CRISPR-based assessment of genomic structure in the conserved SQUAMOSA promoter-binding-like gene clusters in rice.

Although SQUAMOSA promoter-binding-like (SPL) transcription factors are important regulators of development in rice (Oryza sativa), prior assessments of the SPL family have been limited to single genes. A functional comparison across the full gene family in standardized genetic backgrounds has not been reported previously. Here, we demonstrate that the SPL gene family in rice is enriched due to the most recent whole genome duplication (WGD). Notably, 10 of 19 rice SPL genes (52%) cluster in four units that have persisted for at least 50 million years. We show that SPL gene grouping and retention following WGD is widespread in angiosperms, suggesting the conservatism and importance of this gene arrangement. We used Cas9 editing to generate transformation lines for all 19 SPL genes in a common set of backgrounds, and found that knockouts of 14 SPL genes exhibited defects in plant height, 10 exhibited defects in panicle size, and nine had altered grain lengths. We observed subfunctionalization of genes in the paleoduplicated pairs, but little evidence of neofunctionalization. Expression of OsSPL3 was negatively correlated with that of its closest neighbor in its synteny group, OsSPL4, and its sister paired gene, OsSPL12, in the opposing group. Nucleotide diversity was lower in eight of the nine singleton genes in domesticated rice, relative to wild rice, whereas the reverse was true for the paired genes. Together, these results provide functional information on eight previously unexamined OsSPL family members and suggest that paleoduplicate pair redundancy benefits plant survival and innovation.

CRISPR-Based Assessment of Gene Specialization in the Gibberellin Metabolic Pathway in Rice.

Gibberellin (GA) functions as an essential natural regulator of growth and development in plants. For each step of the GA metabolic pathway, different copy numbers can be found in different species, as is the case with the 13 genes across four enzymatic steps in rice (Oryza sativa). A common view is that such gene duplication creates homologs that buffer organisms against loss-of-function (LOF) mutations. Therefore, knockouts of any single homolog might be expected to have little effect. To test this question, we generated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) knockouts for these homologs and measured effects on growth and reproduction. Surprisingly, we report here that there is consistently one or more essential gene at each enzymatic step, for which LOF mutation induces death or sterility-suggesting that the GA pathway does not have a redundancy route and that each gene family is essential for GA metabolism. In most of these genes from the same gene family, we observed defects in plant height and infertility, suggesting that the duplicated members retain functions related to GA synthesis or degradation. We identified both subfunctionalization of the three recently diversified homologs OsKO1, OsKO2, and OsKO5 and neofunctionalization in OsKO3 and OsKO4 Thus, although the function of each step is conserved, the evolution of duplicates in that step is diversified. Interestingly, the CRISPR/Cas9 lines at the SD1 locus were typically sterile, whereas the natural sd1 mutants, related to the "Green Revolution" in rice, show normal setting rates. Collectively, our results identify candidates for control of GA production and provide insight into the evolution of four critical gene families in plants.

Foliar bacteria and soil fertility mediate seedling performance: a new and cryptic dimension of niche differentiation.

The phyllosphere (comprising the leaf surface and interior) is one of the world's largest microbial habitats and is host to an abundant and diverse array of bacteria. Nonetheless, the degree to which bacterial communities are benign, harmful, or beneficial to plants in situ is unknown. We tested the hypothesis that the net effect of reducing bacterial abundance and diversity would vary substantially among host species (from harmful to beneficial) and this would be strongly mediated by soil resource availability. To test this, we monitored tree seedling growth responses to commercial antibiotics among replicated resource supply treatments (N, P, K) in a tropical forest in Panama for 29 months. We applied either antibiotics or control water to replicated seedlings of five common tree species (Alseis blackiana, Desmopsis panamensis, Heisteria concinna, Sorocea affinis, and Tetragastris panamensis). These antibiotic treatments significantly reduced both the abundance and diversity of bacteria epiphytically as well as endophytically. Overall, the effect of antibiotics on performance was highly host specific. Applying antibiotics increased growth for three species by as much as 49% (Alseis, Heisteria, and Tetragastris), decreased growth for a fourth species by nearly 20% (Sorocea), and had no impact on a fifth species (Desmopsis). Perhaps more importantly, the degree to which foliar bacteria were harmful or not varied with soil resource supply. Specifically, applying antibiotics had no effect when potassium was added but increased growth rate by almost 40% in the absence of potassium. Alternatively, phosphorus enrichment caused the effect of bacteria to switch from being primarily beneficial to harmful or vice versa, but this depended entirely on the presence or absence of nitrogen enrichment (i.e., important and significant interactions). Our results are the first to demonstrate that the net effect of reducing the abundance and diversity of bacteria can have very strong positive and negative effects on seedling performance. Moreover, these effects were clearly mediated by soil resource availability. Though speculative, we suggest that foliar bacteria may interact with soil fertility to comprise an important, yet cryptic dimension of niche differentiation, which can have important implications for species coexistence.

Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana.

Plants can defend themselves against a wide array of enemies, from microbes to large animals, yet there is great variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack. A second explanation comes from an evolutionary 'tug of war', in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best explained by costs incurred from constitutive defences in a pest-free environment. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6), underpins marked pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its marked impact on growth.

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines.

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

ATP-dependent binding cassette transporter G family member 16 increases plant tolerance to abscisic acid and assists in basal resistance against Pseudomonas syringae DC3000.

Plants have been shown previously to perceive bacteria on the leaf surface and respond by closing their stomata. The virulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 (PstDC3000) responds by secreting a virulence factor, coronatine, which blocks the functioning of guard cells and forces stomata to reopen. After it is inside the leaf, PstDC3000 has been shown to up-regulate abscisic acid (ABA) signaling and thereby suppress salicylic acid-dependent resistance. Some wild plants exhibit resistance to PstDC3000, but the mechanisms by which they achieve this resistance remain unknown. Here, we used genome-wide association mapping to identify an ATP-dependent binding cassette transporter gene (ATP-dependent binding cassette transporter G family member16) in Arabidopsis (Arabidopsis thaliana) that contributes to wild plant resistance to PstDC3000. Through microarray analysis and ß-glucuronidase reporter lines, we showed that the gene is up-regulated by ABA, bacterial infection, and coronatine. We also used a green fluorescent protein fusion protein and found that transporter is more likely to localize on plasma membranes than in cell walls. Transferred DNA insertion lines exhibited consistent defective tolerance of exogenous ABA and reduced resistance to infection by PstDC3000. Our conclusion is that ATP-dependent binding cassette transporter G family member16 is involved in ABA tolerance and contributes to plant resistance against PstDC3000. This is one of the first examples, to our knowledge, of ATP-dependent binding cassette transporter involvement in plant resistance to infection by a bacterial pathogen. It also suggests a possible mechanism by which plants reduce the deleterious effects of ABA hijacking during pathogen attack. Collectively, these results improve our understanding of basal resistance in Arabidopsis and offer unique ABA-related targets for improving the innate resistance of plants to bacterial infection.

Herbivory and relative growth rates of Pieris rapae are correlated with host constitutive salicylic acid and flowering time.

Treatment of plants with exogenous salicylic acid (SA) improves resistance to many bacterial pathogens, but can suppress resistance to insect herbivores. While plants vary naturally in constitutive SA, whether such differences are predictive of resistance to insect herbivores has not been studied previously. We examined the possible role of this endogenous SA in structuring the interactions between the cabbage white butterfly, Pieris rapae, and ten hosts in the mustard family (Brassicaceae). Because P. rapae has multiple generations that utilize different hosts across the year, we included five spring-flowering mustards and five summer-flowering mustards that co-occur in ruderal habitats in upstate New York. Under common garden conditions, the spring flowering mustards (Capsella bursa-pastoris, Draba verna, Cardamine impatiens, Barbarea vulgaris, and Arabidopsis thaliana) were significantly more resistant to P. rapae, supporting 42 % less herbivory (P = 0.015) and 64 % lower relative growth rates (P = 0.007), relative to the summer flowering mustards (Sisymbrium altissimum, Brassica nigra, Sinapis arvense, Lepidium campestre, and Arabis canadensis). Leaf total constitutive SA explained significant variation in larval herbivory (R (2) = 75.3 %, P = 0.007) and relative growth rates (R (2) = 59.4 %, P = 0.043). The three species with the lowest levels of constitutive SA (Capsella bursa-pastoris, Draba verna, and Cardamine impatiens) were the most resistant to larvae. Barbarea vulgaris and Arabis canadensis were notable exceptions, exhibiting high SA concentrations and intermediate resistance to P. rapae. These results suggest a curvilinear relationship between leaf constitutive SA and the herbivory by P. rapae, and they provide some insight into the ecology and possible management of this economically important crop pest.

