bioRxiv preprint doi: https://doi.org/10.1101/260380; this version posted February 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Modulation of defence and iron homeostasis genes in rice roots by the diazotrophic endophyte 2 Herbaspirillum seropedicae 3 4 Brusamarello-Santos,1 L.C.C, Alberton,2 D., Valdameri,2 G., Camilios-Neto,1,6 D., Covre,3 R., Lopes,3 5 K., Tadra-Sfeir,1 M.Z., Faoro,1,7 H., Monteiro, R. A.1, Silva,3,8 A.B., Broughton4, W. J., Pedrosa,1 F.O., 6 Wassem,5 R., Souza*,1 E.M. 7 1Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, 8 Brasil 9 2Departamento de Patologia Médica, Setor Ciências da Saúde, Curitiba, PR, Brasil 10 3Setor de Educação Profissional e Tecnológica, Universidade Federal do Paraná, Curitiba, PR, Brasil 11 4Federal Institute of Materials Research and Testing, Division 4 Environment, Berlin, Germany 12 5Departamento de Genética, Universidade Federal do Paraná, Curitiba, PR, Brasil 13 6 Present address: Departamento de Bioquímica e Biotecnologia, Universidade Estadual de Londrina, 14 Londrina, PR, Brasil 15 7 Present address: Instituto Carlos Chagas – Fiocruz, Curitiba, PR, Brasil 16 9 Present address: Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 17 Esch-sur-Alzette, Luxembourg. 18 19 * Corresponding author: 20 Address: Universidade Federal do Paraná/UFPR, Rua Coronel Francisco Heráclito dos 21 Santos, s/n Caixa Postal 19046, 81531-980 – Curitiba, 22 E-mail: [email protected] 23 Telephone and fax : +55413361-1667 24 25 E-mail of authors: 26 Brusamarello-Santos, L.C.C: [email protected] 27 Alberton, D.: [email protected] 28 Valdameri, G.: [email protected] 29 Camilios-Neto, D.: [email protected] 30 Covre, R.: [email protected] 31 Lopes, K.: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/260380; this version posted February 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 Tadra-Sfeir,1 M.Z.: [email protected] 33 Faoro, H.: [email protected] 34 Monteiro, R. A.: [email protected] 35 Silva, A.B.: [email protected] 36 Broughton, William J.: [email protected] 37 Pedrosa, F.O.: [email protected] 38 Wassem, R.: [email protected] 39 Date of submission: February 05, 2018 40 Number of tables: 5 41 Number of figures: 6 (Figure 1, 3, 5,6 (colour online-only) , figure 2,4 (black-and-white) 42 Word count: 6421 43 Supplementary data: 3 tables 44 45 Modulation of defence and iron homeostasis genes in rice roots by the diazotrophic endophyte 46 Herbaspirillum seropedicae 47 48 Running title: Modulation of defence-related rice genes by H. seropedicae 49 50 Highlights 51 52 RNASeq of H. seropedicae colonised rice roots showed remarkable regulation of defence, metal 53 transport, stress and signalling genes. Fe-uptake genes were highly induced with implications in plant 54 nutrition and immunity. 55 56 Abstract 57 58 Rice is staple food of nearly half the world’s population. Rice yields must therefore increase to feed 59 ever larger populations. By colonising rice and other plants, Herbaspirillum spp. stimulate plant 60 growth and productivity. However the molecular factors involved are largely unknown. To further 61 explore this interaction, the transcription profiles of Nipponbare rice roots inoculated with 62 Herbaspirillum seropedicae were determined by RNA-seq. Mapping the 104 million reads against the 2 bioRxiv preprint doi: https://doi.org/10.1101/260380; this version posted February 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 63 Oryza sativa cv. Nipponbare genome produced 65 million unique mapped reads that represented 64 13,840 transcripts each with at least two-times coverage. About 7.4 % (1,019) genes were 65 differentially regulated and of these 256 changed expression levels more than two times. Several of the 66 modulated genes encoded proteins related to plant defence (e.g. a putative probenazole inducible 67 protein), plant disease resistance as well as enzymes involved in flavonoid and isoprenoid synthesis. 68 Genes related to the synthesis and efflux of phytosiderophores (PS) and transport of PS-iron 69 complexes were also induced by the bacteria. These data suggest that the bacterium represses the rice 70 defence system while concomitantly activating iron uptake. Transcripts of H. seropedicae were also 71 detected amongst which genes involved in nitrogen fixation, cell motility and cell wall synthesis were 72 the most expressed. 