RESUMEN
Plants in their natural ecosystems interact with numerous microorganisms, but how they influence their microbiota is still elusive. We observed that sulfatase activity in soil, which can be used as a measure of rhizosphere microbial activity, is differently affected by Arabidopsis accessions. Following a genome-wide association analysis of the variation in sulfatase activity we identified a candidate gene encoding an uncharacterized cytochrome P450, CYP71A27 Loss of this gene resulted in 2 different and independent microbiota-specific phenotypes: A lower sulfatase activity in the rhizosphere and a loss of plant growth-promoting effect by Pseudomonas sp. CH267. On the other hand, tolerance to leaf pathogens was not affected, which agreed with prevalent expression of CYP71A27 in the root vasculature. The phenotypes of cyp71A27 mutant were similar to those of cyp71A12 and cyp71A13, known mutants in synthesis of camalexin, a sulfur-containing indolic defense compound. Indeed, the cyp71A27 mutant accumulated less camalexin in the roots upon elicitation with silver nitrate or flagellin. Importantly, addition of camalexin complemented both the sulfatase activity and the loss of plant growth promotion by Pseudomonas sp. CH267. Two alleles of CYP71A27 were identified among Arabidopsis accessions, differing by a substitution of Glu373 by Gln, which correlated with the ability to induce camalexin synthesis and to gain fresh weight in response to Pseudomonas sp. CH267. Thus, CYP71A27 is an additional component in the camalexin synthesis pathway, contributing specifically to the control of plant microbe interactions in the root.
Asunto(s)
Arabidopsis , Sistema Enzimático del Citocromo P-450 , Indoles/metabolismo , Raíces de Plantas , Pseudomonas/metabolismo , Tiazoles/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Mutación , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiologíaRESUMEN
Plants induce systemic acquired resistance (SAR) upon localized exposure to pathogens. Pipecolic acid (Pip) production via AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) is key for SAR establishment. Here, we report a positive feedback loop important for SAR induction in Arabidopsis thaliana We showed that local activation of the MAP kinases MPK3 and MPK6 is sufficient to trigger Pip production and mount SAR. Consistent with this, mutations in MPK3 or MPK6 led to compromised Pip accumulation upon inoculation with the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pto) AvrRpt2, which triggers strong sustained MAPK activation. By contrast, P. syringae pv maculicola and Pto, which induce transient MAPK activation, trigger Pip biosynthesis and SAR independently of MPK3/6. ALD1 expression, Pip accumulation, and SAR were compromised in mutants defective in the MPK3/6-regulated transcription factor WRKY33. Chromatin immunoprecipitation showed that WRKY33 binds to the ALD1 promoter. We found that Pip triggers activation of MPK3 and MPK6 and that MAPK activation after Pto AvrRpt2 inoculation is compromised in wrky33 and ald1 mutants. Collectively, our results reveal a positive regulatory loop consisting of MPK3/MPK6, WRKY33, ALD1, and Pip in SAR induction and suggest the existence of distinct SAR activation pathways that converge at the level of Pip biosynthesis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transaminasas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Resistencia a la Enfermedad , Retroalimentación Fisiológica , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Ácidos Pipecólicos/metabolismo , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Pseudomonas syringae/patogenicidad , Transaminasas/genética , Factores de Transcripción/genéticaRESUMEN
Following a previous microbial inoculation, plants can induce broad-spectrum immunity to pathogen infection, a phenomenon known as systemic acquired resistance (SAR). SAR establishment in Arabidopsis thaliana is regulated by the Lys catabolite pipecolic acid (Pip) and flavin-dependent-monooxygenase1 (FMO1). Here, we show that elevated Pip is sufficient to induce an FMO1-dependent transcriptional reprogramming of leaves that is reminiscent of SAR. In planta and in vitro analyses demonstrate that FMO1 functions as a pipecolate N-hydroxylase, catalyzing the biochemical conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates in plants after microbial attack. When exogenously applied, it overrides the defect of NHP-deficient fmo1 in acquired resistance and acts as a potent inducer of plant immunity to bacterial and oomycete infection. Our work has identified a pathogen-inducible L-Lys catabolic pathway in plants that generates the N-hydroxylated amino acid NHP as a critical regulator of systemic acquired resistance to pathogen infection.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Oxigenasas/metabolismo , Ácidos Pipecólicos/metabolismo , Inmunidad de la Planta/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Cromatografía de Gases y Espectrometría de Masas , Lisina/metabolismo , Oomicetos/patogenicidad , Oxigenasas/genética , Ácidos Pipecólicos/análisis , Ácidos Pipecólicos/farmacología , Hojas de la Planta/enzimología , Hojas de la Planta/inmunología , Hojas de la Planta/metabolismo , Pseudomonas syringae/patogenicidad , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/µ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.
