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Agrobacterium radiobacter K84, used worldwide to biocontrol crown gall disease caused by Agrobacterium tumefaciens, produces an antiagrobacterial compound called agrocin 84. We report the nucleotide sequence of pAgK84, a 44.42-kb plasmid coding for production of this disubstituted adenine nucleotide antibiotic. pAgK84 encodes 36 ORFs, 17 of which (agn) code for synthesis of or immunity to agrocin 84. Two genes, agnB2 and agnA, encode aminoacyl tRNA synthetase homologues. We have shown that the toxic moiety of agrocin 84 inhibits cellular leucyl-tRNA synthetases and AgnB2, which confers immunity to the antibiotic, is a resistant form of this enzyme. AgnA, a truncated homologue of asparaginyl tRNA synthetase could catalyze the phosphoramidate bond between a precursor of the methyl pentanamide side group and the nucleotide. We propose previously undescribed chemistry, catalyzed by AgnB1, to generate the precursor necessary for this phosphoramidate linkage. AgnC7 is related to ribonucleotide reductases and could generate the 3'-deoxyarabinose moiety of the nucleoside. Bioinformatics suggest that agnC3, agnC4, and agnC6 contribute to maturation of the methyl pentanamide, whereas agnC2 may produce the glucofuranose side group bound to the adenine ring. AgnG is related to bacterial exporters. An agnG mutant accumulated agrocin 84 intracellularly but did not export the antibiotic, pAgK84 is transmissible and encodes genes for conjugative DNA processing but lacks a type IV secretion system, suggesting that pAgK84 transfers by mobilization. By sequence analysis, the deletion engineered into pAgK1026 removed the oriT and essential tra genes, confirming the enhanced environmental safety of this modified form of pAgK84. Agrobacterium radiobacter | biological control | pAgK84

Pathogens can be an important selective agent in plant evolution because they can severely reduce plant fitness and growth. However, the role of pathogen selection on plant evolution depends on the extent of genetic variation for resistance traits and their covariance with host fitness. Although it is usually assumed that resistance traits will covary with plant fitness, this assumption has not been tested rigorously in plant-pathogen interactions. Many plant species are tolerant to herbivores, decoupling the relationship between resistance and fitness. Tolerance to pathogens can reduce selection for resistance and alter the effect of pathogens on plant evolution. In this study, we measured three components of Arabidopsis thaliana resistance (pathogen growth, disease symptoms, and host fitness) to the bacteria Pseudomonas syringae and investigated their covariation to determine the relative importance of resistance and tolerance. We observed extensive quantitative variation in the severity of disease symptoms, the bacterial population size, and the effect of infection on host fitness among 19 accessions of A. thaliana infected with P. syringae. The severity of disease symptoms was strongly and positively correlated with bacterial population size. Although the average fitness of infected plants was smaller than noninfected plants, we found no correlation between the bacterial growth or symptoms expressed by different accessions of A. thaliana and their relative fitness after infection. These results indicate that the accessions studied vary in tolerance to P. syringae, reducing the strength of selection on resistance traits, and that symptoms and bacterial growth are not good predictors of host fitness.

Homologues of the yeast ubiquitin ligase-associated protein SGT1 are required for disease resistance in plants mediated by nucleotide-binding site/leucine-rich repeat (NBS-LRR) proteins. Here, by silencing SGT1 in Nicotiana benthamiana, we extend these findings and demonstrate that SGT1 has an unexpectedly general role in disease resistance. It is required for resistance responses mediated by NBS-LRR and other R proteins in which pathogen-derived elicitors are recognized either inside or outside the host plant cell. A requirement also exists for SGT1 in nonhost resistance in which all known members of a host species are resistant against every characterized isolate of a pathogen. Our findings show that silencing SGT1 affects diverse types of disease resistance in plants and support the idea that R protein-mediated and nonhost resistance may involve similar mechanisms.

