Abstract
Rice is staple food of nearly half the world’s population. Rice yields must therefore increase to feed ever larger populations. By colonising rice and other plants, Herbaspirillum spp. stimulate plant growth and productivity. However the molecular factors involved are largely unknown. To further explore this interaction, the transcription profiles of Nipponbare rice roots inoculated with Herbaspirillum seropedicae were determined by RNA-seq. Mapping the 104 million reads against the Oryza sativa cv. Nipponbare genome produced 65 million unique mapped reads that represented 13,840 transcripts each with at least two-times coverage. About 7.4 % (1,019) genes were differentially regulated and of these 256 changed expression levels more than two times. Several of the modulated genes encoded proteins related to plant defence (e.g. a putative probenazole inducible protein), plant disease resistance as well as enzymes involved in flavonoid and isoprenoid synthesis. Genes related to the synthesis and efflux of phytosiderophores (PS) and transport of PS-iron complexes were also induced by the bacteria. These data suggest that the bacterium represses the rice defence system while concomitantly activating iron uptake. Transcripts of H. seropedicae were also detected amongst which genes involved in nitrogen fixation, cell motility and cell wall synthesis were the most expressed.
Highlights RNASeq of H. seropedicae colonised rice roots showed remarkable regulation of defence, metal transport, stress and signalling genes. Fe-uptake genes were highly induced with implications in plant nutrition and immunity.
Introduction
To answer the ever increasing demand for cereals, genetic improvement of rice and the concomitant development of bio-fertilisers are promising, low environmental-impact solutions. After water, the most limiting nutrient in plant development is nitrogen and nitrogenous fertilisers have been heavily used in rice cultivation (Ladha and Reddy, 2003). Heavy use of nitrogen fertilisers causes environmental damage including contamination of ground-water and the release of nitrogen oxides.
An alternative to the use of nitrogenous fertilisers is to employ plant-associated microorganisms that fix nitrogen. Herbaspirillum seropedicae is an endophytic diazotrophic that can colonise many plants and improve their productivity (reviewed by Monteiro et al., 2012; Chubatsu et al., 2012). Inoculation of rice with H. seropedicae increased root and shoot biomass by 38 to 54 % and 22 to 50 % respectively (Gyaneshwar et al., 2002) part of which was attributable to biological nitrogen fixation (James, 2000; Gyaneshwar et al., 2002; James et al, 2002; Roncato-Maccari et al., 2003). Pankievicz et al. (2015) showed that the nitrogen fixed by H. seropedicae and Azospirillum brasilense was rapidly incorporated into Setaria viridis. Other factors, including production of phyto-hormones by the bacteria stimulate plant growth and several authors have observed that the increase in biomass of inoculated plants is dependent on the plant genotype (Gyaneshwar et al., 2002; Sasaki et al., 2010).
Transcriptome based studies are powerful tools to detect differentially expressed genes and discover novel molecular processes (Nobuta et al., 2007; Mizuno et al, 2010; Xu et al, 2012, Xu-H et al. 2015; Wakasa et al, 2014; Magbanua et al, 2014; Shankar et al, 2016). Expression analyses (using EST sequencing and RT-qPCR) of rice roots inoculated with >H> seropedicae suggested that genes related to auxin and ethylene syntheses as well as defence are modulated by the microorganism in a cultivar dependent manner (Brusamarello-Santos et al., 2011). Here we used RNA-seq to profile the transcriptome of rice inoculated with H seropedicae.
Materials and Methods
Plant material and growth conditions
Testas were removed from seeds of rice (Oryza sativa ssp japonica cv. Nipponbare, kindly provided by the Instituto Riograndense do arroz, IRGA - Avenida Missoes 342, Porto Alegre, RS, Brazil), then disinfected with 70 % (v/v) ethanol for 5 min followed by 30 min soaking in 8 % sodium hypochlorite (1 mL per seed) containing 0.1 % v/v Triton-X100. After rinsing 20 times with sterile water, the seeds were treated with 0.025 % (v/v) Vitavax-Thiram (Chentura, Avenida Naçoes Unidas 4777, Alto de Pinheiros, Sâo Paulo, SP, Brazil) fungicide solution and stirred (120 rpm) for 24 h in the dark at 30 °C. The seeds were then transferred to 0.7 % water-agar and left for two days to germinate after which the seedlings were inoculated with 1 mL of Herbaspirillum seropedicae strain SmR1 (108 cells/seedling) for 30 minutes while control seedlings were treated with 1 mL of N-free NFbHP-malate medium (Klassen et al., 1997) (controls). Seedlings were washed with sterile water and transferred to glass tubes (25 cm long, 2.5 cm diameter) containing propylene beads and 25 mL of modified Hoagland’s solution (Hoagland and Arnon, 1950) without nitrogen (1mM KH2PO4, 1mM K2HPO4, 2mM MgSO4.7H2O, 2mM CaCl2.2H2O, 1mL/L micronutrient solution (H3BO3 2.86 g.L−1, MnCl2.4H2O 1.81 g.L−1, ZnSO4.7H2O 0.22 g.L−1, CuSO4.5H2O 0.08 g.L−1, Na2MoO4.2H2O 0.02 g.L−1) and 1mL.L−1 Fe-EDTA solution (Na2H2EDTA.2H2O 13.4 g.L−1 and FeCl3.6H2O 6 g.L−1)), pH 6.5-7.0. Plants were cultivated at 24 °C under 14 h light and 10 h dark for 3 days. H. seropedicae was cultivated in NFbHP malate medium containing 5 mM glutamate as the nitrogen source. Cells were shaken (120 rpm) overnight at 30 °C, then centrifuged, washed once with N-free NFbHP-malate and suspended in the same medium to OD600 = 1 (corresponding to 108 cells.mL−1). Strain SmR1 (Souza et al., 2000) is a spontaneous streptomycin resistant mutant of strain Z78 (Baldani et al., 1986). Root colonisation was monitored by counting the number of colony forming units (CFU) per gram of fresh root. Approximately 100 mg of roots were washed in 70 % v/v ethanol for 1 min, 1% chloramine T for 1 min followed by three washes with sterile water (Dobereiner et al., 1995). The roots were then crushed with a mortar and pestle in 1 mL of NFbHP-malate and serial dilutions (10−1 to 10−5) were plated onto solid NFbHP-malate containing 20 mM NH4Cl. Two days later the cells were counted.
