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Seedlings in Response to Pseudomonas Putida Stain FBKV2 Inoculation Under Drought Stress

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Seedlings in Response to Pseudomonas Putida Stain FBKV2 Inoculation Under Drought Stress Annals of Microbiology (2018) 68:331–349 https://doi.org/10.1007/s13213-018-1341-3 ORIGINAL ARTICLE Transcriptomic profiling of maize (Zea mays L.) seedlings in response to Pseudomonas putida stain FBKV2 inoculation under drought stress Ali SkZ1 & Sandhya Vardharajula1 & Sai Shiva Krishna Prasad Vurukonda2 Received: 29 November 2017 /Accepted: 26 April 2018 /Published online: 4 May 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018 Abstract Several mechanisms have been proposed for plant growth-promoting rhizobacteria (PGPR)-mediated drought stress tolerance in plants, but little is known about the molecular pathways involved in the drought tolerance promoted by PGPR. We, therefore, aim to study the differential gene response between Pseudomonas putida strain FBKV2 and maize interaction under drought stress using Illumina sequencing. RNA Seq libraries were generated from leaf tissue of maize seedlings with and without strain FBKV2 subjected to drought stress. The libraries were mapped with maize genome database for the identification of differentially expressed genes (DEGs). The expression studies confirmed the downregulation of ethylene biosynthesis (ET), abscisic acid (ABA) and auxin signaling, superoxide dismutase, catalase, and peroxidase in FBKV2-inoculated seedlings. On the other hand, genes involved in β-alanine and choline biosynthesis, heat shock proteins, and late embryogenesis abundant (LEA) proteins were upregulated, which could act as key elements in the drought tolerance conferred by P. putida strain FBKV2. Another remarkable expression was observed in genes encoding benzoxazinoid (BX) biosynthesis which act as the chemoattractant, which was further confirmed by gfp-labeled P.putida strain FBKV2 root colonization studies. Overall, these results indicate that secretion of BXs attracted P. putida strain FBKV2 resulted in root colonization and mediated drought tolerance by modulating metabolic, signaling, and stress-responsive genes. Keywords RNA Seq . Transcriptome . Drought tolerance . Maize . Pseudomonas putida Introduction Phytohormone producing Azospirillum sp. enhanced plant growth promotion and induced drought tolerance in wheat Plant growth-promoting rhizobacteria (PGPR) are the rhizo- (Arzanesh et al. 2011). 2R, 3R-butanediol producing sphere bacteria that can offer several benefits to host plants Pseudomonas chlororaphis O6 induced stomatal closure such as increasing the availability of nutrients, elimination of genes and reduced water loss in Arabidopsis thaliana (Cho deleterious pathogens, and tolerance to abiotic stresses, in- et al. 2008). Similarly, 1-aminocyclopane-1-carboxylate cluding drought stress. Several mechanisms have been pro- (ACC)-deaminase producing Achromobacter piechaudii posed for PGPR mediated drought stress tolerance in plants. ARV8 lowered ethylene levels and induced drought tolerance in tomato and pepper seedlings exposed to drought stress Electronic supplementary material The online version of this article (Mayak et al. 2004). PGPR P. putida strain GAP-P45 en- (https://doi.org/10.1007/s13213-018-1341-3) contains supplementary hanced plant biomass, relative water content, and leaf water material, which is available to authorized users. potential by the accumulation of proline in maize seedlings exposed to drought stress (Sandhya et al. 2010). * Ali SkZ Exopolysaccharides producing Rhizobium leguminosarum [email protected] (LR-0), Mesorhizobium ciceri (CR-30 and CR-39), and Rhizobium phaseoli (MR-2) showed beneficial interaction to 1 Department of Microbiology, Agri Biotech Foundation, PJTS wheat (non-legume) under drought stress (Hussain et al. Agricultural University Campus, Rajendranagar, 2014). Furthermore, Bacillus sp. enhanced drought tolerance Hyderabad, Telangana State 500030, India in maize seedlings by reducing the activity of the antioxidant 2 Department of Life Science, Science, Technology and Biotechnology enzymes such as ascorbate peroxidase (APX) and glutathione Agro-Food, University of Modena e Reggio Emilia, 41125 Modena, Emilia-Romagna, Italy peroxidase (GPX) (Vardharajula et al. 2011). 