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Molecular Genetics of Plant Defense Against Insects

Howe, Gregg
Michigan State University
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Our long-term research objective is to elucidate molecular and biochemical mechanisms of plant resistance to arthropod herbivores. The plant hormone JA plays a central role in regulating many, if not most, induced plant defense responses to herbivory. Our research program uses both Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) as experimental model systems in which to address this question.

The specific objectives of this umbrella project are: (A) to identify physiological ligands of the JA receptor; (B) to determine how jasmonate ZIM domain-containing (JAZ) proteins regulate JA signaling; (C) to determine the role of JAs in systemic wound signaling; (D) to identify and characterize plant proteins that exert toxic effects in the insect gut; and (E) to understand how insects adapt to JA-regulated plant defensive compounds.

This research will contribute broadly to an understanding of the molecular mechanisms by which hormones control developmental and immune function in plants. Cross-kingdom conservation of components of the JA signaling cascade indicates that the research will provide insight into signaling processes that are conserved between plants and animals. The paradigm of ligand-mediated SCF-substrate recognition that has emerged from plant hormone research establishes a novel mechanism for sensing small molecules in biological systems. Finally, results obtained from this research are expected to yield new insight into fundamental mechanisms of hormone action, and provide tools to improve pest tolerance in crop plants.

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NON-TECHNICAL SUMMARY: Arthropod herbivores pose a significant threat to agricultural crops in the United States. In addition to yield losses resulting directly from tissue destruction, these pests play a significant role in transmitting pathogens that cause a variety of plant diseases. Nowhere better are these problems echoed than in the state of Michigan, where pests such as the Colorado potato beetle can cause millions of dollars in crop losses in a single year. Despite the effectiveness of chemical pesticides in controlling herbivore populations, it is widely recognized that many of these compounds adversely affect the environment, food safety, and human health. These considerations justify the need develop alternative pest management strategies that reduce the nation's reliance on environmentally persistent chemicals. One way to achieve this goal is to make greater use of "built-in" plant defense systems that are known to provide durable resistance to a broad spectrum of arthropod pests. For example, physical barriers (e.g., trichomes and cuticular waxes) effectively inhibit feeding by many arthropod herbivores. Other defense traits involve toxins that perturb the insect's growth, or volatile compounds that attract natural predators of the pest. Recent studies indicate that the plant hormone jasmonate (JA) plays a central role in regulating the wound-induced expression of defensive processes that confer durable host resistance. This form of induced resistance has been documented in well over 100 plant species throughout the plant kingdom. The widespread occurrence of induced defenses, together with their effectiveness of thwarting insect attacks, suggests potential applications for improving plant resistance to insect pests. Our research is broadly aimed at identifying genes that play an important role in JA-mediated plant anti-insect defense. Results obtained from this work may find application in the development of environmentally sound strategies for crop protection that benefit agricultural productivity in Michigan and elsewhere.

APPROACH: A key objective is to determine the range of JA derivatives that act as ligands for the receptor. To address this question, we have established in vitro pull-down and yeast two-hybrid (Y2H) assays to assess to ability of small molecules to promote COI1-JAZ interaction. Two general classes of JA-Ile derivatives will be tested for activity: (i) derivatives altered in the jasmonoyl moiety; and (ii) derivatives altered in the Ile moiety. The results will provide quantitative information on the activity of each compound relative to control reactions performed with a standard concentration of JA-Ile. Equilibrium competition binding assays with [3H]-JA-Ile will be used as an alternative and complementary approach to determine the relative affinity of various JA-Ile derivatives for the receptor. By defining the range of JA derivatives that promote COI1 interaction with diverse members of the JAZ protein family, we expect to determine the chemical nature of JA derivatives that signal through COI1. This goal will be complemented by parallel LC-MS/MS analysis of JA-amino acid conjugate levels in Arabidopsis tissues. The Y2H assay will also be used to identify JAZ-interacting proteins. A collection of well-defined loss of function jaz mutants will provide the main tool with which to define the physiological function of individual members of the JAZ family. T-DNA insertion "knock-out" lines for various JAZ genes will be obtained from the Arabidopsis Biological Research Center. Affymetrix ATH1 GeneChips will be used to identify genes that are differentially expressed in the mutant lines in comparison to wild-type plants. These experiments will provide important information on the ability of specific JAZ proteins to regulate the expression of distinct (but perhaps overlapping) sets of target genes. A proteomics approach will be used to identify plant proteins that accumulate in the insect digestive tract. As a non-targeted approach to characterize the herbivore's adaptive response to JA-regulated plant defenses, we will perform deep sequencing of transcripts isolated from gut tissue of a generalist lepidopteran pest reared either on wild-type tomato plants or on jai1 mutant plants that lack the JA receptor.

