Moth oviposition shapes the species-specific transcriptional and phytohormonal response of nicotiana attenuata to larval feeding

ABSTRACT Oviposition by lepidopteran herbivores on _Nicotiana attenuata_ primes plant defence responses that are induced by the feeding larvae. While oviposition by both the generalist

_Spodoptera exigua_ and the specialist _Manduca sexta_ primes the production of defensive phenylpropanoids, their larvae are differentially affected. We investigate here the impact of prior

oviposition on the transcriptome and phytohormone levels of plants that were later attacked by larvae to find regulatory signals of this priming. In a full-factorial design, we evaluated the

effects of oviposition and herbivory by both species. Oviposition alone had only subtle effects at the transcriptional level. Laval feeding alone induced species-specific plant responses.

Larvae of the generalist regulated phytohormones and gene expression stronger than larvae of the specialist. A day after larvae started to feed, we detected no significant alterations of the

plant’s response to larval feeding due to prior oviposition by conspecific moths. Yet, oviposition by each of the species profoundly influenced the plant’s transcriptional and phytohormonal

response to feeding larvae of the other species. Remarkably, the species-specific plant responses to larval feeding shifted towards the response normally elicited by larvae of the

ovipositing species. Thus, plants may already recognise an insect’s identity upon its oviposition. SIMILAR CONTENT BEING VIEWED BY OTHERS NYMPHAL FEEDING SUPPRESSES OVIPOSITION-INDUCED

INDIRECT PLANT DEFENSE IN RICE Article Open access 08 January 2025 SPIDER MITE EGG EXTRACT MODIFIES ARABIDOPSIS RESPONSE TO FUTURE INFESTATIONS Article Open access 06 September 2021

_AGASICLES HYGROPHILA_ ATTACK INCREASES NEROLIDOL SYNTHASE GENE EXPRESSION IN _ALTERNANTHERA PHILOXEROIDES_, FACILITATING HOST FINDING Article Open access 12 October 2020 INTRODUCTION Plants

evolved a multitude of adaptations to resist herbivorous insects. In turn, insects evolved adaptations to find and colonise suitable host plants. Plants possess diverse defence traits which

are often inducible upon herbivory1. Insects evolved traits to avoid, resist, or even manipulate a plant’s defence response2. The closer the evolutionary relationship between plants and

insect herbivores, the more sophisticated and specialised defence and counter-defence can be geared to each other. The degree of such coevolution is driven by the insect’s impact on plant

fitness and its host plant range. Specialised herbivores with narrow host plant ranges can often tolerate, detoxify or even co-opt plant defences for their own defence3. In several plants,

generalist and specialist insects elicit different responses4,5,6, which may be adaptive for the plant or the insect7. Plants may render their defence responses most efficient against

different insects that are differentially susceptible, and adapted insects may evolve traits to manipulate plant defence. Inducible plant defences allow plants to limit their investments of

resources into defence to circumstances in which these pay off, i.e. herbivores are present. Plants can even integrate environmental cues that predict upcoming herbivory to accelerate or

enhance their induced defences which is termed as priming8,9. For example volatiles of herbivore-attacked plants can prime herbivory-induced defence in adjacent plants10. The egg-deposition

of herbivorous insects on a host plant can also prime plant defence against the feeding larvae as has been shown for the wild tobacco _Nicotiana attenuata_11,12. Also in tomato, moth

oviposition primes the wound-induced expression of defence-related parameters13. Priming by insect oviposition is likely more common because several plants possess an increased resistance to

insect larvae if previously exposed to its eggs14,15,16,17. Yet, the larvae may also be affected by defence responses that are directly activated upon insect oviposition. Plant responses to

insect oviposition include the formation of neoplasms, necrosis, egg-crushing tissues, ovicidal substances or volatiles that attract egg predators and parasitoids18,19,20,21,22,23,24.

Several plant responses to oviposition as well as the effects on plant resistance to subsequent larval attack can be specific to the insect species. For example, _Brassica nigra_ plants are

more resistant to feeding larvae in response to a specialist’s but not a generalist’s moth oviposition25, suggesting a differential plant response to oviposition by both insects. Yet, _N.

attenuata_ plants are more resistant to larvae of only the generalist _Spodoptera exigua_ but not the specialist _Manduca sexta_ in response to oviposition by either of the two species12.

Oviposition by both moth species equally primes the feeding-induced plant production of phenylpropanoid-polyamine conjugates (PPCs), which reduces performance of _S. exigua_ larvae feeding

on oviposited plants11. This suggests a differential susceptibility of the larvae but no species-specific plant response to oviposition, though _N. attenuata_ may show other, still

undiscovered, responses that may differ for the two moth species. Plant responses to herbivory are multifaceted, which is reflected in a large reprogramming of a plant’s transcriptome and

various shifts in its primary and secondary metabolism26. This reprogramming is regulated by a network of signalling pathways in which the phytohormone jasmonic acid (JA) plays a central

role27. Differential activation of these signalling pathways in response to different elicitors, e.g. damage- and herbivore associated molecular patterns, allows species-specific responses

to different herbivores28,29,30. For example, larval feeding of _M. sexta_ and _S. exigua_ elicits distinct transcriptional profiles in _N. attenuata_4, likely due to differential