RNA isolation and construction of libraries
The roots were separated from the aerial part and immediately stored in RNA later™ (Life Technologies, Foster City, CA, USA) Total RNA was extracted from roots of five rice plants using a RNAqueos kit (Ambion, Austin, TX, USA). Contaminating genomic DNA was eliminated with RNase-free DNase I (Ambion) for 30 min at 37 °C. Total RNA (7 to 10 μg) was depleted of ribosomal RNA by treatment with a RiboMinus™ Plant Kit for RNA-Seq (Invitrogen, Carlsbad, CA, USA). The integrity and quality of the total RNA was checked spectrophotometrically and by agarose gel electrophoresis. Whole Transcriptome Analysis RNA Kits™ (Life Technologies) were used on 500 ng purified RNA to construct the libraries and sequencing was performed in a SOLiD4 (Life Technologies) sequencer.
Sequencing and analysis of short reads
SOLiD sequencing produced 161 million 50 bp reads that were analysed by SAET software (Applied Biosystems - Foster City, CA, EUA) to improve base calling, followed by quality trimming using the CLC Genomics Workbench (CLC bio, a QIAGEN Company, Silkeborgvej 2, DK-8000 Aarhus C, Denmark) (quality scores higher than 0.05 and reads with less than 20 bp were discarded). Then the reads were mapped to the rice genome database from the MSU Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) using the following parameters: a minimum length fraction of 95 %, minimum similarity of 90 % and only one hit. Differential expression was analysed using DESeq (Anders and Huber, 2010) from RobiNA software (Lohse et al., 2012). Genes covered at least twice were considered expressed and regulated when expression changed two times and the P-value was lower than 0.05.
Quantification of mRNA levels using RT-qPCR
Reverse transcription quantitative PCR (RT-qPCR) analyses were used to evaluate gene expression under the conditions described above. Total RNA was isolated from rice roots using the TRI Reagent (Sigma, St. Louis, MO, USA) and contamination with genomic DNA was removed with DNase I (Life Technologies). The integrity and quality of the total RNA was confirmed by spectrophotometric analyses and electrophoresis. cDNA was produced from 1 g DNase-treated total RNA using high-capacity cDNA reverse transcription kits (Life Technologies). The cDNA reaction was diluted 60 times before quantitative PCR using Power SYBR-Green PCR Master Mix on a Step One Plus Real Time-PCR System (both from Life Technologies). Primer sequences are listed in Table S1 and were designed with the Primer express 3.0 software (Applied Biosystems) and the NCBI primer designing tool using the genome sequence of O. sativa ssp. japonica cv. Nipponbare. Calibration curves for all primer sets were linear over four orders of magnitude (R2 = 0.98 to 0.99) and efficiencies were 90 % or higher. mRNA expression levels were normalised using the expression levels of actin 1, tubulin beta-2 chain (beta-2 tubulin) (Jain et al., 2006) and a hypothetical protein (protein kinase) (Narsai et al., 2010) using geNorm 3.4 software (Vandesompele et al., 2002). The relative expression level was calculated according to Pfaffl (2001). Three independent samples were analysed for each condition and each sample was assayed in triplicate.
Results and Discussion
Transcriptional analyses
>H> seropedicae enters rice roots via cracks at the points of lateral root emergence and later (three to 15 days) colonises the intercellular spaces, aerenchyma, cortical cells and vascular tissue (Elbeltagy et al., 2001; Gyaneshwar et al., 2002; James et al., 2002; Roncato-Maccari ei al., 2003). 14 days after inoculation we observed an increase of weight of roots and leaf but this increase was not statistically significant. The number of endophytic >H> seropedicae reached approximately 105 to 106 CFU per gram of fresh root weight one to two days after inoculation (DAI), with a peak at three DAI (data not shown). For this reason roots were collected for RNA-seq analyses three DAI when the population of H. seropedicae had stabilised in the intercellular spaces and xylem (Roncato-Maccari et al., 2003). Rice plants inoculated in parallel with the samples used for RNA extraction contained 4.2 × 105 endophytic CFU.g−1 of fresh roots and 4.4 × 108 epiphytic CFU.g−1 of root at three DAI.
Sixty-four percent of the reads (103,563,118) were finally used for mapping to the reference rice (www.rice.plantbiology) and H. seropedicae genomes. Illustration of RNA-seq analyses is shown in Figure 1 and the numbers of reads mapped to each reference genome are listed in Table 1. Mapping on the rice genome database (http://rice.plantbiology.msu.edu/) produced 22 million unique mapped reads representing 13,837 expressed transcripts.
