Epigenetic Mechanisms Are Involved in Sex-Specific Trans-Generational Immune Priming in the Lepidopteran Model Host Manduca sexta

Parents invest in their offspring by transmitting acquired resistance against pathogens that only the parents have encountered, a phenomenon known as trans-generational immune priming (TGIP). Examples of TGIP are widespread in the animal kingdom. Female vertebrates achieve TGIP by passing antibodies to their offspring, but the mechanisms of sex-specific TGIP in invertebrates are unclear despite increasing evidence suggesting that both male-specific and female-specific TGIP occurs in insects. We used the tobacco hornworm (Manduca sexta) to investigate sex-specific TGIP in insects because it is a model host for the analysis of insect immunity and the complete genome sequence is available. We found that feeding larvae with non-pathogenic Escherichia coli or the entomopathogen Serratia entomophila triggered immune responses in the infected host associated with shifts in both DNA methylation and histone acetylation. Maternal TGIP was mediated by the translocation of bacterial structures from the gut lumen to the eggs, resulting in the microbe-specific transcriptional reprogramming of genes encoding immunity-related effector molecules and enzymes involved in the regulation of histone acetylation as well as DNA methylation in larvae of the F1 generation. The third-instar F1 larvae displayed sex-specific differences in the expression profiles of immunity-related genes and DNA methylation. We observed crosstalk between histone acetylation and DNA methylation, which mediated sex-specific immune responses in the F1 generation derived from parents exposed to a bacterial challenge. Multiple routes for TGIP seem to exist in M. sexta and – partially sex-specific – effects in the offspring depend on the microbial exposure history of their parents. Crucially, the entomopathogen S. entomophila appears to be capable of interfering with TGIP in the host

Natural variation in linalool metabolites: One genetic locus, many functions?

The ubiquitous volatile linalool is metabolized in plants to nonvolatile derivatives. We studied Nicotiana attenuata plants which naturally vary in (S)‐(+)‐linalool contents, and lines engineered to produce either (R)‐(‐)‐ or (S)‐(+)‐linalool. Only (S)‐(+)‐linalool production was associated with slower growth of a generalist herbivore, and a large fraction was present as nonvolatile derivatives. We found that variation in volatile linalool and its nonvolatile glycosides mapped to the same genetic locus which harbored the biosynthetic gene, NaLIS, but that free linalool varied more in environmental responses. This study reveals how (S)‐(+)‐linalool and conjugates differ in their regulation and possible functions in resistance

A persistent major mutation in canonical jasmonate signaling is embedded in an herbivory-elicited gene network

When insect herbivores attack plants, elicitors from oral secretions and regurgitants (OS) enter wounds during feeding, eliciting defense responses. These generally require plant jasmonate (JA) signaling, specifically, a jasmonoyl-L-isoleucine (JA-Ile) burst, for their activation and are well studied in the native tobacco Nicotiana attenuata. We used intraspecific diversity captured in a 26-parent MAGIC population planted in nature and an updated genome assembly to impute natural variation in the OS-elicited JA-Ile burst linked to a mutation in the JA-Ile biosynthetic gene NaJAR4. Experiments revealed that NaJAR4 variants were associated with higher fitness in the absence of herbivores but compromised foliar defenses, with two NaJAR homologues (4 and 6) complementing each other spatially and temporally. From decade-long seed collections of natural populations, we uncovered enzymatically inactive variants occurring at variable frequencies, consistent with a balancing selection regime maintaining variants. Integrative analyses of OS-induced transcriptomes and metabolomes of natural accessions revealed that NaJAR4 is embedded in a nonlinear complex gene coexpression network orchestrating responses to OS, which we tested by silencing four hub genes in two connected coexpressed networks and examining their OS-elicited metabolic responses. Lines silenced in two hub genes (NaGLR and NaFB67) co-occurring in the NaJAR4/6 module showed responses proportional to JA-Ile accumulations; two from an adjacent module (NaERF and NaFB61) had constitutively expressed defenses with high resistance. We infer that mutations with large fitness consequences can persist in natural populations due to compensatory responses from gene networks, which allow for diversification in conserved signaling pathways and are generally consistent with predictions of an omnigene model