phytohormone induction patterns that result from different ratios of elicitors in the larval oral secretions of both species31. The phytohormonal and transcriptional plant responses to

insect oviposition are less examined than those to insect feeding but an involvement of salicylic acid (SA) and JA signalling pathways has been inferred32. _Arabidopsis thaliana_ leaves

oviposited by _Pieris brassicae_ accumulate high levels of SA33 and SA-responsive transcripts34. Consistent with the role of SA as a key regulator of plant responses to phytopathogens, the

transcriptional profile in response to _P. brassicae_ oviposition is more similar to that of plants harbouring a bacterial infection than to plants fed by _P. brassicae_ larvae34. The SA

signal is also detected in systemic leaves of oviposited plants that thereby even acquire resistance (SAR) to bacterial pathogens35. JA may also play a role in plant responses to oviposition

as indicated by an increased hatching rate of spider mite eggs on JA-deficient tomato mutants36. Moreover, JA-biosynthesis genes are induced upon oviposition that involves wounding of plant

tissue37,38 and plant responses to such oviposition can be mimicked with JA treatments39,40. Very few studies examined the influence of an earlier oviposition on plant signalling in

response to the feeding larvae. Transcriptome analyses revealed no significant effects of _P. brassicae_ oviposition on _A. thaliana_’s response to larval feeding41 or very few _B. nigra_

genes that were altered in larval attacked plants due to a pre-treatment with _P. brassicae_ egg extract42. In tomato, the increase of wound-induced expression of a defensive protease

inhibitor (PI) gene in response to a moth’s oviposition coincides with a stronger JA-burst13. Yet, in _N. attenuata_, the priming effect of moth oviposition on the induction of PI activity

and PPC production in response to larval feeding could not be explained by increased induction of JA11,43. Overall, our current knowledge on the signalling of oviposition-induced responses

is anecdotal, especially regarding the question how oviposition affects plant responses to subsequently feeding larvae. Here we explore _N. attenuata_’s signalling response to oviposition

and herbivory by two of its major herbivores. Specifically, we examine the plant for i) systemic imprints of oviposition by _M. sexta_ and _S. exigua_, ii) how the moth’s oviposition affects

the feeding-induced plant response to its larvae, and iii) whether these effects of oviposition on plant responses to feeding are specific for the insect species. In a full-factorial design

(Fig. 1), we explore the effects of larval feeding and prior oviposition by _S. exigua_ and _M. sexta_ on _N. attenuata_’s transcriptional and phytohormonal profiles in leaves that were

systemic to oviposition but local for the larval attack. RESULTS PLANT RESPONSES TO OVIPOSITION IN LEAVES SYSTEMIC TO THE OVIPOSITION SITE We evaluated the direct effects of oviposition on

plants without larval feeding. These leaves were harvested at the same time and from the same leaf position as the leaves of feeding-induced plants and were therefore systemic to the

oviposited leaf (see methods). We found no differences in the phytohormone profiles between oviposited and non-oviposited control plants. Levels of JA and JA-Ile generally varied around the

limit of quantification (ca. 7 and 3 ng/g FW, respectively) and are therefore not displayed. Levels of SA and ABA did not differ between plants oviposited by _S. exigua_ or _M. sexta_ and

control plants (see Supplementary Fig. S1). However, the transcriptome profiles of _M. sexta_-oviposited plants (EmsL0) separated from those of control plants (E0L0) in a principal component

analysis (PCA) along the second principal component (Fig. 2a). In contrast, transcriptome profiles of _S. exigua_-oviposited plants (EseL0) grouped within those of control plants.

Oviposition by _M. sexta_, but not by _S. exigua_, significantly altered the expression of 59 genes, several of which are related to ethylene and auxin signalling as well as regulators of

transcription (Supplementary Table S1). _N. ATTENUATA_ RESPONDS SPECIES-SPECIFIC TO LARVAL FEEDING Altogether, transcriptomes of plants attacked by larvae clearly separated from the

non-attacked plants along the first principal component in a PCA (Fig. 2a). Along the second principal component, explaining about 10% of the variation, _S. exigua_-fed plants (E0Lse; filled

squares) clearly separated from _M. sexta-_fed plants (E0Lms; filled triangles). Herbivory by any of the two species significantly altered the expression of 4749 genes (11% of all genes on

the array) relative to control plants (E0L0). Whereas feeding by _S. exigua_ altered the expression of almost 92% (4361) of these genes, _M. sexta_ feeding regulated only about half (2302)

of them (Fig. 2b). As a consequence, the feeding-induced plant responses to both species overlapped just for 40% (1914). Of all feeding-inducible transcripts, only about 8% (388) were

specific to _M. sexta_ but almost 52% (2447) were unique for feeding _S. exigua_. In a direct comparison between _S. exigua_-fed (E0Lse) and _M. sexta_-fed (E0Lms) plants, 20% of the

feeding-induced genes differed significantly. The transcriptional changes (log2-fold changes; log2-FC) relative to untreated controls correlated with an R2 of 0.72 between plants fed by

larvae of the two different species (Fig. 2c), which is about 20% lower than correlations between plants fed by larvae of the same species (either or not previously oviposited by

conspecifics; see below and Fig. 2d). The difference in the transcriptional response to the two herbivore species was accompanied by a different induction of phytohormones. Feeding by both

species increased levels of JA, JA-Ile, SA and ABA. In _S. exigua_-fed plants, levels of SA and the ratio of JA-Ile to JA were about twofold higher compared to _M. sexta_-fed plants (see

outer bars in Fig. 3), while ABA was altered similarly by both species (Supplementary Fig. S1). EFFECTS OF OVIPOSITION ON _N. ATTENUATA’S_ RESPONSE TO HERBIVORY BY CONSPECIFIC LARVAE

