An effector–reporter system to study cellular signal transduction in strawberry fruit (Fragaria ananassa)

An effector–reporter system is a powerful tool used to study cellular signal transduction, but this technique has been traditionally used in protoplasts. A similar system to study cellular signal transduction in fruits has not yet been established. In this study, we aimed to establish an effector–reporter system for strawberry fruit, a model nonclimacteric fruit. We first investigated the characteristics of transient gene expression in strawberry fruits and found marked variation in gene expression levels among individual fruits, and this variation has complicated the establishment of a technical system. To overcome this difficulty, we investigated a sampling strategy based on a statistical analysis of the activity pattern of four different reporters (GUS, GFP, FLuc, and RLuc) among individual fruits and combinations of pairs of reporters (GUS/GFP and RLuc/FLuc). Based on an optimized sampling strategy, we finally established a step-by step protocol for the effector/reporter assay. Using FaMYB10 and FaWRKY71 as the effectors and GUS driven by the FaCHS promoter as the reporter, we demonstrated that this effector/reporter system was practical and reliable. This effector/reporter technique will contribute to an in-depth exploration of the signaling mechanism for the regulation of strawberry fruit ripening.

Sirt2-SUMOylation is essential for its tumor-suppressor function in neuroblastoma

Background SUMOylation is an important post-translational modification and participates in a variety of cellular physiological and pathological processes in eukaryotic cells. Sirt2, as a NAD+-dependent deacetylase, usually exerts tumor-suppressor function. However, its SUMOylation roles in cancer cells have not been illustrated. Methods Ni2+-NTA, GST-pull down and immunoprecipitation in vitro and in vivo were used for the Sirt2-SUMOylation or P38-Acetylation assay. Immunofluorescence and Nuclear/Cytosol Fractionation Kit were performed to identify the localization of Sirt2 with or without SUMOylation. The proliferation, soft-agar colony formation, migration and invasion were performed to detect the phenotypes of neuroblastoma cells in vitro, and the xenograft tumor model was conducted to verify Sirt2-SUMOylation role in tumorigenesis in vivo. R2 online database were used for the clinical analysis, including expression, survival curve and pathology stage. Results SUMOylation can occur in Sirt2 protein at both lysine 183 and lysine 340 sites. SUMOylation of Sirt2 does not affect its localization or stability, but involves in the P38-mTORC2-AKT cellular signal transduction via the direct deacetylation on P38. SUMOylation-deficient Sirt2 losses the capability of suppressing tumor processes and neuroblastoma cell shows a resistance to Sirt2-specific inhibitor AK-7. Conclusion We revealed an important function of SUMOylation on Sirt2, which is closely associated with the cellular signal transduction and is essential for suppressing the tumorigenesis in neuroblastoma.

An effector-reporter system to study cellular cellular signal transduction in strawberry fruit (Fragaria ananassa)

Background: An effector-reporter system is a powerful tool used to study cellular signal transduction; however, this technique has been traditionally adopted in protoplasts. A similar system to study cellular signal transduction in fruits has not yet been established. In this paper, we aim to establish an effector-reporter system for strawberry fruit, a model non-climacteric fruit.Results: We compared four different reporters, including GUS, GFP, FLuc, and RLuc, as well as combination of two pairs of reporters, including GUS/GFP and RLuc/FLuc, by statistically analyzing the pattern of reporter activities exhibited among individual fruits. Among the reporters examined, GUS was the best choice. Further sampling indicated that a minimum of five mixed specimens with five fruits each is necessary for an assay. With the so-established system, FaCHS promoter demonstrated a sensitive response to FaMYB10 effector as well as to the external and internal cues implicated in the regulation of fruit ripening.Conclusion: A step-by step protocol was established to study cellular signal transduction in strawberry fruits. This finding will contribute to the molecular investigation of fruit ripening.