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