Maize Defense Research Articles

Disruption of OPR7 and OPR8 Reveals the Versatile Functions of Jasmonic Acid in Maize Development and Defense

Here, multiple functions of jasmonic acid (JA) in maize (Zea mays) are revealed by comprehensive analyses of JA-deficient mutants of the two oxo-phytodienoate reductase genes, OPR7 and OPR8. Single mutants produce wild-type levels of JA in most tissues, but the double mutant opr7 opr8 has dramatically reduced JA in all organs tested. opr7 opr8 displayed strong developmental defects, including formation of a feminized tassel, initiation of female reproductive buds at each node, and extreme elongation of ear shanks; these defects were rescued by exogenous JA. These data provide evidence that JA is required for male sex determination and suppression of female reproductive organ biogenesis. Moreover, opr7 opr8 exhibited delayed leaf senescence accompanied by reduced ethylene and abscisic acid levels and lack of anthocyanin pigmentation of brace roots. Remarkably, opr7 opr8 is nonviable in nonsterile soil and under field conditions due to extreme susceptibility to a root-rotting oomycete (Pythium spp), demonstrating that these genes are necessary for maize survival in nature. Supporting the importance of JA in insect defense, opr7 opr8 is susceptible to beet armyworm. Overall, this study provides strong genetic evidence for the global roles of JA in maize development and immunity to pathogens and insects.

A Maize Cystatin Suppresses Host Immunity by Inhibiting Apoplastic Cysteine Proteases

Ustilago maydis is a biotrophic pathogen causing maize (Zea mays) smut disease. Transcriptome profiling of infected maize plants indicated that a gene encoding a putative cystatin (CC9) is induced upon penetration by U. maydis wild type. By contrast, cc9 is not induced after infection with the U. maydis effector mutant Δpep1, which elicits massive plant defenses. Silencing of cc9 resulted in a strongly induced maize defense gene expression and a hypersensitive response to U. maydis wild-type infection. Consequently, fungal colonization was strongly reduced in cc9-silenced plants, while recombinant CC9 prevented salicylic acid (SA)-induced defenses. Protease activity profiling revealed a strong induction of maize Cys proteases in SA-treated leaves, which could be inhibited by addition of CC9. Transgenic maize plants overexpressing cc9-mCherry showed an apoplastic localization of CC9. The transgenic plants showed a block in Cys protease activity and SA-dependent gene expression. Moreover, activated apoplastic Cys proteases induced SA-associated defense gene expression in naïve plants, which could be suppressed by CC9. We show that apoplastic Cys proteases play a pivotal role in maize defense signaling. Moreover, we identified cystatin CC9 as a novel compatibility factor that suppresses Cys protease activity to allow biotrophic interaction of maize with the fungal pathogen U. maydis.

Natural Variation in Maize Defense against Insect Herbivores

Genetic mapping projects with maize (Zea mays) have resulted in the identification of numerous quantitative trait loci (QTL) that influence resistance to insect herbivores. However, the underlying genetic basis of these QTL has been confirmed in only a small number of cases. Recent advances in genome sequencing, the development of large mapping populations, and advances in reverse genetic approaches will accelerate the discovery of novel herbivore resistance genes in maize. Areas that will merit particular research emphasis are natural variation in maize resistance to rootworms and phloem-feeding insects as well as the identification of previously unknown loci involved in the biosynthesis of maize defensive secondary metabolites.

Fungal and oomycete effectors – strategies to subdue a host

Molecular studies focusing on the interface between microbes and plant hosts have provided major insights into the basis underlying pathogenesis, symbiosis and plant defence and resistance mechanisms. A more recent focus on microbes, facilitated by the generation of complete genome sequences, has uncovered the sheer number of protein effectors microbes deliver in this interface as well as inside host cells to manipulate the plant immune system. Although studies on the characterization and roles of bacterial effectors are further advanced, in this review we focus on the current knowledge of fungal and oomycete effectors and their roles. Examples are given of effectors disarming plant defence enzymes, such as the apoplastic effector AVR2 from Cladosporium fulvum which inhibits the tomato defence cysteine protease. Other effectors interfere with the perception by the host of microbes exposing molecular determinants such as Phytophthora infestans INF1 protein. Many effectors alter gene expression induced by the host during defence, exemplified in fungi by Ustilago maydis Pit2 suppressing maize defence genes. Effectors recognized by resistance gene products, either directly or indirectly, and eliciting defence, represent the classical avirulence genes and almost 50 have now been cloned from fungi and oomycetes. Evolutionary adaptations and arms races have produced diversification in both pathogen and host, and in pathogens, are the cause of breaking crop resistance in agricultural settings. Molecular insight provides valuable information for applications. For example, some effectors are crucial for pathogenesis, thereby revealing targets for disease control and others interact with host resistance gene products and could be used to screen germplasm for novel sources of disease resistance. Variation among effectors will likely yield diagnostic tools for pathogen race identification. The study of model systems is providing insight into avenues by which other, major plant diseases can potentially be controlled.