The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an [alpha]-glucosidase present in the midgut of Culex pipiens larvae. In this work, we studied the molecular basis of the resistance to Bin developed by a strain (GEO) of C. pipiens. Immunohistochemical and in situ hybridization experiments showed that Cpm1 was undetectable in the midgut of GEO larvae, although the gene was correctly transcribed. The sequence of the [cpm1.sub.GEO] cDNA differs from the sequence we previously reported for a susceptible strain ([cpm1.sub.IP]) by seven mutations: six missense mutations and a mutation leading to the premature termination of translation. When produced in insect cells, [Cpm1.sub.IP] was attached to the membrane by a glycosylphosphatidylinositol (GPI). In contrast, the premature termination of translation of [Cpm1.sub.GEO] resulted in the targeting of the protein to the extracellular compartment because of truncation of the GPI-anchoring site. The interaction between Bin and [Cpm1.sub.GEO] and the enzyme activity of the receptor were not affected. Thus, Bin is not toxic to GEO larvae because it cannot interact with the midgut cell membrane, even though its receptor site is unaffected. This mechanism contrasts with other known resistance mechanisms in which point mutations decrease the affinity of binding between the receptor and the toxin.

Plant disease epidemics resulting from introductions of exotic fungal plant pathogens are a well known phenomenon. An associated risk - that accelerated pathogen evolution may be occurring as a consequence of genetic exchange between introduced, or introduced and resident, fungal pathogens - is largely unrecognized. This is, in part, because examples of natural, interspecific hybridization in fungi are very rare. Potential evolutionary developments range from the acquisition of new host specificities to emergence of entirely new pathogen taxa. We present evidence from cytological behavior, additive nucleotide bases in repetitive internal transcribed spacer regions of the rRNA-encoding DNA (rDNA), and amplified fragment length polymorphisms of total DNA that a new, aggressive Phytophthora pathogen of alder trees in Europe comprises a range of heteroploid-interspecific hybrids involving a Phytophthora cambivora-like species and an unknown taxon similar to Phytophthora fragariae. The hybrids' marked developmental instabilities, unusual morphological variability, and evidence for recombination in their internal transcribed spacer profiles indicates that they are of recent origin and that their evolution is continuing. The likelihood of such evolutionary events may be increasing as world trade in plants intensifies. However, routine diagnostic procedures currently in use are insufficiently sensitive to allow their detection.

The [Ca.sup.2+] signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CAM) isoforms are activated by infection or pathogen-derived elicitors and participate in [Ca.sup.2+]-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 rain by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular [Ca.sup.2+] level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA.independent signal transduction chain leading to disease resistance.

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBSLRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPMI protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.

Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with [approximately equal to]81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.

The Arabidopsis genes EDS1 and NDR1 were shown previously by mutational analysis to encode essential components of race-specific disease resistance. Here, we examined the relative requirements for EDS1 and NDR1 by a broad spectrum of Resistance (R) genes present in three Arabidopsis accessions (Columbia, Landsberg-erecta, and Wassilewskija). We show that there is a strong requirement for EDS1 by a subset of R loci (RPP2, RPP4, RPP5, RPP21, and RPS4), conferring resistance to the biotrophic oomycete Peronospora parasitica, and to Pseudomonas bacteria expressing the avirulence gene avrRps4. The requirement for NDR1 by these EDS1-dependent R loci is either weak or not measurable. Conversely, three NDR1-dependent R loci, RPS2, RPM1, and RPS5, operate independently of EDS1. Another RPP locus, RPP8, exhibits no strong exclusive requirement for EDS1 or NDR1 in isolate-specific resistance to P. parasitica, although resistance is compromised weakly by eds1. Similarly, resistance conditioned by two EDS1-dependent RPP genes, RPP4 and RPP5, is impaired partially by ndr1, implicating a degree of pathway cross-talk. Our results provide compelling evidence for the preferential utilization of either signaling component by particular R genes and thus define at least two disease resistance pathways. The data also suggest that strong dependence on EDS1 or NDR1 is governed by R protein structural type rather than pathogen class.

Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.

Disease resistance in transgenic plants has been improved, for the first time, by the insertion of a gene from a biocontrol fungus. The gene encoding a strongly antifungal endochitinase from the mycoparasitic fungus Trichoderma harzianum was transferred to tobacco and potato. High expression levels of the fungal gene were obtained in different plant tissues, which had no visible effect on plant growth and development. Substantial differences in endochitinase activity were detected among transformants. Selected transgenic lines were highly tolerant or completely resistant to the foliar pathogens Alternaria alternata, A. solani, Botrytis cinerea, and the soilborne pathogen Rhizoctonia solani. The high level and the broad spectrum of resistance obtained with a single chitinase gene from Trichoderma overcome the limited efficacy of transgenic expression in plants of chitinase genes from plants and bacteria. These results demonstrate a rich source of genes from biocontrol fungi that can be used to control diseases in plants.

