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 null 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.
Highlights
The conserved SNF1/AMP-activated protein kinase (AMPK) family is well known to serve as the cellular energy sensor and regulator of carbon metabolism in eukaryotes [1,2,3]
We sought for other M. oryzae orthologs involved in SNF1 pathway to obtain a further understanding of its function
Domains identified by the InterPro database of these M. oryzae proteins exhibited high conservation (Table S1), including GBD and ID in the C-terminal region of MoSip2, two pairs of cystathionine-beta-synthase (CBS) repeats integrated in MoSnf4, and kinase domains in MoSak1 and MoTos3
Summary
The conserved SNF1/AMP-activated protein kinase (AMPK) family is well known to serve as the cellular energy sensor and regulator of carbon metabolism in eukaryotes [1,2,3]. The best documented function of SNF1 kinase is to respond to glucose limitation and enable yeast cells to utilize non-preferred carbon sources when glucose is deprived [3,5]. Sak is the major kinase in this activation, only simultaneous absence of the three Snf1activating kinases confers completely abolished growth on nonpreferred carbon sources, indicating a partially redundant function among them [7,8,9]. One of the best-studied targets of yeast SNF1 is the transcriptional repressor Mig, which represses the expression of pivotal enzymes involved in the utilization of alternative sugars [10]. SNF1 is activated by its upstream kinases and thereafter phosphorylates the repressor Mig, resulting in the translocation of Mig from the nucleus to the cytoplasm and the relief of transcriptional repression imposed by Mig1 [3,10]. In addition to nutrient stress, the yeast SNF1 pathway participates in environmental stress resistance, aging, invasive and pseudohyphal growth [3,14,15]
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