73 74 Keywords: Herbaspirillum seropedicae, rice, biotic stresses, flavonoids, nitrogen fixation, pathogenic 75 responses, pathogen-associated molecular patterns (PAMPs) 76 77 Introduction 78 To answer the ever increasing demand for cereals, genetic improvement of rice and the 79 concomitant development of bio-fertilisers are promising, low environmental-impact solutions. After 80 water, the most limiting nutrient in plant development is nitrogen and nitrogenous fertilisers have been 81 heavily used in rice cultivation (Ladha and Reddy, 2003). Heavy use of nitrogen fertilisers causes 82 environmental damage including contamination of ground-water and the release of nitrogen oxides. 83 An alternative to the use of nitrogenous fertilisers is to employ plant-associated micro- 84 organisms that fix nitrogen. Herbaspirillum seropedicae is an endophytic diazotrophic that can 85 colonise many plants and improve their productivity (reviewed by Monteiro et al., 2012; Chubatsu et 86 al., 2012). Inoculation of rice with H. seropedicae increased root and shoot biomass by 38 to 54 % and 87 22 to 50 % respectively (Gyaneshwar et al., 2002) part of which was attributable to biological nitrogen 88 fixation (James, 2000; Gyaneshwar et al., 2002; James et al., 2002; Roncato-Maccari et al., 2003). 89 Pankievicz et al. (2015) showed that the nitrogen fixed by H. seropedicae and Azospirillum brasilense 90 was rapidly incorporated into Setaria viridis. Other factors, including production of phyto-hormones 91 by the bacteria stimulate plant growth and several authors have observed that the increase in biomass 92 of inoculated plants is dependent on the plant genotype (Gyaneshwar et al., 2002; Sasaki et al., 2010). 3 bioRxiv preprint doi: https://doi.org/10.1101/260380; this version posted February 5, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 Transcriptome based studies are powerful tools to detect differentially expressed genes and 94 discover novel molecular processes (Nobuta et al., 2007; Mizuno et al., 2010; Xu et al., 2012, Xu-H et 95 al. 2015; Wakasa et al., 2014; Magbanua et al., 2014; Shankar et al., 2016). Expression analyses 96 (using EST sequencing and RT-qPCR) of rice roots inoculated with H. seropedicae suggested that 97 genes related to auxin and ethylene syntheses as well as defence are modulated by the microorganism 98 in a cultivar dependent manner (Brusamarello-Santos et al., 2011). Here we used RNA-seq to profile 99 the transcriptome of rice inoculated with H. seropedicae. 100 101 Materials and Methods 102 Plant material and growth conditions 103 Testas were removed from seeds of rice (Oryza sativa ssp japonica cv. Nipponbare, kindly provided 104 by the Instituto Riograndense do arroz, IRGA – Avenida Missões 342, Porto Alegre, RS, Brazil), then 105 disinfected with 70 % (v/v) ethanol for 5 min followed by 30 min soaking in 8 % sodium hypochlorite 106 (1 mL per seed) containing 0.1 % v/v Triton-X100. After rinsing 20 times with sterile water, the seeds 107 were treated with 0.025 % (v/v) Vitavax-Thiram (Chentura, Avenida Nações Unidas 4777, Alto de 108 Pinheiros, São Paulo, SP, Brazil) fungicide solution and stirred (120 rpm) for 24 h in the dark at 30 ºC. 109 The seeds were then transferred to 0.7 % water-agar and left for two days to germinate after which the 110 seedlings were inoculated with 1 mL of Herbaspirillum seropedicae strain SmR1 (108 cells/seedling) 111 for 30 minutes while control seedlings were treated with 1 mL of N-free NFbHP-malate medium 112 (Klassen et al., 1997) (controls). Seedlings were washed with sterile water and transferred to glass 113 tubes (25 cm long, 2.5 cm diameter) containing propylene beads and 25 mL of modified Hoagland’s 114 solution (Hoagland and Arnon, 1950) without nitrogen (1mM KH2PO4, 1mM K2HPO4, 2mM -1 115 MgSO4.7H2O, 2mM CaCl2.2H2O, 1mL/L micronutrient solution (H3BO3 2.86 g.L , MnCl2.4H2O 1.81 -1 -1 -1 -1 -1 116 g.L , ZnSO4.7H2O 0.22 g.L , CuSO4.5H2O 0.08 g.L , Na2MoO4.2H2O 0.02 g.L ) and 1mL.L Fe- -1 -1 117 EDTA solution (Na2H2EDTA.2H2O 13.4 g.L and FeCl3.6H2O 6 g.L )), pH 6.5-7.0. Plants were 118 cultivated at 24 ºC under 14 h light and 10 h dark for 3 days. H. seropedicae was cultivated in NFbHP 119 malate medium containing 5 mM glutamate as the nitrogen source. Cells were shaken (120 rpm) 120 overnight at 30 °C, then centrifuged, washed once with N-free NFbHP-malate and suspended in the 8 -1 121 same medium to OD600 = 1 (corresponding to 10 cells.mL ). Strain SmR1 (Souza et al., 2000) is a 122 spontaneous streptomycin resistant mutant of strain Z78 (Baldani et al., 1986). Root colonisation was 123 monitored by counting the number of colony forming units (CFU) per gram of fresh root. 4 bioRxiv preprint doi: https://doi.org/10.1101/260380; this version posted February 5, 2018.