Limits...
Crosstalk between SNF1 pathway and the peroxisome-mediated lipid metabolism in Magnaporthe oryzae.

Zeng XQ, Chen GQ, Liu XH, Dong B, Shi HB, Lu JP, Lin F - PLoS ONE (2014)

Bottom Line: And the upstream kinases, MoSak1 and MoTos3, play unequal roles in SNF1 activation with a clear preference to MoSak1 over MoTos3.Meanwhile, the mutant lacking both of them exhibited a severe phenotype comparable to ΔMosnf1, uncovering a cooperative relationship between MoSak1 and MoTos3.Taken together, our data indicate that the SNF1 pathway is required for fungal development and facilitates pathogenicity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, Hangzhou, China.

ABSTRACT
The SNF1/AMPK pathway has a central role in response to nutrient stress in yeast and mammals. Previous studies on SNF1 function in phytopathogenic fungi mostly focused on the catalytic subunit Snf1 and its contribution to the derepression of cell wall degrading enzymes (CWDEs). However, the MoSnf1 in Magnaporthe oryzae was reported not to be involved in CWDEs regulation. The mechanism how MoSnf1 functions as a virulence determinant remains unclear. In this report, we demonstrate that MoSnf1 retains the ability to respond to nutrient-free environment via its participation in peroxisomal maintenance and lipid metabolism. Observation of GFP-tagged peroxisomal targeting signal-1 (PTS1) revealed that the peroxisomes of ΔMosnf1 were enlarged in mycelia and tended to be degraded before conidial germination, leading to the sharp decline of peroxisomal amount during appressorial development, which might impart the mutant great retard in lipid droplets mobilization and degradation. Consequently, ΔMosnf1 exhibited inability to maintain normal appressorial cell wall porosity and turgor pressure, which are key players in epidermal infection process. Exogenous glucose could partially restore the appressorial function and virulence of ΔMosnf1. Toward a further understanding of SNF1 pathway, the β-subunit MoSip2, γ-subunit MoSnf4, and two putative Snf1-activating kinases, MoSak1 and MoTos3, were additionally identified and characterized. Here we show the mutants ΔMosip2 and ΔMosnf4 performed multiple disorders as ΔMosnf1 did, suggesting the complex integrity is essential for M. oryzae SNF1 kinase function. And the upstream kinases, MoSak1 and MoTos3, play unequal roles in SNF1 activation with a clear preference to MoSak1 over MoTos3. Meanwhile, the mutant lacking both of them exhibited a severe phenotype comparable to ΔMosnf1, uncovering a cooperative relationship between MoSak1 and MoTos3. Taken together, our data indicate that the SNF1 pathway is required for fungal development and facilitates pathogenicity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae.

Show MeSH

Related in: MedlinePlus

Effects of SNF1 pathway mutations on GFP-PTS1 distribution.(A) Confocal microscopic observation of mutant strains expressing GFP-PTS1. Images shown were representative of the majority of vegetative hyphae. Enlarged peroxisomes were more frequently observed in ΔMosnf1 and ΔMosak1ΔMotos3 than WT. Arrows point to peroxisomes. Bar  = 5 µm. (B) Colocalization of GFP-PST1-positive peroxisomes and CMAC-stained vacuoles. The amount of cytoplasmic peroxisomes was decreased dramatically in the conidia of ΔMosnf1, ΔMosak1, and ΔMosak1ΔMotos3, while in WT and ΔMotos3, numerous peroxisomal puncta were observed with the absence of vacuolar GFP fluorescence. The localization patterns of GFP-PTS1 in ΔMosip2 and ΔMosnf4 conidia were indistinguishable from that in ΔMosnf1 conidia. Bars  = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4121083&req=5

pone-0103124-g005: Effects of SNF1 pathway mutations on GFP-PTS1 distribution.(A) Confocal microscopic observation of mutant strains expressing GFP-PTS1. Images shown were representative of the majority of vegetative hyphae. Enlarged peroxisomes were more frequently observed in ΔMosnf1 and ΔMosak1ΔMotos3 than WT. Arrows point to peroxisomes. Bar  = 5 µm. (B) Colocalization of GFP-PST1-positive peroxisomes and CMAC-stained vacuoles. The amount of cytoplasmic peroxisomes was decreased dramatically in the conidia of ΔMosnf1, ΔMosak1, and ΔMosak1ΔMotos3, while in WT and ΔMotos3, numerous peroxisomal puncta were observed with the absence of vacuolar GFP fluorescence. The localization patterns of GFP-PTS1 in ΔMosip2 and ΔMosnf4 conidia were indistinguishable from that in ΔMosnf1 conidia. Bars  = 5 µm.

