Nicotiana Roots Recruit Rare Rhizosphere Taxa As Major Root-Inhabiting Microbes

11 SUPPLEMENTARY INFORMATION FOR2 3 Nicotiana roots recruit rare rhizosphere taxa as major root-inhabiting microbes4 Muhammad Saleem1, Audrey D. Law1 and Luke A. Moe1*5 1Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, USA, 40546-03126 Muhammad Saleem: [email protected]; Audrey Law: [email protected]; Luke Moe: 7 [email protected] 89 Materials and Methods10 Plant growth and root sampling11 Two commercial burley tobacco (Nicotiana tabacum) varieties (KT204LC and NCBH129LC) 12 were grown at the University of Kentucky Spindletop Farm (Lexington, KY) in the summer of 13 2013 using standard agronomic practices (Pearce et al., 2015). Briefly, seeds were germinated in 14 a dedicated greenhouse and seedlings were transplanted to field plots in June 2013. The plants 15 were grown in a plot that was used for burley tobacco in the previous growing season. At 16 tobacco harvest, the vegetative portion of the plants was removed for curing, and the rhizosphere17 and root samples (n=6) were taken from six plants of each variety. The root and associated soil 18 was removed using a spading fork. After gently shaking the roots to remove loosely adhered soil,19 the soil remaining attached to the root (rhizosphere soil) was physically removed by hand, using 20 sterile latex gloves, into a sterile bag. The remaining root mass was put into a sterile bag and all 21 samples were subsequently stored at -80C until processing. Soil data is listed in Table S1.22 Isolation of bacteria from plant roots 223 Roots were washed with distilled water to eliminate any remaining soil. The larger roots linked 24 with the stem were removed and are considered primary roots. Roots emerging from the primary 25 roots were removed and are considered secondary roots. Fine roots were collected from 26 secondary roots. Cutting of the roots was done using a sterilized blade. Bacteria were isolated 27 from plant tissues following a previously established method (Ikeda et al., 2009) with some 28 modifications. About 20 g of fresh root composite sample was taken from each root portion 29 (main root, secondary roots and fine roots). The root samples were cut into small pieces and 30 added to to a 1 L blender jar (Kinematica Microtron MB 550) with 400 ml BCE buffer (50 mM 31 Tris-HCl, pH 7.5, 1% Triton X-100, 2 mM 2-mercaptoethanol added right before blending). 32 Samples were blended for 2 min at full speed, followed by 5 min incubation on ice, for a total of 33 three cycles. The homogenized samples were filtered through a sterile Mira cloth using a 34 Buchner funnel. The resulting filtrate was centrifuged at 500  g for 5 min at 10C. The 35 supernatant was centrifuged again at 5,500  g for 20 min at 10C. The supernatant was then 36 discarded and the pellet resuspended in 250 ml BCE, and centrifuged at 10,000  g for 10 min at 37 10C, for a total of two times. The final pellet was resuspended in 6 ml Tris Buffer (50 mM, pH 38 7.5). The suspension was gently overlayed onto 4 ml nycodenz (8g nycodenz in 10 ml Tris 39 buffer) in a 15 ml glass tube. The glass tube was centrifuged at 10,000  g for 40 min at 10C. A40 band containing the bacterial cells formed at the interface of buffer and nycodenz was removed 41 with a pipette and transferred into a 2 ml microcentrifuge tube with an equal volume of deionized42 sterile water. After centrifugation at 21,130  g for 5 min, the supernatant was removed and the 43 pellet was stored at -20C.44 Genomic DNA purification 345 Genomic DNA purification followed the protocol of Wilson (2001). The frozen microbial 46 pellets isolated from plant roots were dissolved in 567 l of TE buffer, 30 l of 10% SDS and 3 47 l of proteinase K (20mg/ml) were added, and the mixture was incubated at 37C for one hour. 48 100 l of 5 M NaOH and 80 l of a CTAB/NaCl solution (41 g NaCl and 100 g CTAB per liter 49 of solution) was added before another incubation for 10 min at 65C. An equal volume of 50 chloroform/isoamyl alcohol (24:1) was used to extract the lysate, followed by extraction with an 51 equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). Nucleic acids were precipitated 52 using 0.6 volumes of isopropanol. The precipitate was centrifuged at 12,000  g for 25 min at 53 4C, and the pellet was rinsed twice with 1 ml of 70% ethanol. The DNA pellet was dissolved in 54 TE buffer and quantified using a Qubit fluorometer. DNA from the