In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 3 ARTICLE NUMBER: 16207 Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses Neena Mitter 1 *, Elizabeth A. Worrall 1, Karl E. Robinson 1, Peng Li 2, Ritesh G. Jain 1, Christelle Taochy 1,3, Stephen J. Fletcher 1,3, Bernard J. Carroll 3, G. Q. (Max) Lu 2,4 and Zhi Ping Xu 2 * Topical application of pathogen speci c double-stranded RNA (dsrna) for virus resistance in plants represents an 1 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. 2 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. 3 School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia. 4 University of Surrey, Guildford GU2 7XH, UK. *e-mail: n.mitter@uq.edu.au; gordonxu@uq.edu.au NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 1
Suplementary Figure 1: Schematic of BioClay. LEFT: Negatively charged dsrna is loaded onto the LDH nanosheet surface via exchange with Cl - to form dsrna:ldh complex (BioClay). MIDDLE: Facilitated by transpiration, condensation and respiration, LDH is broken down by combination of water and CO2 on the leaf surface, resulting in slow release of dsrna over several weeks. RIGHT: Released dsrna is taken up by plant cells and provides RNAi-based protection to homologous viruses. NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 2
a Relative Intensity (a.u) d 1 10 100 1000 Particle Diameter (nm) 8 b Absorbance (a.u) 3380 1615 1354 730 590 4000 3000 2000 1000 Wavenumber (cm -1 ) e 0 c 100 nm ph solution ph reading 6 4 2 3.9 4.9 5.36 6.89 7.54 7.52 0 0 1 3 10 30 60 Minutes since addition of ph 2.0 to LDH solution Suplementary Figure 2: Characterisation of LDH and release of dsrna from LDH nanosheets. (a) Particle size distribution of LDH. LDH has a moderate size distribution predominantly in the range of 15-120 nm with a Z-average diameter of 45 nm. (b) The layered structure characterized by the FTIR spectrum, indicates a characteristic broad band at 3380 cm - 1 ( O-H in hydroxide layer and H2O molecule), a peak at 1615 cm -1 (the bending vibration of H2O molecules) and peaks at around 590 and 730 cm -1 (M-O and M-O-H stretching vibrations). The peak at 1354 cm -1 is assigned to CO3 2- due to the contamination of aerial CO2 during the preparation. (c) TEM image of PMMoVIR54-dsRNA:LDH, shows that the dsrna chain (circled) can be observed between LDH nanosheets. (d) Increase in ph from acidic to basic over 60 min from the introduction of diluted nitric acid (ph 3.0) to LDH solution. The initial ph of 3.0 quickly changed in the first several minutes and balanced out at ph 7.52 after 1 hr, indicating dissolution of LDH under acidic conditions. (e) Control-dsRNA:LDH resuspended in a range of initial ph solutions (2.0 12.0) and incubated for 24 hr. Release of dsrna can be observed in ph 2.0 and 3.0 at this time point. No dsrna release is observed in ph solutions greater than ph 4 at 24 hr as can be seen by the florescence being retained in the well. M = 1 Kb+ ladder. NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 3
BF Cy3 Chlorophyll Merged a b c d Cy3 only e f g h Cy3:LDH CMV2b-dsRNA -Cy3 Untreated Treated i j k l m n o p CMV2b-dsRNA -Cy3:LDH Treated Treated q r s t u v w x Supplementary Figure 3: Confocal micrographs of Arabidopsis leaves show uptake of Cy3 fluorophore attached to CMV2b-dsRNA. Bright field image (1 st column), Cy3 florescence image (2 nd column), natural florescence of chlorophyll (3 rd column) and merged imaged of all three (4 th column) are shown. The treatments include (a-d) Cy3 only, (e-h) Cy3:LDH (i-p) CMV2b-dsRNA-Cy3 and (q-x) CMV2b-dsRNA-Cy3:LDH. Cy3 and Cy3:LDH control plants clearly show no florescence (b, d, f and h). CMV2b-dsRNA-Cy3 treated leaves showed uptake in the xylem (j, l) as well as moving into the apical untreated leaves (n, p). CMV2b-dsRNA-Cy3:LDH treated leaves showed uptake in the xylem (r, t) and spongy mesophyll (v, x). NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 4
Supplementary Figure 4: Fate of topically applied dsrna:ldh in sprayed plants. (a-b) N. tabacum plants with masking tape covering the apical meristem were sprayed on day 0 with LDH, CMV2b-dsRNA and CMV2b-dsRNA:LDH. RNA was extracted from new unsprayed leaves that emerged 20 days post treatment. (a) Polyacrylamide gel electrophoresis of low molecular weight RNA probed with CMV2b specific 24 nt DIG-labelled oligonucleotide. M= Zymo Research small RNA ladder (b) Agarose gel electrophoresis of total RNA probed with full length CMV2b 330 bp DIG-labelled probe. M = DIG labelled RNA molecular weight marker III. Lower panels show equal loading of RNA. NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 5
