Elsevier

Journal of Proteomics

Volume 212, 10 February 2020, 103580
Journal of Proteomics

Comparative quantitative proteomics of osmotic signal transduction mutants in Botrytis cinerea explain mutant phenotypes and highlight interaction with cAMP and Ca2+ signalling pathways

https://doi.org/10.1016/j.jprot.2019.103580Get rights and content

Highlights

  • Comparative proteomics of two kinase mutants in the fungus Botrytis cinerea.

  • 628 differential proteins indicate independent & shared regulatory functions.

  • Changes in proteins related to plant infection correlate with virulence defects.

  • New link between hyper-osmolarity MAPK, cAMP and Ca2+ signalling.

Summary

Signal transduction (ST) is essential for rapid adaptive responses to changing environmental conditions. It acts through rapid post-translational modifications of signalling proteins and downstream effectors that regulate the activity and/or subcellular localisation of target proteins, or the expression of downstream genes. We have performed a quantitative, comparative proteomics study of ST mutants in the phytopathogenic fungus Botrytis cinerea during axenic growth under non-stressed conditions to decipher the roles of two kinases of the hyper-osmolarity pathway in B. cinerea physiology. We studied the mutants of the sensor histidine kinase Bos1 and of the MAP kinase Sak1.

Label-free shotgun proteomics detected 2425 proteins, 628 differentially abundant between mutants and wild-type, 270 common to both mutants, indicating independent and shared regulatory functions for both kinases. Gene ontology analysis showed significant changes in functional categories that may explain in vitro growth and virulence defects of both mutants (secondary metabolism enzymes, lytic enzymes, proteins linked to osmotic, oxidative and cell wall stress). The proteome data also highlight a new link between Sak1 MAPK, cAMP and Ca2+ signalling.

This study reveals the potential of proteomic analyses of signal transduction mutants to decipher their biological functions.

Text-Vulgarisation

The fungus Botrytis cinerea is responsible for grey mold disease of hundreds of plant species. During infection, the fungus has to face important changes of its environment. Adaptation to these changing environmental conditions involves proteins of such called signal transduction pathways that regulate the production, activity or localisation of cellular components, mainly proteins. While the components of such signal transduction pathways are well known, their role globally understood, the precise impact on protein production remains unknown.

In this study we have analysed and compared the global protein content of two Botrytis cinerea signal transduction mutants - both avirulent – to the pathogenic parental strain. The data of 628 differential proteins between mutants and wild-type, showed significant changes in proteins related to plant infection (secondary metabolism enzymes, lytic enzymes, proteins linked to osmotic, oxidative and cell wall stress) that may explain the virulence defects of both mutants. Moreover, we observed intracellular accumulation of secreted proteins in one of the mutants suggesting a potential secretion defect.

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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      Histidine kinase (bos1) and MAPK (sak1) participate in signal transduction pathways for fungal oxidative stress response (Liu et al., 2008) (Figure 3). In B. cinerea, mutants of bos1 and sak1 have low levels of mannitol dehydrogenase, BcSOD, CAT7, BcTRX, PRX1, and PRX9, which are necessary for intracellular ROS neutralization (Kilani et al., 2020). Upon silencing of the stress-associated MAPK PsMPK7, Phytophthora sojae exhibits increased sensitivity to H2O2 and accumulates a higher level ROS at the infection site, similar to Δvdpbs2 mutants of Verticillium dahliae (Gao et al., 2015; Tian et al., 2016) (Figure 3).

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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]

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