The Arabidopsis thaliana disease resistance genes RPS2 and RPM1 belong to a class of plant disease resistance genes that encode proteins that contain an N-terminal tripartite nucleotide binding site (NBS) and a C-terminal tandem array of leucine-rich repeats. RPS2 and RPM1 confer resistance to strains of the bacterial phytopathogen Pseudomonas syringae carrying the avirulence genes avrRpt2 and avrB, respectively. In these gene-for-gene relationships, it has been proposed that pathogen avirulence genes generate specific ligands that are recognized by cognate receptors encoded by the corresponding plant resistance genes. To test this hypothesis, it is crucial to know the site of the potential molecular recognition. Mutational analysis of RPS2 protein and in vitro translation/translocation studies indicated that RPS2 protein is localized in the plant cytoplasm. To determine whether avirulence gene products themselves are the ligands for resistance proteins, we expressed the avrRpt2 and avrB genes directly in plant cells using a novel quantitative transient expression assay, and found that expression of avrRpt2 and avrB elicited a resistance response in plants carrying the corresponding resistance genes. This observation indicates that no bacterial factors other than the avirulence gene products are required for the specific resistance response as long as the avirulence gene products are correctly localized. We propose that molecular recognition of P. syringae in RPS2- and RPM1-specified resistance occurs inside of plant cells.

Genomic mapping has been used to identify a region of the host genome that determines resistance to fusiform rust disease in loblolly pine where no discrete, simply inherited resistance factors had been previously found by conventional genetic analyses over four decades. A resistance locus, behaving as a single dominant gene, was mapped by association with genetic markers, even though the disease phenotype deviated from the expected Mendelian ratio. The complexity of forest pathosystems and the limitations of genetic analysis, based solely on phenotype, had led to an assumption that effective long-term disease resistance in trees should be polygenic. However, our data show that effective long-term resistance can be obtained from a single qualitative resistance gene, despite the presence of virulence in the pathogen population. Therefore, disease resistance in this endemic coevolved forest pathosystem is not exclusively polygenic. Genomic mapping now provides a powerful tool for characterizing the genetic basis of host pathogen interactions in forest trees and other undomesticated organisms, where conventional genetic analysis often is limited or not feasible.

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of [H.sub.2][O.sub.2], which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxidase (APX), the other key enzyme for scavenging [H.sub.2][O.sub.2]. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 [mu]M ascorbic acid (AsA), substrate-dependent [IC.sub.50] values of 78 [mu]M and 95 [mu]M were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated [H.sub.2][O.sub.2] levels or coupled perturbations of the cellular redox state.

A mutant strain nim1 (non-inducible immunity phenotype) of Arabidopsis is unable to induce the expression of the systemic acquired resistance (SAR) genes in response to salicyclic acid (SA) or 2,6-dichloroisonicotinic acid. However the increase in the amount of SA which is an endogenous signal for SAR is seen in nim1 which also support the growth of normally incompatible fungal pathogens. Nim1 gene probably encodes a substance required for signal transduction in the pathway which controls the expression of the SAR genes and confers resistance to the plant.

The locus NDR1 (non-race-specific disease resistance) present in Arabidopsis thaliana is necessary for the disease resistance of the plant to the bacteria, Pseudomonas syringae pv. tomato strain DC3000 containing non virulent genes and the fungus Peronospora parasitica. A mutant Arabidopsis strain ndr1-1 in which the mutation segregates as a single recessive gene on chromosome III is susceptible to these pathogens. NDR1 is necessary for the recognition of an avirulent pathogen and the resistance to these unrelated pathogens involves a common step in the molecular pathway.

