Electronic supplementary material Planta

Title

Identification and molecular characterization of a trans-acting small

interfering RNA producing locus regulating leaf rust responsive gene

expression in wheat (Triticum aestivum L.)

Summi Dutta1, Dhananjay Kumar1#, Shailendra Jha2, Manish Kumar1, Kumble Vinod Prabhu2,

Kunal Mukhopadhyay1

1 Department of Bio-Engineering, Birla Institute of Technology, Mesra, Ranchi 835215

Jharkhand, India

2 Division of Genetics, Indian Agricultural Research Institute, New Delhi 110012

#Present address: Department of Botany, PDM University, Bahadurgarh, Haryana 124507 India

Corresponding Author

Dr. Kunal Mukhopadhyay

Mobile: +919431382720

Fax: +916512275401

Email: kmukhopadhyay@bitmesra.ac.in

Online Resource S1 List of primers and their sequences used in this study

S.No. Primer name Sequence (5'-3')1 s1 TACCGTGTGCCGGATTATGAC2 s2 AATTGGTCATCGCGCTTGGTT3 s3 AGAGGAACCGTTGATTCACAC4 s4 CCTACTGATGACAGTGTCGCG5 s1r GTCATAATCCGGCACACGGTA6 s2r AACCAAGCGCGATGACCAATT7 s3r GTGTGAATCAACGGTTCCTCT8 s4r CGCGACACTGTCATCAGTAGG9 gli F GCAATGTCTACATCCCTCCAC10 gli R TGGTACCAAAGATGCCAAATG11 LRR F CAGGAGTTCACTGGAATCATCA12 LRR R TAGAGCCCTCTCTCCTTCATTC13 GstF CCGGTTCAAGAGAAGAGACCT14 GstR ACTCGAACTAGACGGGCAGA15 fad F GCTCCCAATGCACCTCAT16 fad R CTGACAAAGTGATCGACTCTCAA17 tmp F CTCCCTCATCGTGACCATCT18 tmp R GCTTCCTCTTGAGCGTCTTG19 TamiR16(-) CGGCACACGGTAGCTTCGCGCC20 mRQ3' primer provided in the Mir-X miRNA First

Strand Synthesis kit)21 RACE NPI GGGTTCGCATGCTAGACTGG

Online Resource S2 Distribution of number of reads with 18-24nt size in the four sRNA libraries S-Mmi (a), S-PImi (b), R-Mmi(c), R-PImi (d)

Online Resource S3 GC percentage of reads of sRNAs in the four sRNA libraries.) S-Mmi (a), S-PImi (b), R-Mmi (c), R-PImi (d)

Online Resource S4 Numbers and percentages of 18-26 nucleotides sRNAs in the S-Mmi, S-PImi, R-Mmi, R-PImi libraries showing similarity with different categories of sequences (wheat ESTs, miRNA precursor, matured miRNA, non-coding, coding, Pucciniatranscript sequences).

Attributes

Library code

S-Mmi S-PImi R-Mmi R-PImi

Total read counts 2520419 2889484 4227780 2578748

18-26nt seq. 1987400 1520821 1595153 1435876

Wheat EST 1500270 (75.5%) 1048028(68.9%) 1034782(64.8%) 932000 (64.9%)

miRNA

precursor

124060 (6.2%) 190179 (12.5%) 212309 (13.3%) 260406 (18.1%)

Matured miRNA 105700 (5.3%) 162771 (10.7%) 182306(11.4%) 225910(15.7%)

Non-coding

RNAs

1205780 (60.7%) 752986 (49.5%) 747545(46.9%) 623164(43.4%)

Coding

sequences

362500 (18.2%) 248081 (16.3%) 238431 (14.9%) 219330 (15.3%)

Puccinia

transcript

52880 (2.7%) 32832 (2.2%) 36281 (2.3%) 28625 (1.9%)

Percentage (%) in parenthesis represent percentage of 18-26 nt reads

Online Resource S6 Snapshotsof the predicted TAS sequence as visualized using the VisSR tool of UEA sRNA Workbench showing predicted loci with positions of sRNAs mapped onto it which includes both phased and unphasedsRNAs.

(a)

(b)

(c)

(d)

Online Resource S7 A total of 55, 299, 86 and 213 loci were detected in S-Mmi, S-PImi, R-Mmi and R-PImirespectively using non-stringent criteria (p-value threshold= 0.001). Multiple sequence alignmentof detected TAS from each library was performed to construct phylogenetic trees to know the relatedness of the sequences TAS identified from S-Mmi (a), S-Pmi (b), R-Mmi (c), R-PImi. (d)

Online Resource S8 Circular cladogram showing number of TAS detected in the four libraries

using non-stringent criteria (P-value threshold= 0.001). Shaded areas represent coding

TAS. S-Mmi (a), S-Pmi (b), R-Mmi (c), R-PImi (d)

Online Resource S9 WEGO plot showing GO based comparison of predicted TAS derived from