Asunto(s)
Arabidopsis/inmunología , Ácidos Pipecólicos/inmunología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Pseudomonas syringae/inmunología , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Interacciones Huésped-Patógeno/inmunología , Cetoácidos/inmunología , Cetoácidos/metabolismo , Leucina/inmunología , Leucina/metabolismo , Lisina/inmunología , Lisina/metabolismo , Metionina/inmunología , Metionina/metabolismo , Ácidos Pipecólicos/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/fisiología , Transaminasas/genética , Transaminasas/inmunología , Transaminasas/metabolismoRESUMEN
We investigated the relationships of the two immune-regulatory plant metabolites, salicylic acid (SA) and pipecolic acid (Pip), in the establishment of plant systemic acquired resistance (SAR), SAR-associated defense priming, and basal immunity. Using SA-deficient sid2, Pip-deficient ald1, and sid2 ald1 plants deficient in both SA and Pip, we show that SA and Pip act both independently from each other and synergistically in Arabidopsis thaliana basal immunity to Pseudomonas syringae. Transcriptome analyses reveal that SAR establishment in Arabidopsis is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by preactivating multiple stages of defense signaling and that SA accumulation upon SAR activation leads to the downregulation of photosynthesis and attenuated jasmonate responses systemically within the plant. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. During SAR, Pip orchestrates SA-dependent and SA-independent priming of pathogen responses in a FLAVIN-DEPENDENT-MONOOXYGENASE1 (FMO1)-dependent manner. We conclude that a Pip/FMO1 signaling module acts as an indispensable switch for the activation of SAR and associated defense priming events and that SA amplifies Pip-triggered responses to different degrees in the distal tissue of SAR-activated plants.
Asunto(s)
Arabidopsis/inmunología , Inmunidad Innata , Ácidos Pipecólicos/metabolismo , Enfermedades de las Plantas/inmunología , Ácido Salicílico/metabolismo , Transducción de Señal , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Vías Biosintéticas , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Modelos Biológicos , Oxilipinas/metabolismo , Fotosíntesis , Enfermedades de las Plantas/microbiología , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Transpiración de Plantas , Pseudomonas syringae/fisiología , Transcripción GenéticaRESUMEN
We recently characterized a highly dynamic fungal disease outbreak in native populations of Nicotiana attenuata in the southwestern United States. Here, we explore how phytohormone signalling contributes to the observed disease dynamics. Single inoculation with three native Fusarium and Alternaria fungal pathogens, isolated from diseased plants growing in native populations, resulted in disease symptoms characteristic for each pathogen species. While Alternaria sp.-infected plants displayed fewer symptoms and recovered, Fusarium spp.-infected plants became chlorotic and frequently spontaneously wilted. Jasmonic acid (JA) and salicylic acid (SA) levels were differentially induced after Fusarium or Alternaria infection. Transgenic N. attenuata lines silenced in JA production or JA conjugation to isoleucine (JA-Ile), but not in JA perception, were highly susceptible to infection by F. brachygibbosumâ Utah 4, indicating that products derived from the JA-Ile biosynthetic pathway, but not their perception, is associated with increased Fusarium resistance. Infection assays using ov-nahG plants which were silenced in pathogen-induced SA accumulations revealed that SA may increase N. attenuata's resistance to Fusarium infection but not to Alternaria. Taken together, we propose that the dynamics of fungal disease symptoms among plants in native populations may be explained by a complex interplay of phytohormone responses to attack by multiple pathogens.
Asunto(s)
Alternaria/fisiología , Ciclopentanos/metabolismo , Fusarium/fisiología , Nicotiana/inmunología , Oxilipinas/metabolismo , Enfermedades de las Plantas/inmunología , Reguladores del Crecimiento de las Plantas/metabolismo , Resistencia a la Enfermedad , Interacciones Huésped-Patógeno , Isoleucina/análogos & derivados , Isoleucina/metabolismo , Ácido Salicílico/metabolismo , Transducción de Señal , Nicotiana/genéticaRESUMEN
The wild tobacco species Nicotiana attenuata has been intensively used as a model plant to study its interaction with insect herbivores and pollinators in nature, however very little is known about its native pathogen community. We describe a fungal disease outbreak in a native N. attenuata population comprising 873 plants growing in an area of about 1500 m2. The population was divided into 14 subpopulations and disease symptom development in the subpopulations was monitored for 16 days, revealing a waxing and waning of visible disease symptoms with some diseased plants recovering fully. Native fungal N. attenuata pathogens were isolated from diseased plants, characterized genetically, chemotaxonomically and morphologically, revealing several isolates of the ascomycete genera Fusarium and Alternaria, that differed in the type and strength of the disease symptoms they caused in bioassays on either detached leaves or intact soil-grown plants. These isolates and the bioassays will empower the study of N. attenuata-pathogen interactions in a realistic ecological context.
