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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.

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Intracellular mobilization of lipid droplets in WT and SNF1 pathway mutants during appressorium morphogenesis.Conidial suspensions were incubated on the surfaces of hydrophobic films and stained with Nile red to observe the status of lipid droplets movement and distribution at the indicated time points under epifluorescence microscope. (A) ΔMosnf1 and ΔMosak1ΔMotos3 showed significant delays in lipid mobilization and degradation with the presence of Nile red-stained lipid bodies even at 96 hpi, while fluorescent signals were almost invisible in WT at 48 hpi. Bars  = 5 µm. (B) Percentages of conidia (left) or appressoria (right) that contained lipid droplets. Varied degrees of defect in lipid mobilization were observed among the mutants.
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pone-0103124-g006: Intracellular mobilization of lipid droplets in WT and SNF1 pathway mutants during appressorium morphogenesis.Conidial suspensions were incubated on the surfaces of hydrophobic films and stained with Nile red to observe the status of lipid droplets movement and distribution at the indicated time points under epifluorescence microscope. (A) ΔMosnf1 and ΔMosak1ΔMotos3 showed significant delays in lipid mobilization and degradation with the presence of Nile red-stained lipid bodies even at 96 hpi, while fluorescent signals were almost invisible in WT at 48 hpi. Bars  = 5 µm. (B) Percentages of conidia (left) or appressoria (right) that contained lipid droplets. Varied degrees of defect in lipid mobilization were observed among the mutants.

Mentions: It was observed that lipid droplets had been entirely translocated from conidia to appressoria within 24 h and the majority was degraded at 48 hpi in Guy11 (Figure 6). In ΔMotos3, no distinguishable difference was detected in the transfer efficiency of lipid bodies as compared to WT, but the degradation rate was slightly decreased, with about 20% appressoria stained by Nile red compared to only 1.67% in Guy11 at 96 hpi (Figure 6B). On the contrary, the mobilization of lipid reserves was significantly retarded in ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, and ΔMosak1ΔMotos3, ranging from 64.0% to 85.2% conidia still containing large lipid deposits after 48 h of incubation (Figure 6A, 6B left). The degradation rates were also severely influenced, with large merged lipid droplets still observed in more than 74% appressoria of the mutants at 96 hpi (Figure 6A, 6B right). We therefore conclude that the SNF1 pathway is indispensible for lipid droplets translocation and degradation.


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)

Intracellular mobilization of lipid droplets in WT and SNF1 pathway mutants during appressorium morphogenesis.Conidial suspensions were incubated on the surfaces of hydrophobic films and stained with Nile red to observe the status of lipid droplets movement and distribution at the indicated time points under epifluorescence microscope. (A) ΔMosnf1 and ΔMosak1ΔMotos3 showed significant delays in lipid mobilization and degradation with the presence of Nile red-stained lipid bodies even at 96 hpi, while fluorescent signals were almost invisible in WT at 48 hpi. Bars  = 5 µm. (B) Percentages of conidia (left) or appressoria (right) that contained lipid droplets. Varied degrees of defect in lipid mobilization were observed among the mutants.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103124-g006: Intracellular mobilization of lipid droplets in WT and SNF1 pathway mutants during appressorium morphogenesis.Conidial suspensions were incubated on the surfaces of hydrophobic films and stained with Nile red to observe the status of lipid droplets movement and distribution at the indicated time points under epifluorescence microscope. (A) ΔMosnf1 and ΔMosak1ΔMotos3 showed significant delays in lipid mobilization and degradation with the presence of Nile red-stained lipid bodies even at 96 hpi, while fluorescent signals were almost invisible in WT at 48 hpi. Bars  = 5 µm. (B) Percentages of conidia (left) or appressoria (right) that contained lipid droplets. Varied degrees of defect in lipid mobilization were observed among the mutants.
Mentions: It was observed that lipid droplets had been entirely translocated from conidia to appressoria within 24 h and the majority was degraded at 48 hpi in Guy11 (Figure 6). In ΔMotos3, no distinguishable difference was detected in the transfer efficiency of lipid bodies as compared to WT, but the degradation rate was slightly decreased, with about 20% appressoria stained by Nile red compared to only 1.67% in Guy11 at 96 hpi (Figure 6B). On the contrary, the mobilization of lipid reserves was significantly retarded in ΔMosnf1, ΔMosip2, ΔMosnf4, ΔMosak1, and ΔMosak1ΔMotos3, ranging from 64.0% to 85.2% conidia still containing large lipid deposits after 48 h of incubation (Figure 6A, 6B left). The degradation rates were also severely influenced, with large merged lipid droplets still observed in more than 74% appressoria of the mutants at 96 hpi (Figure 6A, 6B right). We therefore conclude that the SNF1 pathway is indispensible for lipid droplets translocation and degradation.

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