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Overlapping Podospora anserina Transcriptional Responses to Bacterial and Fungal Non Self Indicate a Multilayered Innate Immune Response.

Lamacchia M, Dyrka W, Breton A, Saupe SJ, Paoletti M - Front Microbiol (2016)

Bottom Line: Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI.Most functions encoded in response to bacteria favor survival of the fungus while most functions up regulated during VI would lead to cell death.These differences are discussed in the frame of a multilayered response to non self in fungi.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie et Génétique Cellulaire, UMR 5095, Centre National de la Recherche Scientifique et Université de Bordeaux Bordeaux, France.

ABSTRACT
Recognition and response to non self is essential to development and survival of all organisms. It can occur between individuals of the same species or between different organisms. Fungi are established models for conspecific non self recognition in the form of vegetative incompatibility (VI), a genetically controlled process initiating a programmed cell death (PCD) leading to the rejection of a fusion cell between genetically different isolates of the same species. In Podospora anserina VI is controlled by members of the hnwd gene family encoding for proteins analogous to NOD Like Receptors (NLR) immune receptors in eukaryotes. It was hypothesized that the hnwd controlled VI reaction was derived from the fungal innate immune response. Here we analyze the P. anserina transcriptional responses to two bacterial species, Serratia fonticola to which P. anserina survives and S. marcescens to which P. anserina succumbs, and compare these to the transcriptional response induced under VI conditions. Transcriptional responses to both bacteria largely overlap, however the number of genes regulated and magnitude of regulation is more important when P. anserina survives. Transcriptional responses to bacteria also overlap with the VI reaction for both up or down regulated gene sets. Genes up regulated tend to be clustered in the genome, and display limited phylogenetic distribution. In all three responses we observed genes related to autophagy to be up-regulated. Autophagy contributes to the fungal survival in all three conditions. Genes encoding for secondary metabolites and histidine kinase signaling are also up regulated in all three conditions. Transcriptional responses also display differences. Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI. Most functions encoded in response to bacteria favor survival of the fungus while most functions up regulated during VI would lead to cell death. These differences are discussed in the frame of a multilayered response to non self in fungi.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization of the interaction between P. anserina and S. fonticola or S. marcescens. (A) Confrontation assay against S. fonticola and S. marcescens. Fungal growth toward the bacterial colonies stops before contact is made. Fungal colony edge appears linear in confrontation to S. fonticola and is altered in confrontation to S. marcescens. The white line delineates the fungal colony edge. (B) Fungal cell morphology appears altered on the side of the bacterial colony, especially at apices, with cells swelling in confrontation with both bacterial species, here S. marcescens. (C) Cell death is observed by Evans Blue staining in confrontation with both bacteria. (D) Cell morphology and (E) Evans Blue staining in absence of bacteria. (F–K) Large vacuoles (F,H,J) and expression of IDI1-GFP (G,I,K) are observed in presences S. fonticola(F,G) or S. marcescens(H,I), or during the VI reaction (J,K) but not in cells growing away from the bacterial colony (L,M). Scale bars = 10 μm. The same induction of expression and localization is observed for Pa-GFP-ATG8 and IDI2-GFP proteins (not shown). (N) In the transfer assay, bacteria and P. anserina (on cellophane stripes) are grown for 48 h on separate plates, and the fungus is then transferred onto the bacteria seeded plates, setting the initial time point of the reaction. (O) Estimation of the fungal cell death level after transfer to S. fonticola or S. marcescens seeded plates.
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Figure 1: Phenotypic characterization of the interaction between P. anserina and S. fonticola or S. marcescens. (A) Confrontation assay against S. fonticola and S. marcescens. Fungal growth toward the bacterial colonies stops before contact is made. Fungal colony edge appears linear in confrontation to S. fonticola and is altered in confrontation to S. marcescens. The white line delineates the fungal colony edge. (B) Fungal cell morphology appears altered on the side of the bacterial colony, especially at apices, with cells swelling in confrontation with both bacterial species, here S. marcescens. (C) Cell death is observed by Evans Blue staining in confrontation with both bacteria. (D) Cell morphology and (E) Evans Blue staining in absence of bacteria. (F–K) Large vacuoles (F,H,J) and expression of IDI1-GFP (G,I,K) are observed in presences S. fonticola(F,G) or S. marcescens(H,I), or during the VI reaction (J,K) but not in cells growing away from the bacterial colony (L,M). Scale bars = 10 μm. The same induction of expression and localization is observed for Pa-GFP-ATG8 and IDI2-GFP proteins (not shown). (N) In the transfer assay, bacteria and P. anserina (on cellophane stripes) are grown for 48 h on separate plates, and the fungus is then transferred onto the bacteria seeded plates, setting the initial time point of the reaction. (O) Estimation of the fungal cell death level after transfer to S. fonticola or S. marcescens seeded plates.