S-Mmi and S-PImi libraries (a), R-Mmi and R-PImi libraries (b)

Online Resource S10 Ethidium bromide stained gel image showing PCR amplification of approximately 500bp. Lane 1: PCR using gene specific primer (RACE NP1 and mRQ3' primer;details in Supporting Information Table S1); Lane 2: PCR using M13 universal forward and reverse primer pair; Lane 3: Molecular weight marker (NEX-GEN DNA Ladder; 100bp; PUREGENE )

Online Resource S11 Localization of SMK539 sequence on wheat chromosome arms

Online Resource S12 (a) Expression ratios (log10) and standard deviation values for ta-siRNA and target genes

Sample name

Target nameGLI TMP GST FAD LRR

SM0 hpi 0 0 0±0.07 0 0SPI12 hpi 1.99±0.09 7.19±0.40 2.36±1.5 0.04±0.03 0.97±0.02SPI24 hpi 0.26±0.04 4.25±0.05 1.79±0.02 0.17±0.00 -0.57±0.05SPI48 hpi -0.68±0.04 0.5±0.40 2.21±0.49 0.12±0.27 0.47±0.21SPI72 hpi -0.76±0.05 1.6±0.05 1.37±0.35 0.46±0.30 -0.26±0.05SPI168 hpi -1.22±0.10 0.64±0.71 1±0.5 0.1±0.18 -0.09±0.00RM0 hpi 5.85±0.42 5.52±1.06 4.55±0.9 0.5±0.18 1.42±0.37RPI12 hpi -0.69±0.13 3.6±0.05 8.6±0.03 1.28±0.25 -4.69±0.37RPI24 hpi 0 3.88±0.61 3.63±0.59 -0.17±0.06 1.01±0.31RPI48 hpi 0 1.6±0 1.52±0.31 -0.47±0.03 0.29±0.10RPI72 hpi 0 3.17±0.33 2.89±1.15 0±0 1.14±0.21RPI168 hpi 0 1.71±0.30 1.77±0.21 0.26±0.02 0.66±0.08SM: Susceptible mock-inoculated; SPI: Susceptible pathogen-inoculated; RM: Resistant mock-inoculated; RPI: Resistant pathogen-inoculated; hpi: hours post inoculation; ±values are standard deviations; n=3

Online Resource S12 (b) Expression ratios (log10) and standard deviation values for TAS,ta-siRNAs and TamiR16(-)

Sample name TAS s1 s2 s4(+) s4(-) TamiR16(-)

SM0 hpi 0 0 0 0 0 0

SPI12 hpi 0.66±0.08 -0.01±0.005 -0.26±0.04-

0.04±0.03 -0.02±0.10.46±0.40

SPI24 hpi 1.67 ±0.03 1.04±0.1 -0.31±0.1 0.3±0.02 -0.1±0.065 0.38±0.05SPI48 hpi 3.4±0.08 1.43±0.01 0.25±0.03 0.4±0.1 -0.82±0.18 0.48±0.40SPI72 hpi 1.6±0.51 4.47±0.07 0.23±0.03 1.89±0.12 -0.35±-0.09 0.96±0.05SPI168 hpi 0.5±0.28 4.89±0.4 0.28±-0.2 2.73±0.09 -3.25±0.30 0.94±0.7

RM0 hpi -1.5±0.42 -5±0.89 -1.7±0.04-

0.82±0.08 -0.7±0.320.78±0.07

RPI12 hpi 3.89±0.006 -2.88±0.25 -0.74±0.12 1.07±0.23 0.6±0.04 0.54±0.05RPI24 hpi 3.63±0.03 3.15±0.71 -0.42±0.03 1.72±0.08 0±0 0.94±0.60RPI48 hpi -0.13±0.06 3.2±1.09 -0.28±0 2.07±0.04 -2.6±0.09 0.73±0RPI72 hpi -0.82±0.09 6.11±0.6 0±0.3 2.76±0.44 -3.3±0.31 0.12±0.35RP I68 hpi 0±0.21 6.46±0.18 0.86±0.07 2.87±0.37 -3.1±0.36 0.58±0.20SM: Susceptible mock-inoculated; SPI: Susceptible pathogen-inoculated; RM: Resistant mock-inoculated; RPI: Resistant pathogen-inoculated; hpi: hours post inoculation; ±values are standard deviations; n=3

Online Resource S12 (c) P-values obtained through one-sample t-test:

Sample name

S-Mmi vs S-PImi RM vs R-PImi S-PImi vs R-PImi

TAS 0.035 0.289 0.606GLI 0.539 0.008 0.371TMP 0.202 0.012 0.937GST 0.133 0.01 0.781LRR 0.108 0.082 0.567FAD 0.386 0.028 0.053S1 0.237 0.567 0.060S2 0.018 0.430 0.049S4 0.106 0.123 0.052S4r 0.154 0.430 0.049

Online Resource S12 (d) Ct values all reference, target genes and ta-siRNAs obtained from real time PCR data