Oviposition by neither _M. sexta_ nor _S. exigua_ altered any of the assessed phytohormone accumulations in response to herbivory by conspecific larvae (Fig. 3, see Supplementary Fig. S1 and

Supplementary Table S2). For each of the two species, non-oviposited and previously oviposited plants shared about 75% of the transcriptional regulation in response to feeding by

conspecific larvae (relative to control plants; see Supplementary Fig. S2). To test whether the previous oviposition changed the plant’s transcriptional response to the subsequent feeding by

conspecific larvae, we evaluated the effects of previous oviposition and the larval feeding in two-factorial analyses (including E0L0/EmsL0/E0Lms/EmsLms in case of _M. sexta_ and

E0L0/EseL0/E0Lse/EseLse plants in case of _S. exigua_), but we found no interactions between oviposition and larval feeding for neither of the two species. Similarly, we found no significant

differences in direct comparisons of gene expression in plants only exposed to larval feeding and plants that were oviposited prior to larval herbivory by the same species (E0Lms versus

EmsLms and E0Lse versus EseLse). In accordance with that, the feeding-induced transcriptional changes (log2-FC) relative to control plants correlated very strongly for previously oviposited

and non-oviposited plants (Fig. 2d). Also in the PCA, _M. sexta-_fed plants with a prior oviposition by conspecifics (EmsLms) cluster closely together with plants only fed by _M. sexta_

(E0Lms) and similarly do _S. exigua-_fed plants with a prior oviposition by conspecifics (EseLse) and plants only fed by _S. exigua_ (E0Lse) cluster together (Fig. 2a). OVIPOSITION SHAPES

THE SPECIES-SPECIFIC RESPONSE OF _N. ATTENUATA_ TO LARVAL FEEDING We investigated furthermore, whether the plant response to feeding larvae is altered in plants previously oviposited by

heterospecific moths. In the PCA, transcript profiles of the EseLms cross-species treatment (blue stars in Fig. 2a) clearly separated from the other two _M. sexta_-fed plant treatments (blue

filled triangles) and aggregated with the _S. exigua_-fed treatments (red filled squares). Similarly, transcript profiles of the other EmsLse cross-species treatment (red stars) shifted

towards plants fed by _M. sexta_. This pattern was further supported by a clustering analysis of the six feeding induced treatments, in which EseLms plants clustered together with _S.

exigua-_fed plants that were either oviposited by conspecific moths or not (Fig. 2e). _Vice versa_, EmsLse clustered with _M. sexta-_fed plants. The numbers of genes regulated by the

cross-species treatments fall in-between those regulated in plants that only experienced one of the species (Fig. 4a). For example, the number of genes regulated in EseLms plants increased

by more than 1000 compared to the other two _M. sexta_-fed plant treatments (E0Lms, EmsLms). As a consequence, EseLms plants shared only 51% of its regulated genes with E0Lms plants, while

these overlapped with 82% of the genes regulated in EmsLms (Fig. 4b). Instead, genes regulated in EseLms plants overlap with 62% of the genes regulated in E0Lse plants, which is considerably

more than the 44–48% that the other _M. sexta_-fed plants overlapped with the E0Lse plants. Similarly, plants of the _S. exigua_-fed cross-species treatment (EmsLse) had about 500 genes

less regulated than the other two _S. exigua_-fed plant treatments. While, E0Lse and EseLse plants overlapped in 85–88% of their regulated genes, EmsLse plants overlapped only with 68% of

the genes regulated in E0Lse plants. Conversely, the proportion of _M. sexta_-responsive genes (E0Lms) that are also altered in response to _S. exigua_ feeding increased from 83% (E0Lse) to

91% (EmsLse) when _M. sexta_ oviposition preceded _S. exigua_ feeding. Thus, the comparisons of the regulated gene sets support that the plant response in cross-species treatments is

intermediate to the species-specific responses to larval feeding. Finally, the shift in the species-specific response to feeding was also reflected in pairwise regression analyses of the

log2-FC relative to untreated controls (Fig. 4c, see Supplementary Fig. S3). The strong correlations between E0Lse and EseLse as well as between E0Lms and EmsLms with an R2 around 0.9 were

not found between the cross-species treatments (EmsLse and EseLms) and the E0Lms and E0Lse treatments. Instead, the expression pattern of EmsLse plants correlated similarly with E0Lms and

E0Lse (R2 of 0.82 and 0.83) and EseLms plants correlated even slightly stronger with E0Lse than with E0Lms plants. Lastly, the cross-species treatment for which phytohormones were analysed

