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Genome-Wide Identification of Azospirillum Brasilense Sp245

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Genome-Wide Identification of Azospirillum Brasilense Sp245 Koul et al. BMC Genomics (2020) 21:821 https://doi.org/10.1186/s12864-020-07212-7 RESEARCH ARTICLE Open Access Genome-wide identification of Azospirillum brasilense Sp245 small RNAs responsive to nitrogen starvation and likely involvement in plant-microbe interactions Vatsala Koul1,2†, Divya Srivastava2†, Pushplata Prasad Singh2* and Mandira Kochar2* Abstract Background: Small RNAs (sRNAs) are non-coding RNAs known to regulate various biological functions such as stress adaptation, metabolism, virulence as well as pathogenicity across a wide range of bacteria, mainly by controlling mRNA stabilization or regulating translation. Identification and functional characterization of sRNAs has been carried out in various plant growth-promoting bacteria and they have been shown to help the cells cope up with environmental stress. No study has been carried out to uncover these regulatory molecules in the diazotrophic alpha-proteobacterium Azospirillum brasilense Sp245 to date. Results: Expression-based sRNA identification (RNA-seq) revealed the first list of ~ 468 sRNA candidate genes in A. brasilense Sp245 that were differentially expressed in nitrogen starvation versus non-starved conditions. In parallel, in silico tools also identified 2 of the above as candidate sRNAs. Altogether, putative candidates were stringently curated from RNA-seq data based on known sRNA parameters (size, location, secondary structure, and abundance). In total, ~ 59 significantly expressed sRNAs were identified in this study of which 53 are potentially novel sRNAs as they have no Rfam and BSRD homologs. Sixteen sRNAs were randomly selected and validated for differential expression, which largely was found to be in congruence with the RNA-seq data. Conclusions: Differential expression of 468 A. brasilense sRNAs was indicated by RNA-seq data, a subset of which was confirmed by expression analysis. Four of the significantly expressed sRNAs were not observed in nitrogen starvation while 16 sRNAs were found to be exclusively expressed in nitrogen depletion. Putative candidate sRNAs identified have potential mRNA targets primarily involved in stress (abiotic and biotic) adaptability; regulation of bacterial cellular, biological and molecular pathways such as nitrogen fixation, polyhydroxybutyrate synthesis, chemotaxis, biofilm formation and transcriptional regulation. In addition to directly influencing bacteria, some of these sRNAs also have targets influencing plant-microbe interactions through adhesion of bacteria to plant roots directly, suppressing host response, inducing plant defence and signalling. Keywords: Sstress response, sRNA, A. brasilense Sp245, RNA-seq, PGPB, sRNAscanner * Correspondence: [email protected]; [email protected] †Vatsala Koul and Divya Srivastava contributed equally to this work. 2TERI Deakin Nanobiotechnology Centre, Sustainable Agriculture Division, The Energy and Resources Institute, Gurugram-Faridabad Road, Gwal Pahari, Haryana 122003, India Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Koul et al. BMC Genomics (2020) 21:821 Page 2 of 16 Background 550 bp, usually located in the intergenic regions and The Alphaproteobacterial class of Azospirillum include demonstrate a characteristic stem-loop secondary struc- physiologically distinct, rhizosphere competent, plant ture [18–22], though some exceptions exist [23]. A sin- growth-promoting bacteria (PGPB) that promote the gle bacterial genome is estimated to encode 200–300 growth of a wide range of plants through diverse, direct sRNAs, possessing diverse functions such as plasmid and indirect mechanisms [1, 2]. Nitrogen fixation is not replication [23], stress adaptation [24], regulating the ex- the most significant field function of this bacterial group, pression of outer membrane proteins [25], iron homeo- and it is the production of the plant growth regulators stasis [26], quorum sensing [27] chemotaxis and biofilm and their crosstalk which are the key signals for plant formation [28], among others. Different studies have growth promotion effects [3–7]. Genome sequencing been carried out to discover sRNAs in plant-associated and