PROGRESS: 2007/01 TO 2007/12
OUTPUTS: The lipid-derived signaling compound jasmonic acid (JA) regulates a wide variety of defense-related processes in higher plants. The F-box protein COI1 plays an essential role in jasmonate signaling. It was proposed that JA signaling involves ubiquitination of specific target proteins by COI1 and their subsequent degradation by the 26S proteasome. We discovered a family of COI1 substrates, named JAZ (Jasmonate ZIM domain-containing) proteins, through transcriptional profiling of stamen development in response to JA treatment. The JAZ family of proteins in Arabidopsis consists of 12 members, which have been classified as a subgroup of the larger family of tify proteins that share a conserved TIFYxG sequence within the ZIM domain. A second defining feature of JAZs is the highly conserved Jas motif near the C-terminus. Several JAZ proteins have been localized to the nucleus. Expression of a truncated JAZ1 protein (JAZ1d3A) lacking the Jas motif yielded plants that exhibited male sterility and other JA-insensitive phenotypes. Deletion of the C-terminal region of JAZ prevents the protein's degradation and allows continued suppression of JA-responsive genes, suggesting that the C-terminal region of JAZ contains the sequence determinants for jasmonate-dependent interaction with COI1. Direct evidence for COI1-JAZ1 interaction was obtained from both yeast two-hybrid experiments and in vitro protein pull-down assays. Strikingly, these experiments showed that the JA conjugate JA-Ile is highly active in promoting COI1-JAZ1 interaction. These findings provide direct evidence that JA-Ile is an active form of the hormone. Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of JA, and expression of defense genes. To test the function of MAPKs in the response to systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the systemin precursor prosystemin . These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the systemin signaling pathway and most likely function upstream of JA biosynthesis. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2, indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced systemin-induced defense responses. We show that cosilencing of MPK1 and MPK2 compromised prosystemin-mediated resistance to Manduca sexta herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.
PARTICIPANTS: Individuals in the PI's lab who worked on the project include Gregg Howe (PI), Abe Koo (Postdoc), Sastry Jayanty (Postdoc), Hoo Sun Chung (Graduate student), Leron Katsir (Graduate student), and Guanghui Liu (technician). Partner Organizations that collaborated on the research project include Washington State University (John Browse lab) and University of South Carolina (Johannes Stratmann lab). Project collaborators at Michigan State University include Sheng Yang He. The research project provided training or professional development undergraduate and graduate students, as well as post-doctoral fellows.

IMPACT: 2007/01 TO 2007/12
The involvement of jasmonate signaling in plant defense against insects and pathogens has been investigated. The discovery of JAZ proteins as substrates for COI1 marks a major advance in our understanding of the molecular mechanism of jasmonate signaling, and provides new fundamental knowledge about how plant hormones work. This finding will permit experiments to determine whether COI1 is a component of the jasmonate receptor. The discovery of specific kinases involved in systemin-mediated defense responses further increases our understanding of plant defense responses to insect attack. Knowledge gained from these studies may be applied to the development of new and environmentally friendly strategies for crop protection.

