Facilitation of Symplastic Effector Protein Mobility by Paired Effectors Is Conserved in Different Classes of Fungal Pathogens.

It has been discovered that plant pathogens produce effectors that spread via plasmodesmata (PD) to allow modulation of host processes in distal uninfected cells. Fusarium oxysporum f. sp. lycopersici (Fol) facilitates effector translocation by expansion of the size-exclusion limit of PD using the Six5/Avr2 effector pair. How other fungal pathogens manipulate PD is unknown. We recently reported that many fungal pathogens belonging to different families carry effector pairs that resemble the SIX5/AVR2 gene pair from Fol. Here, we performed structural predictions of three of these effector pairs from Leptosphaeria maculans (Lm) and tested their ability to manipulate PD and to complement the virulence defect of a Fol SIX5 knockout mutant. We show that the AvrLm10A homologs are structurally related to FolSix5 and localize at PD when they are expressed with their paired effectors. Furthermore, these effectors were found to complement FolSix5 function in cell-to-cell mobility assays and in fungal virulence. We conclude that distantly related fungal species rely on structurally related paired effector proteins to manipulate PD and facilitate effector mobility. The wide distribution of these effector pairs implies Six5-mediated effector translocation to be a conserved propensity among fungal plant pathogens. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Quantification of Cell-to-Cell Connectivity Using Particle Bombardment.

Plant cells are connected by cytoplasmic bridges called plasmodesmata. Plasmodesmata are lined by the plasma membrane, essentially forming tunnels that directly connect the cytoplasm of adjacent cells through which soluble molecules can move from cell to cell. This cell-to-cell mobility is underpinned by cytoplasmic advection and diffusion in a manner dependent on molecular size. This movement of molecules is regulated by the aperture of plasmodesmata. GREEN FLUORESCENT PROTEIN (GFP) is a 27 kDa soluble protein that can move passively between cells via plasmodesmata. Thus, it serves as an ideal probe to assess plasmodesmal aperture. GFP can be transgenically produced in single cells by microprojectile bombardment-mediated transformation, and its cell-to-cell mobility can be measured by live-cell imaging and counting the number of cells (or cell layers) to which it has moved. Thus, the number of cells in which GFP is visible serves as a measure of plasmodesmal aperture and functional cell-to-cell connectivity. Here we present methods for microprojectile bombardment of GFP into leaf epidermal cells and statistical analysis of resulting data.