(EmsLse) showed a phytohormonal induction pattern in-between of E0Lms and E0Lse plants (Fig. 3a,b). The intermediate levels of SA and JA-Ile/JA in EmsLse plants did not significantly differ

from both treatments with _M. sexta_ feeding and JA-Ile/JA levels in EmsLse plants even differed significantly from E0Lse plants. VALIDATION OF MICROARRAY ANALYSIS BY REAL-TIME QPCR OF

CANDIDATE GENES We quantified transcripts in 9-10 biological replicates for a set of genes that we selected based on their regulation in the microarray analysis or that are known to be key

regulators in different plant signalling pathways. We selected (i) genes that were significantly regulated by the oviposition alone (Supplementary Table S3) or that indicated an altered

regulation after larval feeding due to prior oviposition by the same species (Fig. 5), (ii) signalling-related genes that may reflect the species-specific response to the larval feeding of

both species (Fig. 6), and (iii) genes that signify the shift of the species-specific feeding-induced response by the other species’ oviposition (Fig. 7). Overall, we validated the

expression patterns determined by the microarray analysis for all 17 genes by real-time qPCR (Supplementary Figs S4 and S5). Our analyses verified the up-regulation of the oligo peptide

transporter _PTR1_-like in response to the oviposition by both species whereas up-regulation of a _defensin_-like protein by _M. sexta_ oviposition was only indicated by trend (Fig. 5).

Plants with _M. sexta_ oviposition had significantly reduced transcript levels of a predicted _WRKY71_ transcription factor and an L-aspartate oxidase gene (_AO_). While plants with _S.

exigua_ oviposition had increased transcript levels of the trypsin protease inhibitor gene _NaPI_ and the geranylgeranyl diphosphate synthase gene _NaGGPPS2_. Transcripts of _PTR1_ and

_defensin_ also increased in response to larval feeding by both species, which tended to be stronger when the plants were previously oviposited by conspecifics. Though not statistically

significant, this could point towards additive effects of oviposition and feeding. The increased transcript accumulation of the _NaPI_ gene in response to _S. exigua_ oviposition did also

not result in increased _NaPI_ transcripts after larval feeding compared to only feeding-damaged plants (Fig. 5e). In contrast, dual comparisons between feeding-induced plants revealed

significantly increased transcripts of _NaGGPPS2_ in _S. exigua_-oviposited compared to only feeding-induced plants (Fig. 5f). Transcript levels of the _WRKY71_-like transcription factor and

the _AO_ gene were similar in feeding-induced plants whether the plants were oviposited or not. But since these genes were down-regulated in only-oviposited plants, they show a significant

interaction between oviposition and larval feeding by _S. exigua_ and _M. sexta_ respectively (Fig. 5b,d). In summary, 24 hours after onset of larval feeding, we could still determine some

genes affected by the previous oviposition _per se_ but we could not pin down the effect of prior oviposition by conspecific moths on feeding-induced gene expression. We additionally

analysed a set of well characterised _N. attenuata_ genes that play a role in different plant signalling pathways related to herbivory. These genes are involved in the regulation of the JA

pathway (_NaLOX3_, _NaTD_, _NaWRKY6_), biosynthesis of ethylene (_NaACO2_) and of SA signalling (_NaNPR1_). All of these genes were significantly induced by larval feeding but not by

oviposition and oviposition did not affect the level of induction after feeding (Fig. 6). They all showed a higher transcript accumulation in response to _S. exigua_ than to _M. sexta_,

except for _NaLOX3_, which was not differently regulated between the two species (Supplementary Fig. S4). As oviposition shifted the species-specific transcriptomic imprint of the larval

feeding, we also verified such a transcriptional shift for a selected set of genes. Three of them, the _mitogen-activated protein kinase kinase NaMKK1_, the _SA methyl transferase SAMT_ and

the _1-aminocyclopropane-1-carboxylic acid oxidase NaACO2_ were stronger upregulated in response to _S. exigua_ feeding than in response to _M. sexta_ feeding but showed an intermediate

expression to feeding by both larvae when they were previously oviposited by the other species (Fig. 7, Supplementary Fig. S5, Supplementary Table S4). Three other genes, the circadian clock

gene _timing of CAB expression NaTOC1_, an _early flowering-like_ gene and a gene homologous to _Jumonji C-domain-containing proteins, JmjC30-like_ showed a higher expression to _M. sexta_

than to _S. exigua_ feeding, which was switched when the plant was previously oviposited by the other species. DISCUSSION We examined whether the oviposition _per se_ left imprints in the

plant’s phytohomonal and transcriptional regulation in a systemic leaf. In line with previous studies11,12, we found no differences in phytohormone levels between oviposited and control

plants. The transcript profile in plants oviposited by _S. exigua_ did also not differ from that of control plants, but our microarray analysis revealed an imprint of _M. sexta_ oviposition

on _N. attenuata_’s transcriptome. Even a day after the end of the egg incubation time, expression of some genes was significantly altered and many of them were annotated to signalling and

regulatory functions (e.g. transcription factors, hormone biosynthesis, see Supplementary Table S1). Moreover, about half of these genes were also altered in response to _M. sexta_ feeding

