Herbivory in the previous generation primes plants for enhanced insect resistance

Inducible defenses, which provide enhanced resistance after initial attack, are nearly universal in 35 plants. This defense signaling cascade is mediated by the synthesis, movement, and perception of 36 jasmonic acid (JA) and related plant metabolites. To characterize the long-term persistence of 37 plant immunity, we challenged Arabidopsis thaliana (Arabidopsis) and Solanum lycopersicum 38 (tomato) with caterpillar herbivory, application of methyl jasmonate, or mechanical damage 39 during vegetative growth and assessed plant resistance in subsequent generations. Here we show 40 that induced resistance was associated with transgenerational priming of jasmonic acid- 41 dependent defense responses in both species, causes caterpillars to grow up to 50% smaller than 42 on control plants, and persists for two generations in Arabidopsis. Arabidopsis mutants that are 43 deficient in jasmonate perception ( coi1 ) or in the biogenesis of small interfering RNA (siRNA; 44 dcl2 dcl3 dcl4 and nrpd2a nrpd2b ) do not exhibit inherited resistance. The observation of 45 inherited resistance in both the Brassicaceae and Solanaceae suggests that this trait may be more 46 widely distributed in plants. Epigenetic resistance to herbivory thus represents a phenotypically 47 plastic mechanism for enhanced defense across generations .


Introduction
Since Arabidopsis dies shortly after seed set, it is unlikely that plants are "saving" resources for 1 4 3 later growth and reproduction, as might be expected from a perennial plant. We determined whether parental-generation herbivory inherently alters known defense traits in 1 4 7 the seeds or the progeny generation. Seed content of the plant hormones JA, salicylic acid (SA), 1 4 8 abscisic acid, and indole-3-acetic acid was not significantly affected by caterpillar feeding (Fig.   1  4  9 4), showing that H1 plants are not primed for insect resistance through the storage of these 1 5 0 defense signaling molecules in the seeds. Glucosinolates, a crucifer-specific class of defensive 1 5 1 secondary metabolites, also did not differ between seeds of control and P. rapae-treated plants ylmethylglucosinolate (1MI3M) was increased relative to C1 plants without P. rapae caterpillar 1 5 4 feeding in the parental generation (Fig. 5B). Furthermore, the overall phenotype of the progeny 1 5 5 plants was similar, irrespective the parental treatment ( Figure S1), suggesting that plant size is 1 5 6 not growth-limiting for the caterpillars. Rosette leaf trichome density, which is associated with 1 5 7 insect resistance (Levin, 1973;Mauricio and Rausher, 1997), was not significantly altered by  JA-isoleucine conjugates and therefore fail to initiate defense-related gene expression changes in 1 6 6 response to herbivory (Chini et al., 2007;Thines et al., 2007;Howe and Jander, 2008). Consistent with this known defense signaling function of COI1, homozygous coi1-1 mutant 1 6 8 parent plants, which were induced by P. rapae feeding and pollinated with wild-type pollen, did 1 6 9 not show increased resistance in the H1 generation ( Fig. 6B; test for treatment effect; F 1,53 = 1 7 0 0.023, p = 0.880). Functionally wild-type heterozygous COI1/coi1-1 plants were also subjected 1 7 1 to P. rapae herbivory. Segregating H1 progeny from P. rapae-induced parents were more 1 7 2 resistant to herbivory, irrespective of their genotype ( Fig. 6C; for treatment effect, F 1,37 = 13.143, p = 0.001, for progeny genotype effect; F 1,37 = 0.656, p = 0.423, and for the interaction between 1 7 4 treatment and progeny genotypes; F 1,37 = 0.025, p = 0.875). Therefore, in contrast to the parental 1 7 5 generation ( Fig. 6B), perception of JA-isoleucine by COI1 in the progeny generation ( Fig 6C) is 1 7 6 not required for inherited P. rapae resistance. generation caused JA levels to be two-fold higher in H1 plants compared to C1 plants in the 1 8 0 absence of caterpillar damage (dashed lines in Figure 6D, and parental treatment effect below). other hand, SA, a phytohormone that is primarily associated with pathogen defense, did not 1 8 6 exhibit parental treatment effects. In fact, P. rapae herbivory reduced SA levels by 15% over a increased in plants subjected to caterpillar feeding in the prior generation (Fig. 6E, for LOX2 1 9 0 parental treatment effect, F 1,18 = 3.135, p = 0.094, for induction effect, F 1,18 = 5.389, p = 0.032, 1 9 1 and for interaction, F 1,18 = 4.078, p = 0.058). Allene oxide syntheses (AOS), a JA biosynthesis 1 9 2 gene (Howe and Jander, 2008), was also more highly induced by P. rapae if the previous induced to a higher level by wounding if the previous generation had been exposed to MeJA  The elevated caterpillar resistance ( Fig. 1) and JA-mediated defenses (Fig. 6D methoxyindol-3-ylmethylglucosinolate (4MI3M), which has been associated with pathogen correlated with lower abundance of glucosinolates, which are P. rapae feeding stimulants (Barth Our results demonstrate that both Arabidopsis and tomato plants that were subjected to herbivory 2 2 9 are more resistant to subsequent attack in the next generation. In the case of Arabidopsis, this defense responses and requires siRNA biogenesis. orchestrating plant responses to tissue damage or wounding (Howe, 2004;De Vos et al., 2005).

