UPLC-MS/MS analysis and biological activity of the potato cyst nematode hatching stimulant, solanoeclepin A, in the root exudate of Solanum spp.

Main conclusion Solanoeclepin A is a hatching stimulant for potato cyst nematode in very low (pM) concentrations. We report a highly sensitive method for the analysis of SolA in plant root exudates using UHPLC-MS/MS and show that there is considerable natural variation in SolA production in Solanum spp. corresponding with their hatching inducing activity. Abstract Potato cyst nematode (PCN) is a plant root sedentary endoparasite, specialized in the infection of solanaceous species such as potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Earlier reports (Mulder et al. in Hatching agent for the potato cyst nematode, Patent application No. PCT/NL92/00126, 1996; Schenk et al. in Croat Chem Acta 72:593–606, 1999) showed that solanoeclepin A (SolA), a triterpenoid metabolite that was isolated from the root exudate of potato, induces the hatching of PCN. Its low concentration in potato root exudate has hindered progress in fully understanding its hatching inducing activity and exploitation in the control of PCN. To further investigate the role of SolA in hatching of PCN, the establishment of a highly sensitive analytical method is a prerequisite. Here we present the efficient single-step extraction and UHPLC-MS/MS based analysis for rapid determination of SolA in sub-nanomolar concentrations in tomato root exudate. This method was used to analyze SolA production in different tomato cultivars and related solanaceous species, including the trap crop Solanum sisymbriifolium. Hatching assays with PCN, Globodera pallida, with root exudates of tomato genotypes revealed a significant positive correlation between SolA concentration and hatching activity. Our results demonstrate that there is natural variation in SolA production within solanaceous species and that this has an effect on PCN hatching. The analytical method we have developed can potentially be used to support breeding for crop genotypes that induce less hatching and may therefore display reduced infection by PCN.

RNA TRIzol isolation of Datura spp. v1

We present the TRIzol protocol for RNA extraction. We employed leaf tissue from vegetative and reproductive plants from four solanaceous species of the genus Datura; D. stramonium, D. pruinosa, D. inoxia and D. wrightii.

Primary restriction of S-RNase cytotoxicity by a stepwise ubiquitination and degradation pathway in Petunia hybrida

ABSTRACTIn self-incompatible Solanaceous species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific non-self S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Here, we first show that Petunia hybrida (Ph) S3-RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, II and III. R I is ubiquitinated in unpollinated, self- and cross-pollinated pistils, indicating its occurrence prior to PhS3-RNase uptake into pollen tubes, whereas R II and III are exclusively ubiquitinated in cross-pollinated pistils. Second, removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and III in less extents, indicating their increased cytotoxicity. In consistent, the mutated R II of PhS3-RNase resulted in marked reduction of its degradation, whereas that of R I and III in less reductions. Taken together, our results demonstrate that PhS3-RNase R II functions as a major ubiquitination region for its destruction and R I and III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.ONE SENTENCE SUMMARYBiochemical and transgenic analyses reveal that Petunia hybrida S3-RNase cytotoxicity is largely restricted by a stepwise ubiquitination and degradation pathway during cross-pollination.