Mentions: Peroxisomal matrix proteins usually include specific motifs known as peroxisomal targeting signals (PTSs), which could be recognized by the import machinery and targeted to peroxisome. PTS1 is a conserved tripeptide sequence (S/A/C) (H/R/K) (I/L/M) at the C terminus of most known peroxisomal matrix proteins [34], [40]. In this study, PTS1 (SKL) signal was employed to visualize peroxisome by introducing GFP-PTS1 vector (kindly provided by Dr. Jiaoyu Wang) into ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, ΔMotos3, ΔMosak1ΔMotos3, and the wild type, respectively. Subcellular localization of GFP-PTS1 was then investigated in the transformed strains. During vegetative growth phase, both Guy11 and the SNF1 pathway mutants performed punctate GFP fluorescence, indicative of peroxisomal structures, however, the puncta size seemed to be different among them. In ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, and ΔMosak1ΔMotos3 background, enlarged peroxisomes were more frequently observed than in WT and ΔMotos3, suggesting some aberrant changes therein (Figure 5A). Likewise, numerous GFP-PTS1 labeled peroxisomes were observed as punctate spots in the conidia of WT and ΔMotos3 (Figure 5B), and began to enter the vacuolar lumens during appressorial differentiation (Figure S2); in the meantime, their incipient appressoria were peroxisome-rich (Figure S2). While in other mutants, GFP-PTS1 had already been mis-localized to the CMAC-stained vacuoles even before conidial germination, leaving sharp decline of fluorescent spots in the conidial cytoplasm (77.6±5.1%, 64.3±4.6%, 62.2±5.7%, 35.5±5.3%, 76.9±6.6% spores with fluorescent vacuoles in ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, and ΔMosak1ΔMotos3, respectively, vs. 1.6±0.7%, 1.4±0.6% in WT and ΔMotos3, respectively) (Figure 5B). Further investigation of the GFP-PTS1 sequential localization revealed that the fluorescent signals were almost invisible in ΔMosnf1 appressoria but arrested in conidial spherical vacuoles (Figure S2).


Crosstalk between SNF1 pathway and the peroxisome-mediated lipid metabolism in Magnaporthe oryzae.

Zeng XQ, Chen GQ, Liu XH, Dong B, Shi HB, Lu JP, Lin F - PLoS ONE (2014)

Effects of SNF1 pathway mutations on GFP-PTS1 distribution.(A) Confocal microscopic observation of mutant strains expressing GFP-PTS1. Images shown were representative of the majority of vegetative hyphae. Enlarged peroxisomes were more frequently observed in ΔMosnf1 and ΔMosak1ΔMotos3 than WT. Arrows point to peroxisomes. Bar  = 5 µm. (B) Colocalization of GFP-PST1-positive peroxisomes and CMAC-stained vacuoles. The amount of cytoplasmic peroxisomes was decreased dramatically in the conidia of ΔMosnf1, ΔMosak1, and ΔMosak1ΔMotos3, while in WT and ΔMotos3, numerous peroxisomal puncta were observed with the absence of vacuolar GFP fluorescence. The localization patterns of GFP-PTS1 in ΔMosip2 and ΔMosnf4 conidia were indistinguishable from that in ΔMosnf1 conidia. Bars  = 5 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4121083&req=5

