Elsevier

Journal of Plant Physiology

Volume 182, 15 June 2015, Pages 79-94
Journal of Plant Physiology

Molecular Biology
Comparative proteomic analysis in pea treated with microbial consortia of beneficial microbes reveals changes in the protein network to enhance resistance against Sclerotinia sclerotiorum

https://doi.org/10.1016/j.jplph.2015.05.004Get rights and content

Abstract

Microbial consortia may provide protection against pathogenic ingress via enhancing plant defense responses. Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 were used either singly or in consortia in the pea rhizosphere to observe proteome level changes upon Sclerotinia sclerotiorum challenge. Thirty proteins were found to increase or decrease differentially in 2-DE gels of pea leaves, out of which 25 were identified by MALDI-TOF MS or MS/MS. These proteins were classified into several functional categories including photosynthesis, respiration, phenylpropanoid metabolism, protein synthesis, stress regulation, carbohydrate and nitrogen metabolism and disease/defense-related processes. The respective homologue of each protein identified was trapped in Pisum sativum and a phylogenetic tree was constructed to check the ancestry. The proteomic view of the defense response to S. sclerotiorum in pea, in the presence of beneficial microbes, highlights the enhanced protection that can be provided by these microbes in challenged plants.

Introduction

The molecular mechanisms involved in plant response to biotic stress are of fundamental importance to plant sciences. Knowledge about these mechanisms is critical for improving stress tolerance in crops. Altered gene expression during pathogenic ingress causes up- and/or down-regulation of large number of early responsive proteins involved in signal transduction, in pathways maintaining homeostasis, inducing resistance, regulating metabolic pathways and protein processing. These proteins can be powerful tools in monitoring biotic stress as they can provide valuable information about the host physiological state.

In plant–pathogen interactions, extracellular/apoplastic proteins generally play important roles in disease suppression (Yang et al., 2011). An increase in extracellular proteins involved in the reduction of bacterial multiplication in leaves was observed in an incompatible interactions between resistant rice cultivars and the vascular pathogen Xanthomonas oryzae pv. oryza (Guo et al., 1993). Similarly, in another report, analysis of the grape tissues affected by herbicides also showed differential expression of photosynthesis-related and pathogenesis-related proteins from chemically stressed tissues, suggesting the stimulation of plant defense system in alteration of the carbon flux due to impaired photosynthesis and an increased need for osmotic adjustment in affected tissues (Castro et al., 2005). Along with this, if the proteome level changes induced by biocontrol agents (BCAs) (either singly or in consortium mode) are also identified, one can better understand their underlying mode of action. The type of the proteins over- and/or under-expressed may provide the actual cellular response generated by the incoming pathogen and the BCAs present in the rhizosphere. Also, using microbes in consortium may enhance the effectiveness in managing the upcoming biotic stress (Jain et al., 2013a), by providing augmented defense responses.

Earlier studies based on transcriptome analysis have shown induction of defense responses in Arabidopsis by plant growth-promoting microorganisms like Pseudomonas putida (Ahn et al., 2007), or with Bradyrhizobium sp. (Cartieaux et al., 2008), or with Trichoderma asperellum (Segarra et al., 2009). These studies have reported expression of a large number of ISR involved genes after pathogen infection. Proteome studies have further revealed that most of the proteins showing differential response have essential functions either in the regulation of the response (Dóczi et al., 2007, Jones et al., 2006) or in stress adaptation and management process (Delauré et al., 2008, Xiong et al., 2007). Thus, it is expected that proteomic approaches will therefore be helpful in providing useful information to study the protein expression, function and interactions, while characterizing the physiological processes induced under biotic stress (Speicher, 2002). This would also help to identify the role of post-translational modifications of proteins in the development of cellular stress.

In our previous reports, we evaluated the efficacy of three BCAs, viz. isolates of Trichoderma harzianum ARS culture collection number NRRL 30596, Bacillus subtilis JN099686 and Pseudomonas aeruginosa JN099685 in a microbial consortium for management of Sclerotinia rot of pea under greenhouse conditions and found decrease in plant mortality, increase in defense-related, antioxidant enzymes and suppression of oxalic acid-induced cell death and increased plant growth (Jain et al., 2012, Jain et al., 2013b, Jain et al., 2014a, Jain et al., 2015). These microbes in consortium also enhanced nutritional quality of pea seeds and pericarp (Jain et al., 2014b). Pathogenic ingress can be linked with signal perception, transduction and involvement of cellular and morphological response network to combat the stress. This may involve accumulation of certain metabolites and may lead to increase or decrease in certain proteins. However, no such report is available on Sclerotinia sclerotiorum stress-responsive proteins using molecular mechanisms. The present study was thus carried out with the aim of evaluating proteome level changes induced by the rhizosphere colonizing beneficial microbes either singly and/or in consortium in providing resistance against S. sclerotiorum.

Section snippets

Inoculum preparation

Pseudomonas aeruginosa PJHU15 (GenBank accession: JN099685) and Bacillus subtilis BHHU100 (GenBank accession: JN099686), the two bacterial isolates used in the present study, were isolated from rhizosphere of Pisum sativum (Jaipur and Hyderabad, respectively), as described in our previous study (Jain et al., 2012). Trichoderma isolate TNHU27 was isolated from an agricultural farm (Pantnagar) and was previously identified as Trichoderma harzianum (ATCC No. PTA-3701). These three microbial

Results

The representative gels of control pathogen unchallenged, control pathogen challenged and BCA treated (either singly or in the form of consortium) pathogen challenged are shown in Fig. 1, Fig. 2. More than 600 spots were detected using PD Quest software. Comparative proteomic profiles showed 30 reproducibly significant spots with intensities >1.5-fold up- or down-regulation in maximum of the treatments. All the protein spots were excised from the gels and subjected to MALDI-TOF MS or MS/MS

Discussion

The interaction between primary and secondary metabolic pathways is still poorly understood in aspects of plant–microbe interaction. It is a well-established fact that the plants treated with beneficial microorganisms showed increase in their primary metabolism upon pathogenic interaction, which may be possibly to allocate more resources to plants for defending the pathogenic challenge. This response allows the energy reallocation, with enhancement of photosynthesis in comparison to untreated

Acknowledgements

Akansha Jain is grateful to Department of Science and Technology, Govt. of India, New Delhi, for financial assistance under AORC scheme as INSPIRE-SRF. The authors are also thankful to Dr. C.S. Nautiyal, Director at CSIR-National Botanical Research Institute, Lucknow, India, for providing MALDI TOF analysis facility.

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      Proteins are structural and functional components in all living cells. Studies at proteome level can ideally provide information about functional molecules in the cell and changes induced in expression patterns of proteins in various states and under different stresses (Rakwal and Agrawal, 2003; Jain et al., 2015). Proteomics help us to identify not only the gene products with pivotal functions as pathogenic and virulence factors in the pathogens but also the ones with a decisive role in defense and stress regulation in plants (Fernández and Novo, 2011).

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