Cloning of the disease-resistance gene RPS2 of Arabidopsis thaliana makes it possible to use it as a model host to study the genetic aspects of plant defense mechanisms to microbial pathogens. RPS2 corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship, is similar to the tobacco N resistance gene and encodes a 105-kDa protein comprising a leucine zipper, a nucleotide binding site and 14 imperfect leucine-rich repeats.

Salicylic acid (SA) plays a major role in inducing systemic acquired resistance, a natural plant defense mechanism against pathogen attack. This is evident in the high concentration levels of SA in a tobacco plant infected with tobacco mosaic virus. Besides this, SA assists in developing genetic resistance to diseases. H2O2, unlike SA, apparently does not induce systemic acquired resistance, even though SA inhibition of catalase gives rise to accumulation of H2O2.

The structure and evolution of two chromosome sites of flax (Linum usitatissimum) are different, although the rust resistance genes against pathogens at each site have sequence similarities. While the L locus is simple and has a single gene with 13 alleles, the M locus is complex and contains a tandem array of genes of similar sequence.

Resistance to plant diseases may be guided by gene-for-gene interaction in which a single resistance gene (Pto) responds to the expression of a specific avirulence gene in the pathogen. In a tomato plant affected by bacterial speck disease, the Pto gene is associated with a hypersensitive response upon exposure to the organophosphorous insecticide fenthion. The Pto gene and fenthion-sensitivity gene Fen encode similar protein kinases indicating that both participate in signal transduction involved in disease resistance.

The hypersensitive response, product of an interaction between resistance genes and a pathogen avirulence gene product, is a defense mechanism in plants to combat pathogen invasions. In tobacco plants afflicted by the mosaic virus, the resistance gene (N) functions in a signal transduction pathway by stimulating rapid defense responses. A sequence analysis of the N gene reveals that it encodes a protein with an amino-terminal domain.

The plant-insect interaction benefits plants as parasitic and predatory insects are natural enemies of herbivores. Parasitoids eliminate herbivores that attack the plants. A herbivore-induced emission of volatile from plants sends signals to these insects. Emission of odors is well timed and emitted within hours of herbivore attack. They are mostly produced during daytime, when parasitoids are foraging. Volatile signals are clear, large in number and can be distiguished from background odors, but these signals are not specific when various herbivores attack the same plant.

Grand fir (Abies grandis) saplings and cell cultures can be useful in understanding the regulation of defensive oleoresinosis in conifers. The chemical ecological relationship between conifers and bark beetles may cause injury and oleoresin biosynthesis can be useful to improve tree resistance. Studies with grand fir proved more useful than with pine or spruce.

The resultant H2O2 from an oxidative reaction to an avirulent pathogen strain plays an essential role in the formation of a localized hypersensitive response. This hypersensitive response produces many defenses including cell wall formation, lytic enzymes and the localized host cell death that inhibits the spread of infection. An integrated model of the hypersensitive response to an avirulent pathogen attack can be developed.

Plant cell walls have regulatory molecules, oligogalacturonides (OGAs), that stimulate defense gene expression during the disease process. In vitro, the phosphorylation of plant protein 34 is stimulated by OGAs of 13 to 26 residues long, whereas in vivo, fragments of only 10 to 15 residues long stimulate defense responses. A damaged cell wall, however, can allow access to larger fragments.

The elicitor/suspension-cultured parsley cell system is a good model for studying elicitor recognition, signal transduction and defense gene activation. Elicitor binding by a receptor in the plasma membrane causes H2O2 cell responses, which are followed by transient changes in phosphorylation of proteins and activation of defense genes. With some limitations, each individual defense response was seen to be closely mimicked in elicitor/culture parsley cells, thus upholding the validity of the system.

Salicylic acid signaling appears to activate plant defense responses against pathogens and play an important role in binding catalase and impeding its enzymatic activity. Increased salicylic acid levels correlate with infection of tobacco with tobacco mosaic virus and induction of defense-related genes such as that which encodes pathogenesis-related protein 1 (PR-1). The PR gene expression and disease resistance inhibit catalytic activity. The results indicate that catalase can also act as receptor for salicylic acid.

Protein phosphorylation maintains the oxidative burst which is a defense mechanism to pathogen invasion in plants. Phosphatase inhibitors such as calyculin A and okadaic acid cause the burst whereas protein-kinase inhibitors such as K-252a and staurosporine prevent the process. Such a phosphorylation indicates the possibility of the role of phosphatases and kinases in the mechanisms of disease-resistance in plants.

