains seems to be due to differences in the activity levels of the salivary enzyme phospholipase C. The levels of plant defense responses were negatively correlated with caterpillar growth, indicating a fitness effect. Our results indicate that specific elicitors in the saliva of fall armyworm stains trigger differential levels of plant defense responses that affect caterpillar growth and thus may influence host plant associations in field conditions. The composition and secretion of plant defense elicitors may have a strong influence in the host plant association of insect herbivores., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Insect resistance against root herbivores like the western corn rootworm (WCR, Diabrotica virgifera virgifera) is not well understood in non-transgenic maize. We studied the responses of two American maize inbreds, Mp708 and Tx601, to WCR infestation using biomechanical, molecular, biochemical analyses, and laser ablation tomography. Previous studies performed on several inbreds indicated that these two maize genotypes differed in resistance to pests including fall armyworm (Spodoptera frugiperda) and WCR. Our data confirmed that Mp708 shows resistance against WCR, and demonstrates that the resistance mechanism is based in a multi-trait phenotype that includes increased resistance to cutting in nodal roots, stable root growth during insect infestation, constitutive and induced expression of known herbivore-defense genes, including ribosomal inhibitor protein 2 (rip2), terpene synthase 23 (tps23) and maize insect resistance cysteine protease-1 (mir1), as well high constitutive levels of jasmonic acid and production of (E)-β-caryophyllene. In contrast, Tx601 is susceptible to WCR. These findings will facilitate the use of Mp708 as a model to explore the wide variety of mechanisms and traits involved in plant defense responses and resistance to herbivory by insects with several different feeding habits.

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Lepidopteran larvae secrete saliva on plant tissues during feeding. Components in the saliva may aid in food digestion, whereas other components are recognized by plants as cues to elicit defense responses. Despite the ecological and economical importance of these plant-feeding insects, knowledge of their saliva composition is limited to a few species. In this study, we identified the salivary proteins of larvae of the fall armyworm (FAW), Spodoptera frugiperda; determined qualitative and quantitative differences in the salivary proteome of the two host races-corn and rice strains-of this insect; and identified changes in total protein concentration and relative protein abundance in the saliva of FAW larvae associated with different host plants. Quantitative proteomic analyses were performed using labeling with isobaric tags for relative and absolute quantification followed by liquid chromatography-tandem mass spectrometry. In total, 98 proteins were identified (>99% confidence) in the FAW saliva. These proteins were further categorized into five functional groups: proteins potentially involved in (1) plant defense regulation, (2) herbivore offense, (3) insect immunity, (4) detoxification, (5) digestion, and (6) other functions. Moreover, there were differences in the salivary proteome between the FAW strains that were identified by label-free proteomic analyses. Thirteen differentially identified proteins were present in each strain. There were also differences in the relative abundance of eleven salivary proteins between the two FAW host strains as well as differences within each strain associated with different diets. The total salivary protein concentration was also different for the two strains reared on different host plants. Based on these results, we conclude that the FAW saliva contains a complex mixture of proteins involved in different functions that are specific for each strain and its composition can change plastically in response to diet type., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Plant defenses to insect herbivores have been studied in response to several insect behaviors on plants such as feeding, crawling, and oviposition. However, we have only scratched the surface about how insect feces induce plant defenses. In this study, we measured frass-induced plant defenses in maize, rice, cabbage, and tomato by chewing herbivores such as European corn borer (ECB), fall armyworm (FAW), cabbage looper (CL), and tomato fruit worm (TFW). We observed that caterpillar frass induced plant defenses are specific to each host-herbivore system, and they may induce herbivore or pathogen defense responses in the host plant depending on the composition of the frass deposited on the plant, the plant organ where it is deposited, and the species of insect. This study adds another layer of complexity in plant-insect interactions where analysis of frass-induced defenses has been neglected even in host-herbivore systems where naturally frass accumulates in enclosed feeding sites over extended periods of time.

The vasculature of plants act as a channel for transport of signal(s) that facilitate long-distance intraplant communication. In maize, Maize insect resistance1-Cysteine Protease (Mir1-CP), which has homology to papain-like proteases, provides defense to different feeding guilds of insect pests. Furthermore, accumulation of Mir1-CP in the vasculature suggests that Mir1-CP can potentially function as a phloem-mobile protein. In a recent study, we provided evidence that Mir1-CP can curtail the growth of phloem-sap sucking insect, corn leaf aphid (CLA; Rhopalosiphum maidis). Our current study further examined whether aboveground feeding by CLA can induce resistance to subsequent herbivory by belowground feeding western corn rootworm (WCR; Diabrotica virgifera virgifera). Aboveground feeding by CLA systemically induced the accumulation of Mir1-CP in the roots. Furthermore, foliage feeding by CLA provided enhanced resistance to subsequent herbivory by belowground feeding of WCR. Taken together, our previous findings and results presented here indicate that long-distance transport of Mir1-CP is critical for providing enhanced resistance to insect attack in maize.