PROGRESS: 2006/01/01 TO 2006/12/31
The lipid-derived signaling compound jasmonic acid (JA) regulates a wide variety of defense-related processes in higher plants. The peroxisomal stage of JA synthesis involves reduction of 12-oxo-phytodienoic acid (OPDA) to OPC-8:0, which is subsequently converted to JA by three rounds of beta-oxidation. The initial step of beta-oxidation is catalyzed by acyl-CoA oxidase (ACX). Among the five expressed members (ACX1-5) of the ACX gene family in Arabidopsis, only ACX1 is known to serve a role in JA production. We used transgenic promoter-reporter lines to show that ACX1 is highly expressed in mature and germinating pollen, and stem epidermal cells. Wound-induced JA accumulation was reduced in a mutant that is defective in ACX1, and was abolished in a mutant that is impaired in both ACX1 and its closely related paralog, ACX5. The severe JA deficiency in acx1/5 double mutants was accompanied by decreased resistance to leaf-eating insects. Treatment of acx1/5 plants with JA restored protection to insects. Unexpectedly, acx1/5 plants accumulated JA in response to infection by the fungal pathogen Alternaria brassicicola. JA produced during this interaction was correlated with host resistance to the pathogen. These results indicate that ACX1/5-mediated JA synthesis is essential for resistance to insects, and further suggest that other ACX isozymes contribute to JA production in response to A. brassicicola challenge. Thus, different types of biotic stress may induce JA synthesis via distinct enzymatic routes. It is widely assumed that ACX metabolizes JA precursors that are activated to their CoA derivatives. However, acyl-activating enzymes (AAEs) that catalyze this reaction in vivo have remained elusive. We reported the use of co-expression analysis to identify an AAE gene that is coordinately regulated with known JA biosynthetic components in Arabidopsis. A combination of genetic, biochemical, and cellular approaches were used to demonstrate that this gene, called OPC-8:0 CoA ligase1, has a physiological role in activating JA precursors in the peroxisome. Similar approaches may be useful for identifying additional components of the jasmonate pathway, as well as AAEs that participate in the synthesis of other plant signaling compounds. The JA signaling pathway regulates the synthesis of secondary metabolites in a wide range of plant species. We showed that exogenous JA elicits massive accumulation of caffeoylputrescine (CP) in tomato leaves via the jasmonate signaling pathway. A transgenic tomato line that exhibits constitutive JA signaling accumulated high levels of leaf CP in the absence of jasmonate treatment. RNA blot analysis showed that genes encoding enzymes in the phenylpropanoid and polyamine pathways for CP biosynthesis are up-regulated in JA-treated wild-type plants. These results indicate that CP accumulation in tomato is tightly controlled by the jasmonate signaling pathway.

IMPACT: 2006/01/01 TO 2006/12/31
The involvement of the jasmonic acid (JA) signal transduction pathway in plant defense against insects and pathogens has been investigated. The results support a role for peroxisomes in the production of bioactive jasmonates that promote plant defense responses, and show that JA is an essential signal for induced plant resistance to biotic stress. Knowledge gained from these studies may be applied to the development of new and environmentally friendly strategies for crop protection. The results provide proof-of-concept that the production of plant secondary metabolites can be enhanced by transgenic manipulation of endogenous JA levels.

PROGRESS: 2005/01/01 TO 2005/12/31
Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores. We characterized a JA-deficient mutant of tomato that lacks local and systemic expression of defensive proteinase inhibitors (PIs) in response to wounding. Map-based cloning studies demonstrated that this phenotype results from loss of function of an acyl-CoA oxidase (ACX1A) that catalyzes the first step in the peroxisomal beta-oxidation stage of JA biosynthesis. Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs, and was also was active in the metabolism of C18 cyclopentanoid-CoA precursors of JA. The overall growth, development, and reproduction of acx1 plants were similar to wild-type plants. However, the mutant was compromised in its defense against tobacco hornworm attack. Grafting experiments showed that loss of ACX1A function disrupts the production of the transmissible signal for wound-induced PI expression, but does not affect the recognition of this signal in undamaged responding leaves. We conclude that ACX1A is essential for the beta-oxidation stage of JA biosynthesis, and that JA or its derivatives is required both for anti-herbivore resistance and the production of the systemic wound signal. These findings support a role for peroxisomes in the production of lipid-based signaling molecules that promote systemic defense responses. Although it is well established that JA controls the expression of a large set of target genes in response to tissue damage, very few gene products have been shown to play a direct role in reducing herbivore performance. To test the hypothesis that JA-inducible proteins (JIPs) thwart attack by disrupting digestive processes in the insect gut, we used a mass spectrometry-based approach to identify host proteins that accumulate in the midgut of Manduca sexta larvae reared on tomato plants. We showed that two JIPs, arginase (ARG) and threonine deaminase (TD), act in the M. sexta midgut to catabolize the essential amino acids Arg and Thr, respectively. Transgenic plants that overexpress ARG were more resistant to M. sexta larvae, and this effect was correlated with reduced levels of midgut Arg. We present evidence indicating that the ability of TD to degrade Thr in the midgut is enhanced by herbivore-induced proteolytic removal of the C-terminal regulatory domain of TD that, in planta, confers negative feedback regulation by isoleucine. Our results demonstrate that the JA signaling pathway strongly influences the midgut protein content of phytophagous insects, and support the hypothesis that catabolism of amino acids in the insect digestive tract by host enzymes plays a role in plant antiherbivore defense.