(see Supplementary Fig. S2). This is consistent with the idea that also subtle changes in gene regulation can be biologically relevant44. We identified several genes responsive to

oviposition by both _M. sexta_ and _S. exigua_ in our better replicated qPCR analysis (Fig. 5). Among them are genes involved in plant defence, for example _NaGGPPS_ and _NaPI_45,46. These

were induced by _S. exigua_- but not by _M. sexta_-oviposition. Interestingly, PI activity in response to larval feeding was also increased in _S. exigua_- but not in _M. sexta_-oviposited

plants11,12. However, compared to the effect of larval feeding, oviposition only slightly induced _NaPI_ expression and whether that could explain the priming of feeding-induced PI activity

remains an open question. Generally, it is not surprising that one day after egg-removal, systemic _N. attenuata_ leaves did not display the large extent of changes in gene expression as

described for the local leaves of _A. thaliana_ 24–72 hours after oviposition by _Pieris_ moths34. Even in local _A. thaliana_ leaves, no transcriptional imprint was detected anymore, one

day after egg-removal41. Yet, both plant species differ in their physiological response to insect oviposition. Other than _N. attenuata_, _A. thaliana_ responds to oviposition with HR-like

symptoms34 which can result in SAR against phytopathogenic bacteria33,35. Whereas oviposition by the specialist _P. brassicae_ induces HR-like necrosis in many brassicaceous plants17,21,

oviposition by the generalist _Mamestra brassicae_ does not, nor does the latter affect larval performance25. However, in _N. attenuata_, oviposition by both a specialist and a generalist

moth species prime feeding-induced defences and lower the performance of _S. exigua_ larvae11,12. In line with that, all defence-related candidate genes that were affected by oviposition in

our qPCR analysis showed similar tendencies for the oviposition by both moth species even if the effect was only significant for one of the two species. As expected, _N. attenuata_ exhibited

diverging responses to the feeding larvae of both species. Larvae of _S. exigua_ induced more SA than those of _M. sexta_, just as had been previously described31. Contrasting this earlier

study31, _M. sexta_ feeding also induced some SA and _S. exigua_-fed plants showed no lower JA induction but a greater JA-Ile/JA ratio than _M. sexta_-fed plants, which may even suggest a

higher conversion of JA to the biologically active conjugate. However, in line with our results, a recent study47 also describes SA induction after _M. sexta_ feeding and an equal

JA-elicitation for _N. attenuata_’s response to _M. sexta_ and _Spodoptera littoralis_. Consistent with our phytohormone analysis, transcripts of the Ile-biosynthesis gene _NaTD_ as well as

the SA-receptor _NaNPR1_ were differentially activated by larval feeding of both species, whereas that of the JA-biosynthesis gene _NaLOX3_ was not (Fig. 6, Supplementary Fig. S4). Thus, our

results support that SA signalling plays a role in tuning _N. attenuata_’s species-specific response to larval feeding, but this tuning is not necessarily linked to JA-SA antagonism as had

been suggested earlier31. The distinguished species-specific response to the feeding larvae was reflected in the number of regulated genes as well as in PCA, regression and cluster analyses

of the plant’s transcriptome (Figs 2–4). Our results match well with those of a study that pioneered _N. attenuata_’s species-specific transcriptional response to different herbivores4 using

cDNA microarrays displaying only 240 genes with a bias for herbivory-related genes. Like this earlier study4, our unbiased transcriptome microarray, showed that a large fraction of

feeding-responsive genes was specifically regulated by feeding of the generalist _S. exigua_ and this fraction was greater among down- than among up-regulated genes. In line with this, the

shared transcriptional response to two generalist herbivores _Heliothis virescens_ and _S. exigua_ differentiating from the response to _M. sexta_ is also dominated by down-regulated genes4.

Differential plant responses to herbivores of the same feeding guild but different diet breadth are also apparent in other plant-insect interactions, which could be either adaptive for the

plant or the result of host manipulation by insects5,6,7,26. Presumably, generalist herbivores are more likely to evolve mechanisms to suppress plant defences than specialists as they have

to cope with diverse plant defences7. However, _S. exigua_ feeding altered more than 2000 genes more than _M. sexta_ feeding. Although, this was more pronounced for down-regulated genes,

defence-related genes and signalling pathways were often stronger induced in response to the generalist (Figs 2–7) and the majority of genes down-regulated after herbivory are usually

related to primary plant metabolism48. _N. attenuata_ effectively defends itself against _S. exigua_11,46,49, whereas _M. sexta_ is relatively tolerant to many of its direct defences47,50.