332 Ann Microbiol (2018) 68:331–349 Gene expression profiling has also been used to characterize a protein (gfp) gene under the control of lac promoter with variety of genes expressed under drought stress in plants inocu- gentamycin (Gm) resistance, was obtained from Plant lated with PGPR (Kandasamy et al. 2009; Yuwono et al. 2005). Microbe Interaction lab, Centre for Cellular and Molecular Increased mRNA transcripts of a drought-response gene, Biology (CCMB), Telangana State, India. The wild-type EARLY RESPONSE TO DEHYDRATION 15 (ERD15) in P. putida strain FBKV2 was grown in 5 mL of Luria Bertani Paenibacillus polymyxa B2-inoculated A. thaliana enhanced (LB) medium at 28 °C under continuous shaking (120 rpm) drought tolerance (Timmusk and Wagner 1999). Six differential- until the optical density of 0.6 at λ 600 nm. The cells were ly expressed stress proteins were identified in pepper plants in- harvested by centrifugation at 5000 rpm from 5 min, re- oculated with Bacillus licheniformis K11 under drought stress suspended in 5 mL of cold sterile water, and centrifuged at (Lim and Kim 2013). Stress-related genes APX1, SAMS1, and 5000 rpm for 5 min. The supernatant was discarded, and the HSP17.8 were induced in wheat inoculated with Bacillus pellet was washed twice (5000 rpm for 5 min) with 10% amyloliquefaciens 5113 and Azospirillum brasilense NO40 glycerol. Transformation was performed by electroporation (Kasim et al. 2013) respectively. Jasmonic acid marker genes, (Gene Pulser, Bio-Rad) in an electroporation cuvette salicylic acid-regulated genes, and the ethylene-response genes (0.2 cm) containing 100 μL of competent cells (P. putida were upregulated in A. thaliana colonized with P. chlororaphis strain FBKV2), plus 2 μL plasmid DNA (100 ng μL−1). The O6 (Cho et al. 2013). Recent studies with Illumina sequencing following pulse conditions were applied: 12.5 kV cm−1, (HiSeq 2000 system) revealed abscisic acid (ABA) signaling 25 μF, and 200 Ω (Krzyzanowska et al. 2012a, b;Choietal. genes in sugarcane cv. SP70-1143 inoculated with 2006). After transformation, 1 mL of LB medium was added; Gluconacetobacter diazotrophicus conferred drought tolerance the mixture was incubated for 1 h at 28 °C and plated on to LB (Vargas et al. 2014). agar medium supplemented with gentamycin (40 μgmL−1) Our previous work has shown that the potential of P.putida and incubated for 48 h at 28 °C. The identification and selec- strain FBKV2 and GAPP45 colonization-enhanced drought tion of clones carrying the gfp gene were carried out under UV tolerance in maize by the accumulation of cellular metabolites light. reduced stomatal conductance and antioxidant enzymes (Vurukonda et al. 2016; Vardharajula et al. 2011; Sandhya Plant material and growth conditions et al. 2010). Till date, little is known about the molecular pathways involved in the drought tolerance promoted by A pot experiment was conducted under greenhouse condition PGPR that colonize the maize. We, therefore, hypothesized to compare the efficacy of selected bacterial strain P. putida that rhizobacteria inoculation alter the expression of metabol- strain FBKV2 (gfp labeled) for promoting the growth of maize ic, signaling, and stress-protective genes in maize leaves. To under drought stress conditions. The soil used for pot experi- address this hypothesis and to advance our understanding of ments belongs to the Chalkas series and has been classified as maize response to drought stress with and without bacterial Bred earths with loamy subsoil^ in the Indian soil classifica- inoculation, we adopted RNA Seq transcriptome analysis tion system, which falls under the order Alfisols using Illumina deep sequencing of maize seedlings. (Bhattacharyya et al. 2007). The soil was collected from the homogeneous horizon (0–20 cm) of College Farm (Field soil), PJTSAU Campus, Rajendranagar, Hyderabad, India, a semi- Material and methods arid region under rain-fed production system. The soil was air- dried and sieved (<2 mm) before being analyzed for the phys- Bacterial strain icochemical properties. The soil contained 73.2% sand, 5.6% silt, 21.2% clay with 1.43 Mg m−3 bulk density, 2.52 Mg m−3 In our previous studies, Pseudomonas spp. strain FBKV2, particle density, 38.4% total porosity, and 39.2% water hold- isolated from eggplant (Solanum melongeana L.) rhizosphere, ing capacity; it had a pH of 6.8 with an electrical conductivity showed multiple PGP traits under both non-stress and drought of 0.13 dms; the soil contain Ca2+,Mg2+,K+,andNa+ in the stress conditions and enhanced drought tolerance in maize. ratio of 3.2, 2.1, 0.31, and 0.25 cmol (p+)/kg with CEC of The strain was identified as Pseudomonas putida by 16.2 cmol (p+)/kg. The organic C, total N, and total P content 16SrRNA sequence analysis, and the sequence was submitted of soil were 0.89, 0.13, and 0.09 g/kg, respectively to GenBank under the accession number KT311002.1 (Vurukonda et al. 2016). (Vurukonda et al. 2016). Maize (Zea mays L. var. DHM 117) seeds were surface sterilized with 0.1% HgCl2 (30 s) followed by 70% ethanol gfp labeling of P. putida strain FBKV2 for 1 min and six times of repeated washing with sterile water (1 min each time) (Vurukonda et al. 2016). The effectiveness The derivative of broad-host-range vector pBBR1MCS-5 of surface sterilization was checked by plating the seeds and (Stuurman et al. 2000), which contains the green fluorescent
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