Finally, a special thank you to my co-authors for their contribution to the success of this project.
I am grateful for the unwavering support and guidance of my supervisor, @talbotlabtsl.bsky.social
This project was supported💰 by the Halpin Scholarship for Rice Blast Research (13/13)

In summary, this study uncovers a remarkable spatio-temporal coordination in fungal infection. One cell lives, two die, and together they build a weaponized appressorium. (12/13)

These observations provide further evidence that distal and middle conidial cells are programmed for autophagic degradation, and organelle trafficking and de novo mitochondrial synthesis occurs only in the germinating conidium cell and appressorium at the onset of appressorium melanisation. (11/13)

At 7h post-inoculation, we observed a mixed population of red and green mitochondria in the appressorium and apical cell, while photoconverted mitochondrial decreases in the distal and middle cells. (10/13)

Next, we determine timing of de novo organelle synthesis, we fused Scad2 with the photoconvertible protein mEos3.2, which shifts fluorescence from green to red upon UV activation. We tracked red (pre-existing) and green (newly synthesized) mitochondria from germ tube swelling to appressorium.

We found the volume of mitochondria in the appressorium was greater than the starting average mitochondrial the apical conidial cell at 0h. This observation suggests de novo biogenesis during appressorium maturation. (9/13)

Having established that the apical cell is the source of organelles in the appresssorium, we set out to determine whether organelle biogenesis occurs within the infection cell prior to plant infection. We carried out 3D volumetric analysis of mitochondria using Imaris (Bitplane).

We investigated whether autophagy is required for organelle breakdown in conidial cells. Using a ∆atg8:Scad2-paGFP strain lacking autophagy, we found that only the apical cell transported mitochondria to the appressorium, while those in distal and middle cells remained intact. (8/13)

We next asked whether the three-celled conidium of M. oryzae uniformly contributes organelles to the appressorium. Using paGFP-tagged mitochondria, we found that only the apical cell transfers intact mitochondria to the appressorium. The distal and middle cells retain their photoactivated signal 7/

Our observations provide evidence that the three-celled conidium is an open system that facilitates trafficking from all cells to the appressorium. However, conidium-appressorium connectivity is transient. By 6 hpi, cytoplasmic streaming into the appressorium ceases.(6/13)

Given that filamentous fungi possess septal pores for intercellular exchange, we asked whether the conidium behaves as an open system. Photoactivation experiments confirmed rapid cytoplasmic diffusion across cells and into the appressorium, indicating open septal pores early in development. (5/13)

To quantify these changes, we modeled fluorescence intensity over time. The distal and middle cells showed a rapid linear decrease in organelle signal, while the apical cell retained organelles longer, correlating with a steady increase in the appressorium—suggesting directional trafficking. (4/13)

Using 4D live-cell imaging, we observed a continuous influx of organelles from the conidium into the growing appressorium, accompanied by a decline in organelle abundance within the conidial cells (3/13)

To carry out the investigation, first we fluorescently tagged 10 organelle proteins with eGFP in M. oryzae to track their fate, and confirmed that vector integration didn't affect pathogenicity (2/13)