Rapidly Induced Chemical Defenses in Maize Stems and Their Effects on Short-term Growth of Ostrinia nubilalis

Plants damaged by insect herbivory often respond by inducing a suite of defenses that can negatively affect an insect's growth and fecundity. Ostrinia nubilalis (European corn borer, ECB) is one of the most devastating insect pests of maize, and in the current study, we examined the early biochemical changes that occur in maize stems in response to ECB herbivory and how these rapidly induced defenses influence the growth of ECB. We measured the quantities of known maize defense compounds, benzoxazinoids and the kauralexin class of diterpenoid phytoalexins. ECB herbivory resulted in decreased levels of the benzoxazinoid, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-β-D-glucopyranose (DIMBOA-Glc), and a corresponding increase in 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-β-D-glucopyranose (HDMBOA-Glc). Total quantities of benzoxazinoids and kauralexins were increased as early as 24h after the initiation of ECB feeding. The plant hormones, jasmonic acid (JA) and ethylene (ET), and the transcripts encoding their key biosynthetic enzymes also accumulated in response to ECB herbivory, consistent with a role in defense regulation. The combined pharmacological application of JA and the ET precursor, 1-aminocyclopropane-1-carboxylic acid to stem internode tissue likewise resulted in changes in benzoxazinoids similar to that observed with ECB damage. Despite the fact that maize actively mounts a defense response to ECB stem feeding, no differences in percent weight gain were observed between ECB larvae that fed upon non-wounded control tissues compared to tissues obtained from plants previously subjected to 24h ECB stem herbivory. These rapid defense responses in maize stems do not appear to negatively impact ECB growth, thus suggesting that ECB have adapted to these induced biochemical changes.

Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize.

Nonvolatile terpenoid phytoalexins occur throughout the plant kingdom, but until recently were not known constituents of chemical defense in maize (Zea mays). We describe a novel family of ubiquitous maize sesquiterpenoid phytoalexins, termed zealexins, which were discovered through characterization of Fusarium graminearum-induced responses. Zealexins accumulate to levels greater than 800 μg g⁻¹ fresh weight in F. graminearum-infected tissue. Their production is also elicited by a wide variety of fungi, Ostrinia nubilalis herbivory, and the synergistic action of jasmonic acid and ethylene. Zealexins exhibit antifungal activity against numerous phytopathogenic fungi at physiologically relevant concentrations. Structural elucidation of four members of this complex family revealed that all are acidic sesquiterpenoids containing a hydrocarbon skeleton that resembles β-macrocarpene. Induced zealexin accumulation is preceded by increased expression of the genes encoding TERPENE SYNTHASE6 (TPS6) and TPS11, which catalyze β-macrocarpene production. Furthermore, zealexin accumulation displays direct positive relationships with the transcript levels of both genes. Microarray analysis of F. graminearum-infected tissue revealed that Tps6/Tps11 were among the most highly up-regulated genes, as was An2, an ent-copalyl diphosphate synthase associated with production of kauralexins. Transcript profiling suggests that zealexins cooccur with a number of antimicrobial proteins, including chitinases and pathogenesis-related proteins. In addition to zealexins, kauralexins and the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-glucose) were produced in fungal-infected tissue. HDMBOA-glucose accumulation occurred in both wild-type and benzoxazine-deficient1 (bx1) mutant lines, indicating that Bx1 gene activity is not required for HDMBOA biosynthesis. Together these results indicate an important cooperative role of terpenoid phytoalexins in maize biochemical defense.

