Gustavo Bonaventure

Herbivore-associated elicitors: FAC signaling and metabolism

The recognition of insect and pathogen attack requires the plants ability to perceive chemical cues generated by the attacker. In contrast to the recognition of microbe-associated molecular patterns and effectors, little is known about the molecular recognition of herbivore-associated elicitors (HAEs) and the signaling mechanisms operating in plants after their perception. HAE perception depends strongly on the natural history of both plants and insects and it is therefore expected that many of the responses induced by different HAEs are specific to the species involved in the interaction. The interaction between Nicotiana attenuata and the specialist lepidopteran Manduca sexta presents a relevant biological system to understand HAE perception and signal transduction systems in plants.

Nicotiana Attenuata SIPK, WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.

Nicotiana attenuata LECTIN RECEPTOR KINASE1 Suppresses the Insect-Mediated Inhibition of Induced Defense Responses during Manduca sexta Herbivory

This work demonstrates that the oral secretion–inducible LECTIN RECEPTOR KINASE1 is indispensable during Manduca sexta herbivory to suppress the insect-mediated inhibition of defense responses and thereby to stimulate the unfettered jasmonic acid–mediated induction of defense metabolites. Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)–induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.

Empoasca leafhoppers attack wild tobacco plants in a jasmonate-dependent manner and identify jasmonate mutants in natural populations

Choice of host plants by phytophagous insects is essential for their survival and reproduction. This choice involves complex behavioral responses to a variety of physical and chemical characteristics of potential plants for feeding. For insects of the order Hemiptera, these behavioral responses involve a series of steps including labial dabbing and probing using their piercing mouthparts. These initial probing and feeding attempts also elicit a rapid accumulation of phytohormones, such as jasmonic acid (JA), and the induced defense metabolites they mediate. When Nicotiana attenuata plants are rendered JA deficient by silencing the initial committed step of the JA biosynthesis pathway, they are severely attacked in nature by hemipteran leafhoppers of the genus Empoasca. By producing N. attenuata plants silenced in multiple steps of JA biosynthesis and perception and in the biosynthesis of the plant’s three major classes of JA-inducible insecticidal defenses, we demonstrate that the choice of plants for feeding by Empoasca leafhoppers in both nature and the glasshouse is independent of the accumulation of major insecticidal molecules. Moreover, this choice is independent of the presence of Candidatus Phytoplasma spp. and is not associated with detectable changes in plant volatiles but instead depends on the plant´s capacity to mediate JA signaling. We exploited this trait and used Empoasca leafhoppers to reveal genetic variation in JA accumulation and signaling hidden in N. attenuata natural populations.

Jasmonate perception regulates jasmonate biosynthesis and JA-Ile metabolism: the case of COI1 in Nicotiana attenuata

CORONATINE INSENSITIVE 1 (COI1) is a well-known key player in processes downstream of jasmonic acid (JA) biosynthesis: silencing COI1 in Nicotiana attenuata (ir-coi1) makes plants insensitive to JA, prevents the up-regulation of JA-mediated defenses and decreases the plants resistance to herbivores and pathogens. In agreement with previous studies, we observed that regulation of several JA biosynthesis genes elicited by Manduca sexta oral secretions (OS) is COI1 dependent. In response to wounding and application of OS ir-coi1 plants accumulate 75% less JA compared with wild-type plants (WT), resembling JA levels found in plants silenced in the key enzyme in JA biosynthesis LIPOXYGENASE 3 (as-lox). However, while OS-elicited as-lox plants also accumulated lower levels of the JA-conjugate JA-isoleucine (JA-Ile) than did WT plants, JA-Ile accumulation in ir-coi1 was higher, prolonged and peaked with a delay of 30 min. In vivo substrate feeding experiments of N. attenuata demonstrate that the increased and prolonged JA-Ile accumulation pattern in ir-coi1 is not the result of altered substrate availability, i.e. of JA and/or Ile, but is due to an approximately 6-fold decrease in JA-Ile turnover. These results provide the first evidence for a second, novel regulatory feedback function of COI1 in enhancing JA-Ile turnover. Hence, in addition to its control over JA biosynthesis, COI1 might fine-tune the dynamics of the jasmonate response after induction by herbivore elicitors.

Perception of insect feeding by plants

The recognition of phytophagous insects by plants induces a set of very specific responses aimed at deterring tissue consumption and reprogramming metabolism and development of the plant to tolerate the herbivore. The recognition of insects by plants requires the plants ability to perceive chemical cues generated by the insects and to distinguish a particular pattern of tissue disruption. Relatively little is known about the molecular basis of insect perception by plants and the signalling mechanisms directly associated with this perception. Importantly, the insect feeding behaviour (piercing-sucking versus chewing) is a decisive determinant of the plants defence response, and the mechanisms used to perceive insects from different feeding guilds may be distinct. During insect feeding, components of the saliva of chewing or piercing-sucking insects come into contact with plant cells, and elicitors or effectors present in this insect-derived fluid are perceived by plant cells to initiate the activation of specific signalling cascades. Although receptor-ligand interactions controlling insect perception have yet not been molecularly described, a significant number of regulatory components acting downstream of receptors and involved in the activation of defence responses against insects has been reported. Some of these regulators mediate changes in the phytohormone network, while others directly control gene expression or the redox state of the cell. These processes are central in the orchestration of plant defence responses against insects.