analysis revealed that Azospirillum spp. which are bacteria (PAB) like Sinorhizobium meliloti, Bacillus sub- now primarily established as plant-associated bacteria in tilis, Bradyrhizobium japonicum, Azotobacter vinelandii, the terrestrial habitat have actually transitioned from the and many others [29–34]. aquatic environment [8, 9]. For improved adaptation in Azospirilla are the most well-known PGPB and consist the rhizosphere, nearly 50% of its genome has been of the strains Sp245, Sp7, and SM which are known to horizontally acquired from distantly related terrestrial enhance the plant root morphology, specifically due to bacteria [representatives of Rhizobiales (alpha-proteo- their two essential traits: biological nitrogen fixation bacteria) and Burkholderiales (beta-proteobacteria)]. The (BNF) and biosynthesis of plant growth regulators (IAA, horizontally transferred (HGT) genes consist of genes cytokinin, gibberellin, ethylene, and nitric oxide) [6, 35– encoding cellulolytic activity (required for penetration 37]. The genome size of Azospirillum brasilense Sp245 into plant roots), attachment (using Type IV pili), and (GC content: 68.5%) is around 7.5 Mb distributed in chemotaxis operon, among other essential genes re- seven replicons-main chromosome and six plasmids quired for plant-microbe interaction [8]. (AZOBR_p1-p6). Although its genome is sequenced, no Specific genes involved in rhizosphere adaptation sug- study has been carried out to identify the sRNAs in this gest niche-specific responses of the strains [9]. It is ex- organism. The genome sequencing of Azospirillum sp. tremely important to study the extent of influence and B510 revealed putative genes for two types of small regulation of these microbial plant growth regulators so RNAs: the B subunit of RNaseP (rnpB) and signal recog- that their effect on plants can be judged for their appro- nition particle RNA (ffs)[8], albeit no regulatory role has priate action and application. Basic knowledge about the been associated with them so far. modulation of key physiological properties of such In this study, we attempt to identify the sRNAs of A. PGPBs is crucial for understanding diverse aspects re- brasilense Sp245 and quantify their transcription under lated to rhizosphere performance, modes of action and nitrogen-limited conditions. Alongside genome-wide successful interactions with plant roots. Bacteria possess identification of novel sRNAs with follow-up Northern various regulatory mechanisms for stress adaptation and blots and qPCR, we provide a validated community re- to improve the stress enduring capability, it is important source for future studies. Many of these sRNAs may be to gather in-depth information of the underlying physio- involved in regulating important biological mechanisms logical and molecular mechanisms as well as unravel the such as plant growth regulators action, chemotaxis/mo- participating intermediates especially during stress at- tility, stress-responsive behaviour, nitrogen-metabolism, tacks [10–13]. The ensuing cellular cascade in response transcriptional regulation and plant association. to changing environmental conditions is not always completely understood due to the lack of information Results about regulatory molecules such as small RNAs which Plant growth-promoting trait evaluation play an essential role at the post-transcriptional level Plant growth-promoting traits of A. brasilense Sp245 [14]. This knowledge will also help kindle ideas about were investigated under various physiological conditions how to advance the production and application strat- ranging from non-stressed to stressed nutrient condi- egies in PGPB inoculants. tions (predominantly carbon and nitrogen) to determine In the last couple of decades, genome-wide searches in the variation in these traits which may be relevant in the different bacteria have led to the discovery of a plethora rhizosphere niches for their functional behaviour. The of small, non-coding regulatory RNA molecules results depicted in Table 1 indicate that polyhydroxybu- (sRNAs), which possess the capability to regulate expres- tyrate (PHB) production, NO production and Nitroge- sion of multiple genes, transcriptional factors, sigma fac- nase activity was significantly higher in both nutrient tors, and chaperones,
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