RESUMO

Grapevine, as other woody perennials, has been considered a recalcitrant crop to produce transgenic plants. Since the production of transgenic and/or edited plants requires the ability to regenerate plants from transformed tissues, this step is often the biggest bottleneck in the process. The objective of this work is to review the state of the art technologies and strategies for the improvement of grapevine transformation and regeneration, focusing on three aspects: (i) problems associated with grapevine transformation; (ii) genes that promote grapevine regeneration; and (iii) vehicles for gene delivery. Concerning the first aspect, it is well documented that one of the main factors explaining the low success rate in obtaining transgenic plants is the regeneration process. After transgenic integration into receptor cells, tissue culture is required to regenerate transgenic seedlings from transformed cells. This process is time consuming and often requires the addition of environmentally damaging reagents (antibiotics and herbicides) to the culture medium to select transgenic plants. On the other hand, the expression of genes such as the so-called developmental regulators (DR), which induce specific development programs, can be used to avoid traditional tissue culture methods. The ectopic expression of specific combinations of DR in somatic cells has the potential to induce de novo meristems in diverse crops, including grapevine. Successful genome editing by de novo reprogramming of plant meristems in somatic tissues has been reported. Moreover, it has been shown that the expression of certain transcription factors can increase the regeneration efficiency in wheat, citrus, and rice. Finally, recent reports showed the use of nanoparticles, such as carbon dots (CDs), as an attractive alternative to Agrobacterium- and biolistic-mediated plant genetic transformation. In this way, the use of antibiotics in culture media is avoided, overcoming the loss of viability of plant tissues and accelerating the regeneration processes. It has been shown that CDs can act as a vehicle to transport plasmids to plant cells in transient transformation in several crops without negative impacts on photosynthesis or growth. Based on these advances, it is possible to combine these new available strategies and technologies to overcome the regeneration problems of species such as grapevine and other crops considered as recalcitrant.

RESUMO

The retromer is a pentameric protein complex that mediates the retrograde transport of acid hydrolase receptors between endosomes and the trans-Golgi network and is conserved across all eukaryotes. Unlike other eukaryotes, the endomembrane system of Giardia trophozoite is simple and is composed only of the endoplasmic reticulum and peripheral vesicles (PVs), which may represent an ancient organellar system converging compartments such as early and late endosomes and lysosomes. Sorting and trafficking of membrane proteins and soluble hydrolases from the endoplasmic reticulum to the PVs have been described as specific and conserved but whether the giardial retromer participates in receptor recycling remains elusive. Homologs of the retromer Vacuolar Protein Sorting (Vps35p, Vps26p, and Vps29p) have been identified in this parasite. Cloning the GlVPS35 subunit and antisera production enabled the localization of this protein in the PVs as well as in the cytosol. Tagged expression of the subunits was used to demonstrate their association with membranes, and immunofluorescence confocal laser scanning revealed high degrees of colabeling between the retromer subunits and also with the endoplasmic reticulum and PV compartment markers. Protein-protein interaction data revealed interaction between the subunits of GlVPS35 and the cytosolic domain of the hydrolase receptor GlVps. Altogether our data provide original information on the molecular interactions that mediate assembly of the cargo-selective retromer subcomplex and its involvement in the recycling of the acid hydrolase receptor in this parasite.

Assuntos

RESUMO

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells.

Assuntos

RESUMO

Giardia is a flagellated protozoan parasite that has to face different microenvironments during its life cycle in order to survive. All cells exchange materials with the extracellular medium through the reciprocal processes of endocytosis and secretion. Unlike more evolved cells, Giardia lacks a defined endosomal/lysosomal system, but instead possesses peripheral vacuoles that play roles in endocytosis, degradation, recycling, and secretion of proteins during growth and differentiation of the parasite. This review focuses on recent reports defining the role of different molecules involved in protein trafficking to the peripheral vacuoles, and discusses possible mechanisms of receptor recycling. Since Giardia is an early-branching protist, the study of this parasite may lead to a clearer understanding of the minimal machinery required for protein transport in eukaryotic cells.

Assuntos

RESUMO

As Giardia lamblia is unable to synthesize cholesterol de novo, this steroid might be obtained from the host's intestinal milieu by endocytosis of lipoproteins. In this work, we identified a putative Giardia lamblia low-density lipoprotein receptor-related proteins (GlLRP), a type I membrane protein, which shares the substrate N-terminal binding domain and a FXNPXY-type endocytic motif with human LRPs. Expression of tagged GlLRP showed that it was localized predominantly in the endoplasmic reticulum, lysosomal-like peripheral vacuoles and plasma membrane. However, the FXNPXY-deleted GlLRP was retained at the plasma membrane suggesting that it is abnormally transported and processed. The low-density lipoprotein and chylomicrons interacted with GlLRP, with this interaction being necessary for lipoprotein internalization and cell proliferation. Finally, we show that GlLRP binds directly to the medium subunit of Giardia adaptor protein 2, indicating that receptor-mediated internalization occurs through an adaptin mechanism.

Assuntos