The perception of herbivory by plants is known to be triggered by the deposition of insect-derived factors such as saliva and oral secretions, oviposition materials, and even feces. Such insect-derived materials harbor chemical cues that may elicit herbivore and/or pathogen-induced defenses in plants. Several insect-derived molecules that trigger herbivore-induced defenses in plants are known; however, insect-derived molecules suppressing them are largely unknown. In this study, we identified two plant chitinases from fall armyworm (Spodoptera frugiperda) larval frass that suppress herbivore defenses while simultaneously inducing pathogen defenses in maize (Zea mays). Fall armyworm larvae feed in enclosed whorls of maize plants, where frass accumulates over extended periods of time in close proximity to damaged leaf tissue. Our study shows that maize chitinases, Pr4 and Endochitinase A, are induced during herbivory and subsequently deposited on the host with the feces. These plant chitinases mediate the suppression of herbivore-induced defenses, thereby increasing the performance of the insect on the host. Pr4 and Endochitinase A also trigger the antagonistic pathogen defense pathway in maize and suppress fungal pathogen growth on maize leaves. Frass-induced suppression of herbivore defenses by deposition of the plant-derived chitinases Pr4 and Endochitinase A is a unique way an insect can co-opt the plant's defense proteins for its own benefit. It is also a phenomenon unlike the induction of herbivore defenses by insect oral secretions in most host-herbivore systems., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Caterpillar behaviors such as feeding, crawling, and oviposition are known to induce defenses in maize and other plant species. We examined plant defense responses to another important caterpillar behavior, their defecation. Fall armyworms (FAW, Spodoptera frugiperda), a major threat to maize (Zea mays), are voracious eaters and deposit copious amounts of frass in the enclosed whorl tissue surrounding their feeding site, where it remains for long periods of time. FAW frass is composed of molecules derived from the host plant, the insect itself, and associated microbes, and hence provides abundant cues that may alter plant defense responses. We observed that proteins from FAW frass initially induced wound-responsive defense genes in maize; however, a pathogenesis-related (pr) defense gene was induced as the time after application increased. Elicitation of pathogen defenses by frass proteins was correlated with increased herbivore performance and reduced fungal pathogen performance over time. These responses differ from the typical plant response to oral secretions of the FAW. The results pave the way for identification of protein molecule(s) from the excretion of an herbivore that elicits pathogen defense responses while attenuating herbivore defenses in plants.

Signaling networks among multiple phytohormones fine-tune plant defense responses to insect herbivore attack. Previously, it was reported that the synergistic combination of ethylene (ET) and jasmonic acid (JA) was required for accumulation of the maize insect resistance1 (mir1) gene product, a cysteine (Cys) proteinase that is a key defensive protein against chewing insect pests in maize (Zea mays). However, this study suggests that mir1-mediated resistance to corn leaf aphid (CLA; Rhopalosiphum maidis), a phloem sap-sucking insect pest, is independent of JA but regulated by the ET-signaling pathway. Feeding by CLA triggers the rapid accumulation of mir1 transcripts in the resistant maize genotype, Mp708. Furthermore, Mp708 provided elevated levels of antibiosis (limits aphid population)- and antixenosis (deters aphid settling)-mediated resistance to CLA compared with B73 and Tx601 maize susceptible inbred lines. Synthetic diet aphid feeding trial bioassays with recombinant Mir1-Cys Protease demonstrates that Mir1-Cys Protease provides direct toxicity to CLA. Furthermore, foliar feeding by CLA rapidly sends defensive signal(s) to the roots that trigger belowground accumulation of the mir1, signifying a potential role of long-distance signaling in maize defense against the phloem-feeding insects. Collectively, our data indicate that ET-regulated mir1 transcript accumulation, uncoupled from JA, contributed to heightened resistance to CLA in maize. In addition, our results underscore the significance of ET acting as a central node in regulating mir1 expression to different feeding guilds of insect herbivores., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

In addition to feeding damage, herbivores release cues that are recognized by plants to elicit defenses. Caterpillar oral secretions have been shown to trigger herbivore defense responses in several different plant species. In this study, the effects of two fall armyworm (Spodoptera frugiperda) oral secretions (saliva and regurgitant) on caterpillar defense responses in maize (Zea mays) were examined. Only minute amounts of regurgitant were deposited on the maize leaf during larval feeding bouts and its application to leaves failed to induce the expression of several herbivore defense genes. On the other hand, caterpillars consistently deposited saliva on leaves during feeding and the expression of several maize defense genes significantly increased in response to saliva application and larval feeding. However, feeding by ablated caterpillars with impaired salivation did not induce these defenses. Furthermore, bioassays indicated that feeding by unablated caterpillars significantly enhanced defenses when compared with that of ablated caterpillars. Another critical finding was that the maize genotype and stage of development affected the expression of defense genes in response to wounding and regurgitant treatments. These results demonstrate that fall armyworm saliva contains elicitors that trigger herbivore defenses in maize.

