Regulation of jasmonate metabolism and activation of systemic signaling in Solanum nigrum: COI1 and JAR4 play overlapping yet distinct roles.

• Jasmonates are ubiquitous messengers in land plants essential for the activation of defense responses. However, their signaling properties, accumulation and metabolism vary substantially among species. Solanum nigrum is a wild Solanaceous species developed as a model to study defense responses. • Solanum nigrum plants transformed to silence the expression of key genes in jasmonate production (SnLOX3), conjugation (SnJAR4) and perception (SnCOI1) were generated to analyze the function of these genes in jasmonate accumulation and metabolism (studied by a combination of LC-MS/MS and (13) C-isotope labeling methods) and in signaling [studied by the systemic elicitation of leucine aminopeptidase (LAP) activity]. • In contrast with the early single jasmonic acid (JA) burst induced by wounding in wild-type (WT) plants, elicitation with insect oral secretions induced a later, second burst that was essential for the induction of systemic LAP activity, as demonstrated by ablation experiments. This induction was dependent on SnLOX3 and SnCOI1, but not on SnJAR4. In addition, the local accumulation of JA-glucose and JA-isoleucine was dependent on SnCOI1, whereas the accumulation of hydroxylated jasmonates was dependent on both SnCOI1 and SnJAR4. • The results demonstrate that SnLOX3, SnCOI1 and SnJAR4 have overlapping yet distinct roles in jasmonate signaling, differentially controlling jasmonate metabolism and the production of a systemic signal.

The structure of the culturable root bacterial endophyte community of Nicotiana attenuata is organized by soil composition and host plant ethylene production and perception.

*A plant's bacterial endophyte community is thought to be recruited from the rhizosphere, but how this recruitment is influenced by the plant's phytohormone signaling is unknown. Ethylene regulates plant-microbe interactions; here, we assess the role of ethylene in the recruitment of culturable endophytic bacteria from native soils. *We grew wild-type Nicotiana attenuata plants and isogenic transformed plants deficient in ethylene biosynthesis (ir-aco1) or perception (35S-etr1) in four native soils and quantified the extent of culturable bacterial endophyte colonization (by plate counting) and diversity (by amplified rDNA restriction analysis and 16S rDNA sequencing). *The endophyte community composition was influenced by soil type and ethylene signaling. Plants grown in organic (vs mineral) soils harbored a more diverse community and plants impaired in ethylene homeostasis harbored a less diverse community than wild-type plants. Wild-type and ethylene signaling-impaired plants fostered distinct bacteria in addition to common ones. In vitro re-colonization by common and genotype-specific isolates demonstrated the specificity of some associations and the susceptibility of 35S-etr1 seedlings to all tested bacterial isolates, suggesting an active process of colonization driven by plant- and microbe-specific genes. *We propose that soil composition and ethylene homeostasis play central roles in structuring the bacterial endophyte community in N. attenuata roots.

Optimized virus-induced gene silencing in Solanum nigrum reveals the defensive function of leucine aminopeptidase against herbivores and the shortcomings of empty vector controls.

Virus-induced gene silencing (VIGS) enables high-throughput analysis of gene function in plants but is not universally applicable and requires optimization for each species. Here a VIGS system is described for Solanum nigrum, a wild relative of tomato and potato and a valuable model species for ecogenomics. The efficiency of the two most widely used Tobacco rattle virus (TRV) vectors to silence phytoene desaturase (PDS) in S. nigrum was tested. Additionally, the infiltration method and growth temperatures for gene silencing were optimized and the suitability of different control vectors evaluated. Using leucine aminopeptidase (LAP), a herbivore-induced protein, silencing efficiency and the applicability of silenced plants for herbivore feeding assays were assessed. Vacuum infiltration of seedlings with Agrobacterium carrying the vector, pYL156, proved the most efficient means of silencing genes. Empty-vector controls decreased plant growth but control vectors carrying a piece of noncoding sequence did not. Silencing LAP significantly increased the larval mass of Manduca sexta that fed on silenced plants. This VIGS protocol proved highly successful for S. nigrum, which should include control vectors carrying noncoding sequence as control treatments. Silencing LAP provided the first experimental evidence that LAP has a defensive function against herbivores.

Native bacterial endophytes promote host growth in a species-specific manner; phytohormone manipulations do not result in common growth responses.

BACKGROUND: All plants in nature harbor a diverse community of endophytic bacteria which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phytohormone homoeostasis by plant-growth-promoting (PGP) rhizobacteria which can decrease ethylene (ET) levels enzymatically by 1-aminocyclopropane-1-carboxylate (ACC) deaminase or produce indole acetic acid (IAA). Whether these common PGP mechanisms work similarly for different plant species has not been rigorously tested. METHODOLOGY/PRINCIPAL FINDINGS: We isolated bacterial endophytes from field-grown Solanum nigrum; characterized PGP traits (ACC deaminase activity, IAA production, phosphate solubilization and seedling colonization); and determined their effects on their host, S. nigrum, as well as on another Solanaceous native plant, Nicotiana attenuata. In S. nigrum, a majority of isolates that promoted root growth were associated with ACC deaminase activity and IAA production. However, in N. attenuata, IAA but not ACC deaminase activity was associated with root growth. Inoculating N. attenuata and S. nigrum with known PGP bacteria from a culture collection (DSMZ) reinforced the conclusion that the PGP effects are not highly conserved. CONCLUSIONS/SIGNIFICANCE: We conclude that natural endophytic bacteria with PGP traits do not have general and predictable effects on the growth and fitness of all host plants, although the underlying mechanisms are conserved.

