Unknown,Transcriptomics,Genomics,Proteomics

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The transcriptional response to the inactivation of the PaMpk1 and PaMpk2 MAP kinase pathways in Podospora anserina

ABSTRACT: To gain insight into the targets of the PaMpk1 and PaMpk2 MAPK, we here determine the transcription profile of the mutants inactivated for PaMpk1, PaMpk2 and PaNox1 during vegetative growth at stages where they are required for proper development, in comparison with wild type. Microarrays representing 10556 coding sequences (CDS) predicted by the P. anserina genome project were used to estimate the transcription profiles (transcriptomes) during vegetative growth of three-day-old homokaryotic cultures of M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and the IDC343 PaNox1 mutants in comparison to wild type. Three zones corresponding to three developmental stages can be defined in such wild-type culture. From the growing edge the colony, the first five milimeters (zone 1) are constituted of hyphae lacking pigment and growing on the substrate. In this zone, very few hyphae are aerial and appressorium-like structures are not differentiated. In the second intermediate zone (zone 2), profuse aerial hyphae and appressorium-like structures are differentiated. This zone is about 7 mm wide and corresponds to the area where most perithecia are differentiated during sexual development of heterokaryotic mycelia. The zone that is most distal from the edge lack aerial hyphae (zone 3) accumulates a larger amount of pigment and differentiates few perithecia during sexual reproduction. It is about 1 cm wide. These three zones are easily visualized by the naked eye for sample recovery. The M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and the IDC343 PaNox1 mutants lack a clear zonation, since the three genes are involved in the signalling of stationary phase . Materials from these mutants were nonetheless recovered as three samples corresponding to the same zones, based mostly on the distance from the growing edge but also from subtle morphological differences. With GPL10116 : reference design, 4 strains (WT, M-bM-^HM-^FPaMpk1, M-bM-^HM-^FPaMpk2 and IDC343 PaNox1 with 3 growth conditions (zone1,2 and 3) each with four biological replicates, the common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5

ORGANISM(S): Podospora anserina

SUBMITTER: Frederique Bidard

PROVIDER: E-GEOD-21331 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Analyzing culture supernatants of yeast and hyphal cells of Candida albicans by mass spectrometry, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome of this human pathogen. By sequence homology, we assigned three yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, in addition to Rbe1p and Rbt4p to a novel family of proteins. Correspondent with our secretome analysis RBE1 was expressed in blastospores and opaque cells, whereas transcription was down-regulated in hyphae. On the contrary, RBT4 was up-regulated in hyphae and down-regulated in opaque cells. Remarkably, transcription of RBT4 and RBE1 was each up-regulated in blastospores of M-bM-^HM-^Frbe1 or hyphae of M-bM-^HM-^Frbt4 deletion strains, respectively, indicating a compensatory function of both proteins. In a M-bM-^HM-^Frbe1/M-bM-^HM-^Frbt4 double deletion strain, genome-wide transcriptional analysis showed differential transcription of a limited set of genes that are also implicated in virulence and oxidative stress response. In this context, deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in the M-bM-^HM-^Frbe1/M-bM-^HM-^Frbt4 double deletion strain, where virulence was strongly affected. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leukocytes (neutrophils). Therfore, our data suggest that the crucial contribution of both C. albicans pathogenesis-related proteins for in vivo virulence results at least partially from reduced survival in phagocytes. Experiments were performed under blastospore (YPD) as well as hyphae (alpha-MEM)-inducing conditions. In total, three biological replicates were performed for each condition. All experiments were performed as dye swaps. Thus, in total six arrays have been hybridzed for each comparison (alpha-MEM and YPD, respectively). Hybridization experiments included a reference strain (C. albicans SC5314) and a double deletion strain (C. albicans MRC27). The array included one technical replicate of each probe.

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The transcriptional response to the inactivation of the PaMpk1 and PaMpk2 MAP kinase pathways in Podospora anserina

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