Some plant-derived anti-herbivore defensive proteins are induced by insect feeding, resist digestion in the caterpillar gut and are eliminated in the frass. We have identified several maize proteins in fall armyworm (Spodoptera frugiperda) frass that potentially play a role in herbivore defense. Furthermore, the toxicity of one of these proteins, ribosome-inactivating protein 2 (RIP2), was assessed and factors regulating its accumulation were determined. To understand factors regulating RIP2 protein accumulation, maize (Zea mays) plants were infested with fall armyworm larvae or treated with exogenous hormones. The toxicity of recombinant RIP2 protein against fall armyworm was tested. The results show that RIP2 protein is synthesized as an inactive proenzyme that can be processed in the caterpillar gut. Also, caterpillar feeding, but not mechanical wounding, induced foliar RIP2 protein accumulation. Quantitative real-time PCR indicated that RIP2 transcripts were rapidly induced (1 h) and immunoblot analysis indicated that RIP2 protein accumulated soon after attack and was present in the leaf for up to 4 d after caterpillar removal. Several phytohormones, including methyl jasmonate, ethylene, and abscisic acid, regulated RIP2 protein expression. Furthermore, bioassays of purified recombinant RIP2 protein against fall armyworm significantly retarded caterpillar growth. We conclude that the toxic protein RIP2 is induced by caterpillar feeding and is one of a potential suite of proteins that defend maize against chewing herbivores., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Plants can recognize the insect elicitors and activate its defense mechanisms. European Corn Borer (ECB; Ostrinia nubilalis) saliva, produced from the labial salivary glands and released through the spinneret, is responsible for inducing direct defenses in host plants. Glucose oxidase (GOX) present in the ECB saliva induced direct defenses in tomato. By contrast, GOX activity in ECB saliva was insufficient to trigger defenses in maize, suggesting that host-specific salivary elicitors are responsible for inducing direct defenses in host plants. Our current study further examined whether ECB saliva can trigger indirect defenses in tomato. Relative expression levels of TERPENE SYNTHASE5 (TPS5) and HYDROPEROXIDE LYASE (HPL), marker for indirect defenses in host plants, were monitored. Quantitative real-time PCR analysis revealed that ECB saliva can induce the expression of TPS5 and HPL, suggesting that salivary signals can induce indirect defenses in addition to the direct defenses. Further experiments are required to identify different ECB elicitors that are responsible for inducing direct and indirect defenses in host plants.

Induced plant defenses in response to herbivore attack are modulated by cross-talk between jasmonic acid (JA)- and salicylic acid (SA)-signaling pathways. Oral secretions from some insect herbivores contain effectors that overcome these antiherbivore defenses. Herbivores possess diverse microbes in their digestive systems and these microbial symbionts can modify plant-insect interactions; however, the specific role of herbivore-associated microbes in manipulating plant defenses remains unclear. Here, we demonstrate that Colorado potato beetle (Leptinotarsa decemlineata) larvae exploit bacteria in their oral secretions to suppress antiherbivore defenses in tomato (Solanum lycopersicum). We found that antibiotic-untreated larvae decreased production of JA and JA-responsive antiherbivore defenses, but increased SA accumulation and SA-responsive gene expression. Beetles benefit from down-regulating plant defenses by exhibiting enhanced larval growth. In SA-deficient plants, suppression was not observed, indicating that suppression of JA-regulated defenses depends on the SA-signaling pathway. Applying bacteria isolated from larval oral secretions to wounded plants confirmed that three microbial symbionts belonging to the genera Stenotrophomonas, Pseudomonas, and Enterobacter are responsible for defense suppression. Additionally, reinoculation of these bacteria to antibiotic-treated larvae restored their ability to suppress defenses. Flagellin isolated from Pseudomonas sp. was associated with defense suppression. Our findings show that the herbivore exploits symbiotic bacteria as a decoy to deceive plants into incorrectly perceiving the threat as microbial. By interfering with the normal perception of herbivory, beetles can evade antiherbivore defenses of its host.

Plants turn on induced defenses upon insect herbivory. In the current study, we evaluated the role of European corn borer (ECB) elicitors (molecules secreted by herbivores) that either induce/suppress defenses in Solanum lycopersicum (tomato) and Zea mays (maize), two very important crop plants that are grown for food and/or fuel throughout the world. We used a combination of molecular, biochemical, confocal and scanning electron microscopy, caterpillar spinneret ablation/cauterization, and conventional insect bioassay methods to determine the role of ECB elicitors in modulating defenses in both tomato and maize crop plants. Our results clearly demonstrate that the components present in the ECB saliva induce defense-related proteinase inhibitors in both tomato (PIN2) and maize (MPI). Presence of glucose oxidase in the ECB saliva induced defenses in tomato, but not in maize. However, ECB saliva induced genes present in the jasmonic acid biosynthesis pathway in both tomato and maize. Although ECB saliva can induce defenses in both tomato and maize, our results suggest that host-specific salivary components are responsible for inducing host plant defenses. Proteomic analysis of ECB salivary elicitors and plant receptors/signaling mechanisms involved in recognizing different ECB elicitors remains to be determined., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Many studies have documented the induction of belowground defenses in plants in response to aboveground herbivory and vice versa, but the genes and signaling molecules mediating systemic induction are not well understood. We performed comparative microarray analysis on maize whorl and root tissues from the insect resistant inbred Mp708 in response to foliar feeding by fall armyworm (Spodoptera frugiperda) caterpillars. Although Mp708 has elevated jasmonic acid (JA) levels prior to herbivory, genes involved in JA biosynthesis were up-regulated in whorls in response to fall armyworm feeding. Alternatively, genes possibly involved in regulating ethylene (ET) perception and signaling were up-regulated in roots following foliar herbivory. Transcript levels of genes encoding proteins involved in direct defenses against herbivores were enhanced both in roots and leaves, but transcriptional factors and genes involved in various biosynthetic pathways were selectively down-regulated in the whorl. The results indicate that foliar herbivory by fall armyworm changes root gene expression pathways suggesting profound long distance signaling. Tissue specific induction and suppression of JA and ET signaling pathway genes provides a clue to their possible roles in signaling between the two distant tissue types that eventually triggers defense responses in the roots in response to foliar herbivory., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Herbivore microbial associates can affect diverse interactions between plants and insect herbivores. Some insect symbionts enable herbivores to expand host plant range or to facilitate host plant use by modifying plant physiology. However, little attention has been paid to the role of herbivore-associated microbes in manipulating plant defenses. We have recently shown that Colorado potato beetle secrete the symbiotic bacteria to suppress plant defenses. The bacteria in oral secretions from the beetle hijack defense signaling pathways of host plants and the suppression of induced plant defenses benefits the beetle's performance. While the defense suppression by the beetle-associated bacteria has been investigated in local damaged leaves, little is known about the effects of the symbiotic bacteria on the manipulation of plant defenses in systemic undamaged leaves. Here, we demonstrate that the symbiotic bacteria suppress plant defenses in both local and systemic tissues when plants are attacked by antibiotic-untreated larvae.

