Crosstalk between SNF1 Pathway and the Peroxisome-Mediated Lipid Metabolism in Magnaporthe oryzae

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.

Mitochondrial fission protein MoFis1 mediates conidiation and is required for full virulence of the rice blast fungus Magnaporthe oryzae

“…In budding yeast, Fis1 cooperates with adaptor proteins Mdv1 and Caf4 to recruit the cytoplasmic dynamin-related GTPase Dnm1 to sites of membrane scission from the cytoplasm during mitochondrial fission (Cerveny and Jensen 2003;Cerveny et al 2001;Jakobs et al 2003;Mozdy et al 2000;Tieu and Nunnari 2000;Tieu et al 2002). The damage of mitochondrial fission in Mofis1 maybe leads to defects in carbon metabolism, which is required for fungal development and virulence (Fernandez et al 2012;Zeng et al 2014). Therefore, MoFis1 functions in the fungal development and virulence through keeping normal mitochondrial fission in M. oryzae.…”

MoARG1, MoARG5,6 and MoARG7 involved in arginine biosynthesis are essential for growth, conidiogenesis, sexual reproduction, and pathogenicity in Magnaporthe oryzae

“…All M. oryzae strains were routinely cultured at 25 • C with a 16 h light/8 h dark-photoperiod on complete medium (CM) agar plates (Petri dish, 9 cm diameter). Strain maintenance protocols and medium compositions as described previously (Talbot et al, 1993;Zeng et al, 2014). Minimal medium (MM: CM lacking peptone, yeast extract, and casamino acids) and oatmeal agar [OMA: 5% oatmeal (w/v), 1.5% agar (w/v)] were used for phenotypic assays.…”

“…After staining, the samples were washed with phosphate-buffered saline (PBS) and then observed by fluorescence microscopy. To examine lipid bodies during appressorial development, Nile Red staining was used, as previously described (Zeng et al, 2014). 3-Amino,4-aminomethyl-2 ,7 -difluorescein, diacetate (DAF-FM DA) (Beyotime Inst.…”

“…Colony diameters were measured at 10 days. Conidial germination and appressorium formation were measured on a hydrophobic surface (Zeng et al, 2014). Conidial germination and appressorium formation were examined at 4, 8, 12, and 24 h post-incubation (hpi); at least 200 conidia or appressoria were counted for each strain.…”

“…Plant pathogenicity assays were performed as described previously (Zeng et al, 2014). Disease symptom development on leaves was examined and photographed 5 days after inoculation.…”