Differentially expressed genes
Statistical analyses were performed using DESeq software (Anders and Huber, 2010) and comparison of non-inoculated with inoculated samples revealed 1,015 differentially expressed genes (P < 0.05). Amongst these, 255 had fold-changes higher than two and they were functionally categorised using MapMan (http://mapman.gabipd.org/) (Figure 2). Considering the number of regulated genes in relation to the number of expressed genes in each category, the main categories down-regulated by H. seropedicae were: metal handling (9.0%; 4/41), polyamine metabolism (9.1%; 1/11), secondary metabolism (9%; 15/167), hormone metabolism (4.7%; 11/233); stress (4.8%; 24/503) and nucleotide metabolism (4.9%; 6/123). Among genes up-regulated by bacteria were those involved in gluconeogenesis/glyoxylate cycles (28.6%; 01/07), polyamine metabolism (27.3%; 03/11), metal handling (24.4%; 10/41), fermentation (23.1%; 03/13) and nitrogen metabolism (18.8%; 03/16) (Figure 2). Some of these categories of regulated genes were analysed in more detail below.
Biotic and abiotic stresses
Among the 255 differentially expressed genes (> 2fold), 59 were stress-related (30 repressed and 29 induced) in the following categories: secondary metabolites, hormone signalling, cell-wall, proteolysis, PR-proteins, signalling, transcription factors, redox-state, abiotic stress and peroxidases (Figure 3 and Table S2).
Secondary metabolism
Amongst the secondary metabolic pathways with higher numbers of regulated genes were those involved in phenylpropanoid and isoprenoid synthesis (Table S2). Phenylpropanoids synthesised by deamination of L-phenylalanine are eventually converted to p-coumaric acid, a precursor of flavonoids and lignin (Ferrer et al., 2008; Hassan and Mathesius, 2012). H. seropedicae modulated expression of four genes involved in flavonoid synthesis. Amongst them, the gene encoding chalcone isomerase (CHI), which catalyses the synthesis of naringenin from tetrahydroxy-chalcone (Naoumkina et al., 2010), was repressed 2.1-fold. Naringenin is a key intermediate in the synthesis of other compounds including flavonol [flavonol synthase (FS), repressed 8.3-fold by H. seropedicae], and anthocyanins [dihydroxiflavonol 4-reductase (DRF), repressed 2.5-fold]. In addition, isoflavone reductase (IFR) gene, involved in isoflavone synthesis, was repressed 1.3-fold (P = 0.01). RT-qPCR of FS confirmed repression by H. seropedicae (Figure 4) but to a much lower extent (1.8-fold). Flavonols such as quercetin (Hassan and Mathesius, 2012) exhibit antimicrobial activity possibly by binding and inhibiting DNA gyrase (Plaper et al., 2003). Previously, Balsanelli et al. (2010) showed that narigenin has antimicrobial activity against H. seropedicae.
Naoumkina et al. (2010) reported that flavonone-3-β-hydroxylase was induced in rice following infection with Xanthomonas oryzae. Nematode cysts stimulate isoflavone synthesis (including chalcone reductases, chalcone isomerase, isoflavon 2’-hydroxylase, isoflavones and isoflavone reductase synthase) in soybeans. Our data thus indicate that down-regulation of flavonoid/isoflavone synthesis by H. seropedicae is part of the attenuation of the defence system in rice roots that is necessary to host an endophyte.
As mentioned above p-coumaric acid is also a precursor of lignin which is assembled from monolignols (Vanholme et al., 2010). In this pathway cinnamoyl-CoA esters are converted into monolignols by two enzymes, cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) (Boerjan et al., 2003). RNA-seq data showed that a gene (L0C_0s02g56700.1) encoding a cinnamoyl CoA reductase was induced 2.8-fold in rice inoculated with H. seropedicae (Table S2) while Kawasaki et al. (2006) reported that cinnamoyl-CoA reductase 1 (0sCCR1 - L0C_0s02g56460.1) is an effector of the small GTPase Rac that is involved in defence responses in rice (Kawasaki et al., 1999; Ono et al., 2001; Suharsono et al., 2002; Akamatsu et al., 2013). Transcriptome analyses of rice inoculated with the endophyte Harpophora oryzae or the pathogen Magnaporthe oryzae showed that a cinnamoyl CoA reductase (L0C_0s08g34280.1) like other enzymes in lignin synthesis was repressed in beneficial interactions and induced in pathogenic ones (Xu et al., 2015). L0C_0s08g34280.1 was also slightly repressed in our experiments (≈1.3 fold). It therefore seems as if a balance between induction and repression of defence-related genes allows H. seropedicae to survive inside rice tissues while concomittantly activating some defence responses to protect the plant from pathogens.
Isoprenoid (or terpenoid) synthesis was also modulated by H. seropedicae (Table S2 and Figure 5). Isoprenoids are derived from isomeric compounds including isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (Chen et al., 2011) via two pathways: the mevalonate pathway (MEV) and the methyl-erythritol phosphate pathway (MEP) (Figure 5). The MEV pathway occurs in the cytoplasm and the MEP pathway in plastids (Vranová et al, 2013) producing an estimated that 55,000 isoprenoid compounds. As a consequence, the biological function of these molecules is diverse and includes precursors of hormones, components of membranes, defence agents and carotenoids.
In rice, H. seropedicae repressed genes of the MEP pathway (Table S2 and Figure 5). The most repressed gene (5.9 times) encodes 1-deoxy-D-xylulose 5-phosphate synthase (DXS) the first enzyme of the pathway that synthesises 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate. DXP is a precursor of antimicrobial compounds including phytoalexins. Chitin induces the MEP pathway in cultured rice cells and as a result phytoalexins accumulate (Okada et al, 2007). Furthermore treatment of the cells with inhibitors of the enzymes DXS and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) impeded chitin-dependent accumulation of phytoalexin. The authors suggest that activation of the MEP is required to meet the demand for isoprenoid and phytoalexin synthesis in infected cells. In our experiments and those of Drogue et al. (2014), the gene encoding stemar-13-ene synthase (0sDTC2), an enzyme involved in synthesis of phytoalexins in rice, was repressed 1.9-fold in H. seropedicae treated rice roots (Figure 5) and twofold when infected with Azospirillum spp. (Drogue et al, 2014). It thus seems as if H. seropedicae represses production of defence-related isoprenoids in rice roots perhaps to allow the bacteria to enter and rapidly colonise the intercellular spaces and xylem.