Ascorbic acid deficiency in arabidopsis induces constitutive priming that is dependent on hydrogen peroxide, salicylic acid, and the NPR1 gene.

The ascorbic acid (AA)-deficient Arabidopsis thaliana vtc1-1 mutant exhibits increased resistance to the virulent bacterial pathogen Pseudomonas syringae. This response correlates with heightened levels of salicylic acid (SA), which induces antimicrobial pathogenesis-related (PR) proteins. To determine if SA-mediated, enhanced disease resistance is a general phenomenon of AA deficiency, to elucidate the signal that stimulates SA synthesis, and to identify the biosynthetic pathway through which SA accumulates, we studied the four AA-deficient vtc1-1, vtc2-1, vtc3-1, and vtc4-1 mutants. We also studied double mutants defective in the AA-biosynthetic gene VTC1 and the SA signaling pathway genes PAD4, EDS5, and NPR1, respectively. All vtc mutants were more resistant to P. syringae than the wild type. With the exception of vtc4-1, this correlated with constitutively upregulated H(2)O(2), SA, and messenger RNA levels of PR genes. Double mutants exhibited decreased SA levels and enhanced susceptibility to P. syringae compared with the wild type, suggesting that vtc1-1 requires functional PAD4, EDS5, and NPR1 for SA biosynthesis and pathogen resistance. We suggest that AA deficiency causes constitutive priming through a buildup of H(2)O(2) that stimulates SA accumulation, conferring enhanced disease resistance in vtc1-1, vtc2-1, and vtc3-1, whereas vtc4-1 might be sensitized to H(2)O(2) and SA production after infection.

Pleiotropic changes revealed by in situ recovery of the semi-dwarf gene sd1 in rice.

The "Green Revolution" that dramatically reduced cultivar heights and sharply boosted rice production mid-century was achieved in large part through introgression of defective alleles of Semi-Dwarf 1 (SD1), which encodes a GA20ox oxidase involved in the final steps of the synthesis of bioactive gibberellin in rice. Here, we ask whether converting the defective sd1 version in a modern semi-dwarf cultivar back to wild-type SD1 in situ recovers ancestral plant traits, and more broadly, what it reveals about pleiotropic effects of this gene. We assess these effects of SD1 restoration in three independent recombinant lines recovered from F2 progeny of a cross between 93-11 and PA64s. We then used RNA-seq to dissect gene network changes that accompanied SD1 restoration. We report that this in situ restoration of wild-type SD1 nearly doubles plant height, increases total grain yield per panicle, and elongates the second-leaf length. Comparison of expression profiles reveals changes in key nodes of the gibberellin pathway, such as OsKO1 and OsGA2ox3, and more broadly in genes related to metabolic networks, defense response, and catabolic processes. Two JA-induced genes, RIR1b and OsPR1b, are extremely down-regulated after SD1 restoration, suggesting that SD1 restoration alters the balance between GA and JA to plant growth, at the cost of degrading the defense response. This in situ approach at the SD1 locus also provides a model example that is applicable to other systems and will further understanding of gene networks underlying high-yield traits in crops.

Low levels of polymorphism in genes that control the activation of defense response in Arabidopsis thaliana.