IMPACT: 2005/01/01 TO 2005/12/31
The involvement of the jasmonic acid (JA) signal transduction pathway in plant defense against insects has been investigated. The results indicate that JA is an essential signal role induced plant resistance to chewing insects, and that numerous JA-regulated proteins exert antinutritional effects in the insect midgut. Knowledge gained from these studies may be applied to the development of new and environmentally friendly strategies for crop protection.

PROGRESS: 2004/01/01 TO 2004/12/31
Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development, but is not essential for other developmental aspects of the plant life cycle. We described the phenotypic and molecular characterization of a sterile mutant of tomato (jai1) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in maternal control of seed maturation, which was associated with loss of accumulation of JA-regulated proteinase inhibitor (PI) proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes including inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrated that these defects are caused by loss of function of the tomato homolog of CORONATINE-INSENSITIVE1, an F-box protein that functions in the ubiquitin-proteosome pathway. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and, further, suggest a role for JA in the promotion of glandular trichome-based defenses. A second project was focused on understanding the regulation and function of plant arginase, which catalyzes the conversion of arginine to urea and ornithine. Although higher plants utilize arginine for the production of polyamines and nitric oxide (NO), the potential role of arginase as a control point for arginine homeostasis has not been investigated. We characterized two genes (LeARG1 and LeARG2) from tomato that encode arginase. Phylogenic analysis showed that LeARG1 and 2, like all other plant arginases, are more similar to agmatinase than to arginases from vertebrates, fungi, and bacteria. Nevertheless, recombinant LeARG1 and 2 exhibited specificity for L-arginine over agmatine and related guanidino substrates. These results indicate that plant arginases define a distinct group of ureohydrolases that function as authentic L-arginases. LeARG1 and LeARG2 transcripts accumulated to their highest levels in reproductive tissues. In leaves, LeARG2 expression and arginase activity were induced in response to wounding and treatment with JA. Wound- and JA-induced expression of LeARG2 was not observed in the tomato jai1 mutant, indicating that this response is strictly dependent on an intact JA signal transduction pathway. Infection of wild-type plants with a virulent strain of Pseudomonas syringae pv. tomato also upregulated LeARG2 expression and arginase activity. This response was mediated by the bacterial phytotoxin coronatine, which exerts its virulence effects by co-opting the host JA signaling pathway. These results highlight striking similarities in the regulation of arginase in plants and animals, and suggest that stress-induced arginase may perform similar roles in diverse biological systems.

IMPACT: 2004/01/01 TO 2004/12/31
The involvement of the jasmonic acid (JA) signal transduction pathway in plant defense against insects and bacterial pathogens has been investigated. The results indicate that JA signaling through COI1 plays an essential role in several processes including induced resistance of tomato to herbivores, development of glandular trichomes, and maternal control of seed maturation. These results provide insight into the molecular basis of agronomically important traits. Our work on plant arginase further suggests that arginine homoestasis may be an important component of these phenotypic traits. Knowledge gained from these studies may be applied to the development of new and environmentally friendly strategies for crop protection.