Chloroplast-localized small heat-shock proteins (Cp-sHSP) protect Photosystem II and thylakoid membranes during heat and other stresses, and Cp-sHSP production levels are related to plant thermotolerance. However, to date, a paucity of Cp-sHSP sequences from C4 or CAM species, or from other extremely heat-tolerant species, has precluded an examination to determine if Cp-sHSP genes or proteins might differ among plants with photosynthetic pathways or between heat-sensitive and heat-tolerant species. To investigate this, we isolated and characterized novel Cp-sHSP genes in four plant species: two moderately heat-tolerant C4 species, Spartina alterniflora (monocot) and Amaranthus retroflexus (eudicot), and two very heat-tolerant CAM species, Agave americana (monocot) and Ferocactus wislizenii (eudicot) (respective genes: SasHSP27.12, ArsHSP26.43, AasHSP26.85 and FwsHSP27.52) by PCR-based genome walking and cDNA RACE. Analysis of these Cp-sHSPs has confirmed the presence of conserved domains common to previously examined species. As expected, the transit peptide was found to be the most variable part of these proteins. Promoter analysis of these genes revealed differences in CAM versus C3 and C4 species that were independent of a general difference between monocots and eudicots observed for the entire protein. Heat-induced gene and protein expression indicated that Cp-sHSP protein levels were correlated with thermotolerance of photosynthetic electron transport, and that in most cases protein and transcript levels were correlated. Thus, available evidence indicates little variation in the amino acid sequence of Cp-sHSP mature proteins between heat-sensitive and -tolerant species, but that variation in Cp-sHSP protein production is related to heat tolerance or photosynthetic pathway (CAM vs. C3 and C4) and is driven by promoter differences. Key message We isolated and characterized four novel Cp-sHSP genes with promoters from wild plants, analysis has shown qualitative and quantitative interspecific variations in Cp-sHSPs of C3, C4, and CAM plant thermotolerance.

Various pests, such as those in the order Lepidoptera, frequently feed on young maize (Zea mays) plants and pose a significant threat to plant development and survival. To manage this problem, maize generates a wide variety of responses to attack by pests, from activation of wound-response pathways to the release of volatile compounds. Mp708, an inbred line resistant to feeding by the larvae of the fall armyworm (Spodoptera frugiperda J.E. Smith Lepidoptera: Noctuidae), has been developed through traditional breeding methods, but its underlying mechanisms of resistance are still not completely understood. Mp708 has been shown to have a moderately high constitutive expression of jasmonic acid (JA) before infestation by fall armyworm. However, Tx601, a genotype susceptible to feeding by fall armyworm, activates JA pathway only in response to feeding, suggesting that Mp708 is "primed" to respond swiftly to an attack. Current research indicates that fall armyworm show a lack of preference to feeding on Mp708, leading to the hypothesis that volatiles constitutively released by the plant may also play an important role in its resistance. Analysis of volatiles released by Mp708 and Tx601 in the presence and absence of fall armyworm larvae identified (E)-beta-caryophyllene, a terpenoid associated with resistance, released constitutively in Mp708. Fall armyworm fed samples of both Mp708 and Tx601 showed high transcript number of tps23, the gene responsible for the synthesis of (E)-beta-caryophyllene. In addition, fall armyworm larvae show a preference for Tx601 whorl tissue over Mp708 tissue, and the dosage of Tx601 whorl with (E)-beta-caryophyllene repels the fall armyworm.

Caterpillars produce oral secretions that may serve as cues to elicit plant defenses, but in other cases these secretions have been shown to suppress plant defenses. Ongoing work in our laboratory has focused on the salivary secretions of the tomato fruitworm, Helicoverpa zea. In previous studies we have shown that saliva and its principal component glucose oxidase acts as an effector by suppressing defenses in tobacco. In this current study, we report that saliva elicits a burst of jasmonic acid (JA) and the induction of late responding defense genes such as proteinase inhibitor 2 (Pin2). Transcripts encoding early response genes associated with the JA pathway were not affected by saliva. We also observed a delayed response to saliva with increased densities of Type VI glandular trichomes in newly emerged leaves. Proteomic analysis of saliva revealed glucose oxidase (GOX) was the most abundant protein identified and we confirmed that it plays a primary role in the induction of defenses in tomato. These results suggest that the recognition of GOX in tomato may represent a case for effector-triggered immunity. Examination of saliva from other caterpillar species indicates that saliva from the noctuids Spodoptera exigua and Heliothis virescens also induced Pin2 transcripts.