Sample name GAPDH (Reference)

TAS GLI TMP GST FAD LRR

SPI0 hpi 17.24 20.58 18.71 6.37 11 13.48 12.57SPI12 hpi 13.84 19.38 8.69 26.85 15.47 10.2 12.4SPI24 hpi 17.63 26.47 19.99 20.88 17.37 14.47 11.05SPI48 hpi 24.66 39.45 23.88 15.45 25.77 21.4 21.56SPI72 hpi 20.16 28.82 19.09 14.63 18.5 17.96 14.61SPI168 hpi 21.15 26.14 18.57 12.42 18.25 17.73 16.16RPI 0hpi 23.67 21.95 5.67 31.15 32.57 21.61 23.71RPI12 hpi 35 49.53 34.18 36.08 28.79 35.48 14.34RPI24 hpi 32.72 35.62 34.19 34.74 38.55 28.38 31.42RPI48 hpi 21.85 38.13 23.32 16.32 20.68 16.54 16.18RPI72 hpi 30.15 30.73 31.62 29.82 33.52 26.39 29.28RPI168 hpi 23.89 27.24 25.36 18.7 23.56 21 21.43

SPI: Susceptible pathogen-inoculated; RPI: Resistant pathogen-inoculated; hpi: hours post inoculation

Sample name GAPDH (Reference)

TAS GLI TMP GST FAD LRR

SM0 hpi(calibrator)

17.24 20.58 18.71 6.37 11 13.48 12.57

SM12 hpi 17.81 21.80 17.62 5.39 10.59 13.39 12.23SM24 hpi 21.63 21.83 17.78 5.89 10.99 13.42 12.41SM48 hpi 21.31 21.31 17.91 5.92 10.83 12.99 12.59SM72 hpi 20.75 21.75 18.69 6.23 11.12 13.27 12.53SM168 hpi 21.09 21.71 18.57 6.18 10.79 13.49 12.22RM0 hpi 23.67 21.95 5.67 31.15 32.57 21.61 23.71RM12 hpi 12.73 20.81 5.23 29.97 31.79 21.11 23.33RM24 hpi 26.62 21.35 5.55 30.23 31.99 20.59 23.61RM48 hpi 21.11 20.23 5.10 30.75 32.33 20.78 23.19RM72 hpi 29.87 21.37 5.39 30.95 33.42 21.65 23.83RM168 hpi 22.32 21.61 5.60 31.00 32.51 21.53 22.91

SM: Susceptible mock-inoculated; RM: Resistant mock-inoculated; hpi: hours post inoculation

Sample name

U6(Reference)

s1 s2 s4(+) s4(-) TamiR16(-)

SPI0 hpi 28.37 28.72 29.47 32.38 24.28 21.57SPI12 hpi 13.74 14.04 13.98 9.87 9.57 8.48SPI24 hpi 20.28 24.08 20.35 17.37 15.85 14.75SPI48 hpi 16.88 21.98 17.15 14.21 10.04 11.68SPI72 hpi 14.45 29.66 14.79 16.72 9.18 10.77SPI168 hpi 16.46 33.03 16.62 21.53 1.57 12.8RPI0 hpi 16.81 7.71 20.34 23.63 18.41 12.61RPI12 hpi 17.5 8.28 16.12 17.07 15.45 12.5RPI24 hpi 15.81 26.63 15.52 17.51 11.72 12.15RPI48 hpi 24.39 35.38 24.55 27.26 28.94 20.04RPI72 hpi 19.78 40.47 20.88 24.97 4.7 8.22RPI168 hpi 18.75 40.62 22.74 24.28 4.21 9.93SPI: Susceptible pathogen-inoculated; RPI: Resistant pathogen-inoculated; hpi: hours post inoculation

Sample name

U6(Reference)

s1 s2 s4(+) s4(-) TamiR16(-)

SM0 hpi(Calibrator)

26.81 28.72 29.47 32.38 24.28 21.57

SM12 hpi 23.00 28.37 29.43 32.33 24.23 8.48SM24 hpi 20.41 28.61 29.52 31.98 24.39 14.75SM48 hpi 26.20 28.75 28.79 32.36 22.38 11.68SM72 hpi 29.43 28.42 28.98 32.39 23.38 10.77SM168 hpi 25.51 29.11 29.99 32.42 24.83 12.8RM0 hpi 14.73 7.71 20.34 23.63 18.18 12.61RM12 hpi 16.85 6.97 20.62 23.53 18.20 12.5RM24 hpi 15.50 7.23 20.44 23.51 18,19 12.15RM48 hpi 22.10 7.76 21.01 23.56 18.17 20.04RM72 hpi 11.20 7.21 20.11 23.54 18.12 8.22RM168 hpi 15.50 7.69 21.24 23.55 18.15 9.93SM: Susceptible mock-inoculated; RM: Resistant mock-inoculated; hpi: hours post inoculation