PROGRESS: 2003/01/01 TO 2003/12/31
Wound-induced expression of defensive proteinase inhibitor (PI) genes in tomato requires the action of systemin, a peptide signal, and the fatty acid-derived hormone, jasmonic acid (JA). Although it is known that systemin induces PI expression by activating JA biosynthesis, relatively little is known about how systemin and JA interact to promote long-distance signaling between damaged and undamaged leaves. We addressed this question by characterizing a systemin-insensitive mutant of tomato called spr1. We showed that the spr1 mutation abolished JA accumulation in response to systemin and abrogated the systemic wound response. Grafting experiments showed that spr1 impedes systemic PI expression by blocking the production of the long-distance wound signal in damaged leaves. These findings indicate that Spr1 is involved in a signaling step that couples systemin perception to activation of JA biosynthesis. A second project was focused on molecular cloning the Spr2 gene that is required for wound-induced synthesis of JA and production of the systemic wound signal. Using a map-based cloning approach, we showed that Spr2 encodes a fatty acid desaturase that converts linoleic acid to linolenic acid, the metabolic precursor of JA. spr2 mutant plants exhibited normal growth, development, and reproduction, but were compromised in defense against attack by tobacco hornworm larvae. These results indicate that JA synthesis from chloroplast pools of linolenic acid is required for wound- and systemin-induced defense responses. A third project was aimed at understanding the role of JA in the interaction of tomato with Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000), the casual agent of bacterial speck disease. This research was conducted in collaboration with Dr. Sheng Yang He (MSU). The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of JA. We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type plants. Treatment of wild-type plants with exogenous JA complemented the virulence defect of a bacterial mutant deficient in COR production. Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA- and wound-response genes that have been implicated in defense against herbivores. Concomitant with the induction of JA/wound-response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected wild-type plants. Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound-response genes. These results indicate that COR promotes bacterial virulence by activating the host JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway.

IMPACT: 2003/01/01 TO 2003/12/31
The involvement of jasmonic acid (JA) in plant defense against insects and bacterial pathogens has been investigated. The results indicate that JA plays a central role in systemic induced resistance of tomato to insect pests, and suggest a greater role for this hormone in plant protection than was previously thought. Our results also demonstrate that JA plays a role in promoting susceptibility of tomato to bacterial speck disease. Knowledge gained from these studies may be applied to the development of new and environmentally friendly strategies for crop protection.

PROGRESS: 2002/01/01 TO 2002/12/31
This research is aimed at understanding the molecular basis of induced plant defense against herbivores, using tomato as a model system. We are particularly interested in determining the role of jasmonic acid (JA) and other oxylipins in plant defense. One project was to determine the role of JA biosynthesis and JA perception in wound-induced activation of systemic defense responses. Using a JA biosynthetic mutant (spr2) and a JA-insensitive mutant (jai1) that are both defective in the wound-induced systemic response, we conducted grafting experiments to determine whether these mutants are defective in the production of a long-distance wound signal in rootstock leaves, or the recognition of that signal in undamaged scion leaves. The results showed that JA biosynthesis and perception, while both required for long-distance signaling, operate at distinct spatial positions along the systemic signaling pathway. More specifically, a functional JA biosynthetic pathway in damaged leaves is required for the production of a long-distance signal whose recognition in distal leaves requires JA perception. A second subproject investigated the role of JA in defense of tomato against cell content-feeding herbivores. We found that infestation of cultivated tomato plants with the two-spotted spider mites (Tetranychus urticae) or western flower thrips (Frankliniella occidentalis) caused induction of host plant defense responses. These responses were not observed in a tomato mutant (def1) that is deficient in JA biosynthesis. Moreover, def1 plants were much more susceptible to herbivore attack than wild-type plants. We mapped the Def1 gene to the long end of chromosome 3 as a prelude to identifying the gene. We also found that transgenic tomato plants engineered for constitutive JA synthesis were highly resistant to herbivore attack. A third subproject involved the molecular cloning and characterization of a novel allene oxide synthase (AOS)-encoding cDNA from tomato. The recombinant protein (called AOS3) showed spectral characteristics of a cytochrome P450. The enzyme transformed 9- and 13-hydroperoxides of linoleic and linolenic acid to ketol fatty acids and cyclopentenones. Kinetic assays demonstrated that AOS3 was 10-fold more active against 9-hydroperoxides than the corresponding 13-isomers. AOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants. These findings suggest that AOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.

IMPACT: 2002/01/01 TO 2002/12/31
Results obtained indicate that JA is active as a systemic signal for plant defense responses, and suggest a greater role for oxylipins in defense signaling than was previously thought. Our results also provide the first demonstration of genetically engineered plant resistance to economically important cell content-feeding herbivores. The information gained from these studies may find application in the development of new and environmentally friendly strategies for crop protection.