Plants use signaling pathways involving salicylic acid, jasmonic acid, and ethylene to defend against pathogen and herbivore attack. Many defense response genes involved in these signaling pathways have been characterized, but little is known about the selective pressures they experience. A representative set of 27 defense response genes were resequenced in a worldwide set of 96 Arabidopsis thaliana accessions, and patterns of single nucleotide polymorphisms (SNPs) were evaluated in relation to an empirical distribution of SNPs generated from either 876 fragments or 236 fragments with >400 bp coding sequence (this latter set was selected for comparisons with coding sequences) distributed across the genomes of the same set of accessions. Defense response genes have significantly fewer protein variants, display lower levels of nonsynonymous nucleotide diversity, and have fewer nonsynonymous segregating sites. The majority of defense response genes appear to be experiencing purifying selection, given the dearth of protein variation in this set of genes. Eight genes exhibit some evidence of partial selective sweeps or transient balancing selection. These results therefore provide a strong contrast to the high levels of balancing selection exhibited by genes at the upstream positions in these signaling pathways.

Glucosinolates and trichomes track tissue value in two sympatric mustards.

Glucosinolates, trichomes, nitrogen, and carbon are not distributed uniformly through the canopies of mustards. In this study, we asked whether glucosinolate concentrations and trichome densities in two sympatric mustards, Brassica kaber and B. nigra, are highest in tissues of greatest value to the plant. We also asked whether nitrogen or carbon content is the stronger predictor of tissue value, and what fraction of each resource is incorporated in glucosinolates. To quantify tissue values, we removed three equal-area fractions (lower, middle, and upper) from the canopies of B. kaber and B. nigra in the greenhouse, as well as whole canopies of naturally growing B. nigra in the field, at two times during growth and measured reductions in their performance relative to controls. We also measured trichome density in both experiments, as well as glucosinolate, nitrogen, and carbon concentrations for the equal-area fractions in the greenhouse. We found that upper leaves had the highest glucosinolate concentrations, trichome densities, and tissue values. Furthermore, young plants in the field had higher trichome densities and tissue values than did older plants. Collectively, these data provide strong support for optimal defense theory and are among the first such evidence for glucosinolates and for physical defenses. The positive relationship between trichome density and tissue value was strong even after we accounted for the effects of leaf expansion. While nitrogen and carbon have both received attention as currencies for trade-offs, our data suggest that nitrogen concentration is a significantly better predictor of tissue value in these two mustard species. Interestingly, <1% of the nitrogen or carbon in leaves was incorporated in glucosinolates, which may explain why glucosinolates lack a consistent response to nitrogen fertilization.

Salicylic acid and jasmonic acid signaling defense pathways reduce natural bacterial diversity on Arabidopsis thaliana.

Terrestrial plants serve as large and diverse habitats for a wide range of pathogenic and nonpathogenic microbes, yet these communities are not well described and little is known about the effects of plant defense on microbial communities in nature. We designed a field experiment to determine how variation in two plant defense signaling pathways affects the size, diversity, and composition of the natural endophytic and epiphytic bacterial communities of Arabidopsis thaliana. To do this, we provide an initial characterization of these bacterial communities in one population in southwestern Michigan, United States, and we compare these two communities among A. thaliana mutants deficient in salicylic acid (SA) and jasmonic acid (JA) signaling defense pathways, controls, and plants with artificially elevated levels of defense. We identified 30 distinct bacterial groups on A. thaliana that differ in colony morphology and 16S rRNA sequence. We show that induction of SA-mediated defenses reduced endophytic bacterial community diversity, whereas plants deficient in JA-mediated defenses experienced greater epiphytic bacterial diversity. Furthermore, there was a positive relationship between total community size and diversity, indicating that relatively susceptible plants should, in general, harbor higher bacterial diversity. This experiment provides novel information about the ecology of bacteria on A. thaliana and demonstrates that variation in two specific plant-signaling defense pathways can influence bacterial diversity on plants.

SAR increases fitness of Arabidopsis thaliana in the presence of natural bacterial pathogens.