Mentions: In a confrontation assay against S. marcescens or S. fonticola, P. anserina grew normally away from the bacterial colony, but growth toward the bacterial colony soon stopped before contact was made. Where the edge of the fungal colony appears almost linear in confrontation to S. fonticola, it appears altered in confrontation to S. marcescens (Figure 1A). After 2–3 days growth resumed in the confrontation to S. fonticola and the fungal colony eventually covered the bacterial colony. In the confrontation to S. marcescens growth did not resume. Fungal growth arrest in confrontation with Serratia species has already been reported (Li et al., 2015). Fungal cell morphology was altered in confrontation to both bacteria, with an intense vacuolization, apical cell swelling, and occasional cell death (Figures 1B–E). These phenotypes appeared more pronounced in confrontation to S. marcescens. We also observed the induction of autophagy as indicated by the vacuolar localization of Pa-GFP-ATG8, along with the expression of IDI1-GFP and IDI-2-GFP, two small secreted proteins induced during VI and believed to act as defensins (Figures 1F–K; Bourges et al., 1998; Dementhon et al., 2003). During VI IDI1-GFP and IDI2-GFP are localized to the membrane, while in response to bacteria they appear vacuolar, which could be a consequence of bacterial toxins altering the P. anserina secretory pathway (Guichard et al., 2014). These phenotypes decrease in intensity as observations are made further away from the colony edge, and are not observed in fungal cells growing opposite from the bacterial colony (Figures 1L,M).


Overlapping Podospora anserina Transcriptional Responses to Bacterial and Fungal Non Self Indicate a Multilayered Innate Immune Response.

Lamacchia M, Dyrka W, Breton A, Saupe SJ, Paoletti M - Front Microbiol (2016)

Phenotypic characterization of the interaction between P. anserina and S. fonticola or S. marcescens. (A) Confrontation assay against S. fonticola and S. marcescens. Fungal growth toward the bacterial colonies stops before contact is made. Fungal colony edge appears linear in confrontation to S. fonticola and is altered in confrontation to S. marcescens. The white line delineates the fungal colony edge. (B) Fungal cell morphology appears altered on the side of the bacterial colony, especially at apices, with cells swelling in confrontation with both bacterial species, here S. marcescens. (C) Cell death is observed by Evans Blue staining in confrontation with both bacteria. (D) Cell morphology and (E) Evans Blue staining in absence of bacteria. (F–K) Large vacuoles (F,H,J) and expression of IDI1-GFP (G,I,K) are observed in presences S. fonticola(F,G) or S. marcescens(H,I), or during the VI reaction (J,K) but not in cells growing away from the bacterial colony (L,M). Scale bars = 10 μm. The same induction of expression and localization is observed for Pa-GFP-ATG8 and IDI2-GFP proteins (not shown). (N) In the transfer assay, bacteria and P. anserina (on cellophane stripes) are grown for 48 h on separate plates, and the fungus is then transferred onto the bacteria seeded plates, setting the initial time point of the reaction. (O) Estimation of the fungal cell death level after transfer to S. fonticola or S. marcescens seeded plates.
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Related In: Results  -  Collection