PROGRESS: 2001/01/01 TO 2001/12/31
This research is aimed at understanding the molecular basis of induced plant defense against herbivores, using tomato as a model system. One sub-project involved the biochemical and genetic characterization of a mutant called spr2 that lacks wound-induced expression of proteinase inhibitor (PI) encoding genes and, as a consequence, is more susceptible to insects. Plants homozygous for spr2 have a phenotype very similar to that of the jasmonic acid (JA)-deficient def1 mutant that we characterized previously. However, genetic analysis showed that def1 and srp2 define different genes. A gas chromatography-mass spectrometry assay was established to measure endogenous JA in plant tissues. The experiments demonstrated that wounded spr2 plants accumulate less than 10% of the JA levels observed in wounded wild-type plants. This finding indicates that the wound response phenotype of spr2 results from a defect in jasmonate biosynthesis. Mapping experiments showed that genes encoding known JA biosynthetic enzymes in tomato are not genetically linked to Spr2. Accordingly, a map-based cloning approach was initiated to identify Spr2. Bulked segregant analysis of a segregating population was used to identify AFLP markers linked to the target gene. One of these markers was mapped to the long arm of chromosome 6 using tomato introgression lines. Fine scale mapping indicated that the Spr2 locus is tightly linked to RFLP marker TG590. This marker was used to screen a bacterial artificial chromosome (BAC) library of tomato DNA, and positive clones were order into a BAC contig. The data indicate that Spr2 is likely located on a single BAC clone. A second project was aimed at characterizing tomato mutants that are impaired in JA perception. Genetic complementation tests showed that two independently isolated JA-insensitive mutants define the same locus, called Jai1. Jai1 activity was shown to be essential for wound- and systemin-induced expression of PI and other defense-related genes. Consistent with this, jai1 mutant plants are compromised in resistance to various herbivores including tobacco hornworm (Manducta sexta) and the two-spotted spider mite (Tetranychus urticae Koch). These results indicate that resistance of tomato to a broad spectrum of herbivores requires both JA biosyntheis and subsequent JA signaling. Plants homozygous for jai1 produce ripe fruit but do not seed mature seed. Reciprocal crosses showed that this phenotype results from a defect in female fertility. This finding contrasts the well-documented male sterility of JA-deficient mutants of Arabidopsis, and suggests that jasmonates serve diverse physiological roles in different plant species. The sterility of jai1 plants could result from a defect in ovule development, embryogenesis, or another maternal process that affects seed set. Conclusive proof that JA signaling is required for female reproduction in tomato will require cloning of Jai1 and functional complementation of the mutant.

IMPACT: 2001/01/01 TO 2001/12/31
Results obtained indicate that the biosynthesis and perception of the plant hormone jasmonic acid is essential for induced resistance of tomato plants to a broad-spectrum of herbivores. The information gained from these studies may find application in the development of new and environmentally friendly strategies for crop protection.

PROGRESS: 2000/01/01 TO 2000/12/31
This research is aimed at understanding the molecular basis of induced plant defense against herbivores, using tomato as a model system. One sub-project studied induced resistance of tomato to spider mites (Tetranychus urticae). Mite infestation of wild-type leaves resulted in the expression of several defensive proteinase inhibitor (PIN) genes within 24 hours of the challenge. This response was correlated with a subsequent decrease in mite feeding and fecundity. In contrast, the tomato def1 mutant was both susceptible to mite attack and deficient in mite-induced PIN expression. Consistent with the proposed role of Def1 in wound-inducible jasmonic acid (JA) accumulation, exogenous JA restored resistance in def1 plants. Transgenic plants that overexpress prosystemin, the precursor of the systemin wound signal, were highly resistant to spider mites. A second project was aimed at mapping the Def1 gene. Bulk segregant analysis was used to identify AFLP markers that are linked to Def1. Two such markers were mapped to the distal end of the long arm of chromosome 3. RFLP markers located in this region showed tight linkage to Def1, thus confirming the AFLP results. The frequency of recombination between chromosome 3L-specific RFLP markers and Def1 was suppressed, particularly for markers at the extreme end of the chromosome. A third sub-project involved characterization of the tomato spr1 mutant that is compromised in the systemin branch of the wound response pathway. In wounded spr1 plants, PIN genes were expressed to about 50% of wild-type levels in the damaged leaf. However, systemic expression of these genes in spr1 was less than 10% of that observed in wild-type. This phenotype suggests that prosystemin works through Spr1 to facilitate systemic activation of PIN genes. A fourth sub-project investigated the role of two isoforms of prosystemin (A and B) that are generated by alternative splicing of prosystemin pre-mRNA. The results showed that prosysB is the major prosystemin-encoding transcript in tomato, and that both isoforms of the protein are active as wound response signals. Finally, genetic screens were initiated to identify tomato mutants that are insensitive JA. Approximately 24,000 fast-neutron mutagenized seedlings were screened for individuals that fail to express both polyphenol oxidase and PIN2 in response to exogenous JA. One mutant, called jai1, was found to be completely deficient in the accumulation of both marker proteins. jai1 mutant plants showed normal vegetative growth but produced fruit that lacked mature seed. The mutation was maintained in Jai1/jai1 heterozygotes. F2 populations derived from these plants segregated 3:1 for the JA-insensitive phenotype. Northern blot analysis showed that jai1 completely abolishes the plant's ability to activate PIN gene expression in response to exogenous JA.

