Assaying Effector Cell-to-Cell Mobility in Plant Tissues Identifies Hypermobility and Indirect Manipulation of Plasmodesmata.

In plants, plasmodesmata establish cytoplasmic continuity between cells to allow for communication and resource exchange across the cell wall. While plant pathogens use plasmodesmata as a pathway for both molecular and physical invasion, the benefits of molecular invasion (cell-to-cell movement of pathogen effectors) are poorly understood. To establish a methodology for identification and characterization of the cell-to-cell mobility of effectors, we performed a quantitative live imaging-based screen of candidate effectors of the fungal pathogen Colletotrichum higginsianum. We predicted C. higginsianum effectors by their expression profiles, the presence of a secretion signal, and their predicted and in planta localization when fused to green fluorescent protein. We assayed for cell-to-cell mobility of nucleocytosolic effectors and identified 14 that are cell-to-cell mobile. We identified that three of these effectors are "hypermobile," showing cell-to-cell mobility greater than expected for a protein of that size. To explore the mechanism of hypermobility, we chose two hypermobile effectors and measured their impact on plasmodesmata function and found that even though they show no direct association with plasmodesmata, each increases the transport capacity of plasmodesmata. Thus, our methods for quantitative analysis of cell-to-cell mobility of candidate microbe-derived effectors, or any suite of host proteins, can identify cell-to-cell hypermobility and offer greater understanding of how proteins affect plasmodesmal function and intercellular connectivity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Precise levels of the Drosophila adaptor protein Dreadlocks maintain the size and stability of germline ring canals.

Intercellular bridges are essential for fertility in many organisms. The developing fruit fly egg has become the premier model system to study intercellular bridges. During oogenesis, the oocyte is connected to supporting nurse cells by relatively large intercellular bridges, or ring canals. Once formed, the ring canals undergo a 20-fold increase in diameter to support the movement of materials from the nurse cells to the oocyte. Here, we demonstrate a novel role for the conserved SH2/SH3 adaptor protein Dreadlocks (Dock) in regulating ring canal size and structural stability in the germline. Dock localizes at germline ring canals throughout oogenesis. Loss of Dock leads to a significant reduction in ring canal diameter, and overexpression of Dock causes dramatic defects in ring canal structure and nurse cell multinucleation. The SH2 domain of Dock is required for ring canal localization downstream of Src64 (also known as Src64B), and the function of one or more of the SH3 domains is necessary for the strong overexpression phenotype. Genetic interaction and localization studies suggest that Dock promotes WASp-mediated Arp2/3 activation in order to determine ring canal size and regulate growth. This article has an associated First Person interview with the first author of the paper.

The Arp2/3 complex and the formin, Diaphanous, are both required to regulate the size of germline ring canals in the developing egg chamber.

Intercellular bridges are an essential structural feature found in both germline and somatic cells throughout the animal kingdom. Because of their large size, the germline intercellular bridges, or ring canals, in the developing fruit fly egg chamber are an excellent model to study the formation, stabilization, and growth of these structures. Within the egg chamber, the germline ring canals connect 15 supporting nurse cells to the developing oocyte, facilitating the transfer of materials required for successful oogenesis. The ring canals are derived from a stalled actomyosin contractile ring; once formed, additional actin and actin-binding proteins are recruited to the ring to support the 20-fold growth that accompanies oogenesis. These behaviors provide a unique model system to study the actin regulators that control incomplete cytokinesis, intercellular bridge formation, and growth. By temporally controlling their expression in the germline, we have demonstrated that the Arp2/3 complex and the formin, Diaphanous (Dia), coordinately regulate ring canal size and growth throughout oogenesis. Dia is required for successful incomplete cytokinesis and the initial stabilization of the germline ring canals. Once ring canals have formed, the Arp2/3 complex and Dia cooperate to determine ring canal size and maintain stability. Our data suggest that nurse cells must maintain a precise balance between the activity of these two nucleators during oogenesis.

Mechanistic insight into the Cdc28-related protein kinase Ime2 through analysis of replication protein A phosphorylation.

In budding yeast, the meiosis-specific protein kinase Ime2 is required for normal meiotic progression. Current evidence suggests that Ime2 is functionally related to Cdc28, the major cyclin-dependent kinase in yeast that is essential for both cell cycle and meiosis. We have previously reported that a natural target of Ime2 activity is replication protein A (RPA), the cellular single-stranded DNA-binding protein that performs critical functions during DNA replication, repair and recombination. Ime2-dependent RPA phosphorylation first occurs early in meiosis and targets the middle subunit of the RPA heterotrimeric complex (Rfa2). We now demonstrate that Rfa2 serine 27 (S27) is required for Ime2-dependent Rfa2 phosphorylation in vivo. S27 is also required for Rfa2 phosphorylation in vitro catalyzed by immunoprecipitated Ime2. In addition, Ime2 mediates in vitro phosphorylation of a short peptide containing Rfa2 amino acids 23 through 29, thereby providing evidence that S27 itself is the phosphoacceptor. Phosphorylation site mapping supports this conclusion, as mass spectrometry analysis has revealed that at least three residues within Rfa2 amino acids 2 through 35 become phosphorylated specifically during meiosis. Although S27 is embedded in a motif that is recognized by several protein kinases, this sequence is not a typical target of cyclin-dependent kinases. Therefore, the mechanism underlying Ime2 substrate recognition could differ from that of Cdc28.