Herbivore exploits orally secreted bacteria to suppress plant defenses

Abstract

Induced plant defenses in response to herbivore attack are modulated by cross-talk between jasmonic acid (JA)- and salicylic acid (SA)-signaling pathways. Oral secretions from some insect herbivores contain effectors that overcome these antiherbivore defenses. Herbivores possess diverse microbes in their digestive systems and these microbial symbionts can modify plant-insect interactions; however, the specific role of herbivore-associated microbes in manipulating plant defenses remains unclear. Here, we demonstrate that Colorado potato beetle (Leptinotarsa decemlineata) larvae exploit bacteria in their oral secretions to suppress antiherbivore defenses in tomato (Solanum lycopersicum). We found that antibiotic-untreated larvae decreased production of JA and JA-responsive antiherbivore defenses, but increased SA accumulation and SA-responsive gene expression. Beetles benefit from down-regulating plant defenses by exhibiting enhanced larval growth. In SA-deficient plants, suppression was not observed, indicating that suppression of JA-regulated defenses depends on the SA-signaling pathway. Applying bacteria isolated from larval oral secretions to wounded plants confirmed that three microbial symbionts belonging to the genera Stenotrophomonas, Pseudomonas, and Enterobacter are responsible for defense suppression. Additionally, reinoculation of these bacteria to antibiotic-treated larvae restored their ability to suppress defenses. Flagellin isolated from Pseudomonas sp. was associated with defense suppression. Our findings show that the herbivore exploits symbiotic bacteria as a decoy to deceive plants into incorrectly perceiving the threat as microbial. By interfering with the normal perception of herbivory, beetles can evade antiherbivore defenses of its host.

Fig. 1.

(A) Scanning electron microscopy images of bacteria secreted onto leaves during feeding by CPB larvae after first feeding on an artificial diet with antibiotic (Right) or without antibiotic (Left). Damaged leaf tissues were prepared for SEM images 1 h after larval feeding. (Scale bar, 1 μm.) (B) Larval growth and (C) polyphenol oxidase (PPO) activities in plants damaged by larvae that fed on AB-treated or untreated leaves. Neonates were allowed to feed on excised leaflets from each treatment for 5 d and larval mass was determined. Values are means ± SEM. Different letters represent significant differences [ANOVA, P < 0.05; followed by LSD test; F_(2,71) = 6.63, P = 0.0023, n = 23–26]. PPO activities were measured on subsamples from each treatment 48 h after insect feeding [F_(2,9) = 27.13, P = 0.0002, n = 4]. To collect subsamples, two leaf discs from each of two leaves were pooled as one replicate. AB(−), plants damaged by untreated larvae; AB(+), plants damaged by AB-treated larvae; Con, undamaged plants.

Fig. 2.

Expression levels of JA- and SA-regulated genes in plants damaged by larvae that fed on AB-treated or untreated leaves. Gene expression was measured 24 h after initiation of insect feeding. Values are untransformed means ± SEM (n = 4–5). Different letters represent significant differences [ANOVA, P < 0.05; followed by LSD test; CysPI, F_(2,11) = 214.7, P < 0.0001; PPOF, F_(2,11) = 185.5, P < 0.0001; PPOB, F_(2,12) = 36.7, P < 0.0001; PR-1(P4), F_(2,11) = 19.7, P = 0.0002]. AB(−), plants damaged by untreated larvae; AB(+), plants damaged by AB-treated larvae; Con, undamaged plants. CysPI, cysteine proteinase inhibitor; PPOF/B, polyphenol oxidase F/B; PR-1(P4), pathogenesis-related protein 1 (P4).

Fig. 3.

Expression levels of JA- and SA-regulated genes in wild-type Moneymaker and SA-deficient NahG plants damaged by larvae that fed on AB-treated or untreated leaves. Values are untransformed means ± SEM (n = 4–5). Gene expression was measured 24 h after initiation of insect feeding. Different letters represent significant differences [ANOVA, P < 0.05; followed by LSD test; for Moneymaker, CysPI, F_(2,12) = 176, P < 0.0001; PPOF, F_(2,12) = 49.7, P < 0.0001; PPOB, F_(2,1) = 14.7, P = 0.0006; PR-1(P4), F_(2,11) = 17.5, P = 0.0004; for NahG, CysPI, F_(2,12) = 233, P < 0.0001; PPOF, F_(2,12) = 64.6, P < 0.0001; PPOB, F_(2,12) = 33.97, P < 0.0001; PR-1(P4), F_(2,11) = 2.83, P > 0.05]. AB(−), plants damaged by untreated larvae; AB(+), plants damaged by AB-treated larvae; Con, undamaged plants. CysPI, cysteine proteinase inhibitor; PPOF/B, polyphenol oxidase F/B; PR-1(P4), pathogenesis-related protein 1 (P4).

Fig. 4.

PPO activities in plants damaged by AB-treated or untreated larvae following reinoculation of the larvae with three bacterial isolates (Stenotrophomonas sp., Pseudomonas sp., and Enterobacter sp.) cultured from CPB larvae and found to suppress JA-mediated plant defenses. Larvae were allowed to feed on AB-treated or untreated leaves for 2 d and then fed on leaves that were reinoculated with suspension buffer (10 mM MgCl₂) or the bacterial isolates in suspension buffer for 2 d. PPO activities were measured 48 h after initiation of insect feeding. Values are means ± SEM (n = 6). Different letters represent significant differences [ANOVA, P < 0.05; followed by LSD test; (A) Stenotrophomonas sp., F_(4,25) = 33.8, P < 0.0001; (B) Pseudomonas sp., F_(4,25) = 22.6, P < 0.0001; (C) Enterobacter sp., F_(4,25) = 128, P < 0.0001]. Buffer, 10 mM MgCl₂; En, Enterobacter sp.; Ps, Pseudomonas sp.; St, Stenotrophomonas sp. AB(−), plants damaged by untreated larvae; AB(+), plants damaged by AB-treated larvae; Con, undamaged plants.