Small GTP-binding proteins regulate growth, differentiation and intracellular transportation in plants. These proteins play an important role in cytokinin biosynthesis and appear to be involved in plant signal-transduction pathways. Experiments indicate that the defense signal pathway consists of a complex but well regulated network of factors including GTP-binding proteins, cytokinins and salicylic acid.

Octadecanoic acid-derived compounds may play an important role in the synthesis of natural antibiotic compounds required for the defense of plants against pathogen invasion and herbivores. The cis-jasmonic acid is synthesized just after a plant is attacked by insects. A lipid-based transduction system yields jasmonic acid and its methyl ester. 12-oxyphytodienoic acid appears to be involved in signal transduction necessary for plant defenses.

Jasmonic acid plant-derived oligogalacturonides and fungal-derived chitosan oligosaccharides stimulate defensive genes in plants through the octadecanoid pathway. Wounding causes a rapid increase in jasmonic acid in leaf cells. The proteinase inhibitor-inducing activity of plant-derived oligouronides and fungal-derived chitosan oligomers like systemin is inhibited by sodium diethyldithiocarbamate and sodium salicylate, justifying a role for the octadecanoid pathway in defense responses to pathogen attack.

Phytohormones such as abscisic acid (ABA), jasmonic acid (JA) and the peptide systemin constitute the chemical signals that stimulate wound-induced proteinase inhibitor II (Pin2) gene expression in tomato and potato plants. Also, heat treatment and mechanical injury to plants induce electrical signals which contribute to Pin2 gene expression providing defense against pathogen attack. The location of JA's action site is downstream to that of ABA in the signal transduction pathway.

Elicitins are proteins secreted by species of Phytophthora and are responsible for stimulating defense responses in tobacco plants against pathogens. They cause tissue death and protect the plant from subsequent pathogen attack. Elicitins are divided into two classes, alpha and beta. Beta class is more effective in terms of causing necrosis on tobacco leaves and thus protecting against pathogens.

Salicylic acid (SA), a product of phenylpropanoid metabolism formed by decarboxylation of trans-cinnamic acid to benzoic acid and subsequent 2-hydroxylation, is responsible for systemic acquired resistance in plants to subsequent infection. Benzoic acid 2-hydroxylase and UDPglucose:SA glucosyltransferase are the two enzymes involved in the biosynthesis of SA. In a tobacco plant infected with tobacco mosaic virus, newly formed SA is quickly metabolized to SA O-beta-D-glucoside and methyl salicylate.

Experimental investigations reveal that tomato lines having Mi, a nematode-resistance gene, exhibits resistance to Macrosiphum euphorbiae, a potato aphid. This aphid resistance is connected to the nematode resistance, as revealed by their association in near-isogenic lines. A single major locus, constituting the aphid resistance, is linked to Mi genetically, as revealed by F2 population analysis.

Collection and analysis of the volatile compounds released by cotton plants attacked by beet armyworm larvae enable establishment of the diurnal pattern of volatile terpenoid emission. Monitoring the emission over 3 days of attack reveals the emission of terpene hydrocarbon and other volatile compounds derived from lipoxygenase in high concentrations. Other acyclic terpenes exhibit a diurnal pattern of emission with progression of the damage, with maximal emissions in the afternoons. While the diurnal emission of acyclic terpenes continues even after the removal of the caterpillars, cyclic terpene emission stops.

The genome of pea early browning virus (PEBV) consists of of two positive-sense RNAs separately encapsidated in rod-sphaped particles. The PEBV RNA1 sequence contains an open reading frame (ORF) that codes for a 54-kDa protein. Nicotiana benthamiana plants were transformed with the sequence representing the 54-kDa ORF. The resulting transgenic plants were resistant to infection to an uncharacterized isolate of British PEBV as well as to two other tobraviruses. Furthermore, two variants of PEBV that overcame thee 54-kDa-mediated resistance were isolated. Further characterization of these variants will provide clues on the mechanism of resistance to PEBV.

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXL[Phi] or YXX[Phi] endocytosis signals ([Phi] is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434-438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

Production of reactive oxygen intermediates (ROI) and a form of programmed cell death called hypersensitive response (HR) are often associated with disease resistance of plants. We have previously shown that the Rac homolog of rice, OsRac1, is a regulator of ROI production and cell death in rice. Here we show that the constitutively active OsRac1 (i) causes HR-like responses and greatly reduces disease lesions against a virulent race of the rice blast fungus; (ii) causes resistance against a virulent race of bacterial blight; and (iii) causes enhanced production of a phytoalexin and alters expression of defense-related genes. The dominant-negative OsRac1 suppresses elicitor-induced ROI production in transgenic cell cultures, and in plants suppresses the HR induced by the avirulent race of the fungus. Taken together, our findings strongly suggest that OsRac1 has a general role in disease resistance of rice.

To facilitate plant colonization, some pathogenic fungi produce phytotoxic metabolites that damage tissues; plants may be resistant to a particular pathogen if they produce an enzyme(s) that catalyzes detoxification of this metabolite(s). Alternaria blackspot is one of the most damaging and significant fungal diseases of brassica crops, with no source of resistance known within the Brassica species. Destruxin B is the major phytotoxin produced by the blackspot-causing fungus, Alternaria brassicae (Berkley) Saccardo. We have established that a blackspot-resistant species (Sinapis alba) metabolized [sup.14]C-labeled destruxin B to a less toxic product substantially faster than any of the susceptible species. The first metabolite, hydroxydestruxin B ([sup.14]C-labeled), was further biotransformed to the [Beta]-D-glucosyl derivative at a slower rate. The structures of hydroxydestruxin B and [Beta]-D-glucosyl hydroxydestruxin B were deduced from their spectroscopic data [NMR, high resolution (HR)-MS, Fourier transform infrared (FTIR)] and confirmed by total chemical synthesis. Although these hydroxylation and glucosylation reactions occurred in both resistant (S. alba) and susceptible (Brassica napus, Brassica juncea, and Brassica rapa) species, hydroxylation was the rate limiting step in the susceptible species, whereas glucosylation was the rate limiting step in the resistant species. Remarkably, it was observed that the hydroxydestruxin B induced the biosynthesis of phytoalexins in blackspot-resistant species but not in susceptible species. This appears to be a unique example of phytotoxin detoxification and simultaneous phytoalexin elicitation by the detoxification product. Our studies suggest that S. alba can overcome the fungal invader through detoxification of destruxin B coupled with production of phytoalexins.

The enhanced disease resistance 1 (edr1) mutation of Arabidopsis confers resistance to powdery mildew disease caused by the fungus Erysiphe cichoracearum. Resistance mediated by the edr1 mutation is correlated with induction of several defense responses, including host cell death. Double mutant analysis revealed that all edr1-associated phenotypes are suppressed by mutations that block salicylic acid (SA) perception (nim1) or reduce SA production (pad4 and eds1). The NahG transgene, which lowers endogenous SA levels, also suppressed edr1. In contrast, the ein2 mutation did not suppress edr1-mediated resistance and associated phenotypes, indicating that ethylene and jasmonic acid-induced responses are not required for edr1 resistance. The EDR1 gene was isolated by positional cloning and was found to encode a putative MAP kinase kinase kinase similar to CTR1, a negative regulator of ethylene responses in Arabidopsis. Taken together, these data suggest that EDR1 functions at the top of a MAP kinase cascade that negatively regulates SA-inducible defense responses. Putative orthologs of EDR1 are present in monocots such as rice and barley, indicating that EDR1 may regulate defense responses in a wide range of crop species.

