Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea.

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

Phosphorylation of CAD1, PLDdelta, NDT1, RPM1 Proteins Induce Resistance in Tomatoes Infected by Ralstonia solanacearum.

Ralstonia solanacaerum is one of the most devastating bacteria causing bacterial wilt disease in more than 200 species of plants, especially those belonging to the family Solanaceae. To cope with this pathogen, plants have evolved different resistance mechanisms depending on signal transduction after perception. Phosphorylation is the central regulatory component of the signal transduction pathway. We investigated a comparative phosphoproteomics analysis of the stems of resistant and susceptible tomatoes at 15 min and 30 min after inoculation with Ralstonia solanacearum to determine the phosphorylated proteins involved in induced resistance. Phosphoprotein profiling analyses led to the identification of 969 phosphoproteins classified into 10 functional categories. Among these, six phosphoproteins were uniquely identified in resistant plants including cinnamyl alcohol dehydrogenase 1 (CAD1), mitogen-activated protein kinase kinase kinase 18 (MAPKKK18), phospholipase D delta (PLDDELTA), nicotinamide adenine dinucleotide transporter 1 (NDT1), B3 domain-containing transcription factor VRN1, and disease resistance protein RPM1 (RPM1). These proteins are typically involved in defense mechanisms across different plant species. qRT-PCR analyses were performed to evaluate the level of expression of these genes in resistant and susceptible tomatoes. This study provides useful data, leading to an understanding of the early defense mechanisms of tomatoes against R. solanacearum.