Thus, it is more likely that the differential plant response is driven either by the specialist herbivore or the plant itself. The distinct response of _N. attenuata_ to both herbivores is

likely due to the divergent composition of elicitors in their oral secretions31. The receptors recognizing these elicitors have not been identified so far but they presumably activate

mitogen-activated protein (MAP) kinases, usually three in a cascade, that mediate the transduction of the signal. We found that one of the MAP kinases (_NaMKK1_) that are involved in the

regulation of _N. attenuata_’s defence response to herbivory51, reflected the species-specific expression pattern (Fig. 7). Even though _NaMKK1_ is not required for feeding-induced JA and

ethylene production, it fine-tunes _N. attenuata_’s defence response52 and thus it may also be involved in adjusting the plant’s response to the attacking herbivore species. To examine plant

responses that may be associated with _N. attenuata_’s oviposition-primed defence response to larval feeding11, we compared only feeding-induced plants with plants that had been

additionally oviposited by conspecific moths. Yet, we could not determine how insect oviposition on _N. attenuata_ alters feeding-induced plant defence metabolites and the performance or

immune state of _S. exigua_ and _M. sexta_ larvae, respectively11,12. The transcriptional responses between only feeding-induced plants and plants with prior oviposition by conspecifics

correlated to 90%, which only emphasizes the high reproducibility of the herbivory treatments within our microarray analysis. However, despite their biological relevance, subtle changes in

only few genes can be easily overlooked due to the large number of variables that are analysed with a very limited number of replicates44. For example, pre-treatments with phytopathogenic

bacteria or egg extract that both have a negative impact on larval performance of _P. brassicae_, show just very subtle effects on the transcriptional response of _B. nigra_ to larval

feeding42. That we could not determine signalling components which could explain these effects may be due to one or a combination of the following reasons: The regulation of the

oviposition-priming of plant defence may (i) occur at different levels, e.g. post-transcriptionally, (ii) involve only few key components at an effect-size that is too small to be revealed

in a low-replicated multi-variate approach, and (iii) occur at other time points after the onset of larval feeding. Priming of feeding-induced responses may result in an earlier, faster,

stronger, more sensitive or even qualitatively different response on the molecular level9. In order to minimize variation between plant-replicates, that may be driven by random differences

in the feeding behaviour of the larvae on a shorter time scale, we examined a time point at which the feeding-induced response on the transcriptional and phytohormonal level is well settled.

However, thereby we may have missed the time point at which the plant defence priming by oviposition is regulated, for instance if it would cause an earlier or faster plant response just

upon the larvae started feeding. Opposite to prior oviposition by conspecifics, our results revealed that oviposition by one species altered the species-specific feeding response to larvae

of the other species. In both cross-species treatments, the plant’s response to feeding larvae became intermediate in those characteristics that were specific for the larval feeding of both

species. This was consistently apparent in all analyses of _N. attenuata_’s transcriptional response (PCA, cluster and regression analyses as well as the numbers and overlaps of

significantly regulated genes) as well as in the phytohormone data for SA and JA-Ile/JA (Figs 2–4). The oviposition-dependent shift of the species-specific gene expression in response to

larval feeding was further confirmed by qPCR on six selected genes. Half of them were higher expressed in response to _S. exigua_ than to _M. sexta_ and showed an intermediate expression in

the cross-species treatments (Fig. 7, Supplementary Fig. S5). These genes (_ACO_, _SAMT_, _NaMKK1_) are related to signalling pathways that mediate plant defence. Again, as _NaMKK1_ is

fine-tuning _N. attenuata_’s defence response52, it may also play a role in the oviposition-mediated shift of the plant response to larval feeding. Three circadian clock genes were stronger

expressed after _M. sexta_- than after _S. exigua_-feeding but showed the reversed pattern in the cross-species treatments. Two of them were also induced by _M. sexta_ oviposition at levels

comparable to feeding (Supplementary Table S1). Previous studies suggested that diurnal regulation plays a role in orchestrating plant anti-herbivore defences53,54 and this may be the case

for the oviposition-mediated responses we found in _N. attenuata_ as well. Attack by multiple herbivores modifies plant responses in a non-additive manner and consequently can strongly

affect the outcome of plant-insect interactions55. Our study shows that such interactions between different insects can begin before actual herbivory occurs. That insect oviposition alters

plant resistance to the ovipositing species is well-established today56 while the notion that also other biotic stressors can be affected is just emerging12,35. Our study revealed that

insect oviposition can shape the species-specificity of a plant’s response to feeding larvae. What are possible explanations of this surprising phenomenon? On the one hand, _N. attenuata_

may prepare its feeding-induced response to defend most effectively against the herbivore that announced itself by its oviposition. On the other hand, herbivores may start to manipulate

their host plant right upon oviposition. The latter scenario may be more likely for the specialist, yet the shift of the species-specific plant response was more pronounced in response to

the generalist’s oviposition. Despite its wide host plant range, _S. exigua_ is a frequent herbivore in the native habitat of _N. attenuata_49 and as such it may exert considerable selection

pressure on the plant. If the plant would benefit from rendering its response specific to the insect species, the effect that oviposition can determine this species-specificity connotes a

novel mechanism of defence priming. METHODS PLANTS AND INSECTS Seeds of a _Nicotiana attenuata_ Torr. ex Watson inbred line (15x) from a field collection were sterilized and smoke-germinated

on agar plates and seedlings were transferred to potting soil and grown in 1.5 L pots in a greenhouse (24 °C (±10):15 °C; 16:8 h L:D) as described earlier11. _Manduca sexta_ L. and

_Spodoptera exigua_ Hübner were cultured in a climate chamber (24 °C; 16:8 L:D). Insect cultures and the wheat germ based artificial diets of the larvae were described earlier57.