3 6
Previous defense induction can cause plants to be primed for a more robust or rapid defense 2 3 7 response upon subsequent attack (van Hulten et al., 2006), and treatment of seeds with JA primes demonstrate that perception of JA is required in mother plants for increased resistance in the next 2 4 2 generation. Further work using JA signaling-deficient mutants, e.g. jai1, will be needed to we observed priming for faster and higher JA induction in H1 generation, but we did not observe  Small interfering RNA and transgenerational resistance 2 6 0 siRNA is phloem-mobile (Chitwood and Timmermans, 2010) and could provide a signal that is 2 6 1 passed from vegetative tissue to developing seeds in response biotic or abiotic stress. In the 2 6 2 developing seeds and/or progeny plants, siRNA could alter gene expression through targeted DCL2-dependent siRNA production and can be inherited through meiosis, is a possible 2 6 6 mechanism for transgenerational inheritance. Increased resistance over two generations also 2 6 7 indicates that the signal is likely to be propagated in the embryo, rather than maternal tissue that 2 6 8 makes up the Arabidopsis seed coat. plants for these SA-mediated defense responses. Like all forms of phenotypic plasticity, transgenerational resistance to herbivory will only be Therefore, rapid-cycling Arabidopsis genotypes would benefit from priming of insect resistance 2 8 6 in progeny of plants that were subjected to herbivory. A testable prediction is that defense 2 8 7 priming should be reduced in response to longer seed dormancy, which would expose progeny  testing first-generation progeny from P. rapae-treated and control plants, seven showed variable 2 9 7 levels of increased resistance, two showed no significant differences, and none showed a 2 9 8 negative effect, i.e. caterpillar growth was never reduced on C1 plants compared to H1 plants 2 9 9 (Fig. 9). Similar to the protected existence of our laboratory-grown plants, some plants in crops that are particularly vulnerable to herbivory at the seedling stage. Columbia-0 (Col-0) was obtained from the Arabidopsis Biological Resource Center caterpillars were allowed to feed for three days on each plant, and seeds were harvested five to single neonate lepidopteran larva per plant was allowed to feed for 7 days before being collected 3 3 7 and lyophilized for one day. Larval dry weight was determined using a precision balance.

8
For experiments with H. zea, neonates were fed a wheat germ and casein-based artificial four days (one larva per plant). Larval fresh weight was determined using a precision balance. plants. Half of all plants were induced with P. rapae for three days as described above.

5 5
Plants were allowed to self-pollinate, and seeds were collected 5 to 6 weeks after initial planting.

5 6
Harvested seeds were grown as described above and divided into four treatments (with and 3 1 4 without P. rapae feeding in the parental and progeny generations). In the progeny generation, to feed continuously for four days. Full-grown, visibly damaged leaves were harvested after 0, Canada) were added as internal standards and samples were homogenized in a FastPrep® Waltham, MA, USA). Analytes were separated on a C18 reversed phase HPLC column (Gemini- Glucosinolate assays -Arabidopsis leaves were collected, frozen in liquid nitrogen, and The following materials are available in the online version of this article 4 3 0 Supplementary Table S1. Primers used for quantitative RT-PCR in this study. We thank E. Richards, G. Howe, and B. Meyers for Arabidopsis seeds and experimental advice. recombination frequency in vascular tissue of Arabidopsis plants exposed to salt stress. provides evidence for the role of natural enemies in the evolution of plant defense. Differential effects of indole and aliphatic glucosinolates on lepidopteran herbivores. J The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA