Reconstructing the global migration history of Phytophthora infestans towards Colombia.

The evolution of new variants of plant pathogens is one of the biggest challenges to controlling and managing plant diseases. Of the forces driving these evolutionary processes, global migration events are particularly important for widely distributed diseases such as the potato late blight, caused by the oomycete Phytophthora infestans (Mont.) de Bary. However, little is known about its migration routes outside North America and Europe. This work used genotypic data from population studies to elucidate the migration history originating the Colombian P. infestans population. For this purpose, a dataset of 1706 P. infestans genotypes was recollected, representing North and South America, Europe, and Asia. Descriptive analysis and historical records from North America and Europe were used to propose three global migration hypotheses, differing on the origin of the disease (Mexico or Peru) and the hypothesis that it came back to South America from Europe. These scenarios were tested using Approximate Bayesian Computation. According to this analysis, the most probable scenario (posterior probability = 0.631) was the one proposing a Peruvian origin for P. infestans, an initial migration towards Colombia and Mexico, and a later event from Mexico to the United States, and then to Europe and Asia, with no return to northern South America. In Colombia, the scenario considering a single migration from Peru and posterior migrations within Colombia was the most probable with a posterior probability of 0.640. The obtained results support the hypothesis of a Peruvian origin for P. infestans followed by rare colonization events worldwide.

Cylindrical Inclusion Protein of Turnip Mosaic Virus Serves as a Docking Point for the Intercellular Movement of Viral Replication Vesicles.

Plant viruses move from the initially infected cell to adjacent cells through plasmodesmata (PDs). To do so, viruses encode dedicated protein(s) that facilitate this process. How viral proteins act together to support the intercellular movement of viruses is poorly defined. Here, by using an infection-free intercellular vesicle movement assay, we investigate the action of CI (cylindrical inclusion) and P3N-PIPO (amino-terminal half of P3 fused to Pretty Interesting Potyviridae open reading frame), the two PD-localized potyviral proteins encoded by Turnip mosaic virus (TuMV), in the intercellular movement of the viral replication vesicles. We provide evidence that CI and P3N-PIPO are sufficient to support the PD targeting and intercellular movement of TuMV replication vesicles induced by 6K2, a viral protein responsible for the generation of replication vesicles. 6K2 interacts with CI but not P3N-PIPO. When this interaction is impaired, the intercellular movement of TuMV replication vesicles is inhibited. Furthermore, in transmission electron microscopy, vesicular structures are observed in connection with the cylindrical inclusion bodies at structurally modified PDs in cells coexpressing 6K2, CI, and P3N-PIPO. CI is directed to PDs through its interaction with P3N-PIPO. We hypothesize that CI serves as a docking point for PD targeting and the intercellular movement of TuMV replication vesicles. This work contributes to a better understanding of the roles of different viral proteins in coordinating the intercellular movement of viral replication vesicles.

The Vesicle-Forming 6K2 Protein of Turnip Mosaic Virus Interacts with the COPII Coatomer Sec24a for Viral Systemic Infection.

UNLABELLED: Positive-sense RNA viruses remodel host cell endomembranes to generate quasi-organelles known as "viral factories" to coordinate diverse viral processes, such as genome translation and replication. It is also becoming clear that enclosing viral RNA (vRNA) complexes within membranous structures is important for virus cell-to-cell spread throughout the host. In plant cells infected by turnip mosaic virus (TuMV), a member of the family Potyviridae, peripheral motile endoplasmic reticulum (ER)-derived viral vesicles are produced that carry the vRNA to plasmodesmata for delivery into adjacent noninfected cells. The viral protein 6K2 is responsible for the formation of these vesicles, but how 6K2 is involved in their biogenesis is unknown. We show here that 6K2 is associated with cellular membranes. Deletion mapping and site-directed mutagenesis experiments defined a soluble N-terminal 12-amino-acid stretch, in particular a potyviral highly conserved tryptophan residue and two lysine residues that were important for vesicle formation. When the tryptophan residue was changed into an alanine in the viral polyprotein, virus replication still took place, albeit at a reduced level, but cell-to-cell movement of the virus was abolished. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation experiments showed that 6K2 interacted with Sec24a, a COPII coatomer component. Appropriately, TuMV systemic movement was delayed in an Arabidopsis thaliana mutant line defective in Sec24a. Intercellular movement of TuMV replication vesicles thus requires ER export of 6K2, which is mediated by the interaction of the N-terminal domain of the viral protein with Sec24a. IMPORTANCE: Many plant viruses remodel the endoplasmic reticulum (ER) to generate vesicles that are associated with the virus replication complex. The viral protein 6K2 of turnip mosaic virus (TuMV) is known to induce ER-derived vesicles that contain vRNA as well as viral and host proteins required for vRNA synthesis. These vesicles not only sustain vRNA synthesis, they are also involved in the intercellular trafficking of vRNA. In this investigation, we found that the N-terminal soluble domain of 6K2 is required for ER export of the protein and for the formation of vesicles. ER export is not absolutely required for vRNA replication but is necessary for virus cell-to-cell movement. Furthermore, we found that 6K2 physically interacts with the COPII coatomer Sec24a and that an Arabidopsis thaliana mutant line with a defective Sec24a shows a delay in the systemic infection by TuMV.

Identification of the iduronate-2-sulfatase proteome in wild-type mouse brain.

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme involved in the metabolism of the glycosaminoglycans heparan (HS) and dermatan (DS) sulfate. Mutations on IDS gene produce mucopolysaccharidosis II (MPS II), characterized by the lysosomal accumulation of HS and DS, leading to severe damage of the central nervous system (CNS) and other tissues. In this study, we used a neurochemistry and proteomic approaches to identify the brain distribution of IDS and its interacting proteins on wild-type mouse brain. IDS immunoreactivity showed a robust staining throughout the entire brain, suggesting an intracellular reactivity in nerve cells and astrocytes. By using affinity purification and mass spectrometry we identified 187 putative IDS partners-proteins, mainly hydrolases, cytoskeletal proteins, transporters, transferases, oxidoreductases, nucleic acid binding proteins, membrane traffic proteins, chaperons and enzyme modulators, among others. The interactions with some of these proteins were predicted by using bioinformatics tools and confirmed by co-immunoprecipitation analysis and Blue Native PAGE. In addition, we identified cytosolic IDS-complexes containing proteins from predicted highly connected nodes (hubs), with molecular functions including catalytic activity, redox balance, binding, transport, receptor activity and structural molecule activity. The proteins identified in this study would provide new insights about IDS physiological role into the CNS and its potential role in the brain-specific protein networks.

Production of human recombinant phenylalanine hydroxylase in Lactobacillus plantarum for gastrointestinal delivery.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by a defective phenylalanine hydroxylase (PAH), which catalyzes the hydroxylation of l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr) in presence of the cofactor tetrahydrobiopterin (BH4). Defective PAH causes accumulation of phenylalanine, which has neurotoxic effects and leads to dermatological, behavioral, and neurocognitive problems. Treatments for this disease consist in life-long diets that are hard for patients to keep, or supplementation with BH4. In this study, we propose a system where a probiotic lactic acid bacteria (LAB) can be used as vehicle to express in situ an engineered human PAH. Engineered PAHs contain a secretion peptide, a gastrointestinal signal (GI), the human PAH, and a flexible glycine linker followed by the fluorescence protein mEGFP. Engineered constructs were successfully transformed, expressed, and secreted in Lactobacillus plantarum CM_PUJ411. PAH construct containing either the signal peptide GI1 or GI2 were transported through a Caco-2 cell monolayer. Nevertheless, the one containing GI1 allowed the highest transport through the cell monolayer. Co-culture of L. plantarum and Caco-2 cells showed that engineered PAH is produced in-situ and transported through the cell monolayer. Finally, the activity test showed that the engineered PAH secreted by L. plantarum CM_PUJ411 is active, leading to a reduction in l-Phe and an increase in l-Tyr levels, respectively. These results show the potential of this system as a new therapeutic alternative for the treatment of PKU patients.

Hijack it, change it: how do plant viruses utilize the host secretory pathway for efficient viral replication and spread?

The secretory pathway of eukaryotic cells has an elaborated set of endomembrane compartments involved in the synthesis, modification, and sorting of proteins and lipids. The secretory pathway in plant cells shares many features with that in other eukaryotic cells but also has distinct characteristics important for fundamental cell and developmental processes and for proper immune responses. Recently, there has been evidence that the remodeling of this pathway, and often the formation of viral-induced organelles, play an important role in viral replication and spread. The modification of the host secretory pathway seems to be a common feature among most single-stranded positive ss(+)RNA and even some DNA viruses. In this review, we will present the recent advances in the understanding of the organization and dynamics of the plant secretory pathway and the molecular regulation of membrane trafficking in the pathway. We will also discuss how different plant viruses may interact with the host secretory pathway for their efficient replication and spread, with a focus on tobacco mosaic virus and turnip mosaic virus.