rhizosphere soil samples was 55 extracted using the PowerSoil® DNA Isolation Kit according to manufacturer’s instructions. The 56 V4 region of the 16S ribosomal RNA gene was amplified and sequenced on an Illumina MiSeq 57 machine following the protocol of Kozich et al. (2013). MiSeq sequencing was performed at the 58 University of Kentucky Advanced Genetic Technology Center and at the Host Microbiome 59 Initiative (HMI) facility (http://medicine.umich.edu/medschool/research/research-strengths/host-60 microbiome-initiative) at the University of Michigan, Ann Arbor, MI, USA. 61 Bioinformatics and statistical analysis62 MiSeq community sequence data was processed in mothur (http://www.mothur.org/) according 63 to the MiSeq SOP as of September 2014 (http://www.mothur.org/wiki/MiSeq_SOP) (Kozich et 64 al. 2013). Briefly, paired-end reads were assembled into contigs, sequences were filtered for 65 length, ambiguous bases, and homopolymer regions, and aligned to the SILVA reference 66 alignment (release 102) to remove sequences that do not align to the V4 region of the 16S rRNA 467 gene. Pre-clustering to merge highly similar (2 bp or less mismatch) sequences was followed by 68 the removal of chimeras. Sequences were classified based on the RDP reference version 9 and 69 16S rRNA sequences derived from mitochondrial and chloroplast DNA as well as unidentified 70 sequences were removed. The average number of remaining sequences per sample was 13,478 71 (range 2,598–39,474). We randomly selected 2,500 sequences from each sample, and binned 72 them to OTUs based on sequence similarity with a 97% similarity cut-off.73 Since many of the bacterial taxa were rare, data was normalized to 995 OTUs to reduce stress for74 performing Non-Metric Multidimensional Scaling ordination (NMDS) in PC-ORD 6 (Figure 75 S1). The NMDS was applied to assess the beta-diversity based on the Bray–Curtis measure of 76 dissimilarity (Bray and Curtis, 1957; Walke et al., 2014) in OTUs relative abundances across 77 rhizosphere and root samples. To test the selection of dominant and rare taxa by the rhizosphere 78 and root system, we performed Student's t-test and linear regression. In this particular analysis, 79 the top rhizosphere OTUs (with abundance greater than 10) were sorted, and their abundance 80 was compared across fine roots, secondary roots and primary roots using Student's t-tests (Figure81 2a). Similarly, the top fine roots OTUs (n>10) were sorted, and their abundance was compared 82 across rhizosphere, secondary and primary roots using Student's t-tests (Figure 2b). The top 83 secondary root OTUs (n>10) were sorted, and their abundance was compared across rhizosphere,84 fine roots and primary roots using Student's t-tests (Figure 2c). The top primary root OTUs 85 (n>10) were sorted, and their abundance was compared across rhizosphere, fine roots and 86 secondary roots using Student's t-tests (Figure 2d). Both Student's t-test and linear regression 87 were used to test whether the abundance of dominant OTUs differs across fine roots, secondary 88 and primary roots. Wherever the results were the same in terms of statistical significance, we 89 present only linear regression results. In cases in which both tests did not reveal any differences, 590 the OTUs were defined as generalist colonizers (listed in separate excel file in Supplementary 91 Material) of different root portions, and the converse is true for specialist colonizers (Figure S2-92 3). 93 References 94 95 Bray JR, Curtis JT. (1957). An ordination of the upland forest communities of Southern 96 Wisconsin. Ecolog Monogr 27:325–349. 97 98 Ikeda S, Kaneko T, Okubo T, Rallos LEE, Eda S, Mitsui H, et al. (2009). Development of a 99 bacterial cell enrichment method and its application to the community analysis in soybean stems. 100 Microb Ecol 58:703–714. 101 102 Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. (2013). Development of a dual- 103 index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the 104 MiSeq Illumina sequencing platform. Appl Environ Microbiol 79:5112–5120.105 Pearce B, Bailey A, Walker E, eds. 2015. Burley and dark tobacco production guide. University 106 of Kentucky Extension Publication. 107 http://www2.ca.uky.edu/agc/pubs/id/id160/id160.pdf (Accessed May 5, 2015).108 Walke JB, Becker MH, Loftus SC, House LL, Cormier G, Jensen RV, et al. (2014). Amphibian 109 skin may select for rare environmental microbes. ISME J 8:2207–2217. 