Comparative quantitative proteomics of osmotic signal transduction mutants in Botrytis cinerea explain mutant phenotypes and highlight interaction with cAMP and Ca2+ signalling pathways
Graphical abstract
Introduction
Botrytis cinerea is a necrotrophic, polyphageous plant pathogenic fungus responsible for grey mold disease of nearly 600 plant genera [1], many of which have significant agronomic importance. B. cinerea can infect different plant organs such as leaves, flowers or fruits and causes extensive pre- and post-harvest damage leading to yield losses. The resulting economic impact may then be considerable [2,3].
The infection process of B. cinerea involves the secretion of lytic enzymes, production of toxins, reactive oxygen species and small RNAs [4]. In return, B. cinerea has to face to many stresses caused by its environment and in particular by the infected plant such as osmotic, oxidative and cell wall stress, phytoalexins and other plant defence compounds [5]. Response and adaptation to these stresses is therefore crucial for the survival of the fungus. To be able to persist in this hostile environment and to counter the plant defence, the fungus develops rapid adaptive responses. It perceives these stresses and transmits them to the cell in order to give an adequate response via signalling pathways [6].
One of the main signalling pathways in eukaryotes involves MAPKs (Mitogen Activated Protein Kinase), evolutionary conserved signalling pathways. Depending on their phosphorylation status, the activated MAPKs act by regulating numerous cellular processes, notably by activating transcription factors [7]. MAPK cascades are preserved in ascomycetes, but their roles differ according to the fungal species [8]. In Saccharomyces cerevisiae, five MAPK pathways have been described, controlled respectively by Fus3, Kss1, Slt2, Hog1 and Smk1 [9]. The functionally redundant MAPKs Fus3 and Kss1 are involved in sexual and asexual reproduction. Slt2 regulates cell cycle progression and cell wall integrity, while Hog1 regulates the response to osmotic and oxidative stresses. The less studied MAPK Smk1 is involved in the assembly of the ascospore wall. Some MAPK pathways have partially overlapping functions or are interconnected [10]. With the exception of Smk1, homologues of these MAPKs are found in filamentous fungi [8]. Thus, the B. cinerea MAPKs Bmp1, Bmp3 and Sak1 are respectively the homologues of the yeast proteins Fus3/Kss1, Slt2 and Hog1 (reviewed in [6]).
In B. cinerea, the MAPK Bmp1 is involved in vegetative growth, hydrophobic surface perception, pathogenicity, spore and sclerotium formation [[11], [12], [13]]. The MAPK Bmp3 is involved in adaptation to hypo-osmotic and oxidative stress and to the fungicide fludioxonil [14].
The Sak1 MAPK pathway controls fungal development such as growth and macroconidium formation. It is involved in the adaptation to various stresses such as osmotic (ionic), oxidative and cell wall stress but also in infection [15,16]. Indeed, the ∆sak1 mutant is unable to form infection structures and thus to penetrate its host unless the latter is injured [15]. Cross-talk between the Bmp3 and Sak1 pathways has been detected during oxidative stress [16].
Sak1 targets transcription factors such as BcReg1 and BcAtf1 [17,18]. BcReg1 is involved in the regulation of secondary metabolite synthesis, sporulation and host tissue colonization [17] while BcAtf1 is involved in sporulation and the regulation of genes involved in stress response [18].
Upstream of the osmoregulatory MAPK pathway, the perception and transmission of signals pass through a network of signalling proteins referred to as two-component systems. In fungi, the histidine kinase sensor is a hybrid protein (HHK) that contains both a sensor domain and a response regulatory (RR) domain [19]. Thus, following the perception of a signal, the histidine kinase catalyses its autophosphorylation on the conserved histidine residue, then the transfer of the phosphate group to a conserved aspartate residue of the RR domain. The signal is then transmitted via a phosphotransfer protein (PTH) to a RR protein [19]. In B. cinerea, there are two histidine kinases upstream of the Sak1 pathway, Bos1 and BcHK5. The class VI histidine kinase, BcHK5, is the homologue of the unique HHK in S. cerevisiae Sln1. Indispensable in yeast because of its role in regulating stress response, this histidine kinase is not essential for development and pathogenicity in B. cinerea (reviewed in [6]). In several fungi, the deletion of class III histidine kinase leads to resistance to phenylpyrroles and dicarboximides but also to sensitivity to osmotic, oxidative and cell wall stresses [[20], [21], [22]]. This holds true also in B. cinerea [23]. An original finding was the the constitutive phosphorylation of the MAPK Sak1 in the ∆bos1 mutant, indicating that the two-component system negatively controls the activation of the osmoregulatory pathway [24].