Oxidative stress responses
In this study H. seropedicae modulated expression of five redox state genes including those for non-symbiotic hemoglobin 2 and peroxiredoxin. 0ne peroxiredoxin gene (L0C_0s07g44440.1) induced 3.7 and the other L0C_0s01g16152.1 was repressed 2.1 fold. Peroxiredoxins are a group of H202-decomposing antioxidant enzymes related to the redox state. In addition to the reduction of H202, peroxiredoxin proteins also detoxify alkyl hydroperoxides and peroxinitrite (Rhee et al., 2005; Dietz et al, 2006).
Plants respond to attacks by pathogens with rapid increases in reactive oxygen species (R0S) such as superoxide and H202 (Apostol et al., 1989). Peroxidases produce R0S that could cause oxidative damage to proteins, DNA, and lipids. Many defects in the immune system of mature Arabidopsis thaliana plants with reduced expression of two key peroxidase genes, PRX33 or PRX34, were observed (Daudi et al., 2012). Silencing the French-bean class III peroxidase (FBP1) in A. thaliana impaired the oxidative burst and rendered plants more susceptible to bacterial and fungal pathogens (Bindschedler et al., 2006). Proteomic studies of rice roots seven days after inoculation with H. seropedicae showed induction of ascorbate peroxidases (Alberton et al., 2013), though the genes encoding these enzymes were not affected in our RNA-seq analyses. Moreover, seven days after inoculation, R0S levels in Herbaspirillum rubrisubalbicans attached to rice roots had increased suggesting that the bacteria were subject to oxidative stresses (Valdameri et al., 2017). In this work, two peroxidase genes were induced by H. seropedicae in inoculated roots.
Cell wall
A variety of diazotrophic microorganisms such as H. seropedicae Z67, H> rubrisubalbicans and A. brasilense produce cell-wall degrading enzymes (Elbeltagy et al., 2001; James et al, 2002). Interestingly, the H. seropedicae SmR1 genome did not reveal genes coding for known cellulases, pectinases or any other cell-wall degrading enzymes (Pedrosa et al., 2011). Nevertheless, rice roots inoculated with H. seropedicae induced a gene (2.0-fold) encoding a β-D-xylosidase and repressed 2.4-fold a gene coding for a polygalacturonase suggesting re-modeling of plant cell wall. In A. thaliana, β-D-xylosidase has been shown to be involved in secondary cell-wall hemi-cellulose metabolism and plant development (Goujon et al., 2003), but little is known about the function of this enzyme.
0ther differentially expressed genes involved in cell-wall metabolism code for proteins similar to an expansin11 (induced 2.6-fold). Expansins have a loosening effect on plant cell-walls and function in cell enlargement as well as in diverse developmental processes in which cell-wall modification occurs including elongation. In addition, they promote elongation of root-hairs (Lin et al., 2011; ZhiMing et al, 2011) and root-hair initiation (Kwasniewski and Szarejko, 2006).
Expansins have been correlated with plant-bacteria interactions. In tobacco, Bacillus subtilis G1, a plant growth promoting bacterium, induced the expression of two expansins NtEXP2 and NtEXP6 (Wang et al, 2009). Also, inoculation of Melilotus alba with Sinorhizobium meliloti lead to enhanced MaEXP1 mRNA levels in roots and nodules (Giordano and Hirsch, 2004). Together, these results suggest that inoculation with H. seropedicae also led to modification of plant cell wall which may facilitate bacterial colonization of inner tissue by loosening cell wall.
Plant immune responses
The plant immune system can sense and respond to pathogen attacks in two manners: the first involves recognition of pathogen/microbe associated molecular patterns (PAMPs) by surface pattern-recognition receptors (PRRs) resulting in pattern-triggered immunity (PTI). Second, resistance proteins (R) that recognise pathogen effectors are expressed leading to effector-triggered immunity (ETI) (Abramovitch et al, 2006; Jones and Dangl, 2006; McDowell and Simon, 2008). Among the regulated genes that have functions involved in defence (in dark gray squares in Figure 3), as determined by MapMan, three genes encoding PR-proteins were repressed (L0C_0s10g25870.1, L0C_0s11g07680.1, L0C_0s02g38392.1, 2.5, 3.4 and 2.7-fold respectively) while one transcript that encoded a lipase called EDS1 (enhanced disease susceptibility 1) was induced 2-fold.
The role of EDS1 in defence is well described in Arabidopsis and is required for resistance conditioned by R genes which encode proteins that contain nucleotide-binding sites and leucine-rich repeats (NBS-LLR) (Aarts et al., 1998; McHale et al., 2006; Bartsch et al., 2006; Jones and Dangl, 2006; Venugopal et al., 2009). Mutant edsl seedlings exhibited enhanced susceptibility to the biotrophic oomycete Peronospora parasifica (Parker et al, 1996). Analysis of EDS1> and PR-gene expression showed induction of both genes after inoculation with Pseudomonas syringae or treatment with salicylic acid (SA) (Falk et al, 1999). In addition, in the Arabidopsis edsl mutant, the expression of PR-proteins was undetectable. When the edsl mutant was treated with SA however, expression of PR was detected. The authors suggest that EDS1 functions upstream of PRl-mRNA accumulation. Among the 3 genes for PR-proteins repressed, one (L0C_0s02g38392.1) is a NBS-LRR disease resistance protein. Therefore in H. seropedicae-rice interaction an inverse correlation between PR-protein and EDS1 expression was observed, perhaps suggesting a fine regulation of these defence systems by H. seropedicae.