Insect oviposition on plants frequently precedes herbivory. Accumulating evidence indicates that plants recognize insect oviposition and elicit direct or indirect defenses to reduce the pressure of future herbivory. Most of the oviposition-triggered plant defenses described thus far remove eggs or keep them away from the host plant or their desirable feeding sites. Here, we report induction of antiherbivore defense by insect oviposition which targets newly hatched larvae, not the eggs, in the system of tomato Solanum lycopersicum L., and tomato fruitworm moth Helicoverpa zea Boddie. When tomato plants were oviposited by H. zea moths, pin2, a highly inducible gene encoding protease inhibitor2, which is a representative defense protein against herbivorous arthropods, was expressed at significantly higher level at the oviposition site than surrounding tissues, and expression decreased with distance away from the site of oviposition. Moreover, more eggs resulted in higher pin2 expression in leaves, and both fertilized and unfertilized eggs induced pin2 expression. Notably, when quantified daily following deposition of eggs, pin2 expression at the oviposition site was highest just before the emergence of larvae. Furthermore, H. zea oviposition primed the wound-induced increase of pin2 transcription and a burst of jasmonic acid (JA); tomato plants previously exposed to H. zea oviposition showed significantly stronger induction of pin2 and higher production of JA upon subsequent simulated herbivory than without oviposition. Our results suggest that tomato plants recognize H. zea oviposition as a signal of impending future herbivory and induce defenses to prepare for this herbivory by newly hatched neonate larvae.

The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5'-triphosphate (ATP) in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5'-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.

Production of chloroplast-localized small heat-shock proteins (Cp-sHSP) is correlated with increased thermotolerance in plants. Ecotypic variation in function and expression of Cp-sHSPs was analyzed in two Chenopodium album ecotypes from cool vs. warm-temperate USA habitats [New York (NY) and Mississippi (MS) respectively]. P(et) was more heat tolerant in the MS than the NY ecotype, and MS ecotype derived proportionally greater protection of P(et) by Cp-sHSP during high temperatures. Four genes encoding Cp-sHSPs were isolated and characterized: CaHSP25.99n (NY-1) and CaHSP26.23n (NY-2) from NY ecotype, and CaHSP26.04m (MS-1) and CaHSP26.26m (MS-2) from MS ecotype. The genes were nearly identical in predicted amino-acid sequence and hydrophobicity. Gene expression analysis indicated that MS-1 and MS-2 transcripts were constitutively expressed at low levels at 25 °C, while no NY-1 and NY-2 transcripts were detected at this temperature. Maximum accumulation of NY-1 and NY-2 transcripts occurred at 33 °C and 40 °C for MS-1 and MS-2. Immunoblot analysis revealed that (1) protein expression was highest at 37 °C in both ecotypes, but was greater in MS than NY ecotype at 40 °C; and (2) import of Cp-sHSP into chloroplasts was more heat-labile in NY ecotype. The higher expression of one isoform in MS ecotype may contribute to its enhanced thermotolerance. Absence of correlation between protein and transcript levels, suggests the post-transcriptional regulation is occurring. Promoter analysis of these genes revealed significant variations in heat-shock elements (HSE), core motifs required for heat-shock-factor binding. We propose a correlation between unique promoter architecture, Cp-sHSP expression and thermotolerance in both ecotypes., (Published by Elsevier Masson SAS.)

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles of vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.

Several maize, Zea mays L., inbred lines developed from an Antiguan maize population have been shown to exhibit resistance to numerous aboveground lepidopteran pests. This study shows that these genotypes are able to significantly reduce the survival of two root feeding pests, western corn rootworm, Diabrotica virgifera virgifera LeConte, and southern corn rootworm, Diabrotica undecimpunctata howardi Barber. The results also demonstrated that feeding by the aboveground herbivore fall armyworm, Spodoptera frugiperda (J. E. Smith), before infestation by western corn rootworm reduced survivorship of western corn rootworm in the root tissues of some, but not all, genotypes. Likewise, the presence of western corn rootworm in the soil seemed to increase resistance to fall armyworm in the whorl in several genotypes. However, genotypes derived from the Antiguan germplasm with genetic resistance to lepidopterans were still more resistant to the fall armyworm and both rootworm species than the susceptible genotypes even after defense induction. These results suggest that there may be intraplant communication that alters plant responses to aboveground and belowground herbivores.

Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Insect pests that attempt to feed on the caterpillar-resistant maize genotype Mp708 encounter a potent, multipronged defense system that thwarts their invasion. First, these plants are on "constant alert" due to constitutively elevated levels of the phytohormone jasmonic acid that signals the plant to activate its defenses. The higher jasmonic acid levels trigger the expression of defense genes prior to herbivore attack so the plants are "primed" and respond with a faster and stronger defense. The second defense is the rapid accumulation of a toxic cysteine protease called Mir1-CP in the maize whorl in response to caterpillar feeding. When caterpillars ingest Mir1-CP, it damages the insect's midgut and retards their growth. In this article, we discuss a third possible defense strategy employed by Mp708. We have shown that foliar caterpillar feeding causes Mir1-CP and defense gene transcripts to accumulate in its roots. We propose that caterpillar feeding aboveground sends a signal belowground via the phloem that results in Mir1-CP accumulation in the roots. We also postulate that the roots serve as a reservoir of Mir1-CP that can be mobilized to the whorl in response to caterpillar assault.