Supplementary Figure 5: CMV2b-aligned read abundance from CMV2b-dsRNA and CMV2b-BioClay sprayed and newly emerged N. tabacum leaves without any CMV inoculation. Small RNA sequencing was done on (a) sprayed leaves collected 5 days post treatment and (b) newly emerged unsprayed leaves collected 20 days post treatment. Small RNA reads from 18 to 32 nt were aligned to the CMV2b sense arm reference sequence and quantified by size. Normalized read abundance is shown for each discrete srna size as the mean of two biological replicates (± s.e.), except for LDH (day 5), which was a single sample. The abundance of reads outside of sizes typical of dsrna processing (i.e. 21, 22 and 24 nt) suggests the presence of degraded dsrna on the surface of CMV2b-dsRNA and CMV2b- BioClay sprayed leaves at day 5. For systemic (non-sprayed) leaves at day 20, processing of the CMV2b-dsRNA sequence into 21, 22 and 24 nt small RNAs is not detectable. NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 6
a 350 Number of local lesions b 300 250 200 150 100 50 0 50 Water PMMoVIR54- dsrna *** CMV2bdsRNA Number of local lesions 40 30 20 10 0 Water PMMoVIR54- dsrna * CMV2bdsRNA Supplementary Figure 6: Sequence specificity of viral protection provided by topical application of CMV2b-dsRNA. Cowpea plants were sprayed at two leaf stage with water, CMV2b-dsRNA and PMMoVIR54-dsRNA (non-specific control). The plants were challenged with CMV 1 day after the spray and local lesions were counted 10 days post viral challenge, (a) Trial 1 (n=13 plants per treatment group), (b) Trial 2 (n=8 plants per treatment). Significant p-values: <0.05*, <0.01** and <0.001***, Kruskal-Wallis test with post hoc Nemenyi-test for multiple comparisons between samples, compared to water. Data represent mean ± standard error. NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 7
Supplementary Figure 7: CMV infection on sprayed tobacco plants when challenged with the virus 20 days post treatment (trial 1). Plants were sprayed on day 0 and inoculated with CMV on sprayed leaves 20 days post treatment. DAS-ELISA absorbance readings of the two most apical leaves 10 days pvc (a) LDH (n=8), (b) CMV2b-dsRNA (n=16) and (c) CMV2b- BioClay (n=16). Columns represent the average of two wells per sample ± standard error. (+) is untreated CMV inoculated plant. (-) is un-inoculated control. CMV positive threshold is four times the DAS-ELISA absorbance readings of the (-) un-inoculated control, shown by the solid line. (a) 8/8 LDH treated plants tested positive for CMV (positive threshold 0.156). (b) 9/16 CMV2b-dsRNA treated plants tested positive for CMV (positive threshold 0.124). (c) 1/16 CMV2b-BioClay treated plants tested positive for CMV (positive threshold 0.826). NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 8
Supplementary Figure 8: CMV infection on sprayed tobacco plants when challenged with the virus 20 days post treatment (trial 2). Plants were sprayed on day 0 and inoculated with CMV on sprayed leaves 20 days post treatment. DAS-ELISA absorbance readings of the two most apical leaves 10 days pvc (a) LDH (n=8), (b) CMV2b-dsRNA (n=8) and (c) CMV2b- BioClay (n=8). Columns represent the average of two wells per sample ± standard error. (+) is untreated CMV inoculated plant. (-) is un-inoculated control. CMV positive threshold is four times the DAS-ELISA absorbance readings of the (-) un-inoculated control, shown by the solid line. (a) 5/8 LDH treated plants tested positive for CMV (positive threshold 0.199). (b) 4/8 CMV2b-dsRNA treated plants tested positive for CMV (positive threshold 0.212). (c) 2/8 CMV2b-BioClay treated plants tested positive for CMV (positive threshold 0.178). NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 9
Supplementary Figure 9: CMV infection on sprayed tobacco plants when challenged with the virus 20 days post treatment (trial 3). Plants were sprayed on day 0 and inoculated with CMV on sprayed leaves 20 days post treatment. DAS-ELISA absorbance readings of the two most apical leaves 10 days pvc (a) LDH (n=10), (b) CMV2b-dsRNA (n=10) and (c) CMV2b- BioClay (n=10). Columns represent the average of two wells per sample ± standard error. (+) is untreated CMV inoculated plant. (-) is un-inoculated control. CMV positive threshold is four times the DAS-ELISA absorbance readings of the (-) un-inoculated control, shown by the solid line. (a) 7/10 LDH treated plants tested positive for CMV (positive threshold 0.286). (b) 9/10 CMV2b-dsRNA treated plants tested positive for CMV (positive threshold 0.306). (c) 3/10 CMV2b-BioClay treated plants tested positive for CMV (positive threshold 0.286). NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 10