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators

Sequestration of Defenses against Predators Drives Specialized Host Plant Associations in Preadapted Milkweed Bugs (Heteroptera: Lygaeinae)

AbstractHost plant specialization across herbivorous insects varies dramatically, but while the molecular mechanisms of host plant adaptations are increasingly known, we often lack a comprehensive understanding of the selective forces that favor specialization. The milkweed bugs (Heteroptera: Lygaeinae) are ancestrally associated with plants of the Apocynaceae from which they commonly sequester cardiac glycosides for defense, facilitated by resistant NaNa+/K+-ATPases and adaptations for transport, storage, and discharge of toxins. Here, we show that three Lygaeinae species independently colonized four novel nonapocynaceous hosts that convergently produce cardiac glycosides. A fourth species shifted to a new source of toxins by tolerating and sequestering alkaloids from meadow saffron (Colchicum autumnale, Colchicaceae). Across three milkweed bug species tested, feeding on seeds containing toxins did not improve growth or speed of development and even impaired growth and development in two species, but sequestration mediated protection of milkweed bugs against two natural predators: lacewing larvae and passerine birds. We conclude that physiological preadaptations and convergent phytochemistry facilitated novel specialized host associations. Since toxic seeds did not improve growth but either impaired growth or, at most, had neutral effects, selection by predators on sequestration of defenses, rather than the exploitation of additional profitable dietary resources, can lead to obligatory specialized host associations in otherwise generalist insects

Blumenols as shoot markers of root symbiosis with arbuscular mycorrhizal fungi.

High-through-put (HTP) screening for functional arbuscular mycorrhizal fungi (AMF)-associations is challenging because roots must be excavated and colonization evaluated by transcript analysis or microscopy. Here we show that specific leaf-metabolites provide broadly applicable accurate proxies of these associations, suitable for HTP-screens. With a combination of untargeted and targeted metabolomics, we show that shoot accumulations of hydroxy- and carboxyblumenol C-glucosides mirror root AMF-colonization in Nicotiana attenuata plants. Genetic/pharmacologic manipulations indicate that these AMF-indicative foliar blumenols are synthesized and transported from roots to shoots. These blumenol-derived foliar markers, found in many di- and monocotyledonous crop and model plants (Solanum lycopersicum, Solanum tuberosum, Hordeum vulgare, Triticum aestivum, Medicago truncatula and Brachypodium distachyon), are not restricted to particular plant-AMF interactions, and are shown to be applicable for field-based QTL mapping of AMF-related genes

The potato R locus codes for dihydroflavonol 4-reductase

The potato R locus is required for the production of red pelargonidin-based anthocyanin pigments in potato (Solanum tuberosum L.). Red color also requires tissue-specific regulatory genes, such as D (for expression in tuber skin) and F (expression in flowers). A related locus, P, is required for production of blue/purple anthocyanins; P is epistatic to R. We have previously reported that the dihydroflavonol 4-reductase gene (dfr) co-segregates with R. To test directly whether R corresponds to dfr, we placed the allele of dfr associated with red color under the control of the CaMV 35S promoter and introduced it into the potato cultivar Prince Hairy (genotype dddd rrrr P-), which has white tubers and pale blue flowers. Transgenic Prince Hairy tubers remained white, but flower color changed to purple. Three independent transgenic lines, as well as a vector-transformed line, were then crossed with the red-skinned variety Chieftain (genotype D-R-pppp), to establish populations that segregated for D, R, P, and the dfr transgene or empty vector. Markers were used to genotype progeny at D and R. Progeny carrying the empty vector in the genetic background D-rrrr produced white or purple tubers, while progeny with the same genotype and the dfr transgene produced red or purple tubers. HPLC and LC–MS/MS analyses of anthocyanins present in Chieftain and in a red-skinned progeny clone with the dfr transgene in a D-rrrr background revealed no qualitative differences. Thus, dfr can fully complement R, both in terms of tuber color and anthocyanin composition

Mechanisms of jasmonate-induced activation of defense responses in Nicotiana attenuata