Background: Aspergillus flavus Link:Fr, an opportunistic fungus that produces aflatoxin, is pathogenic to maize and other oilseed crops. Aflatoxin is a potent carcinogen, and its presence markedly reduces the value of grain. Understanding and enhancing host resistance to A. flavus infection and/or subsequent aflatoxin accumulation is generally considered an efficient means of reducing grain losses to aflatoxin. Different proteomic, genomic and genetic studies of maize (Zea mays L.) have generated large data sets with the goal of identifying genes responsible for conferring resistance to A. flavus, or aflatoxin., Results: In order to maximize the usage of different data sets in new studies, including association mapping, we have constructed a relational database with web interface integrating the results of gene expression, proteomic (both gel-based and shotgun), Quantitative Trait Loci (QTL) genetic mapping studies, and sequence data from the literature to facilitate selection of candidate genes for continued investigation. The Corn Fungal Resistance Associated Sequences Database (CFRAS-DB) (http://agbase.msstate.edu/) was created with the main goal of identifying genes important to aflatoxin resistance. CFRAS-DB is implemented using MySQL as the relational database management system running on a Linux server, using an Apache web server, and Perl CGI scripts as the web interface. The database and the associated web-based interface allow researchers to examine many lines of evidence (e.g. microarray, proteomics, QTL studies, SNP data) to assess the potential role of a gene or group of genes in the response of different maize lines to A. flavus infection and subsequent production of aflatoxin by the fungus., Conclusions: CFRAS-DB provides the first opportunity to integrate data pertaining to the problem of A. flavus and aflatoxin resistance in maize in one resource and to support queries across different datasets. The web-based interface gives researchers different query options for mining the database across different types of experiments. The database is publically available at http://agbase.msstate.edu.

In this study, we performed the first high-throughput proteomic analysis of developing rachis (cob) from maize genotype Mp313E. Using two proteomic approaches, 2-DE and 2-D LC, we identified 967 proteins. A 2-D proteome reference map was established. Functional classification of identified proteins revealed that proteins involved in various cellular metabolisms, response to stimulus and transport, were the most abundant.

This study was conducted to determine if constitutive levels of jasmonic acid (JA) and other octadecanoid compounds were elevated prior to herbivory in a maize genotype with documented resistance to fall armyworm (Spodoptera frugiperda) and other lepidopteran pests. The resistant inbred Mp708 had approximately 3-fold higher levels of jasmonic acid (JA) prior to herbivore feeding than the susceptible inbred Tx601. Constitutive levels of cis-12-oxo-phytodienoic acid (OPDA) also were higher in Mp708 than Tx601. In addition, the constitutive expression of JA-inducible genes, including those in the JA biosynthetic pathway, was higher in Mp708 than Tx601. In response to herbivory, Mp708 generated comparatively higher levels of hydrogen peroxide, and had a greater abundance of NADPH oxidase transcripts before and after caterpillar feeding. Before herbivore feeding, low levels of transcripts encoding the maize insect resistance cysteine protease (Mir1-CP) and the Mir1-CP protein were detected consistently. Thus, Mp708 appears to have a portion of its defense pathway primed, which results in constitutive defenses and the ability to mount a stronger defense when caterpillars attack. Although the molecular mechanisms that regulate the constitutive accumulation of JA in Mp708 are unknown, it might account for its enhanced resistance to lepidopteran pests. This genotype could be valuable in studying the signaling pathways that maize uses to response to insect herbivores.

The dramatic movements of some carnivorous plants species are triggered by sensory structures derived from trichomes. While unusual plant species such as the Venus fly trap and sundews may be expected to have elaborate sensors to capture their insect prey, more modest plant species might not be expected to have similar sensory capabilities. Our recent work, however, has revealed that glandular trichomes on tomato (Solanum lycopersicum) appear to have a function similar to trigger hairs of carnivorous species, acting as "early warning" sensors. Using a combination of behavioral, molecular, and biochemical techniques, we determined that caterpillars, moths and mechanical disruption upregulate signaling molecules and defensive genes found in glandular trichomes. Importantly, we discovered that plants whose trichomes have been broken respond more vigorously when their defenses were induced. Taken together, our results suggest that glandular trichomes can act as sensors that detect activity on the leaf surface, and ready plants for herbivore attack.

In plants, ethylene and jasmonate control the defense responses to multiple stressors, including insect predation. Among the defense proteins known to be regulated by ethylene is maize insect resistance 1-cysteine protease (Mir1-CP). This protein is constitutively expressed in the insect-resistant maize (Zea mays) genotype Mp708; however, its abundance significantly increases during fall armyworm (Spodoptera frugiperda) herbivory. Within 1 h of herbivory by fall armyworm, Mir1-CP accumulates at the feeding site and continues to increase in abundance until 24 h without any increase in its transcript (mir1) levels. To resolve this discrepancy and elucidate the role of ethylene and jasmonate in the signaling of Mir1-CP expression, the effects of phytohormone biosynthesis and perception inhibitors on Mir1-CP expression were tested. Immunoblot analysis of Mir1-CP accumulation and quantitative reverse-transcriptase polymerase chain reaction examination of mir1 levels in these treated plants demonstrate that Mir1-CP accumulation is regulated by both transcript abundance and protein expression levels. The results also suggest that jasmonate functions upstream of ethylene in the Mir1-CP expression pathway, allowing for both low-level constitutive expression and a two-stage defensive response, an immediate response involving Mir1-CP accumulation and a delayed response inducing mir1 transcript expression.

