Whiteflies glycosylate salicylic acid and secrete the conjugate via their honeydew

Abstract

During insect feeding, a complex interaction takes place at the feeding site, with plants deciphering molecular information associated with the feeding herbivore, resulting in the upregulation of the appropriate defenses, and the herbivore avoiding or preventing these defenses from taking effect. Whiteflies can feed on plants without causing significant damage to mesophyll cells, making their detection extra challenging for the plant. However, whiteflies secrete honeydew that ends up on the plant surface at the feeding site and on distal plant parts below the feeding site. We reasoned that this honeydew, since it is largely of plant origin, may contain molecular information that alerts the plant, and we focused on the defense hormone salicylic acid (SA). First, we analyzed phloem sap from tomato plants, on which the whiteflies are feeding, and found that it contained salicylic acid (SA). Subsequently, we determined that in honeydew more than 80% of SA was converted to its glycoside (SAG). When whiteflies were allowed to feed from an artificial diet spiked with labeled SA, labeled SAG also was produced. However, manually depositing honeydew on undamaged plants resulted still in a significant increase in endogenous free SA. Accordingly, transcript levels of PR1a, an SA marker gene, increased whereas those of PI-II, a jasmonate marker gene, decreased. Our results indicate that whiteflies manipulate the SA levels within their secretions, thus influencing the defense responses in those plant parts that come into contact with honeydew.

Fig. 1

Relative salicylic acid (SA) levels in phloem and honeydew. Honeydew (a) and phloem sap (b) were collected, treated with and without β-glucosidase, and analyzed by LC-MS/MS for SA and sugar (hexose + dihexose) content. N = 4; * indicates a significant difference (Student’s T-test, P < 0.05); error bars represent standard errors

Fig. 2

Deuterated salicylic acid (D4-SA) in honeydew after feeding on artificial diet laced with D4-SA. Whiteflies were fed an artificial diet containing D4-SA for 2 d, honeydew (HD) was collected, β-glucosidase treatment performed and subsequently analyzed for D4-SA by LC-MS/MS. The solid, black line shows D4-SA in honeydew treated with beta-glucosidase, the dashed grey line without

Fig. 3

Salicylic acid (SA) levels after honeydew (HD) treatment and recovery of SA. a Induction of SA after honeydew treatment. 20 μl honeydew or ddH₂O (both containing 0.02 % Tween-20) were pipetted onto the adaxial side of a tomato leaf, the leaves were harvested and analyzed for SA and quantified with a deuterated salicylic acid (D4-SA) internal standard. b Recovery of SA from the leaf surface. 50 ng SA were applied to the leaf surface and extracted after 1 h. As a control, a non-treated leaf was extracted with the extraction solution containing 50n g D4-SA. N = 4 * indicates a significant difference (Student’s T-test, P < 0.05); error bars represent standard errors

Fig. 4

Transcript levels of defense-related genes after honeydew treatment. Plants were treated with 20 μl honeydew or ddH₂O (both containing 0.02 % Tween-20) and after 24 h the leaf material was harvested for RT-qPCR analyses. Data were normalized to Actin transcript levels. N = 3, * indicates a statistical difference (Student T-test, P < 0.05); error bars represent standard errors

Fig. 5

Honeydew from artificial medium laced with salicylic acid (SA) increases SA levels in planta. Two thousand whiteflies were fed artificial diet (AD) with or without 100 μg/ml SA for 72 h. The honeydew was subsequently collected, and 20 μl applied to undamaged leaves for 1 h. SA levels were subsequently measured by LC-MS/MS. Asterisk indicates a significant difference (ANOVA, followed by Scheffé posthoc test P < 0.01); error bars represent standard errors (N = 4)