Plants can defend themselves from herbivorous insects by emitting volatile chemical signals that attract natural enemies of the herbivore. For example, maize seedlings attacked by beet armyworm larvae (Spodoptera exigua) produce a mixture of terpenoid and indole volatiles that serve to attract parasitic wasps. A key step in terpenoid biosynthesis is the conversion of acyclic prenyl diphosphates to terpenoid compounds by specific terpenoid synthases (cyclases). We have cloned a maize sesquiterpene cyclase gene, stc1, by transposon tagging and have identified two deletion mutations of the gene. The stc1 gene is located on chromosome 95 and does not seem to have a closely related ortholog in the maize genome. It is induced 15- to 30-fold in maize leaves by beet armyworm larvae feeding or by application of purified volicitin, the insect-derived elicitor, at a mechanically wounded site. stc1 induction is systemic, because undamaged leaves of the same plant show a similar increase in stc1 transcription. Analysis of volatiles from volicitin-treated seedlings revealed that a major naphthalene-based sesquiterpene was present in wild-type seedlings but absent in the Ac-insertion and x-ray-deletion mutants. Therefore, we have identified a maize gene that responds to caterpillar herbivory by producing a chemical defense signal that most likely serves to attract natural enemies of the herbivore.

Maize and a variety of other plant species release volatile compounds in response to herbivore attack that serve as chemical cues to signal natural enemies of the feeding herbivore. N-(17-hydroxy-linolenoyl)-L-glutamine is an elicitor component that has been isolated and chemically characterized from the regurgitant of the herbivore-pest beet armyworm. This fatty acid derivative, referred to as volicitin, triggers the synthesis and release of volatile components, including terpenoids and indole in maize. Here we report on a previously unidentified enzyme, indole-3-glycerol phosphate lyase (IGL), that catalyzes the formation of free indole and is selectively activated by volicitin. IGL's enzymatic properties are similar to BX1, a maize enzyme that serves as the entry point to the secondary defense metabolites DIBOA and DIMBOA. Genesequence analysis indicates that Igl and Bx1 are evolutionarily related to the tryptophan synthase alpha subunit.

The plant-intracellular interaction of the avirulence protein AvrPto of Pseudomonas syringae pathovar tomato, the agent of bacterial speck disease, and the corresponding tomato resistance protein Pto triggers responses leading to disease resistance. Pto, a serine/threonine protein kinase, also interacts with a putative downstream kinase, Pto-interactor 1, as well as with members of a family of transcription factors Pto-interactors 4, 5, and 6. These proteins are likely involved, respectively, in a phosphorylation cascade resulting in hypersensitive cell death, and in defense gene activation. The mechanism by which the interaction of AvrPto and Pto initiates defense response signaling is not known. To pursue the hypothesis that tertiary interactions are involved, we modified the yeast two-hybrid protein interaction trap and conducted a search for tomato proteins that interact with Pto only in the presence of AvrPto. Five classes of AvrPto-dependent Pto interactors were isolated, and their interaction specificity confirmed. Also, to shed light on a recently demonstrated virulence activity of AvrPto, we conducted a standard two-hybrid screen for tomato proteins in addition to Pto that interact with AvrPto: i.e., potential virulence targets or modifiers of AvrPto. By constructing an N-terminal rather than a C-terminal fusion of AvrPto to the LexA DNA binding domain, we were able to overcome autoactivation by AvrPto and identify four classes of specific AvrPto-interacting proteins.

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the [H.sub.2][O.sub.2]-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to I[Kappa]B[Alpha] and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-l). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-l). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals. MAP kinases | [Ca.sup.+2] | cADP ribose | cGMP | disease resistance

Genetic analysis of plant-pathogen interactions has demonstrated that resistance to infection is often determined by the interaction of dominant plant resistance (R) genes and dominant pathogen-encoded avirulence (Avr) genes. It was postulated that R genes encode receptors for Avr determinants. A large number of R genes and their cognate Avr genes have now been analyzed at the molecular level. R gene loci are extremely polymorphic, particularly in sequences encoding amino acids of the leucine-rich repeat motif. A major challenge is to determine how Avr perception by R proteins triggers the plant defense response. Mutational analysis has identified several genes required for the function of specific R proteins. Here we report the identification of Rcr3, a tomato gene required specifically for Cf-2-mediated resistance. We propose that Avr products interact with host proteins to promote disease, and that R proteins 'guard' these host components and initiate Avr-dependent plant defense responses.

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

Research shows that the gene-for-gene relationships in plant-insect interactions have fundamentally similar plant defense responses for pests ranging from viruses, bacteria and fungi to nematodes and insects. Pathogens have the ability to adapt to different varieties of crop plants due to its capacity to elude recognition by plant resistance genes. Thus, genetic engineering is the most viable option for producing pest-resistant varieties of crops.