IMPACT: 2000/01/01 TO 2000/12/31
Results obtained indicate that wound induced responses in tomato confer resistance to a broad-spectrum of herbivore pests. Mapping of Def1 represents the first step toward molecular cloning of this gene and determination of its role in induced resistance. Analysis of JA- and systemin-insensitive mutants is providing new insight into the molecular mechanism of induced resistance. The information gained from these studies may find application in the development of novel and environmentally sound approaches to crop protection.

PROGRESS: 1999/01/01 TO 1999/12/31
This research is aimed at understanding the biochemical and molecular basis of plant defense against herbivores, using tomato as a model system. One sub-project investigated wound-inducible gene expression in the tomato def1 mutant that is deficient in the octadecanoid signaling pathway and, as a result, is unable to produce defensive proteinase inhibitors or mount resistance to herbivore pests. We showed that a subset of wound responsive genes, including allene oxide synthase and lipoxygenase, are expressed to near wild-type levels in wounded def1 plants. These results demonstrate the existence of two classes of genes whose requirements for wound induction can be defined as being either Def1-dependent or Def1-independent. Given the involvement of Def1 in wound-inducible jasmonic acid (JA) accumulation, we suggest that endogenous JA is a signal for Def1-dependent, but not Def1-independent, wound responses. A second project studied the biochemical basis of the def1 mutant phenotype. Results indicated that def1 plants are not affected in the activity of enzymes that act early in the pathway for JA biosynthesis. Rather, our results suggest that def1 affects a late step in JA biosynthesis, or the transport or stability of JA. To gain more insight into the function of Def1, we initiated a project to map the chromosomal location of the gene, as a prelude to cloning it. A mapping population was constructed from a cross between def1 and L. pennellii. Segregating progeny are currently being scored for RFLP and AFLP markers that are linked to the target locus. A third project involved the characterization of a new group of mutants that are suppressed in prosystemin-mediated expression of proteinase inhibitor and other defense-related genes. These mutants define four new genetic complementation groups called Spr. Physiological studies indicate that the spr1 mutation affects responses to prosystemin and systemin but not other chemical elicitors of the wound response. These results suggest that Spr1 plays a role in the prosystemin branch of the wound response pathway. A final project studied alternative splicing of the tomato prosystemin gene. We discovered that alternative splicing of two adjacent, conserved splice acceptor sites in intron 3 results in the production of two forms of prosystemin mRNA. The deduced effect of this polymorphism is substitution of Arg57 with a Thr and a Gly. Splice site selection was not regulated during plant development, or in response to environmental cues. Experiments are in progress to determine whether alternative splicing affects the ability of prosystemin to transduce systemic wound signals.

IMPACT: 1999/01/01 TO 1999/12/31
Analysis of prosystemin expression and function will provide greater insight into the mechanism of systemin-mediated signal transduction and its role in induced resistance to herbivores. Molecular mapping of the Def1 locus provides the first step toward isolation of this gene and analysis of its biochemical function. The information gained from these studies may find application in the development of novel and environmentally sound approaches to crop protection.

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Nat'l. Inst. of Food and Agriculture
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