Two linked genes encoding a secreted effector and a membrane protein are essential for Ustilago maydis‐induced tumour formation

Ustilago maydis is a biotrophic fungal pathogen that colonizes living tissue of its host plant maize. Based on transcriptional upregulation during biotrophic development we identified the pit (proteins important for tumours) cluster, a novel gene cluster comprising four genes of which two are predicted to encode secreted effectors. Disruption of the gene cluster abolishes U. maydis-induced tumour formation and this phenotype can be caused by deleting either pit1 encoding a transmembrane protein or pit2 encoding a secreted protein. Pit1 localizes to the fungal plasma membrane at hyphal tips, endosomes and vacuoles while Pit2 is secreted to the biotrophic interface. Both Δpit1 and Δpit2 mutants are able to penetrate maize epidermis and grow intracellularly at sites of infection but fail to spread in the infected leaf. Microarray analysis shows an indistinguishable response of the plant to infection by Δpit1 and Δpit2 mutant strains. Transcriptional activation of maize defence genes in infections with Δpit1/2 mutant strains indicates that the mutants have a defect in suppressing plant immune responses. Our results suggest that the activity of Pit1 and Pit2 during tumour formation might be functionally linked and we discuss possibilities for a putative functional connection of the two proteins.

Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize.

Phytoalexins constitute a broad category of pathogen- and insect-inducible biochemicals that locally protect plant tissues. Because of their agronomic significance, maize and rice have been extensively investigated for their terpenoid-based defenses, which include insect-inducible monoterpene and sesquiterpene volatiles. Rice also produces a complex array of pathogen-inducible diterpenoid phytoalexins. Despite the demonstration of fungal-induced ent-kaur-15-ene production in maize over 30 y ago, the identity of functionally analogous maize diterpenoid phytoalexins has remained elusive. In response to stem attack by the European corn borer (Ostrinia nubilalis) and fungi, we observed the induced accumulation of six ent-kaurane-related diterpenoids, collectively termed kauralexins. Isolation and identification of the predominant Rhizopus microsporus-induced metabolites revealed ent-kaur-19-al-17-oic acid and the unique analog ent-kaur-15-en-19-al-17-oic acid, assigned as kauralexins A3 and B3, respectively. Encoding an ent-copalyl diphosphate synthase, fungal-induced An2 transcript accumulation precedes highly localized kauralexin production, which can eventually exceed 100 μg · g(-1) fresh weight. Pharmacological applications of jasmonic acid and ethylene also synergize the induced accumulation of kauralexins. Occurring at elevated levels in the scutella of all inbred lines examined, kauralexins appear ubiquitous in maize. At concentrations as low as 10 μg · mL(-1), kauralexin B3 significantly inhibited the growth of the opportunistic necrotroph R. microsporus and the causal agent of anthracnose stalk rot, Colletotrichum graminicola. Kauralexins also exhibited significant O. nubilalis antifeedant activity. Our work establishes the presence of diterpenoid defenses in maize and enables a more detailed analysis of their biosynthetic pathways, regulation, and crop defense function.

Cell wall composition as a maize defense mechanism against corn borers

European and Mediterranean corn borers are two of the most economically important insect pests of maize (Zea mays L.) in North America and southern Europe, respectively. Cell wall structure and composition were evaluated in pith and rind tissues of resistant and susceptible inbred lines as possible corn borer resistance traits. Composition of cell wall polysaccharides, lignin concentration and composition, and cell wall bound forms of hydroxycinnamic acids were measured. As expected, most of the cell wall components were found at higher concentrations in the rind than in the pith tissues, with the exception of galactose and total diferulate esters. Pith of resistant inbred lines had significantly higher concentrations of total cell wall material than susceptible inbred lines, indicating that the thickness of cell walls could be the initial barrier against corn borer larvae attack. Higher concentrations of cell wall xylose and 8-O-4-coupled diferulate were found in resistant inbreds. Stem tunneling by corn borers was negatively correlated with concentrations of total diferulates, 8-5-diferulate and p-coumarate esters. Higher total cell wall, xylose, and 8-coupled diferulates concentrations appear to be possible mechanisms of corn borer resistance.

The Ustilago maydis Forkhead Transcription Factor Fox1 Is Involved in the Regulation of Genes Required for the Attenuation of Plant Defenses During Pathogenic Development

Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host plant, Zea mays. The pathogenic stage of U. maydis is initiated by the fusion of two haploid cells, resulting in the formation of a dikaryotic hypha that invades the plant cell. The switch from saprophytic, yeast-like cells to the biotrophic hyphae requires the complex regulation of a multitude of biological processes to constitute the compatible host-fungus interaction. Transcriptional regulators involved in the establishment of the infectious dikaryon and penetration of the host tissue have been identified; however, regulators required during the post-penetration stages remained to be elucidated. In this study, we report the identification of a U. maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumor development. The Deltafox1 hyphae induce the accumulation of H(2)O(2) in and around infected cells and a maize defense response phenotypically represented by the encasement of proliferating hyphae in a cellulose-containing matrix. The phenotype can be attributed to the fox1-dependent deregulation of several effector genes that are linked to pathogenic development and host defense suppression.