Given the substantial costs of plant defenses against pathogens, there should be corresponding benefits that prevent resistance from being lost in natural plant populations. Here, we present evidence that systemic acquired resistance (SAR) benefits plants attacked by pathogenic bacteria in nature. In a large field experiment, we found that Arabidopsis thaliana treated with salicylic acid exhibited reduced titers of bacteria in their leaves and elevated fitness relative to controls. Most common members of the culturable bacterial community suffered this decrease, consistent with the role of SAR as a broad spectrum defense. We found no evidence of negative interactions between SAR and jasmonate-dependent resistance. Plants treated with jasmonic acid received significantly lower insect damage to their siliques, but exhibited no differences in bacterial growth or fitness relative to controls. Collectively, these data suggest a likely role of pathogenic bacteria in the maintenance of SAR, but not jasmonate-dependent resistance, in nature.

A geographic cline in leaf salicylic acid with increasing elevation in Arabidopsis thaliana.

Salicylic acid (SA) occupies a key role as a hormone central to both plant resistance to bacterial pathogens and tolerance of abiotic stresses. Plants at high elevation experience colder temperatures and elevated UV levels. While it has been predicted that SA concentrations will be higher in plants from high elevation populations, few studies have addressed this question. Here, we asked how concentrations of SA vary in natural populations of Arabidopsis thaliana collected across an elevational gradient on the Iberian Peninsula. In a series of common garden experiments, we found that constitutive SA concentrations were highest in genotypes from the low elevation populations. This result was in the opposite direction from our prediction and is an exception to the general finding that phenolic compounds increase with increasing elevation. These data suggest that high constitutive SA is not associated with resistance to cold temperatures in these plants. Furthermore, we also found that leaf constitutive camalexin concentrations, an important defense against some bacterial and fungal enemies, were highest in the low elevation populations, suggesting that pathogen pressures may be important. Further examination of this elevational cline will likely provide additional insights into the interplay between phenolic compounds and biotic and abiotic stress.

Is induction response negatively correlated with constitutive resistance in black mustard?

Black mustard, Brassica nigra, is highly variable in both constitutive resistance and induction response following damage by herbivores. A focal population from Ithaca, New York, was used to test the following two predictions of optimal defense theory: (1) that allocation to resistance will reduce plant performance in the absence of herbivores; and (2) that induction response will be negatively correlated with constitutive resistance. The experiment consisted of a half-sib mating design with 47 paternal families and four dams per sire, fully crossed with a damage treatment consisting of 25% leaf removal by the cabbage white butterfly, Pieris rapae, when plants had four leaves. Leaf trichome density, sinigrin concentration, and glucobrassicin concentration were 38%, 19%, and 16% higher, respectively, for the seventh leaf of damaged plants. Paternal families did not vary significantly in their induction response. Narrow-sense heritabilities were h(S)(2) = 0.51, 0.76, and 0.50 for constitutive leaf trichome density, sinigrin concentration, and glucobrassicin concentration, respectively. Positive genetic correlations were found between glucobrassicin concentration and days to first flower, suggesting a genetic cost of resistance. Induction responses were negatively correlated with constitutive allocation for leaf trichome density and sinigrin concentration. The results were therefore consistent with optimal defense theory, offering modest evidence for both predictions.

Effects of CO2 elevation on canopy development in the stands of two co-occurring annuals.