In previous work down-regulation of genes associated with defence was observed in rice plants inoculated with H. seropedicae such as a putative probenazole inducible protein (PBZ1, L0C_0s12g36840.1) that was repressed 3.6-fold as shown by RT-qPCR analysis (Brusamarello-Santos et al, 2011). RNA-seq data showed that this gene (L0C_0s12g36840.1) was repressed 2.9-fold. Another two genes coding proteins similar to PBZ1 (L0C_0s12g36830.1 and L0C_0s12g36850.1) were also repressed (7.2 and 4.1, respectively). Kawahara et al. (2012) using an RNA-seq approach to study the transcriptome of rice inoculated with the blast fungus M> oryzae observed that the same PBZ1 genes detected in our study were induced 273 and 233-fold upon inoculation with the pathogen. The induction of PBZ1 gene by pathogens has been considered as a molecular marker for rice defence response (Kim et al., 2008).
Another gene regulated by H. seropedicae that is related to defence codes for a thionin, a small cysteine-rich protein that occurs in a broad range of plant species (Florack and Stiekema W.J., 1994). Thionins are known for their toxicity to plant pathogens and several studies showed that their over-expression is related to increased resistance to diseases (Iwai et al., 2002; Choi et al, 2008; Muramoto et al., 2012; Ji et al, 2015).
Brusamarello-Santos et al. (2011) showed 5-fold repression of thionin genes from chromosome 6 seven days after inoculation with H. seropedicae but these thionins from chromosome 6 were not regulated in roots three days after inoculation with H. seropedicae. Since there are 15 thionin genes (according to the Rice Genome Annotation Project RGAP7) sharing high identity in chromosome 6, we mapped the unmapped reads to the rice genome as well as to chromosome 6 separately and observed an average of 480 mapped reads for control libraries and 136 for inoculated libraries mapping on thionin genes, a result that is in accordance with repression patterns observed in rice roots seven days after inoculation with H. seropedicae (Brusamarello-Santos et al., 2011). Interestingly, we observed a thionin transcript from chromosome 7 (L0C_0s07g24830.1) that was induced 7.2-fold in the presence of H. seropedicae three DAI. Time-dependent regulation of thionin was also observed by Ji et al. (2015) in rice roots infected with Meloidogyne graminicola. In a by Straub and co-workers study only a few defence-related genes were induced in the transcriptome of Miscanthus sinensis inoculated with Herbaspirillum frisingense helping to explain why this bacterium can effectively invade and colonise plants (Straub et al., 2013).
Signalling
Plant Receptor
The plant immune system employ, at the cell surface, receptors to perceive pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs). Theses receptors have an ectodomain potentially involved in ligand binding, a single transmembrane domain and, most of times, a intracellular kinase domain (Couto and Zipfel, 2016).
Amongst the genes with the highest expression differences seen in RNA-seq data was a wall-associated receptor kinase-like (WAK) 22 precursor gene (L0C_0s10g07556.1), repressed 93-fold (30-fold by RT-qPCR) in H seropedicae inoculated roots. Cell wall-associated receptor kinases (WAKs) contain an extracellular domain composed of one or more epidermal growth factor repeats (EGF). Animal proteins containing these repeats are known to bind small peptides (Hynes and MacDonald, 2009). In A. thaliana WAKs bind to cross-linked pectin cell wall, pathogen- or damage-induced pectin fragments and oligogalacturonides, thus regulating cell expansion or stress response depending on the state of the pectin (Kohorn and Kohorn, 2012; Kohorn, 2015, 2016). Several studies have described the role of WAK genes in rice resistance to pathogens (Li et al., 2009; Cayrol et al., 2016; Harkenrider et al, 2016; Delteil et al, 2016). Plant proteins show a similar domain architecture consisting of cell-wall pectin binding extracellular region, the EGF-like domain, and a kinase domain. Analyses of rice loss-of-function mutants of WAK genes showed that individual genes are important for resistance against M. oryzae. 0sWAK14, 0sWAK91 and 0sWAK92 positively regulate resistance while 0sWAK112d is a negative regulator of blast resistance (Delteil et al., 2016). Cayrol et al. (2016) demonstrated that 0sWAK14, 0sWAK91 and 0sWAK92 can form homo- and hetero-complexes and hypothesized that the loss of function of any of these proteins may destabilized the complex and affect their functioning. In A. thaliana the WAK22 orthologous gene (AtWAKL10) encodes a functional guanylyl cyclase which is co-expressed with pathogen defence related genes (Meier et al., 2010). Thus the wall-associated receptor kinase-like 22 gene of rice could be a candidate for surface pattern recognition receptor and its repression may allow H. seropedicae to evade activation of the plant-defence system.
A cysteine-rich receptor-like protein kinase (CRK) gene (L0C_0s04g56430.1) and a serine/threonine-protein kinase At1g18390 precursor (L0C_0s05g47770.1) were induced 3.2-fold and fold, respectively, while a lectin-like receptor kinase 7 (L0C_0s07g03790.1) and SHR5-receptor-like kinase (L0C_0s08g10320.1) were repressed 2.7 and 2.5-fold respectively in the presence of H. seropedicae (Table S2). The latter protein has 75% identity with sugarcane SHR5 receptor kinase repressed by colonisation with diazotrophic endophytes (Vinagre et al., 2006). The authors suggested that the expression levels of this gene were inversely related to the efficiency of beneficial plant-bacterial interactions (Vinagre et al, 2006). Besides in Arabidopsis cysteine rich receptor-like kinase 5 protein is involved in regulation of growth, development, and acclimatory responses (Burdiak et al., 2015).