Die Zielsetzung dieser Arbeit war die Identifizierung und Charakterisierung der chemischen Signalstoffe im Regurgitat der Raupen des Tabakschwärmers Manduca sexta und der endogenen Signalkaskaden der wilden Tabakpflanze Nicotiana attenuata, die die herbivoren-spezifische Abwehrreaktionen in N. attenuata induzieren. Manuskript I und II beschreiben die Isolierung und Charakterisierung von Fettsäure-Aminosäure-Konjugaten als Elicitoren in den oralen Sekreten von M. sexta Raupen. Diese aktiven Substanzen sind für die Erkennung des Frassbefalls in der wilden Tabakpflanze N. attenuata verantwortlich, amplifizieren die endogene, durch Verwundung induzierte, Jasmonsäurebiosynthese und Herbivoren-spezifische direkte und indirekte Abwehrmechanismen. Durch antisense Transformation wurde die Expression von Genen der durch Raupenfrass oder Behandlung mit Elicitoren in der Pflanze aktivierten Biosynthese von Jasmonsäure und verwandten Signalstoffen manipuliert (Manuscript III) und die Auswirkungen der verminderten Induzierbarkeit auf die Resistenz von N. attenuata in Labor- (Manuskript IV und V) und Freilandversuchen (Manuskript VI) untersucht. Die Laborversuche zeigten eine verminderte Induktion von indirekten (Duftstoffemission) und direkten (Proteinase Inhibitor Aktivität, Nikotin Produktion) Abwehrreaktionen und eine damit verbundende reduzierte Resistenz gegen Raupenfrass durch M. sexta in den transformierten Pflanzen mit verringerter Jasmonsäurebiosynthese. In Freilandversuchen im natürlichen Habitat von N. attenuata wurden die transformierten Pflanzen, zusätzlich zur verminderten Resistenz gegen M. sexta Frass, von zwei oportunistischen Herbivoren (Diabrotica undecimpunctata und Empoasca spec.), die nicht auf Wildtyp N. attenuata Pflanzen beobachtet wurden, attackiert

Mechanisms of jasmonate-induced activation of defense responses in Nicotiana attenuata

Die Zielsetzung dieser Arbeit war die Identifizierung und Charakterisierung der chemischen Signalstoffe im Regurgitat der Raupen des Tabakschwärmers Manduca sexta und der endogenen Signalkaskaden der wilden Tabakpflanze Nicotiana attenuata, die die herbivoren-spezifische Abwehrreaktionen in N. attenuata induzieren. Manuskript I und II beschreiben die Isolierung und Charakterisierung von Fettsäure-Aminosäure-Konjugaten als Elicitoren in den oralen Sekreten von M. sexta Raupen. Diese aktiven Substanzen sind für die Erkennung des Frassbefalls in der wilden Tabakpflanze N. attenuata verantwortlich, amplifizieren die endogene, durch Verwundung induzierte, Jasmonsäurebiosynthese und Herbivoren-spezifische direkte und indirekte Abwehrmechanismen. Durch antisense Transformation wurde die Expression von Genen der durch Raupenfrass oder Behandlung mit Elicitoren in der Pflanze aktivierten Biosynthese von Jasmonsäure und verwandten Signalstoffen manipuliert (Manuscript III) und die Auswirkungen der verminderten Induzierbarkeit auf die Resistenz von N. attenuata in Labor- (Manuskript IV und V) und Freilandversuchen (Manuskript VI) untersucht. Die Laborversuche zeigten eine verminderte Induktion von indirekten (Duftstoffemission) und direkten (Proteinase Inhibitor Aktivität, Nikotin Produktion) Abwehrreaktionen und eine damit verbundende reduzierte Resistenz gegen Raupenfrass durch M. sexta in den transformierten Pflanzen mit verringerter Jasmonsäurebiosynthese. In Freilandversuchen im natürlichen Habitat von N. attenuata wurden die transformierten Pflanzen, zusätzlich zur verminderten Resistenz gegen M. sexta Frass, von zwei oportunistischen Herbivoren (Diabrotica undecimpunctata und Empoasca spec.), die nicht auf Wildtyp N. attenuata Pflanzen beobachtet wurden, attackiert