SERRATE: a new player on the plant microRNA scene.

MicroRNAs (miRNAs) function as sequence-specific guides that control gene expression by post-transcriptional gene silencing. Many miRNAs influence plant development by regulating the accumulation of transcripts that encode transcription factors. Mutants defective in miRNA accumulation, such as dcl1, hen1, hyl1 and ago1, have pleiotropic developmental phenotypes. The serrate-1 (se-1) mutant of Arabidopsis also shows a highly pleiotropic phenotype, which overlaps with the phenotypes of mutants defective in miRNA accumulation. Although it has been proposed that SERRATE (SE) functions specifically in miRNA-mediated repression of the leaf polarity genes PHABULOSA and PHAVOLUTA, microarray analysis shows upregulation of many genes known to be the targets of miRNAs in se-1. We show that SE is a general regulator of miRNA levels affecting the processing of primary miRNA to miRNA.

Specificity in ecological interactions: attack from the same lepidopteran herbivore results in species-specific transcriptional responses in two solanaceous host plants.

Model systems have proven enormously useful in elucidating the biochemical function of plant genes. However their ecological function, having been sculpted by evolutionary forces specific to a species, may be less conserved across taxa. Responses to wounding and herbivore attack differ among plant families and are known to be mediated by oxylipin, ethylene, and systemin-signaling networks. We analyzed transcriptional responses of two native Solanaceous species to the attack of an herbivore whose elicitors are known not to be influenced by diet. With The Institute for Genomic Research 10k-cDNA potato (Solanum tuberosum) microarray, we compared the transcriptional responses of Nicotiana attenuata with those of black nightshade (Solanum nigrum) when both were attacked by the Solanaceous generalist herbivore, Manduca sexta. Based on an NADH dehydrogenase subunit F phylogeny, S. nigrum is more closely related to potato than N. attenuata but responded significantly less to M. sexta attack. Apart from transcriptional differences anticipated from their differences in secondary metabolism, both species showed distinct transcriptional patterns (with only 10% overlap in significantly regulated genes), which point to fundamental differences in the signaling cascades and downstream genes mediating herbivore resistance. The lackluster transcriptional response of S. nigrum could not be attributed to its inability to respond to elicitation, because methyl jasmonate elicitation of S. nigrum resulted in a strong transcriptional response. Given that attack from the same herbivore elicits profoundly different responses in two Solanaceaous taxa, we conclude that blueprints for commonly regulated responses to plant-herbivore interactions appear unlikely.

Solanum nigrum: a model ecological expression system and its tools.

Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum, we describe: (i) an Agrobacterium-based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions. Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants. Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

Molecular interactions between the specialist herbivore Manduca sexta (lepidoptera, sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore-specific transcriptional changes are mediated by fatty acid-amino acid conjugates.

Evidence is accumulating that insect-specific plant responses are mediated by constituents in the oral secretions and regurgitants (R) of herbivores, however the relative importance of the different potentially active constituents remains unclear. Fatty acid-amino acid conjugates (FACs) are found in the R of many insect herbivores and have been shown to be necessary and sufficient to elicit a set of herbivore-specific responses when the native tobacco plant Nicotiana attenuata is attacked by the tobacco hornworm, Manduca sexta. Attack by this specialist herbivore results in a large transcriptional reorganization in N. attenuata, and 161 genes have been cloned from previous cDNA differential display-polymerase chain reaction and subtractive hybridization with magnetic beads analysis. cDNAs of these genes, in addition to those of 73 new R-responsive genes identified by cDNA-amplified fragment-length polymorphism display of R-elicited plants, were spotted on polyepoxide coated glass slides to create microarrays highly enriched in Manduca spp.- and R-induced genes. With these microarrays, we compare transcriptional responses in N. attenuata treated with R from the two most damaging lepidopteran herbivores of this plant in nature, M. sexta and Manduca quinquemaculata, which have very similar FAC compositions in their R, and with the two most abundant FACs in Manduca spp. R. More than 68% of the genes up- and down-regulated by M. sexta R were similarly regulated by M. quinquemaculata R. A majority of genes up-regulated (64%) and down-regulated (49%) by M. sexta R were similarly regulated by treatment with the two FACs. In contrast, few genes showed similar transcriptional changes after H(2)O(2)- and R-treatment. These results demonstrate that the two most abundant FACs in Manduca spp. R can account for the majority of Manduca spp.-induced alterations of the wound response of N. attenuata.