Auxin signalling
Auxins regulate diverse physiological processes such as vascular tissue differentiation, lateral root initiation and have also been linked to defence in plant-pathogen interactions (Rogg and Bartel, 2001; Kazan and Manners, 2009; McSteen, 2010; Adamowski and Friml, 2015). Auxin response elements (AuxREs), when bound to auxin response factors (ARFs), control auxin-dependent gene expression. The Aux/IAA protein family members that inhibit ARFs mediate this regulation (Guilfoyle, 2015).
Four differentially expressed genes related to auxin signalling were identified in rice roots inoculated with H. seropedicae. The gene L0C_0s08g24790.1 encoding an auxin-responsive protein was repressed 2.1-fold (Table 2) whereas the genes coding for auxin-induced proteins, L0C_0s09g25770.1 and L0C_0s05g01570.1, were repressed 1.8 and 2.5-fold, respectively. Repression of genes related to auxin signalling was reported in rice roots inoculated with H. seropedicae (Brusamarello-Santos et al. 2011). Brusamarello-Santos et al. (2011) found that ARF2-like, IAA 11 and IAA18 were repressed, 1.4, 1.5 and 2.8-fold respectively, in the presence of H. seropedicae 7 days after inoculation. These genes were not regulated in our data probably due to the time difference of the cDNA library construction and too low expression levels.
H. seropedicae can produce IAA in the presence of tryptophan, thus suggesting the plant growth promotion effect may be due to bacterial derived auxin (Bastian et al., 1998). In addition exogenous application of auxin lead to increase in lateral root numbers (Inukai et al., 2005). However the number of lateral roots in rice plants inoculated and non-inoculated with H. seropedicae, was determined (data not shown) but no significant difference was observed.
In the absence of clear regulation of auxin-dependent genes and phenotype, the results suggest that auxin does not play an important role in Nipponbare rice roots colonization by H. seropedicae. 0n the other hand the repression of auxin signalling has been correlated to defence responses. Auxin levels have been correlated with susceptibility to pathogens (Kazan and Manners, 2009) that are able to produce high levels of auxin (Jameson, 2000). It has also been shown that pathogen-associated molecular patterns (PAMPs) induce the expression of a miRNA that negatively regulates mRNAs for F-box auxin receptors leading to resistance to P. syringae in Arabidopsis (Navarro et al, 2006). The observed repression in several genes involved with defence in the rice-H> seropedicae interaction opens the question whether auxin could be important for bacteria survival inside the plant by down-regulation defence system. Further studies are needed to elucidate if and how auxin signalling participates in plant-bacterial interactions.
Ethylene signalling
Ethylene is also involved in several biological processes that activate defence responses and adventitious root-growth in rice and other plants (Lorbiecke and Sauter, 1999; Glazebrook, 2005; Robert-Seilaniantz et al., 2011). Ethylene can be synthesised by oxidation of 1-aminocyclopropane-1-carboxylate (ACC) by ACC oxidase. ACC is synthesised from adenosylmethionine (AdoMet) by ACC synthase (ACS). ACS is up regulated 6-fold and ACC oxidase is repressed 2-fold by inoculation with H. seropedicae. An ethylene response factor (ERF) (L0C_0s01g04800.1) gene was also induced 2.2- fold in inoculated roots. These data indicate that ethylene synthesis is attenuated in the presence of bacteria. Moreover Alberton et al. (2013) measured the level of ethylene in inoculated rice roots (seven days after inoculation) and found a decrease of ethylene levels. Furthermore, Valdameri et al. (2017) detected induction of ACC oxidase in rice plants inoculated with the pathogen H rubrisubalbicans. These results suggest that the ethylene pathway is differentially modulated in the presence of pathogens and beneficial endophytic bacteria that promote plant growth.
SA signalling
Salicylic acid is derived from phenolic compounds and is involved in response to attack by pathogens (Vlot et al, 2009; An and Mou, 2011). In rice roots inoculated with H. seropedicae, a SA-dependent carboxyl methyltransferase family protein (L0C_0s11g15340.2) was repressed 40-fold. A member of this family is salicylic acid carboxyl methyltransferase (SAMT) that catalyses the formation of methyl salicylate (MeSA) from SA (Ross et al., 1999). MeSA is an essential signal for systemic acquired resistance (SAR) in tobacco plants. In addition mutations in SAMT showed that this gene is required for SAR (Park et al., 2007).
SA signalling is differentially regulated by members of the WRKY transcription factor family (Eulgem and Somssich, 2007). In inoculated rice roots, two WRKY transcription factors were regulated, one repressed (2.3-fold) and one induced (2.0-fold). The induced gene encodes a WRKY51 similar to WRKY11 of Arabidopsis, whereas the repressed gene encodes WRKY46, which was shown to be induced by SA in Arabidopsis. WRKY11 is a negative regulator of resistance (Journot-Catalino et al, 2006) and Arabidopsis plants in which WRKY46 was over-expressed were more resistant to P. syringae (Hu et al., 2012). These results are in agreement with the hypothesis that the SA signalling and defence system are attenuated in the presence of the H. seropedicae.
Metal ion metabolism
Several genes related to metal transport were differentially expressed, most of them up regulated in the presence of H. seropedicae. Amongst the 20 most highly regulated rice genes, eight were related to metal transport (Table 3; Table 4). Phyto-siderophore synthesis starts with production of nicotianamine (NA) from S-adenosylmethionine, which in turn is derived from 5’-methylthioadenosine of the methionine salvage pathway (MTA cycle) (Figure 6). Transcripts of enzymes of the MTA cycle encoding SAM, MTA nucleosidase, MTR kinase, E1 enolase/phosphatase and acireductone dioxygenase (ARD) were induced 1.9, 2.6, 6.0, 1.9 and 7.5-times, respectively, in roots colonised by H> seropedicae (Figure 6). Using proteomic and RT-qPCR analyses, Alberton (2013) observed similar expression patterns in rice roots inoculated with H. seropedicae.