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Figure 1: Phenotypic characterization of the interaction between P. anserina and S. fonticola or S. marcescens. (A) Confrontation assay against S. fonticola and S. marcescens. Fungal growth toward the bacterial colonies stops before contact is made. Fungal colony edge appears linear in confrontation to S. fonticola and is altered in confrontation to S. marcescens. The white line delineates the fungal colony edge. (B) Fungal cell morphology appears altered on the side of the bacterial colony, especially at apices, with cells swelling in confrontation with both bacterial species, here S. marcescens. (C) Cell death is observed by Evans Blue staining in confrontation with both bacteria. (D) Cell morphology and (E) Evans Blue staining in absence of bacteria. (F–K) Large vacuoles (F,H,J) and expression of IDI1-GFP (G,I,K) are observed in presences S. fonticola(F,G) or S. marcescens(H,I), or during the VI reaction (J,K) but not in cells growing away from the bacterial colony (L,M). Scale bars = 10 μm. The same induction of expression and localization is observed for Pa-GFP-ATG8 and IDI2-GFP proteins (not shown). (N) In the transfer assay, bacteria and P. anserina (on cellophane stripes) are grown for 48 h on separate plates, and the fungus is then transferred onto the bacteria seeded plates, setting the initial time point of the reaction. (O) Estimation of the fungal cell death level after transfer to S. fonticola or S. marcescens seeded plates.
Mentions: In a confrontation assay against S. marcescens or S. fonticola, P. anserina grew normally away from the bacterial colony, but growth toward the bacterial colony soon stopped before contact was made. Where the edge of the fungal colony appears almost linear in confrontation to S. fonticola, it appears altered in confrontation to S. marcescens (Figure 1A). After 2–3 days growth resumed in the confrontation to S. fonticola and the fungal colony eventually covered the bacterial colony. In the confrontation to S. marcescens growth did not resume. Fungal growth arrest in confrontation with Serratia species has already been reported (Li et al., 2015). Fungal cell morphology was altered in confrontation to both bacteria, with an intense vacuolization, apical cell swelling, and occasional cell death (Figures 1B–E). These phenotypes appeared more pronounced in confrontation to S. marcescens. We also observed the induction of autophagy as indicated by the vacuolar localization of Pa-GFP-ATG8, along with the expression of IDI1-GFP and IDI-2-GFP, two small secreted proteins induced during VI and believed to act as defensins (Figures 1F–K; Bourges et al., 1998; Dementhon et al., 2003). During VI IDI1-GFP and IDI2-GFP are localized to the membrane, while in response to bacteria they appear vacuolar, which could be a consequence of bacterial toxins altering the P. anserina secretory pathway (Guichard et al., 2014). These phenotypes decrease in intensity as observations are made further away from the colony edge, and are not observed in fungal cells growing opposite from the bacterial colony (Figures 1L,M).

Bottom Line: Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI.Most functions encoded in response to bacteria favor survival of the fungus while most functions up regulated during VI would lead to cell death.These differences are discussed in the frame of a multilayered response to non self in fungi.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie et Génétique Cellulaire, UMR 5095, Centre National de la Recherche Scientifique et Université de Bordeaux Bordeaux, France.

ABSTRACT
Recognition and response to non self is essential to development and survival of all organisms. It can occur between individuals of the same species or between different organisms. Fungi are established models for conspecific non self recognition in the form of vegetative incompatibility (VI), a genetically controlled process initiating a programmed cell death (PCD) leading to the rejection of a fusion cell between genetically different isolates of the same species. In Podospora anserina VI is controlled by members of the hnwd gene family encoding for proteins analogous to NOD Like Receptors (NLR) immune receptors in eukaryotes. It was hypothesized that the hnwd controlled VI reaction was derived from the fungal innate immune response. Here we analyze the P. anserina transcriptional responses to two bacterial species, Serratia fonticola to which P. anserina survives and S. marcescens to which P. anserina succumbs, and compare these to the transcriptional response induced under VI conditions. Transcriptional responses to both bacteria largely overlap, however the number of genes regulated and magnitude of regulation is more important when P. anserina survives. Transcriptional responses to bacteria also overlap with the VI reaction for both up or down regulated gene sets. Genes up regulated tend to be clustered in the genome, and display limited phylogenetic distribution. In all three responses we observed genes related to autophagy to be up-regulated. Autophagy contributes to the fungal survival in all three conditions. Genes encoding for secondary metabolites and histidine kinase signaling are also up regulated in all three conditions. Transcriptional responses also display differences. Genes involved in response to oxidative stress, or encoding small secreted proteins are essentially expressed in response to bacteria, while genes encoding NLR proteins are expressed during VI. Most functions encoded in response to bacteria favor survival of the fungus while most functions up regulated during VI would lead to cell death. These differences are discussed in the frame of a multilayered response to non self in fungi.

No MeSH data available.


Related in: MedlinePlus