Asunto(s)
Hongos/aislamiento & purificación , Nicotiana/microbiología , Enfermedades de las Plantas/microbiología , Alternaria/genética , Alternaria/aislamiento & purificación , Bioensayo/métodos , Brotes de Enfermedades , Hongos/genética , Fusarium/genética , Fusarium/aislamiento & purificación , Proteínas de Plantas/metabolismo , SueloRESUMEN
Nicotiana attenuata plants silenced in the expression of GLYCEROLIPASEâ A1 (ir-gla1 plants) are compromised in the herbivore- and wound-induced accumulation of jasmonic acid (JA). However, these plants accumulate wild-type (WT) levels of JA and divinyl-ethers during Phytophthora parasitica infection. By profiling oxylipin-enriched fractions with targeted and untargeted liquid chromatography-tandem time-of-flight mass spectrometry approaches, we demonstrate that the accumulation of 9-hydroxy-10E,12Z-octadecadienoic acid (9-OH-18:2) and additional C18 and C19 oxylipins is reduced by ca. 20-fold in P. parasitica-infected ir-gla1 leaves compared with WT. This reduced accumulation of oxylipins was accompanied by a reduced accumulation of unsaturated free fatty acids and specific lysolipid species. Untargeted metabolic profiling of total leaf extracts showed that 87 metabolites accumulated differentially in leaves of P. parasitica-infected ir-gla1 plants with glycerolipids, hydroxylated-diterpene glycosides and phenylpropanoid derivatives accounting together for ca. 20% of these 87 metabolites. Thus, P. parasitica-induced oxylipins may participate in the regulation of metabolic changes during infection. Together, the results demonstrate that GLA1 plays a distinct role in the production of oxylipins during biotic stress responses, supplying substrates for 9-OH-18:2 and additional C18 and C19 oxylipin formation during P. parasitica infection, whereas supplying substrates for the biogenesis of JA during herbivory and mechanical wounding.
Asunto(s)
Lipasa/metabolismo , Nicotiana/enzimología , Nicotiana/inmunología , Oxilipinas/metabolismo , Phytophthora/fisiología , Metabolismo Secundario , Cromatografía Liquida , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Metabolómica , Phytophthora/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Extractos Vegetales , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Fracciones Subcelulares/metabolismo , Nicotiana/microbiologíaRESUMEN
Nicotiana attenuata HSPRO (NaHSPRO) is a negative regulator of seedling growth promoted by the fungus Piriformospora indica. Homologs of NaHSPRO in Arabidopsis thaliana (i.e., AtHSPRO1 and AtHSPRO2) are known to physically interact with the AKINßγ subunit of the SnRK1 complex. To investigate whether NaHSPRO is associated with SnRK1 function during the stimulation of seedling growth by P. indica, we studied N. attenuata plants silenced in the expression of NaGAL83 (as-gal83 plants)--a gene that encodes for the regulatory ß-subunit of SnRK1--and plants silenced in the expression of both NaHSPRO and NaGAL83 (ir-hspro/as-gal83 plants). The results showed that P. indica differentially stimulated the growth of both as-gal83 and ir-hspro/as-gal83 seedlings compared with control seedlings, with a magnitude similar to that observed in ir-hspro seedlings. Thus, we showed that, similar to NaHSPRO, NaGAL83 is a negative regulator of seedling growth stimulated by P. indica. We propose that the effect of NaHSPRO on seedling growth is associated with SnRK1 signaling.
Asunto(s)
Basidiomycota , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Nicotiana/genética , Proteínas de Plantas/genética , Plantones/crecimiento & desarrollo , Simbiosis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Silenciador del Gen , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
In a previous study aimed at identifying regulators of Nicotiana attenuata responses against chewing insects, a 26-nucleotide tag matching the HSPRO (ORTHOLOG OF SUGAR BEET Hs1(pro)(-)(1)) gene was found to be strongly induced after simulated herbivory (Gilardoni et al., 2010). Here we characterized the function of HSPRO during biotic interactions in transgenic N. attenuata plants silenced in its expression (ir-hspro). In wild-type plants, HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv tomato DC3000 and roots with the growth-promoting fungus Piriformospora indica. Reduced HSPRO expression did not affect the regulation of direct defenses against Manduca sexta herbivory or P. syringae pv tomato DC3000 infection rates. However, reduced HSPRO expression positively influenced early seedling growth during interaction with P. indica; fungus-colonized ir-hspro seedlings increased their fresh biomass by 30% compared with the wild type. Grafting experiments demonstrated that reduced HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots. This effect was accompanied by changes in the expression of 417 genes in colonized roots, most of which were metabolic genes. The lack of major differences in the metabolic profiles of ir-hspro and wild-type colonized roots (as analyzed by liquid chromatography time-of-flight mass spectrometry) suggested that accelerated metabolic rates were involved. We conclude that HSPRO participates in a whole-plant change in growth physiology when seedlings interact with P. indica.