HAHB4, a sunflower HD-Zip protein, integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses

The Helianthus annuus (sunflower) HAHB4 transcription factor belongs to the HD-Zip family and its transcript levels are strongly induced when sunflower plants are attacked by herbivores, mechanically damaged or treated with methyl-jasmonic acid (MeJA) or ethylene (ET). Promoter fusion analysis, in Arabidopsis and in sunflower, demonstrated that induction of HAHB4 expression by these treatments is regulated at the transcriptional level. In transiently transformed sunflower plants HAHB4 expression upregulates the transcript levels of several genes involved in JA biosynthesis and defense-related processes such as the production of green leaf volatiles and trypsin protease inhibitors (TPI). In HAHB4 sunflower overexpressing tissue, increased activities of lipoxygenase, hydroperoxide lyase and TPI are detected whereas in HAHB4-silenced tissue these activities are reduced. Transgenic Arabidopsis thaliana and Zea mays plants ecotopically expressing HAHB4 also exhibit higher transcript levels of defense-related genes and when Spodoptera littoralis or Spodoptera frugiperda larvae are placed on each species, respectively, larvae consumed less and gain less mass compared with larvae feeding on control plants. Arabidopsis plants ectopically expressing HAHB4 had higher amounts of JA, JA-isoleucine and ET compared with control plants both before and after wounding, but reduced levels of salicylic acid (SA) after wounding and bacterial infection. We conclude that HAHB4 coordinates the production of phytohormones during biotic stress responses and mechanical damage, specifically by positively regulating JA and ET production and negatively regulating ET sensitivity and SA accumulation.

Phenotypic, genetic and genomic consequences of natural and synthetic polyploidization of Nicotiana attenuata and Nicotiana obtusifolia

BACKGROUND AND METHODSnPolyploidy results in genetic turmoil, much of which is associated with new phenotypes that result in speciation. Five independent lines of synthetic allotetraploid N. x obtusiata (N x o) were created from crosses between the diploid N. attenuata (Na) (male) and N. obtusifolia (No) (female) and the autotetraploids of Na (NaT) and No (NoT) were synthesized. Their genetic, genomic and phenotypic changes were then compared with those of the parental diploid species (Na and No) as well as to the natural allotetraploids, N. quadrivalvis (Nq) and N. clevelandii (Nc), which formed 1 million years ago from crosses between ancient Na and No.nnnKEY RESULTSnDNA fingerprinting profiles (by UP-PCR) revealed that the five N x o lines shared similar but not identical profiles. Both synthetic and natural polyploidy showed a dosage effect on genome size (as measured in seeds); however, only Nq was associated with a genome upsizing. Phenotypic analysis revealed that at the cellular level, N x o lines had phenotypes intermediate of the parental phenotypes. Both allo- and autotetraploidization had a dosage effect on seed and dry biomass (except for NaT), but not on stalk height at first flower. Nc showed paternal (Na) cellular phenotypes but inherited maternal (No) biomass and seed mass, whereas Nq showed maternal (No) cellular phenotypes but inherited paternal (Na) biomass and seed mass patterns. Principal component analysis grouped Nq with N x o lines, due to similar seed mass, stalk height and genome size. These traits separated Nc, No and Na from Nq and N x o lines, whereas biomass distinguished Na from N x o and Nq lines, and NaT clustered closer to Nq and N x o lines than to Na.nnnCONCLUSIONSnBoth allo- and autotetraploidy induce considerable morphological, genetic and genomic changes, many of which are retained by at least one of the natural polyploids. It is proposed that both natural and synthetic polyploids are well suited for studying the evolution of adaptive responses.

SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses

BackgroundPlants 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.ResultsWe 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 ≥ 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.ConclusionsThe 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.

Pectin methylesterase NaPME1 contributes to the emission of methanol during insect herbivory and to the elicitation of defence responses in Nicotiana attenuata

Pectin methylesterases (PMEs) catalyse the demethylation of pectin within plant cell walls, releasing methanol (MeOH) in the process. Thus far, PMEs have been found to be involved in diverse processes such as plant growth and development and defence responses against pathogens. Herbivore attack increases PME expression and activity and MeOH emissions in several plant species. To gain further insights into the role of PMEs in defence responses against herbivores, the expression of a Manduca sexta oral secretion (OS)-inducible PME in Nicotiana attenuata (NaPME1) was silenced by RNA interference (RNAi)-mediated gene silencing. Silenced lines (ir-pme) showed 50% reduced PME activity in leaves and 70% reduced MeOH emissions after OS elicitation compared with the wild type (WT), demonstrating that the herbivore-induced MeOH emissions originate from the demethylation of pectin by PME. In the initial phase of the OS-induced jasmonic acid (JA) burst (first 30 min), ir-pme lines produced WT levels of this hormone and of jasmonyl-isoleucine (JA-Ile); however, these levels were significantly reduced in the later phase (60-120 min) of the burst. Similarly, suppressed levels of the salicylic acid (SA) burst induced by OS elicitation were observed in ir-pme lines even though wounded ir-pme leaves contained slightly increased amounts of SA. This genotype also presented reduced levels of OS-induced trypsin proteinase inhibitor activity in leaves and consistently increased M. sexta larvae performance compared with WT plants. These latter responses could not be recovered by application of exogenous MeOH. Together, these results indicated that PME contributes, probably indirectly by affecting cell wall properties, to the induction of anti-herbivore responses.