Proteome analysis of induced systemic resistance medicated by plant growth promoting Rhizobacteria (PGPR) in rice for biotic stress

Abstract

Rice (Oryza sativa L.), is vulnerable to a number of pest and diseases. Among them sheath blight disease caused by Rhizoctonia solani, and insect pest, brown planthopper (BPH) (Nilapavata lugens) are the most devastating agents and major challenge to rice cultivation. Approximately 30 per cent yield loss has been reported due to above said disease and pest. In this context, the present study entitled „proteome analysis of induced systemic resistance mediated by plant growth promoting rhizobacteria (PGPR) in rice for biotic stress‟ was carry out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2011-2013, with the objective to identify and characterize the interacting proteome in inducing systemic resistance in rice mediated by PGPR during pathogen and insect attack. Plant growth-promoting rhizobacteria (PGPR) are associated with plant roots and augment plant productivity and immunity. However, recent work by several groups shows that PGPR elicit physical or chemical changes related to plant defense, a process referred as „induced systemic resistance‟ (ISR). ISR induced by PGPR has suppressed plant diseases caused by a range of pathogens in both greenhouse and field (Yang et al., 2009). Jyothi (PTB -39), a popular rice variety of Kerala and susceptible to blight and BPH was used as the experimental material. The pot culture experiment was carried out by direct sowing. The biometric observations were taken on control plants and the plants treated with Pseudomonas fluorescens Pf1 (KAU culture) 30 days after sowing. Significant increase was observed in shoot length, root length, number of tillers, fresh weight and dry weight in Pf primed rice plants. Both R. solani and BPH were inoculated forty five days after sowing, to check the efficacy of Pf strain against the pathogen and insect. Protein analysis was carried out to study the molecular mechanisms operating behind the PGPR mediated pest and disease resistance and growth promotion. Total Proteins were isolated from rice plants treated with and without Pf and challenged with pest and pathogen at 0, 6, 24, 48, 72, and 96 hours after inoculation (HAI). The protein samples were analyzed by SDS-PAGE gel system. The comparison analysis of relative abundances of protein bands between inoculated and non inoculated samples were carried out. At 24, 48, and 72h, 16 proteins were expressed in Pf treated and control plants challenged with R. solani. A twenty nine kDa protein was up regulated in all the Pf treated plants challenged with R. solani and this was selected for sequencing. In all the Pf treated and BPH challenged plants 3 different proteins of 19, 23 and 30 kDa were expressed. Two protein bands of 19 kDa from 48h and 30 kDa from 96h were selected for MALDI-TOF sequencing. These three proteins bands were sequenced by MS MALDI-TOF in Sandor Proteomics, Hyderabad. Protein band 1 of 29 kDa, showed, 98 per cent homology to chloroplastic aldolase, fructose-bisphosphate aldolase and 81 per cent homology to peroxidase. The protein band 2 of 19 kDa, showed 84 and 86 per cent homology to 2- cys peroxiredoxin bas and 2-cys peroxiredoxin bas1, respectively. The Protein band 3 of 30 kDa, showed 99 per cent homology to small subunit of ribulose-1, 5-bisphosphate carboxylase and 100 per cent similarity to hypothetical proteins Os12g0291400 and OsI_38046. Western blot analysis was also carried out to further confirm the presence of PR protein chitinase. The analysis confirmed the presence of chitinase of molecular weight 17 kDa, 18 kDa, and 35 kDa in PGPR primed plant challenged with R.solani. The PO assay was also carried out to check the activity of peroxidase enzyme in both control and PGPR primed plants challenged with BPH and pathogen. PO activity was increased from 6 to 96 h after challenge inoculation in Pf primed plants. There was 43.62 and 21.9 per cent increase in PO activity over control plants in pathogen inoculated and BPH challenged Pf primed plants at 96hours. The result obtained gave information for further elucidation of candidate genes operating in signal transduction pathways mediated by PGPR during ISR to biotic stress.