pone-0103124-g005: Effects of SNF1 pathway mutations on GFP-PTS1 distribution.(A) Confocal microscopic observation of mutant strains expressing GFP-PTS1. Images shown were representative of the majority of vegetative hyphae. Enlarged peroxisomes were more frequently observed in ΔMosnf1 and ΔMosak1ΔMotos3 than WT. Arrows point to peroxisomes. Bar  = 5 µm. (B) Colocalization of GFP-PST1-positive peroxisomes and CMAC-stained vacuoles. The amount of cytoplasmic peroxisomes was decreased dramatically in the conidia of ΔMosnf1, ΔMosak1, and ΔMosak1ΔMotos3, while in WT and ΔMotos3, numerous peroxisomal puncta were observed with the absence of vacuolar GFP fluorescence. The localization patterns of GFP-PTS1 in ΔMosip2 and ΔMosnf4 conidia were indistinguishable from that in ΔMosnf1 conidia. Bars  = 5 µm.
Mentions: Peroxisomal matrix proteins usually include specific motifs known as peroxisomal targeting signals (PTSs), which could be recognized by the import machinery and targeted to peroxisome. PTS1 is a conserved tripeptide sequence (S/A/C) (H/R/K) (I/L/M) at the C terminus of most known peroxisomal matrix proteins [34], [40]. In this study, PTS1 (SKL) signal was employed to visualize peroxisome by introducing GFP-PTS1 vector (kindly provided by Dr. Jiaoyu Wang) into ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, ΔMotos3, ΔMosak1ΔMotos3, and the wild type, respectively. Subcellular localization of GFP-PTS1 was then investigated in the transformed strains. During vegetative growth phase, both Guy11 and the SNF1 pathway mutants performed punctate GFP fluorescence, indicative of peroxisomal structures, however, the puncta size seemed to be different among them. In ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, and ΔMosak1ΔMotos3 background, enlarged peroxisomes were more frequently observed than in WT and ΔMotos3, suggesting some aberrant changes therein (Figure 5A). Likewise, numerous GFP-PTS1 labeled peroxisomes were observed as punctate spots in the conidia of WT and ΔMotos3 (Figure 5B), and began to enter the vacuolar lumens during appressorial differentiation (Figure S2); in the meantime, their incipient appressoria were peroxisome-rich (Figure S2). While in other mutants, GFP-PTS1 had already been mis-localized to the CMAC-stained vacuoles even before conidial germination, leaving sharp decline of fluorescent spots in the conidial cytoplasm (77.6±5.1%, 64.3±4.6%, 62.2±5.7%, 35.5±5.3%, 76.9±6.6% spores with fluorescent vacuoles in ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, and ΔMosak1ΔMotos3, respectively, vs. 1.6±0.7%, 1.4±0.6% in WT and ΔMotos3, respectively) (Figure 5B). Further investigation of the GFP-PTS1 sequential localization revealed that the fluorescent signals were almost invisible in ΔMosnf1 appressoria but arrested in conidial spherical vacuoles (Figure S2).

Bottom Line: And the upstream kinases, MoSak1 and MoTos3, play unequal roles in SNF1 activation with a clear preference to MoSak1 over MoTos3.Meanwhile, the mutant lacking both of them exhibited a severe phenotype comparable to ΔMosnf1, uncovering a cooperative relationship between MoSak1 and MoTos3.Taken together, our data indicate that the SNF1 pathway is required for fungal development and facilitates pathogenicity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, Hangzhou, China.

ABSTRACT
The SNF1/AMPK pathway has a central role in response to nutrient stress in yeast and mammals. Previous studies on SNF1 function in phytopathogenic fungi mostly focused on the catalytic subunit Snf1 and its contribution to the derepression of cell wall degrading enzymes (CWDEs). However, the MoSnf1 in Magnaporthe oryzae was reported not to be involved in CWDEs regulation. The mechanism how MoSnf1 functions as a virulence determinant remains unclear. In this report, we demonstrate that MoSnf1 retains the ability to respond to nutrient-free environment via its participation in peroxisomal maintenance and lipid metabolism. Observation of GFP-tagged peroxisomal targeting signal-1 (PTS1) revealed that the peroxisomes of ΔMosnf1 were enlarged in mycelia and tended to be degraded before conidial germination, leading to the sharp decline of peroxisomal amount during appressorial development, which might impart the mutant great retard in lipid droplets mobilization and degradation. Consequently, ΔMosnf1 exhibited inability to maintain normal appressorial cell wall porosity and turgor pressure, which are key players in epidermal infection process. Exogenous glucose could partially restore the appressorial function and virulence of ΔMosnf1. Toward a further understanding of SNF1 pathway, the β-subunit MoSip2, γ-subunit MoSnf4, and two putative Snf1-activating kinases, MoSak1 and MoTos3, were additionally identified and characterized. Here we show the mutants ΔMosip2 and ΔMosnf4 performed multiple disorders as ΔMosnf1 did, suggesting the complex integrity is essential for M. oryzae SNF1 kinase function. And the upstream kinases, MoSak1 and MoTos3, play unequal roles in SNF1 activation with a clear preference to MoSak1 over MoTos3. Meanwhile, the mutant lacking both of them exhibited a severe phenotype comparable to ΔMosnf1, uncovering a cooperative relationship between MoSak1 and MoTos3. Taken together, our data indicate that the SNF1 pathway is required for fungal development and facilitates pathogenicity by its contribution to peroxisomal maintenance and lipid metabolism in M. oryzae.

Show MeSH
Related in: MedlinePlus