The synthesis of NA and MAs involves a set of enzymes including S-adenosylmethionine syntase (SAM) that catalyses the adenylation of L-methionine to S-adenosylmethionine, nicotianamine synthase (NAS) that converts S-adenosylmethionine to nicotianamine, and nicotianamine aminotransferase (NAAT) that catalyses the amino transfer of NA to produce the 3’’-keto intermediate that is reduced by deoxymugineic acid synthase (DMAS) to produce 2’-deoxymugineic acid (DMA) (Shojima et al., 1990; Higuchi et al., 1999; Bashir et al., 2006; Inoue et al., 2008).
Two nicotianamine synthase (NAS) genes of rice - L0C_0s03g19420.2 and L0C_0s03g19427.1 were induced (52- and 32-fold, respectively) in inoculated rice roots. L0C_0s03g19427.1 was also induced in rice roots inoculated with Azospirillum spp. (Drogue et al., 2014). Increased levels of nicotianamine have been shown to increase Fe uptake in rice plants (Lee et al, 2009). Furthermore, in Lotus japonicus inoculated with Mesorhizobium loti, nicotianamine synthase 2 was expressed only in nodules pointing to a role in symbiotic nitrogen fixation (Hakoyama et al., 2009). A tomato mutant defective in the synthesis of nicotianamine was affected in iron metabolism (Ling et al., 1996). In addition NAAT (L0C_0s02g20360.1) and DMAS (L0C_0s03g13390.2) were also induced 10-fold and 19-times respectively in colonised rice roots. Iron deficiency provoked induction of rice gene 0sNAAT1 (Inoue et al., 2008).
Recently, members of a major facilitator super-family have been described as essential to the efflux of MA and NA in rice. T0M1 (transporter of mugineic acid 1) is involved in the efflux of MA, while ENA1 (efflux transporters of nicotianamine 1) and ENA2 in the efflux of NA (Nozoye et al, 2011). T0M1 (L0C_0s 11 g04020.1) and ENA1 (L0C_0s11g05390.1) were induced 40 and 3-fold in colonised roots. Induction of T0M1 was confirmed by RT-qPCR (Figure 4).
Fe+++ ions chelated by PS need to be transported inside the cell. In gramineous plants, two groups of Fe-MA transporters are present: ZmYS1 (Curie et al., 2001) and the YSL (yellow strip-like) transporter family (Inoue et al., 2009). Inoue et al. (2009) analysed the expression of 18 YSL genes in rice and observed induction of 0sYSL15 and 0SYSL16 genes under iron-deficiency. 0ther studies have shown that 0sYSL2 (L0C_0s02g43370) takes up Fe2+-NA (Ishimaru et al., 2010) and 0sYSL16 Fe3+-DMA (L0C_0s04g45900.1) (Kakei et al, 2012). Here we showed induction (3.2-fold) of 0sYSL16 (L0C_0s04g45900.1) and 21-fold increase of transcripts encoding a gene similar to 0SYSL15 (L0C_0s02g43410.1). Induction of 0SYSL15 was confirmed by RT-qPCR (Figure 4). Furthermore, 0sIRT1 (iron-regulated transporter 1) and 0sIRT2 were also induced under low Fe conditions (Ishimaru et al., 2006). We detected induction of 0sIRT2 (L0C_0s03g46454.1) (34-fold in RNA-seq and 93-fold in RT-qPCR) (Figure4).
Other metal transporters such as Nramp6 (L0C_0s07g15460.1) were also up regulated (27fold) in colonised rice roots, a result confirmed by RT-qPCR (Figure 4). Members of the natural resistance-associated macrophage protein (NRAMP) family are transition metal cation/proton cotransporters or anti-porters of broad specificity. AtNRAMP6 of Arabidopsis is up-regulated in response to iron deficiency and is involved with metal mobilization from vacuoles to cytosol (Thomine et al, 2003). Induction of Nramp6 in rice roots colonised by H. seropedicae. In a previous study in rice inoculated with Azospirillum spp the expression of this gene was also induced (Drogue et al. 2014). In addition a gene for an integral membrane protein (L0C_0s09g23300.1) named 0sVIT2 was 17.7-fold repressed by H. seropedicae. This gene is involved in transport of Fe/Zn into vacuoles and is up-regulated in rice roots with excess Fe. Knockout/ knockdown of this gene led to Fe accumulation in seeds (Zhang et al., 2012; Bashir et al., 2013). Together, these results suggest that colonised roots respond in such a manner as to accumulate Fe.
Interestingly the transcript with the highest fold change [104-times - a result confirmed using RT-qPCR (Figure 4)] in colonised roots codes for the non-symbiotic hemoglobin 2 (L0C_0s03g12510.1). High levels of non-symbiotic hemoglobin 2 could help buffer free oxygen and protect bacterial nitrogenase. Arredondo-Peter et al. (1997) described two hemoglobins, Hb1 and Hb2, in rice. The Hb2 induced by H. seropedicae is very similar to the one described by Arredondo-Peter et al. (1997) (coverage 82 % with an identity of 97 %). In addition, Lira-Ruan et al. (2001) studied the synthesis of hemoglobins in rice under normal and stress conditions, coming to the conclusion that Hbs are not part of a generalised stress response. They demonstrated that Hb1 is expressed in different rice organs (root and leaves) during plant development. In etiolated rice plants under 02 limiting conditions the Hb levels increase (Lira-Ruan et al. 2001). This increase suggest that Hb expression maybe due to reduced 02 levels in the presence of the bacteria which make the root environment microaerophilic. The higher requirement for Fe needed for incorporation into Hb may partially explain activation of siderophore synthesis and Fe accumulation. We also found a H. seropedicae bacterioferritin (Hsero_1195) gene induced (2.2-fold), but symptoms of Fe deficiency in colonised rice plants were not observed.