The ability of caterpillar or moth 'footsteps' to elicit defenses in the tomato (Solanum lycopersicum) plant was examined. Although touch responses frequently have been observed in plants, the role of herbivore 'touch' in eliciting antiherbivore defenses has not been adequately examined. A combination of methods, including in situ hybridization, reverse transcriptase-polymerase chain reaction, quantitative real-time polymerase chain reaction and gas chromatography-mass spectrometry, was used to determine the role of trichomes in mediating these touch responses. Mutants compromised in jasmonic acid and glandular trichomes were used to test whether both of these were required for these touch responses. We demonstrated that the rupture of foliar glandular trichomes by caterpillar or moth contact induced the expression of defense transcripts (e.g. proteinase inhibitor 2, or PIN2) regulated by jasmonic acid. Neither chewing nor the release of salivary components was required to initiate this induced response. Jasmonic acid and the genes encoding proteins involved in its biosynthesis were identified in the trichomes. Using mutants, we showed that both jasmonic acid and trichomes were required for the contact-induced expression of PIN2. In addition, hydrogen peroxide, formed on the leaf surface, was required for PIN2 expression. Because these defenses would be activated before egg hatch, this early detection system for herbivores may be of considerable ecological significance.

When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitating the movement of other insecticidal proteins in the diet to the midgut microvilli and thereby enhancing their toxicity. To directly determine the toxicity of Mir1-CP, the purified recombinant enzyme was directly tested against four economically significant Lepidopteran pests in bioassays. Mir1-CP LC(50) values were 1.8, 3.6, 0.6, and 8.0 ppm for corn earworm, tobacco budworm, fall armyworm and southwestern corn borer, respectively. These values were the same order of magnitude as those determined for the Bacillus thuringiensis toxin Bt-CryIIA. In addition to being directly toxic to the larvae, 60 ppb Mir1-CP synergized sublethal concentrations of Bt-CryIIA in all four species. Permeabilization of the PM by Mir1-CP probably provides ready access to Bt-binding sites on the midgut microvilli and increases its activity. Consequently, Mir1-CP could be used for controlling caterpillar pests in maize using non-transgenic approaches and potentially could be used in other crops either singly or in combination with Bt-toxins.

Plant responses to biotic and abiotic stresses are usually accompanied by the release of reactive oxygen species including hydrogen peroxide. Hydrogen peroxide plays a direct role in defense and is involved in many signal transduction pathways that lead to the proliferation of other defenses. Because catalase helps to maintain reactive oxygen homeostasis during biotic and abiotic stress, its activity was measured in various cob tissues during maize ear development. Catalase activity was determined in immature and mature embryos, pericarp, and rachis tissues of maize lines that are resistant and susceptible to Aspergillus flavus infection. The effect of fungal inoculation on catalase activity was also measured. Over two years of field experimentation, a correlation was observed between resistance and the level of catalase-specific activity in immature embryos, which was significantly higher in resistant lines (P < 0.0001). Furthermore, catalase activity in the resistant lines was significantly higher in immature embryos from inoculated ears (P = 0.0199). No correlation was observed between resistance and catalase activity in other ear tissues. Levels of hydrogen peroxide, the catalase substrate, and salicylic acid in the embryo were also determined. The resistant lines showed lower levels of H2O2 (P < 0.0001) and higher levels of salicylic acid (P < 0.0001) as compared with the susceptible lines. Catalase 3 was sequenced from the aflatoxin-resistant (Mp313E) and susceptible (SC212m) inbreds. The predicted amino acid sequence indicated that there was a 20-aa deletion in the resistant inbred that might affect enzymatic activity. Unlike many plant-pathogen interactions, it appears that lowering H2O2 levels helps to prevent A. flavus infection and subsequent aflatoxin accumulation.

Analysis of functional genomics (transcriptomics and proteomics) datasets is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation. To facilitate systems biology in these species we have established the curated, web-accessible, public resource 'AgBase' (www.agbase.msstate.edu). We have improved the structural annotation of agriculturally important genomes by experimentally confirming the in vivo expression of electronically predicted proteins and by proteogenomic mapping. Proteogenomic data are available from the AgBase proteogenomics link. We contribute Gene Ontology (GO) annotations and we provide a two tier system of GO annotations for users. The 'GO Consortium' gene association file contains the most rigorous GO annotations based solely on experimental data. The 'Community' gene association file contains GO annotations based on expert community knowledge (annotations based directly from author statements and submitted annotations from the community) and annotations for predicted proteins. We have developed two tools for proteomics analysis and these are freely available on request. A suite of tools for analyzing functional genomics datasets using the GO is available online at the AgBase site. We encourage and publicly acknowledge GO annotations from researchers and provide an online mechanism for agricultural researchers to submit requests for GO annotations.

Background: Many agricultural species and their pathogens have sequenced genomes and more are in progress. Agricultural species provide food, fiber, xenotransplant tissues, biopharmaceuticals and biomedical models. Moreover, many agricultural microorganisms are human zoonoses. However, systems biology from functional genomics data is hindered in agricultural species because agricultural genome sequences have relatively poor structural and functional annotation and agricultural research communities are smaller with limited funding compared to many model organism communities., Description: To facilitate systems biology in these traditionally agricultural species we have established "AgBase", a curated, web-accessible, public resource http://www.agbase.msstate.edu for structural and functional annotation of agricultural genomes. The AgBase database includes a suite of computational tools to use GO annotations. We use standardized nomenclature following the Human Genome Organization Gene Nomenclature guidelines and are currently functionally annotating chicken, cow and sheep gene products using the Gene Ontology (GO). The computational tools we have developed accept and batch process data derived from different public databases (with different accession codes), return all existing GO annotations, provide a list of products without GO annotation, identify potential orthologs, model functional genomics data using GO and assist proteomics analysis of ESTs and EST assemblies. Our journal database helps prevent redundant manual GO curation. We encourage and publicly acknowledge GO annotations from researchers and provide a service for researchers interested in GO and analysis of functional genomics data., Conclusion: The AgBase database is the first database dedicated to functional genomics and systems biology analysis for agriculturally important species and their pathogens. We use experimental data to improve structural annotation of genomes and to functionally characterize gene products. AgBase is also directly relevant for researchers in fields as diverse as agricultural production, cancer biology, biopharmaceuticals, human health and evolutionary biology. Moreover, the experimental methods and bioinformatics tools we provide are widely applicable to many other species including model organisms.