Role of dehydrodiferulates in maize resistance to pests and diseases.

Phenolic esters have attracted considerable interest due to the potential they offer for peroxidase catalysed cross-linking of cell wall polysaccharides. Particularly, feruloyl residues undergo radical coupling reactions that result in cross-linking (intra-/intermolecular) between polysaccharides, between polysaccharides and lignin and, between polysaccharides and proteins. This review addresses for the first time different studies in which it is established that cross-linking by dehydrodiferulates contributes to maize’s defences to pests and diseases. Dehydrodiferulate cross-links are involved in maize defence mechanisms against insects such as the European, Mediterranean, and tropical corn borers and, storage pest as the maize weevil. In addition, cross-links are also discussed to be involved in genetic resistance of maize to fungus diseases as Gibberella ear and stalk rot. Resistance against insects and fungus attending dehydrodiferulates could go hand in hand. Quantitative trait loci mapping for these cell wall components could be a useful tool for enhancing resistance to pest and diseases in future breeding programs.

Integration of Ethylene and Jasmonic Acid Signaling Pathways in the Expression of Maize Defense Protein Mir1-CP

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.

Isolation, expression and functional analysis of a putative RNA-dependent RNA polymerase gene from maize (Zea mays L.)

RNA-dependent RNA polymerases (RdRPs) in plants have been reported to be involved in post-transcriptional gene silencing (PTGS) and antiviral defense. In this report, an RdRP gene from maize (ZmRdRP1) was obtained by rapid amplification of cDNA ends (RACE) and RT-PCR. The mRNA of ZmRdRP1 was composed of 3785 nucleotides, including a 167 nt 5' untranslated region (UTR), a 291 nt 3'UTR and a 3327 nt open reading frame (ORF), which encodes a putative protein of 1108 amino acids with an estimated molecular mass of 126.9 kDa and a predicated isoelectric point (pI) of 8.37. Real-time quantitative RT-PCR analysis showed that ZmRdRP1 was elicited by salicylic acid (SA) treatment, methyl jasmonate (MeJA) treatment and sugarcane mosaic virus (SCMV) infection. We silenced ZmRdRP1 by constitutively expressing an inverted-repeat fragment of ZmRdRP1 (ir-RdRP1) in transgenic maize plants. Further studies revealed that the ir-RdRP1 transgenic plants were more susceptible to SCMV infection than wild type plants. Virus-infected transgenic maize plants developed more serious disease symptoms and accumulated more virus than wild type plants. These findings suggested that ZmRdRP1 was involved in antiviral defense in maize.

Involvement of Protein Phosphorylation in Water Stress‐induced Antioxidant Defense in Maize Leaves

Using pharmacological and biochemical approaches, the role of protein phosphorylation and the interrelationship between water stress-enhanced kinase activity, antioxidant enzyme activity, hydrogen peroxide (H2O2) accumulation and endogenous abscisic acid in maize (Zea mays L.) leaves were investigated. Water-stress upregulated the activities of total protein phosphorylation and Ca2+-dependent protein kinase, and the upregulation was blocked in abscisic acid-deficient vp5 mutant. Furthermore, pretreatments with a nicotinamide adenine dinucleotide phosphate oxidase inhibitor and a scavenger of H2O2 significantly reduced the increased activities of total protein kinase and Ca2+-dependent protein kinase in maize leaves exposed to water stress. Pretreatments with different protein kinase inhibitors also reduced the water stress-induced H2O2 production and the water stress-enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. The data suggest that protein phosphorylation and H2O2 generation are required for water stress-induced antioxidant defense in maize leaves and that crosstalk between protein phosphorylation and H2O2 generation may occur.

Duration and dosage effect of chemical defense in maize (<I>Zea mays</I>) mediated by exogenous ethephon

为探明外源乙烯利对玉米化学防御作用诱导的浓度和时间效应，本文采用化学和分子生物学相结合的方法，研究了不同浓度乙烯利处理玉米后，叶片乙烯释放量及其合成调控关键基因<i>ACS</i>和<i>ACO</i>表达的变化，典型化感物质丁布的含量及其关键调控基因<i>BX</i>₁和<i>BX</i>₉表达的变化，以及其他调控次生代谢物的关键基因表达的时间动态。结果表明，外源乙烯利处理玉米12 h时，玉米叶片中丁布含量明显增加；24 h后，丁布含量反而大量降低，48 h后对丁布含量影响不大。外源乙烯利对合成丁布的调控基因<i>BX</i>₁没有诱导作用，但处理12 h和24 h对BX₉有明显诱导作用。外源乙烯利处理12 h能够使<i>ACS</i>和<i>ACO</i>表达量上升，浓度为40 μL·L^-1和60 μL·L^-1乙烯利处理使玉米叶片丁布含量大量降低，<i>BX</i>₁和<i>BX</i>₉的表达量有所下调。外源乙烯利能够使蛋白酶抑制剂<i>MPI</i>表达量升高，且乙烯利浓度越高，诱导作用越明显，但对调控挥发物的<i>FPS</i>和<i>TPS</i>基因表达没有影响。说明外源乙烯利能够启动玉米体内的乙烯途径，诱导玉米启动化学防御过程，但这种作用很可能是负反馈型的。