EXPERIMENTAL PROCEDURE We assigned 5-week-old early elongated experimental plants of equal size and elongation state to one of nine treatments for each of 12 biological replicate blocks

(Fig. 1). In a 3 × 3 full-factorial experimental design, we examined the plant responses to oviposition and larval feeding by _M. sexta_ and _S. exigua_ and the interactive effects between

them. The setup of the oviposition and larval feeding treatments were previously described in detail58. In brief, we exposed the second youngest source leaf (2 positions below the

sink-source transition leaf) through slots in the side of a flight cage to _M. sexta_ oviposition. As _S. exigua_ does not accept this setup for oviposition, we exposed whole plants to

oviposition by _S. exigua_ for 12 h. _M. sexta_-oviposited plants received 2.2 ± 0.4 (mean ± SEM) and _S. exigua_-oviposited 37.7 ± 5.6 eggs (dispersed on one to four elder leaves). Within

the range of the natural egg incubation time, 3 and 4 days after oviposition by _S. exigua_ and _M. sexta_ respectively, we removed the eggs of all plants without damaging the leaf surface

either with a soft brush or a featherweight forceps. Within 12 h after egg-removal, we applied either three _M. sexta_ or 20 _S. exigua_ neonates per plant to a standardised leaf position

relative to the sink-source transition leaf. It was the next younger leaf above the _M. sexta_-oviposited leaf and the corresponding leaf position for control or _S. exigua_-oviposited

plants. Due to the size difference of both species, these numbers of larvae inflict comparable amounts of damage. Larvae were kept on the leaf in vented clip cages and plants without

herbivory treatments received empty clip cages at corresponding leaf positions. After 24 hours of larval feeding, the standardised leaf (either exposed to larvae or empty clip cages) was

harvested from all plants, flash-frozen in liquid nitrogen and stored at −80 °C. Aliquots of the leaf material powdered using mortar and pestle under liquid nitrogen were weighed into

separate tubes. EXTRACTION AND ANALYSIS OF PHYTOHORMONES As previously described in more detail59, we extracted about 150 mg of the samples in 1 mL ethyl acetate spiked with deuterated

internal standards of JA, JA-Ile, SA, ABA and re-eluted the dried extracts in 400 µL 70% methanol with 0.1% formic acid before analysis by UPLC-ESI-MS/MS (Synapt G2-S HDMS; Waters®, Milford,

Massachusetts, USA). After separation on a C18-column (Acquinity UPLC BEH-C18) with water and methanol (both containing 0.1% formic acid) as eluents in gradient mode (250 μL min−1),

phytohormones were quantified (negative ionization mode, parent/daughter ion selections for JA: 209/59, JA-Ile: 322/130, SA: 137/93, ABA: 263/153, D6-JA: 215/59, D6-JA-Ile: 328/130, D4-SA:

141/97, D6-ABA: 269/159) according to the peak areas of the respective fragment ions relative to that of the internal standards using MassLynxTM (4.1, Waters). RNA EXTRACTION We extracted

total RNA from 100 mg leaf powder of all individual plants (including those of the EseLms treatment) with TRIzol® Reagent before the precipitated RNA (using 1.2 M sodium chloride and 0.8 M

sodium citrate) was DNase digested using TURBO DNA-free™ (both AmbionTM, Thermo Fisher Scientific, Waltham, MA USA; http://www.thermofisher.com) according to the manufacturer’s instructions.

After a clean-up of the RNA using NucleoSpin® RNA (Macherey-Nagel, Düren, Germany; http://www.mn-net.com/), we adjusted the RNA to 200 ng/µL based on photometric concentration measurements

(μDrop™ Plate; Multiskan™ GO Microplate Spectrophotometer, Thermo Scientific). MICROARRAY ANALYSIS RNA of 3-4 plant replicates per treatment was pooled at equal proportions resulting in 3

biological replicates for each treatment on the microarray (performed by Oaklabs, http://www.oak-labs.com/). All samples had a RNA integrity index between 7.7 and 8.0 as determined with a

2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA, http://www.agilent.com). Fluorescent cRNA was generated using the Low Input QuickAmp Labeling Kit (Agilent Technologies)

and oligo-dT primers following the manufacturer’s protocol. Of the cyanine 3-CTP-labelled cRNA, 600 ng were hybridised using the Agilent Gene Expression Hybridisation Kit (Agilent

Technologies) following the manufacturer’s protocol on the microarray at 65 °C for 17 h. After the microarray was washed twice, the fluorescence signals on microarrays were detected by the

SureScan Microarray Scanner (Agilent Technologies) at a resolution of 3 micron per pixel. The 8 × 60 K microarray harboured the same 60-mer oligonucleotide probes as the Agilent 4 × 44 K

microarray with the GEO accession no. GPL1352760 that was designed based on the _N. attenuata_ transcriptome (BioProject PRJNA223344 at http://www.ncbi.nlm.nih.gov/). Although all RNA

samples were processed at once, a first hybridisation on three arrays (24 samples) included only one cross-species treatment. The EmsLse was chosen because oviposition by _M. sexta_ affects

larval performance of _S. exigua_ but not _vice versa_12. The analysis revealed that, opposite to the effect of prior oviposition of the same species that later fed on the plants, prior

exposure to _M. sexta_ oviposition shifted the response to larval feeding by _S. exigua_. Therefore, a second analysis including the EseLms treatment was performed. REAL-TIME QPCR ANALYSIS

