Vacuolar transporter Mnr2 safeguards mitochondrial integrity in aged cells

Aging is associated with altered mitochondrial function. Mitochondrial function is dependent on the magnesium (Mg+2) ion flux. The molecular mechanism underlying Mg+2 homeostasis, especially during aging has not been well understood. We previously demonstrated that the absence of a vacuolar ion transporter Mnr2 accelerates cell death in the older part of the colony in Magnaporthe oryzae presumably due to an altered Mg+2 homeostasis. Localization of Mnr2 as dynamic puncta at the vacuolar membrane especially in the older Magnaporthe cells further suggests its association with aged cells. Interestingly, such vacuolar Mnr2 puncta colocalized with the filamentous mitochondria in the aged cells. Further, we show that aged mnr2Δ null cells displayed loss of integrity of mitochondria and vacuoles. Remarkably, exogenously added Mg+2 restored the mitochondrial structure as well as improved the lifespan of mnr2Δ null cells. Thus, we uncover a mechanism of maintenance of mitochondrial integrity and function by the ion transporter Mnr2-based Mg+2 homeostasis during aging.


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At a close examination, there seemed to be differences in GFP-Mnr2 expression 175 across the three cells of the conidium. In M. oryzae, conidia formation is marked with 176 three sequential rounds of mitotic events (SHAH et al. 2019). In the chronological order, 177 the basal cell is older than the apical/ terminal cell in a conidium. We quantified the 178 intensity of GFP-Mnr2 in all the three cells and found that indeed the expression of  stress. Indeed, the mnr2∆ mutant was more sensitive to H2O2 when compared to the 218 wild-type ( Figure 2D).

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Next, to study whether the altered vacuolar pH also affected the autophagic 220 turnover of mitochondria, we deleted MNR2 in the Atp1-GFP-expressing strain of M. conditions. We found that overall mitophagy, read as GFP signal intensities in the 224 vacuolar lumen, was impaired in the mnr2∆ strain when compared with the wild-type 225 and was similar to wild-type treated with PMSF (Supplementary Figure S5A). Similarly, 226 we also tested laccase activity as a measure of vacuolar function in both wild-type and mnr2∆ strains. While the specific laccase activity in the wild-type was over 6 U/ mg at 48 228 hours post inoculation (hpi) that in the mnr2∆ mutant was drastically reduced to 0.028 U 229 /mg (Supplementary Figure S5B). Having shown GFP-Mnr2 to be localized as a clustered punctum and with its 249 expression being higher in aged cells of M. oryzae, we next asked whether GFP-Mnr2 showed dynamic sub-cellular localization. Live-cell imaging revealed that GFP-Mnr2 251 puncta were highly dynamic and moved along the vacuolar periphery ( Figure 3A; 252 Supplementary Movie 1). We further found that the number of puncta per cell was 253 dynamic and often associated with the change in the punctum size ( Figure 3B). It is 254 likely that the different sizes of GFP-Mnr2 puncta resulted from differential clustering of 255 more than one punctum/ molecule of Mnr2.

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To assess the pH of the vacuolar lumen, both the wild-type and mnr2∆ strains were 507 grown in complete medium for 48 h, and then transferred to water for 3 h before staining 508 with 1 µg ml -1 quinacrine for 15 min. DiOC6-stained cells was carried out at 488/509 nm excitation/emission wavelengths.

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For live-cell imaging, fungal cultures were inoculated on glass-bottom petri dishes. The 517 maximum projection images were obtained from Z stacks of 0.5 µm-spaced sections 518 and processed and analyzed using ImageJ (https://imagej.nih.gov/ij/download.html) and/or Adobe Photoshop CC 2018 software.

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Quantification of GFP-Mnr2 signal intensity was carried out after the projection of 523 Z-stacks and was represented as the corrected total cell florescence (CTCF). Briefly, 524 after Z projection, while GFP-Mnr2 signal was measured from the puncta, the 525 background noise was calculated from a region surrounding the GFP-Mnr2 puncta, and 526 the CTCF values were calculated as: Integrated density -(Area of selected region X 527 mean fluorescence of background readings) and plotted using GraphPad Prism 6.00.