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

Protein interaction and gene expression analyses of SNF1 kinase complex components and its activating kinases in M. oryzae.(A) Different composition of the heterotrimeric SNF1 kinase complex and upstream kinases between S. cerevisiae and M. oryzae. (B) MoSnf1, MoSip2, and MoSnf4 interacted with each other, while no interaction was observed between MoSnf1 and its activating kinases in yeast two-hybrid assay. Yeast transformants expressing MoSnf1 plus MoSip2, MoSnf1 plus MoSnf4, MoSip2 plus MoSnf4, MoSnf1 plus MoSak1, or MoSnf1 plus MoTos3 were 10-fold serially diluted with a starter culture of 106 cells/ml and then spotted (5 µl) onto SD-Trp-Leu-His-Ade medium. (C) Gene expression profiles of MoSNF1, MoSIP2, MoSNF4, MoSAK1, and MoTOS3 among different developmental stages. Tested fungal tissues included vegetative hyphae (VH), conidia (CO), appressoria 8 hpi (AP), and invasive hyphae (72 hpi), which were within infected plant leaves (IP). Gene expression data, obtained from quantitative RT-PCR analysis, were normalized by using β-tubulin as an internal control and calibrated against the transcript abundances of VH stage.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103124-g001: Protein interaction and gene expression analyses of SNF1 kinase complex components and its activating kinases in M. oryzae.(A) Different composition of the heterotrimeric SNF1 kinase complex and upstream kinases between S. cerevisiae and M. oryzae. (B) MoSnf1, MoSip2, and MoSnf4 interacted with each other, while no interaction was observed between MoSnf1 and its activating kinases in yeast two-hybrid assay. Yeast transformants expressing MoSnf1 plus MoSip2, MoSnf1 plus MoSnf4, MoSip2 plus MoSnf4, MoSnf1 plus MoSak1, or MoSnf1 plus MoTos3 were 10-fold serially diluted with a starter culture of 106 cells/ml and then spotted (5 µl) onto SD-Trp-Leu-His-Ade medium. (C) Gene expression profiles of MoSNF1, MoSIP2, MoSNF4, MoSAK1, and MoTOS3 among different developmental stages. Tested fungal tissues included vegetative hyphae (VH), conidia (CO), appressoria 8 hpi (AP), and invasive hyphae (72 hpi), which were within infected plant leaves (IP). Gene expression data, obtained from quantitative RT-PCR analysis, were normalized by using β-tubulin as an internal control and calibrated against the transcript abundances of VH stage.

Mentions: In Saccharomyces cerevisiae, the SNF1 kinase complex consists of three subunits, the catalytic subunit Snf1, the γ-subunit Snf4, and one of the three β-subunit isoforms, Gal83, Sip1, or Sip2 [4]. There are three upstream Snf1-activating kinases (Sak1, Elm1, and Tos3), each of which is sufficient to activate the SNF1 complex. MoSnf1 (MGG_00803), the catalytic subunit of SNF1 complex in M. oryzae, had been identified and proved functionally homologous to S. cerevisiae Snf1 [23]. In this study, we additionally sought for other M. oryzae orthologs involved in SNF1 pathway to obtain a further understanding of its function. Using protein sequences of S. cerevisiae counterparts for BLASTP searches, we identified only one β subunit MoSip2 (MGG_06930), the γ subunit MoSnf4 (MGG_04005), and two upstream kinases, MoSak1 (MGG_07003) and MoTos3 (MGG_06421) in the M. oryzae genome (http://www.broadinstitute.org/annotation/genome/magnaporthe_comparative/MultiHome.html), named after the best match (Figure 1A and Table S1). Domains identified by the InterPro database (http://www.ebi.ac.uk/interpro/) of these M. oryzae proteins exhibited high conservation (Table S1), including GBD (glycogen-binding domain) and ID (kinase interaction domain) 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.


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)

Protein interaction and gene expression analyses of SNF1 kinase complex components and its activating kinases in M. oryzae.(A) Different composition of the heterotrimeric SNF1 kinase complex and upstream kinases between S. cerevisiae and M. oryzae. (B) MoSnf1, MoSip2, and MoSnf4 interacted with each other, while no interaction was observed between MoSnf1 and its activating kinases in yeast two-hybrid assay. Yeast transformants expressing MoSnf1 plus MoSip2, MoSnf1 plus MoSnf4, MoSip2 plus MoSnf4, MoSnf1 plus MoSak1, or MoSnf1 plus MoTos3 were 10-fold serially diluted with a starter culture of 106 cells/ml and then spotted (5 µl) onto SD-Trp-Leu-His-Ade medium. (C) Gene expression profiles of MoSNF1, MoSIP2, MoSNF4, MoSAK1, and MoTOS3 among different developmental stages. Tested fungal tissues included vegetative hyphae (VH), conidia (CO), appressoria 8 hpi (AP), and invasive hyphae (72 hpi), which were within infected plant leaves (IP). Gene expression data, obtained from quantitative RT-PCR analysis, were normalized by using β-tubulin as an internal control and calibrated against the transcript abundances of VH stage.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103124-g001: Protein interaction and gene expression analyses of SNF1 kinase complex components and its activating kinases in M. oryzae.(A) Different composition of the heterotrimeric SNF1 kinase complex and upstream kinases between S. cerevisiae and M. oryzae. (B) MoSnf1, MoSip2, and MoSnf4 interacted with each other, while no interaction was observed between MoSnf1 and its activating kinases in yeast two-hybrid assay. Yeast transformants expressing MoSnf1 plus MoSip2, MoSnf1 plus MoSnf4, MoSip2 plus MoSnf4, MoSnf1 plus MoSak1, or MoSnf1 plus MoTos3 were 10-fold serially diluted with a starter culture of 106 cells/ml and then spotted (5 µl) onto SD-Trp-Leu-His-Ade medium. (C) Gene expression profiles of MoSNF1, MoSIP2, MoSNF4, MoSAK1, and MoTOS3 among different developmental stages. Tested fungal tissues included vegetative hyphae (VH), conidia (CO), appressoria 8 hpi (AP), and invasive hyphae (72 hpi), which were within infected plant leaves (IP). Gene expression data, obtained from quantitative RT-PCR analysis, were normalized by using β-tubulin as an internal control and calibrated against the transcript abundances of VH stage.
Mentions: In Saccharomyces cerevisiae, the SNF1 kinase complex consists of three subunits, the catalytic subunit Snf1, the γ-subunit Snf4, and one of the three β-subunit isoforms, Gal83, Sip1, or Sip2 [4]. There are three upstream Snf1-activating kinases (Sak1, Elm1, and Tos3), each of which is sufficient to activate the SNF1 complex. MoSnf1 (MGG_00803), the catalytic subunit of SNF1 complex in M. oryzae, had been identified and proved functionally homologous to S. cerevisiae Snf1 [23]. In this study, we additionally sought for other M. oryzae orthologs involved in SNF1 pathway to obtain a further understanding of its function. Using protein sequences of S. cerevisiae counterparts for BLASTP searches, we identified only one β subunit MoSip2 (MGG_06930), the γ subunit MoSnf4 (MGG_04005), and two upstream kinases, MoSak1 (MGG_07003) and MoTos3 (MGG_06421) in the M. oryzae genome (http://www.broadinstitute.org/annotation/genome/magnaporthe_comparative/MultiHome.html), named after the best match (Figure 1A and Table S1). Domains identified by the InterPro database (http://www.ebi.ac.uk/interpro/) of these M. oryzae proteins exhibited high conservation (Table S1), including GBD (glycogen-binding domain) and ID (kinase interaction domain) 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.

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