Mutations in lipopolysaccharide biosynthetic genes impair maize rhizosphere and root colonization of Rhizobium tropici CIAT899

Three transposon mutants of Rhizobium tropici CIAT899 affected in lipopolysaccharide (LPS) biosynthesis were characterized and their maize rhizosphere and endophytic root colonization abilities were evaluated. The disrupted genes coded for the following putative products: the ATPase component of an O antigen ABC-2 type transporter (wzt), a nucleotide-sugar dehydratase (lpsbeta2) and a bifunctional enzyme producing GDP-mannose (noeJ). Electrophoretic analysis of affinity purified LPS showed that all mutants lacked the smooth LPS bands indicating an O antigen minus phenotype. In the noeJ mutant, the rough LPS band migrated faster than the parental band, suggesting a truncated LPS core. When inoculated individually, the wzt and noeJ mutants colonize the rhizosphere and root to a lower extent than the parental strain while no differences were observed between the lpsbeta2 mutant and the parental strain. All mutants were impaired in competitive rhizosphere and root colonization. Pleiotropic effects of the mutations on known colonization traits such as motility and growth rate were observed, but they were not sufficient to explain the colonization behaviours. It was found that the LPS mutants were sensitive to the maize antimicrobial 6-methoxy-2-benzoxazolinone (MBOA). Only the combined effects of altered growth rate and susceptibility to maize antimicrobials could account for all the observed colonization phenotypes. The results suggest an involvement of the LPS in protecting R. tropici against maize defence response during rhizosphere and root colonization.

Endo-β-1,4-xylanase inhibitors in leaves and roots of germinated maize

Extracts of both leaves and roots of germinated maize were found to contain endo–β–1,4–xylanase inhibitors, previously reported only from whole maize meal. The inhibitors seem to be of the xylanase inhibitor protein (XIP) type, since they inhibit endoxylanases of families 10 and 11 and also show some other characteristics similar to XIP inhibitors described in other cereals. Inhibitors from leaves and roots appeared to be similar. A novel property of the inhibitors described in this work is their unusual thermostability. The half-life of inhibitors at pH 4.5 and 100 °C is greater than 10 h. However, the inhibitors are less thermostable at higher pH levels. Because they did not inhibit a plant endoxylanase, the inhibitors may play a role in maize defense against phytopathogens.

Nitric oxide induced by hydrogen peroxide mediates abscisic acid‐induced activation of the mitogen‐activated protein kinase cascade involved in antioxidant defense in maize leaves

* The role of nitric oxide (NO) and the relationship between NO, hydrogen peroxide (H(2)O(2)) and mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Both ABA and H(2)O(2) induced increases in the generation of NO in mesophyll cells of maize leaves, and H(2)O(2) was required for the ABA-induced generation of NO. Pretreatment with NO scavenger and nitric oxide synthase (NOS) inhibitor substantially reduced the ABA-induced production of NO, and partly blocked the activation of a 46 kDa MAPK and the expression and the activities of several antioxidant enzymes induced by ABA. Treatment with the NO donor sodium nitroprusside (SNP) also induced the activation of the MAPK, and enhanced the antioxidant defense systems. * Conversely, SNP treatment did not induce the production of H(2)O(2), and pretreatments with NO scavenger and NOS inhibitor did not affect ABA-induced H(2)O(2) production. * Our results suggest that ABA-induced H(2)O(2) production mediates NO generation, which, in turn, activates MAPK and results in the upregulation in the expression and the activities of antioxidant enzymes in ABA signaling.

Understanding the Mechanisms Employed by Trichoderma virens to Effect Biological Control of Cotton Diseases

Understanding the Mechanisms Employed by <i>Trichoderma virens</i> to Effect Biological Control of Cotton Diseases

Ethylene Signaling Mediates a Maize Defense Response to Insect Herbivory

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.