Many woody perennials, such as poplar (Populus deltoides), are not able to form flower buds during the first several years of their life cycle. They must undergo a transition from the juvenile phase to the reproductive phase to be competent to produce flower buds. After this transition, trees begin to form flower buds in the spring of each growing season. The genetic factors that control flower initiation, ending the juvenile phase, are unknown in poplar. The factors that regulate seasonal flower bud formation are also unknown. Here, we report that poplar FLOWERING LOCUS T2 (FT2), a relative of the Arabidopsis thaliana flowering-time gene FT, controls first-time and seasonal flowering in poplar. The FT2 transcript is rare during the juvenile phase of poplar. When juvenile poplar is transformed with FT2 and transcript levels are increased, flowering is induced within 1 year. During the transition between vegetative and reproductive growth in mature trees, FT2 transcripts are abundant during reproductive growth under long days. Subsequently, floral meristems emerge on flanks of the axillary inflorescence shoots. These findings suggest that FT2 is part of the flower initiation pathway in poplar and plays an additional role in regulating seasonal flower initiation that is integrated with the poplar perennial growth habit.

The signaling pathways that enable plants to mount defenses against insect herbivores are known to be complex. It was previously demonstrated that the insect-resistant maize (Zea mays L.) genotype Mp708 accumulates a unique defense cysteine proteinase, Mirl-CP, in response to caterpillar feeding. In this study, the role of ethylene in insect defense in Mp708 and an insect-susceptible line Tx601 was explored. Ethylene synthesis was blocked with either cobalt chloride or aminoethoxyvinylglycine. Alternatively, ethylene perception was inhibited with 1-methylcyclopropene. Blocking ethylene synthesis and perception resulted in Mp708 plants that were more susceptible to caterpillar feeding. In addition, fall armyworm (Spodoptera frugiperda) larvae that fed on inhibitor-treated Mp708 plants had signifycantly higher growth rates than those reared on untreated plants. In contrast, these responses were not significantly altered in Tx601. The ethylene synthesis and perception inhibitors also reduced the accumulation of Mirl-CP and its transcript mir1 in response to herbivory. These results indicate that ethylene is a component of the signal transduction pathway leading to defense against insect herbivory in the resistant genotype Mp708.

A unique 33-kDa cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM in vitro, dissected PMs were treated with purified, recombinant Mir1-CP and the movement of Blue Dextran 2000 across the PM was measured. Mir1-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and more actively permeabilized the PM than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrinia nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro and is critical to insect defense in maize.

A rapid qualitative protease microassay (RPM) was developed as an alternative to conventional assays of cysteine protease activity in HPLC fractions. Using this technique protease activity in samples could be visually determined within 5 min. The method was sensitive to 3.3x10(-7) U/mL of papain and detected cysteine protease activity in dilute HPLC fractions with activity of 5.4x10(-5) U/mL. Because the method monitors the decolorization of Coomassie Brilliant Blue stained substrate, it can be modified to detect other classes of proteases.

Several heterologous expression systems were tested for their ability to express a unique maize cysteine proteinase Mir1. A baculovirus-based expression system using Trichoplusia ni larvae as host resulted in the expression of Mir1 that was correctly processed and exhibited proteinase activity. Expression in Escherichia coli resulted in accumulation of Mir1, but it had limited solubility and enzymatic activity. Large quantities of Mir1 were produced when Pichia pastoris was used as the host, but the enzyme was insoluble and inactive.

Two variants of creeping bentgrass (Agrostis stolonifera cv palustris), developed using tissue culture, have been used to determine the roles of chloroplast-localized small heat shock proteins (CP-sHSPs) in heat tolerance. Results from previous research indicate that the heat-tolerant variant expressed two additional CP-sHSP isoforms not expressed in the heat-sensitive variant, that accumulation of the additional CP-sHSP isoforms was genetically linked to thermotolerance, and that the presence of the additional isoforms in the heat-tolerant variant provided greater protection to photosystem II during heat stress. To determine the basis of the differential expression, we isolated the genes encoding the CP-sHSPs from both variants and characterized their structure and expression. Two genes, ApHsp26.2 and ApHsp26.7a, were isolated from the heat-tolerant variant, and three genes, ApHsp26.2m, ApHsp26.8, and ApHsp26.7b, were isolated from the heat-sensitive variant. The sequence of ApHsp26.2m from the heat-sensitive variant was identical to ApHsp26.2, except for a point mutation that generated a premature stop codon. Therefore, the protein product of ApHsp26.2m did not accumulate in the heat-sensitive line. Mass spectrometry analysis confirmed that ApHsp26.2 encoded for the CP-sHSP isoforms unique to the heat-tolerant variant. An identical mutation was detected in one of the three parental lines used to develop the creeping bentgrass variants. This suggests that ApHsp26.2m was inherited from this parent and did not arise from a mutation that occurred during tissue culture. The presence of two isoforms encoded by the same gene might be due to differential processing of the N-terminal amino acids during or after import into the chloroplast.