110 111 Wilson K. (2001). Preparation of genomic DNA from bacteria. In: Current protocols in 112 molecular biology, John Wiley & Sons, Inc. 113 http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0204s56/abstract (Accessed January 114 20, 2015).115 116 6117 Supplementary Tables 118 119 Table S1. Analysis of rhizosphere soil Analysis Data K(lb/ac Buffer Location P(lb/ac) pH C(lb/ac) M(lb/ac) ZN(lb/ac) ) pH Spindletop farm 516 209 5.28 6.62 4890 245 4.1 120121KT 204LC NCBH 129LC Bacterial phyla Rhizosph Secondary (%) Rhizosphere Fine roots Secondary roots Primary roots ere Fine roots roots Primary roots Acidobacteria 23.38 2.86 2.12 2.05 20.752 1.89 2.496 2.1066667 Actinobacteria 8.513333333 7.106666667 3.056 3.52 9.928 7.31 5.616 5.4266667 Armatimonade tes 0.813333333 0.173333333 0.136 0.3 0.784 0.13 0.168 0.2 Bacteroidetes 5.506666667 4.733333333 4.84 4.41 5.112 2.89 5.088 4.96 Chlamydiae 0.046666667 0.02 0 0 0.392 0.16 0.048 0.0266667 Chloroflexi 0.486666667 0.046666667 0.008 0.02 0.344 0.08 0.024 0.0133333 Firmicutes 2.7 0.48 0.24 0.69 2.488 1.49 0.864 0.5733333 Gemmatimona detes 1.946666667 0.04 0.024 0.07 2.832 0.06 0.064 0.04 Nitrospira 0.746666667 0.006666667 0.024 0 0.976 0.01 0.016 0 Planctomycete s 2.806666667 2.153333333 1.376 2.25 2.088 3.51 2.696 2.12 Proteobacteria 21.94 74.34 83.04 79.59 23.52 73.25 75.784 77.173333 unclassified 21.39333333 4.04 2.184 2.6 22.416 4.85 3.584 3.92 Verrucomicro bia 9.486666667 3.853333333 2.888 4.48 8.392 4.29 3.488 3.4 7TM7(100) 0.02 0.046666667 0 0.01 0.024 0.07 0.032 0.04 122 Table S2. Composition of rhizosphere and root microbiome in both varieties. 123 8124 Supplementary Figures 125 126 Figure S1. 127 Nonmetric multidimensional scaling (NMDS) ordination based on Bray–Curtis distances 128 between microbial communities of rhizosphere and root samples collected from two Nicotiana 129 varieties. Overall, the ordination axes explain about 95% of the variance in the dissimilarities 130 among microbial communities of different habitats. Each point represents one sample. 131 Figure S2.132 Abundance of specialist OTUs across root architectural traits gradient in the Nicotiana variety 133 KT204LC. Significance for specialists is shown by p-values. Each box represents specialist 134 OTUs from a different phylum (a, Proteobacteria; b, Verrucomicrobia; c, Actinobacteria; d, 135 Bacteroidetes).136 Figure S3.137 Abundance of specialist OTUs across root architectural traits gradient in the Nicotiana variety 138 NCBH129LC. Significance for specialists is shown by p-values. Each box represents specialist 139 OTUs from a different phylum (a, Proteobacteria; b, Verrucomicrobia; c, Bacteroidetes; d, 140 Planctomycetes).141 Figure S4142 Dominant OTUs (abundance >10) in the rhizosphere and their abundance in different root 143 portions of KT204LC. 144 Figure S5 9145 Dominant OTUs (abundance >10) in the rhizosphere and their abundance in different root 146 portions of NCBH129LC.147 Figure S6148 Dominant OTUs (abundance >10) in the fine roots and their abundance in rhizosphere and other 149 root portions of KT204LC.150 Figure S7151 Dominant OTUs (abundance >10) in the fine roots and their abundance in the rhizosphere and 152 other root portions of NCBH129LC.153 Figure S8154 Dominant OTUs (abundance >10) in the secondary roots and their abundance in the rhizosphere 155 and other root portions of KT204LC.156 Figure S9157 Dominant OTUs (abundance >10) in the secondary roots and their abundance in the rhizosphere 158 and other root portions of NCBH129LC.159 Figure S10160 Dominant OTUs (abundance >10) in the primary roots and their abundance in the rhizosphere 161 and other root portions of KT204LC.162 Figure S11 10163 Dominant OTUs (abundance >10) in the primary roots and their abundance in the rhizosphere 164 and other root portions of NCBH129LC.165 Figure S12166 Average abundance of top ten OTUs in the roots of KT 204LC. Lowercase letters indicate 167 statistical significant differences among treatments. The significance was determined using 168 ANOVA followed by Student's t-test.169 Figure S13170 Average abundance of top ten OTUs in the roots of NCBH 129LC. Lowercase letters indicate 171 statistical significant differences among treatments. The significance was determined using 172 ANOVA followed by Student's t-test.

Nicotiana Roots Recruit Rare Rhizosphere Taxa As Major Root-Inhabiting Microbes

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