Elevated CO2 may increase dry mass production of canopies directly through increasing net assimilation rate of leaves and also indirectly through increasing leaf area index (LAI). We studied the effects of CO2 elevation on canopy productivity and development in monospecific and mixed (1:1) stands of two co-occurring C3 annual species, Abutilon theophrasti, and Ambrosia artemisiifolia. The stands were established in the glasshouse with two CO2 levels (360 and 700 µl/l) under natural light conditions. The planting density was 100 per m2 and LAI increased up to 2.6 in 53 days of growth. Root competition was excluded by growing each plant in an individual pot. However, interference was apparent in the amount of photons absorbed by the plants and in photon absorption per unit leaf area. Greater photon absorption by Abutilon in the mixed stand was due to different canopy structures: Abutilon distributed leaves in the upper layers in the canopy while Ambrosia distributed leaves more to the lower layers. CO2 elevation did not affect the relative performance and light interception of the two species in mixed stands. Total aboveground dry mass was significantly increased with CO2 elevation, while no significant effects on leaf area development were observed. CO2 elevation increased dry mass production by 30-50%, which was mediated by 35-38% increase in the net assimilation rate (NAR) and 37-60% increase in the nitrogen use efficiency (NUE, net assimilation rate per unit leaf nitrogen). Since there was a strong overall correlation between LAI and aboveground nitrogen and no significant difference was found in the regression of LAI against aboveground nitrogen between the two CO2 levels, we hypothesized that leaf area development was controlled by the amount of nitrogen taken up from the soil. This hypothesis suggests that the increased LAI with CO2 elevation observed by several authors might be due to increased uptake of nitrogen with increased root growth.

Constitutive camalexin production and environmental stress response variation in Arabidopsis populations from the Iberian Peninsula.

Optimal defense theory predicts that induction of defensive secondary metabolites in plants will be inversely correlated with constitutive expression of those compounds. Here, we asked whether camalexin, an important defense against fungal and bacterial pathogens, support this prediction in structured natural populations of Arabidopsis thaliana from the Iberian Peninsula. In common garden experiments, we found that genotypes from the VIE population constitutively hyper-accumulated camalexin. Camalexin concentrations were not induced significantly when plants were exposed to a temperature of 10°C for 48h. However, they were induced when plants were exposed to 48h of infection by the virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Genotypes from the VIE population with the hyper-accumulation of camalexin were significantly more resistant to bacterial growth. Induction of camalexin was negatively correlated with constitutive camalexin concentrations following log transformation and two different corrections for autocorrelation, thus supporting the tradeoff predicted by optimal defense theory. Constitutive overexpression of camalexin was not explained by the only known natural genetic polymorphism at the Accelerated Cell Death 6, ACD6, locus. Collectively, the results support an important role of camalexin in defense against P. syringae as well as significant structured variation in defense levels within wild populations.

Plant immune system incompatibility and the distribution of enemies in natural hybrid zones.

Natural plant hybrid zones have been described as 'sinks' and 'centers of biodiversity' for herbivores and fungal pathogens. Jasmonic acid is known to be a critical signaling molecule for defense against these enemies. Does inhibition of jasmonic acid perhaps contribute to the susceptibility of hybrid plants to attack by herbivores? Here, we discuss recent evidence that plant immune system incompatibilities are likely to downregulate jasmonic acid (JA)-dependent responses through their effect on expression of the salicylic acid (SA)-dependent pathway. Because these hybrid immune incompatibilities are a function of environmental temperatures, they suggest a dependency between attack rates in hybrid zones and environmental conditions. Hybrid zones in colder environments, for example at higher elevations or latitudes, are more likely to exhibit elevated SA, suppressed jasmonic acid-dependent defenses, and lower resistance to herbivores and other necrotrophic pathogens.

Interactive effects of jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis.

Leaf trichomes protect plants from attack by insect herbivores and are often induced following damage. Hormonal regulation of this plant induction response has not been previously studied. In a series of experiments, we addressed the effects of artificial damage, jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Artificial damage and jasmonic acid caused significant increases in trichome production of leaves. The jar1-1 mutant exhibited normal trichome induction following treatment with jasmonic acid, suggesting that adenylation of jasmonic acid is not necessary. Salicylic acid had a negative effect on trichome production and consistently reduced the effect of jasmonic acid, suggesting negative cross-talk between the jasmonate and salicylate-dependent defense pathways. Interestingly, the effect of salicylic acid persisted in the nim1-1 mutant, suggesting that the Npr1/Nim1 gene is not downstream of salicylic acid in the negative regulation of trichome production. Last, we found that gibberellin and jasmonic acid had a synergistic effect on the induction of trichomes, suggesting important interactions between these two compounds.