Asunto(s)
Basidiomycota/fisiología , Nicotiana/microbiología , Proteínas de Plantas/metabolismo , Plantones/crecimiento & desarrollo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Animales , Muerte Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas , Herbivoria , Manduca , Metaboloma , Enfermedades de las Plantas/microbiología , Reguladores del Crecimiento de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/microbiología , Pseudomonas syringae/patogenicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Plantones/metabolismo , Plantones/microbiología , Análisis de Secuencia de Proteína , Spodoptera , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
The activation of enzymatic oxylipin biosynthesis upon wounding, herbivory and pathogen attack depends on the biochemical activation of lipases that make polyunsaturated fatty acids (PUFAs) available to lipoxygenases (LOXs). The identity and number of the lipases involved in this process remain controversial and they probably differ among plant species. Analysis of transgenic Nicotiana attenuata plants (ir-gla1) stably reduced in the expression of the NaGLA1 gene showed that this plastidial glycerolipase is a major supplier of trienoic fatty acids for jasmonic acid (JA) biosynthesis in leaves and roots after wounding and simulated herbivory, but not during infection with the oomycete Phytophthora parasitica (var. nicotianae). NaGLA1 was not essential for the developmental control of JA biosynthesis in flowers and for the biosynthesis of C(6) volatiles by the hydroperoxide lyase (HPL) pathway; however, it affected the metabolism of divinyl ethers (DVEs) early during infection with P. parasitica (var. nicotianae) and the accumulation of NaDES1 and NaLOX1 mRNAs. Profiling of lysolipids by LC-MS/MS was consistent with a rapid activation of NaGLA1 and indicated that this lipase utilizes different lipid classes as substrates. The results revealed the complexity and specificity of the regulation of lipase-mediated oxylipin biosynthesis, highlighting the existence of pathway- and stimulus-specific lipases.
Asunto(s)
Ciclopentanos/metabolismo , Lipasa/metabolismo , Nicotiana/enzimología , Oxilipinas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/enzimología , Raíces de Plantas/enzimología , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Animales , Secuencia de Bases , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos Insaturados/metabolismo , Flores/enzimología , Flores/genética , Flores/fisiología , Fusarium/fisiología , Regulación de la Expresión Génica de las Plantas , Herbivoria , Insectos , Lipasa/genética , Datos de Secuencia Molecular , Phytophthora/fisiología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Reproducción , Análisis de Secuencia de ADN , Nicotiana/genética , Nicotiana/microbiología , Nicotiana/fisiología , Compuestos de Vinilo/metabolismo , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
Plants stably transformed to manipulate the expression of genes mediating ecological performance have profoundly altered research in plant ecology. Agrobacterium-mediated transformation remains the most effective method of creating plants harbouring a limited number of transgene integrations of low complexity. For ecological/physiological research, the following requirements must be met: (i) the regenerated plants should have the same ploidy level as the corresponding wild-type plant and (ii) contain a single transgene copy in a homozygous state; (iii) the T-DNA must be completely inserted without vector backbone sequence and all its elements functional; and (iv) the integration should not change the phenotype of the plant by interrupting chromosomal genes or by mutations occurring during the regeneration procedure. The screening process to obtain transformed plants that meet the above criteria is costly and time-consuming, and an optimized screening procedure is presented. We developed a flow chart that optimizes the screening process to efficiently select transformed plants for ecological research. It consists of segregational analyses, which select transgenic T1 and T2 generation plants with single T-DNA copies that are homozygous. Indispensable molecular genetic tests (flow cytometry, diagnostic PCRs and Southern blotting) are performed at the earliest and most effective times in the screening process. qPCR to quantify changes in transcript accumulation to confirm gene silencing or overexpression is the last step in the selection process. Because we routinely transform the wild tobacco, Nicotiana attenuata, with constructs that silence or ectopically overexpress ecologically relevant genes, the proposed protocol is supported by examples from this system.
Asunto(s)
Pruebas Genéticas/métodos , Plantas Modificadas Genéticamente/genética , Transformación Genética/genética , Southern Blotting , Ecología/métodos , Citometría de Flujo , Reacción en Cadena de la Polimerasa , Nicotiana/genéticaRESUMEN
BACKGROUND: Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown. RESULTS: We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated >or= 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction. CONCLUSIONS: The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.