To verify microarray results and to test the effects of oviposition and larval feeding on potentially regulated genes with a higher statistical power, we performed real-time qPCR on 9-10

individual plants of each treatment. The verification of the qPCR data was initially performed for 11 genes on the same 8 treatments used in the first microarray hybridisations and was then

extended for 6 genes to all feeding-induced treatments including both cross-species treatments and control plants. We synthesized cDNA from the total RNA with the Reverse Transcriptase Core

kit (Eurogentec, Seraing, Belgium, http://www.eurogentec.com) according to the manufacturer’s instructions. For each gene, 1 µL cDNA was used in 10 µL reactions of SYBR®Green I-based

real-time PCR using a qPCR kit without ROX (Eurogentec) on a StratageneTM Mx3005P® instrument (Agilent Technologies). Real-time PCR was performed with gene-specific primers (see

Supplementary Table S5) in triplicates with all treatments of a replicate on one 96 well plate. We inspected melting curves and used LinRegPCR (http://LinRegPCR.HFRC.nl) to determine

amplification efficiencies and the relative quantities (RQ) of each gene61. The RQ of the genes of interest was normalised to the averaged RQ of two reference genes _β-actin_ and _EF1α_

(NRQ). Transcript accumulations were analysed on the log2-normalized NRQs and depicted relative to the mean of the control treatment. DATA ANALYSIS All statistics were performed in R version

3.2.3 with the packages _lme4, multcomp_, _psych_ and _stats_ (http://www.r-project.org). Phytohormone and qPCR data were checked for normality and variance homogeneity (_QQ_-plots and

Bartlett’s test) before their statistical evaluation. To test for differences in phytohormone levels between oviposited and control plants and for differences among the five groups of plants

exposed to larval feeding, we used either ANOVA followed by paired _t_-tests or Kruskal Wallis test followed by pairwise Wilcoxon rank sum tests. To evaluate the effect of the factors

oviposition and larval feeding as well as their interaction within the 2 × 2 full-factorial priming experiments for each species separately we used linear mixed-effects models (LMMs) and

included the replicate blocks as random factor. We also analysed transcript accumulations determined by qPCR with LMMs to evaluate differences between control and oviposited plants as well

as those between plants of the different larval feeding treatments. We used the _limma_ package62 to analyse the microarray data. To reduce background noise, a detection limit was set at the

10% quantile of fluorescence signal intensity of each array. All structural spots, as well as oligos that were not above this detection limit in any of the arrays, were removed from further

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false discovery rate according to Benjamini-Hochberg. Genes of interest were functionally annotated according to NCBI nucleotide blast and blastx of the corresponding p454 sequences

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ACKNOWLEDGEMENTS We thank Lara Friebe and Julia Felderhoff for help with the experiments and the plant and insect cultures, Vivien Firtzlaff for help with the initial microarray analysis,

Martina Schad and Jim Kallarackal from Oaklabs for great support on the microarray hybridisations and Andreas Springer for excellent service with the UPLC-ESI-MS/MS. We are grateful for the

financial support of the German Research Foundation (DFG; project B2 within the Collaborative Research Centre ‘SFB973’) and the stipend of the German Federal Environmental Foundation (DBU)

for Michele Bandoly. AUTHOR INFORMATION Author notes * Sylvia Drok and Michele Bandoly contributed equally to this work. AUTHORS AND AFFILIATIONS * Freie Universität of Berlin/Institute of

Biology/Dahlem Centre of Plant Sciences, Laboratory of Molecular Ecology, Albrecht-Thaer Weg 6, Berlin, 14195, Germany Sylvia Drok, Michele Bandoly, Sandra Stelzer, Tobias Lortzing & 

Anke Steppuhn Authors * Sylvia Drok View author publications You can also search for this author inPubMed Google Scholar * Michele Bandoly View author publications You can also search for

this author inPubMed Google Scholar * Sandra Stelzer View author publications You can also search for this author inPubMed Google Scholar * Tobias Lortzing View author publications You can

also search for this author inPubMed Google Scholar * Anke Steppuhn View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.B., S.D. and A.S.

designed this study. M.B., S.D. and S.S. conducted the experimental and laboratory work. S.D., M.B. and T.L. analysed the results. S.D., M.B. and A.S. wrote the first draft of the manuscript

that was edited and revised by all authors. CORRESPONDING AUTHOR Correspondence to Anke Steppuhn. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests.

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THIS ARTICLE CITE THIS ARTICLE Drok, S., Bandoly, M., Stelzer, S. _et al._ Moth oviposition shapes the species-specific transcriptional and phytohormonal response of _Nicotiana attenuata_ to

larval feeding. _Sci Rep_ 8, 10249 (2018). https://doi.org/10.1038/s41598-018-28233-z Download citation * Received: 19 January 2018 * Accepted: 13 June 2018 * Published: 06 July 2018 * DOI:

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