Heller et al. [25] conducted a small scale transcriptomic analysis of the B. cinerea ∆sak1 mutant compared to the wild type. Albeit performed on a limited set of genes, this macroarray analysis revealed Sak1 as central element in the overall response to stress and in the infectious process at the transcriptional level.
Another level in systematic analysis is achieved through proteomics. The analysis of the accumulation of intracellular proteins takes into account all regulations during protein synthesis (i.e., transcription and translation) and degradation (mRNA and protein turn-over) [reviewed in [26]]. Although shotgun proteomics have in principle democratized global proteome analyses, such as subcellular proteomics, dynamic proteomic changes and post-translational modification, the required technical equipment and know-how or access to proteomic platforms, as well as costs of analysis are still important limiting factors orientating functional genomics in favour of transcriptomics [27]. Proteomic analyses in Botrytis cinerea have mainly focused on sub-proteomes (e.g., secreted or membrane proteins) or phosphoproteomes [[28], [29], [30], [31], [32], [33], [34], [35], [36], [37]].
In this study, we analysed the role of the osmotic ST pathway in the biology of B. cinerea and particularly the role of the histidine kinase Bos1 and the MAPK Sak1. In order to highlight proteins whose abundance depends on the osmoregulatory pathway, we performed a comparative proteomic analysis of the ∆sak1 and ∆bos1 mutants compared to the wild type strain during exponential growth under axenic growth conditions.
Protein abundance variations corroborated the implication of both protein kinases in biological functions related to the infection process. Moreover, this study highlighted cross-talk between the Sak1 and the cAMP pathway which, on its turn, is involved in pathogenicity as well. Finally, our study revealed different sets of regulation patterns among the detected proteins.
Section snippets
Strains, medium and culture conditions
B. cinerea wild-type strain, B05.10 [38], as well as the mutants ∆sak1 and ∆bos1 [15,24] were cultivated on solid Sisler medium (KH2PO4 2 g l−1, K2HPO4 1.5 g l−1, (NH4)2SO4 1 g l−1, MgSO4 7H2O 0.5 g l−1, glucose 10 g l−1, yeast extract 2 g l−1, agar 15 g l−1).
Pre-cultures were made from calibrated explants (5 mm) deposited on plate with solid Sisler medium previously covered with a sterile cellophane membrane (Bio-Rad, USA). The cultures were maintained 4 days at 20 °C, in darkness. The
Quantitative proteomic analysis of osmotic signal transduction mutants
∆bos1and ∆sak1 mutants' protein contents were compared to the parental wild-type strain B05.10 exponentially grown under axenic in vitro conditions. After tryptic digestion and peptide analysis by LC-MS/MS, protein identification and quantification were performed. 67% of the detected spectra could be assigned to peptides. 2425 proteins out of the 11,701 predicted B. cinerea proteins (20.7% of the theoretical proteome) were detected in the total dataset. XIC analysis allowed to obtain
Comparative proteomics of signal transduction mutants in Botrytis cinerea
In this work, we compared the steady-state protein levels of protein kinase mutants involved in osmosensing in the plant pathogenic fungus Bortytis cinerea. Analysing three different strains grown under axenic conditions led to the identification of roughly 1/5 of the predicted B. cinerea proteome. The abundance of 628 proteins (26% of the detected proteins) differed significantly in either or both mutants (>400 proteins per genotype). These results highlight the strong impact of protein kinase
Funding
JK's doctoral fellowship was supported by University Paris-Sud/University Paris-Saclay. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors are grateful to region “Ile-de-France” and LABEX Saclay Plant Science (SPS) for financial support of proteomics equipment.
Acknowledgements
JK's doctoral fellowship was supported by University Paris-Sud/University Paris-Saclay. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors are grateful to region “Ile-de-France” and LABEX Saclay Plant Science (SPS) for financial support of proteomics equipment. They thank Adeline Simon for helping with enrichment studies.
Author contributions
JK designed and performed all experiments, assisted by MD for the proteomics workflow. MZ performed proteomic data analysis and corresponding statistics. AS performed in silico cluster analysis and functional enrichment studies. SF and MZ conceived the initial project. All authors participated to manuscript writing under the coordination of SF.
Declaration of Competing Interest
The authors disclose any conflict of interest.
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Present address: Jaafar Kilani, EA3142 GEIHP, Institut de Biologie en Santé, C.H.U. d'Angers, F-49933 ANGERS, France [email protected]