Supplementary Figure 10: CMV infection on sprayed tobacco plants when challenged with the virus 20 days post treatment (trial 4). Plants were sprayed on day 0 and inoculated with CMV on sprayed leaves 20 days post treatment. DAS-ELISA absorbance readings of the two most apical leaves 20 days pvc (a) LDH (n=36), (b) CMV2b-dsRNA (n=36) and (c) CMV2b-BioClay (n=36). Columns represent the average of two wells per sample ± standard error. (+) is untreated CMV inoculated plant. (-) is un-inoculated control. CMV positive threshold is four times the DAS-ELISA absorbance readings of the (-) un-inoculated control, shown by the solid line. (a) 24/36 LDH treated plants tested positive for CMV (positive threshold 0.176). (b) 31/36 CMV2b-dsRNA treated plants tested positive for CMV (positive threshold 0.162). (c) 13/36 CMV2b-BioClay treated plants tested positive for CMV (positive threshold 0.174). NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 11
Supplementary Figure 11: CMV infection in unsprayed, new leaves that emerged 20 days post treatment (trial 2). Plants were sprayed on day 0 and inoculated with CMV on unsprayed leaves 20 days post treatment. DAS-ELISA absorbance readings of the two most apical leaves 10, 20 and 30 days pvc (a) LDH (n=10) and (b) CMV2b-BioClay (n=10). CMV positive threshold is four times the un-inoculated control, shown by the solid line ( 0.746). (a) 8/10 LDH treated plants tested positive for CMV. (b) 2/10 CMV2b-BioClay treated plants tested positive for CMV. Columns represent the average of four wells per sample ± standard error. NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 12
Supplementary Table 1: Normalised abundance of 21 and 22 nt reads aligned to the CMV genome. The percentage of aligned 22 nt reads relative to aligned 21 nt reads is also indicated. Sample 21 nt Reads aligned Per Million Reads (mean of 2 replicates) SUPPLEMENTARY INFORMATION 22 nt Reads aligned Per Million Reads (mean of 2 replicates) Percentage 22 nt relative to 21 nt alignments CMV control 255041.3 256643.6 100.63% CMV2b-dsRNA (Day 10) 689.2 711.1 103.2% CMV2b-BioClay (Day 10) 1602.5 1000.2 62.41% CMV2b-dsRNA (Day 15) 15818.9 14142.8 89.4% CMV2b-BioClay (Day 15) 21313.4 20259.0 95.05% NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 13
Supplementary Table 2: Names and description of N. tabacum total RNA samples that underwent small RNA sequencing. Sample No. 1-2 3-4 5-6 7 8 9 10 Sample Name/s Control+CMV-day10-1 Control+CMV-day10-2 11 LDH-day5-1 12-13 14-15 16-17 18 19 20-21 CMV2b-dsRNA-day5-1 CMV2b-dsRNA-day5-2 CMV2b-BioClay-day5-1 CMV2b-BioClay-day5-2 CMV2b-dsRNA+CMVday10-1 CMV2b-dsRNA+CMVday10-2 CMV2b-BioClay+CMVday10-1 CMV2b-BioClay+CMVday10-2 CMV2b-dsRNA+CMVday15-1 CMV2b-dsRNA+CMVday15-2 CMV2b-BioClay+CMVday15-1 CMV2b-BioClay+CMVday15-2 LDH-systemic-day20-1 LDH-systemic-day20-2 CMV2b-systemic-dsRNAday20-1 CMV2b-systemic-dsRNAday20-2 CMV2b-systemic-BioClayday20-1 CMV2b-systemic-BioClayday20-2 Sample Description Leaves sprayed with dh2o Day 0, CMVinoculated Day 5, RNA extracted from washed leaves Day 10 2 biological replicates Leaves sprayed with CMV2b-dsRNA Day 0, CMV-inoculated Day 5, RNA extracted from washed leaves Day 10 2 biological replicates Leaves sprayed with CMV2b-BioClay Day 0, CMV-inoculated Day 5, RNA extracted from washed leaves Day 10 2 biological replicates Leaves sprayed with CMV2b-dsRNA Day 0, CMV-inoculated Day 10, RNA extracted from washed leaves Day 15 2 biological replicates Leaves sprayed with CMV2b-BioClay Day 0, CMV-inoculated Day 10, RNA extracted from washed leaves Day 15 2 biological replicates Leaves sprayed with LDH Day 0, RNA extracted from washed leaves Day 5 Leaves sprayed with CMV2b-dsRNA Day 0, RNA extracted from washed leaves Day 5 2 biological replicates Leaves sprayed with CMV2b-BioClay Day 0, RNA extracted from washed leaves Day 5 2 biological replicates Leaves sprayed with LDH Day 0, RNA extracted from systemic (non-sprayed) leaves Day 20 Leaves sprayed with CMV2b-dsRNA Day 0, RNA extracted from systemic (non-sprayed) leaves Day 20 2 biological replicates Leaves sprayed with CMV2b-BioClay Day 0, RNA extracted from systemic (non-sprayed) leaves Day 20 2 biological replicates NATURE PLANTS DOI: 10.1038/nplants.2016.207 www.nature.com/natureplants 14