Iron homeostasis has been related to plant defence, R0S accumulation and immunity. Also, Fe deficiency triggers accumulation of antimicrobial phenolics compounds. It has been suggested recently that Fe sequestration by bacterial siderophore could be a signal for pathogen infection (Aznar et al., 2015). However, bacterial genes involved in siderophore biosynthesis were not observed among the H. seropedicae genes expressed in rice roots. Also, transcriptomic analysis of H. seropedicae attached to wheat and mayze roots did not show iron metabolism genes up-regulated (Pankievicz et al., 2016; Balsanelli et al., 2016). These results suggest that the effect of bacteria on plant iron mebabolism is more complex than those caused by iron sequestration.
Transcripts expressed in interactions with bacteria
The libraries from inoculated roots were mapped against the H> seropedicae genome (24,278 reads). Amongst the 4,085 annotated genes, 287 had at least one-fold coverage (this set of genes was called H. seropedicae expressed genes). Functional classification of expressed genes was performed using the C0G system. After ribosomal genes, the most abundant functional classes found were “unknown”, “energy production and conversion”, “amino acid transport and metabolism”, “cell motility and cell wall” (Table 5). Comparison of expressed genes of H> seropedicae detected in plants with bacterial genes expressed in culture revealed only 16 differences [p-value < 0.05 using the DESeq statistical package (Table 5)].
Genes involved in nitrogen fixation (nif) were found amongst genes classified as “energy production and conversion” and “amino acid transport and metabolism”. Nif-genes encode proteins involved in the synthesis, maturation and assembly of the nitrogenase complex (Chubatsu et al., 2012). nifD and nifH(coverage 1.7 and 1.2 respectively) were highly expressed in H> seropedicae colonising wheat and maize roots (Pankievicz et al, 2016; Balsanelli et al, 2016). We also found two ferredoxin genes important for nitrogenase activity: fdxA and fdxN(coverage 1.9 and 1.6) (Souza et al., 2010).
The promoters of nif genes are activated by NifA that is regulated by the Ntr system. When comparisons were made between free-living H. seropedicae and those grown in association with rice, glnK and amtB of the Ntr system were induced 43 and 20-times respectively in the plant-bacterial interaction. Pankievicz et al. (2016) also found that amtB was induced in H> seropedicae attached to maize roots. Furthermore, fixNand fixP (both 1.6X coverage) were also detected in planta along with an urtA ABC-type urea transport system (Hsero_4713) (27-fold of induction in planta).
Twenty-three genes related to cell motility were found, four with > 2X coverage. Amongst these were cheW(a positive regulator of CheA), ñhD, fliCand pilZ(Hsero_2062) that encodes a type pilus assembly protein. A methyl-accepting chemotaxis trans-membrane protein (Hsero_2723) was induced 90-fold when compared with expression in culture (Tadra-Sfeir et al., 2015). This gene was also found to be up-regulated in epiphytic H seropedicae colonising wheat and maize (Pankievicz et al., 2016; Balsanelli et al., 2016). Methyl-accepting chemotaxis proteins interact with Che proteins to detect signals from the environment. Although other T4SS genes were not found perhaps these data suggest that type IV secretion plays a role in H-seropedicae-rice interactions. In the nitrogen-fixing, endophytic bacterium Azoarcus spp., mutation of type IV pilus pilAB genes negatively affects colonisation of rice roots (Dörr et al, 1998).
Among the 19 cell-wall related genes, seven were covered at least 2X and an outer-membrane porin (Hsero_4295) was induced 28X. Another membrane-protein (Hsero_0083) of unknown function was induced 14-fold. These could be proteins that H seropedicae uses to recognise rice.
Acknowledgments
We are grateful M.G. Yates for critical reading of the manuscript, Leonardo M. Cruz and Rodrigo A. Cardoso for bioinformatics support. and Instituto Riograndense do Arroz (IRGA) for providing seeds. We are also thankful to Roseli Prado, Marilza Doroti Lamour and Valter A. Baura for technical assistance. This work was supported by National Institute of Science and Technology of Nitrogen Fixation (INCT - Nitrogen Fixation), Council for Scientific and Technological Development (CNPq) and Coordination of Improvement of Higher-Education Personnel (CAPES). Thanks to CNPq for doctoral scholarship.
Footnotes
E-mail of authors: Brusamarello-Santos, L.C.C: lizianebrusa{at}gmail.com, Alberton, D.: dayalberton{at}yahoo.com.br, Valdameri, G.: glaucio_valdameri{at}hotmail.com, Camilios-Neto, D.: camiliosneto{at}yahoo.com.br, Covre, R.: Covrebio06{at}gmail.com, Lopes, K.: katiaplopes{at}gmail.com, Tadra-Sfeir,1 M.Z.: mzitadrasfeir{at}gmail.com, Faoro, H.: hfaoro{at}gmail.com, Monteiro, R. A.: roseadele{at}gmail.com, Silva, A.B.: adriano.bioinfo{at}gmail.com, Broughton, William J.: williamebroughton{at}gmail.com, Pedrosa, F.O.: fpedrosa{at}ufpr.br, Wassem, R.: wassem{at}ufpr.br