Dynamic and Coordinated Expression Changes of Rice Small RNAs in Response to Xanthomonas oryzae pv. oryzae

Accepted Manuscript Dynamic and coordinated expression changes of rice small RNAs in response to Xanthomonas oryzae pv. oryzae Ying-Tao Zhao, Meng Wang, Zhi-Min Wang, Rong-Xiang Fang, Xiu-Jie Wang, YanTao Jia PII:

S1673-8527(15)00137-X

DOI:

10.1016/j.jgg.2015.08.001

Reference:

JGG 389

To appear in:

Journal of Genetics and Genomics

Received Date: 8 June 2015 Revised Date:

4 August 2015

Accepted Date: 7 August 2015

Please cite this article as: Zhao, Y.-T., Wang, M., Wang, Z.-M., Fang, R.-X., Wang, X.-J., Jia, Y.-T., Dynamic and coordinated expression changes of rice small RNAs in response to Xanthomonas oryzae pv. oryzae, Journal of Genetics and Genomics (2015), doi: 10.1016/j.jgg.2015.08.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Dynamic and coordinated expression changes of rice small

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RNAs in response to Xanthomonas oryzae pv. oryzae

3 Ying-Tao Zhao1*, Meng Wang1*, Zhi-Min Wang1, 2*, Rong-Xiang Fang 3, Xiu-Jie

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Wang1#, Yan-Tao Jia3#

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Biology, Chinese Academy of Sciences, Beijing 100101, China

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Academy of Sciences, Beijing 100101, China

State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental University of Chinese Academy of Sciences, Beijing 100049, China

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* These authors contributed equally to this work.

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Corresponding authors:

15 Yan-Tao Jia

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Tel: 86-10-64861838

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Fax: 86-10-64858245

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E-mail: [email protected]

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State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese

Xiu-Jie Wang

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Tel: 86-10-64806590

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Fax: 86-10-64873428

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E-mail: [email protected]

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The number of text pages: 29

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The number of figures: 6

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The number of tables: 1

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The number of words: 7745

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Abbreviations

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Xoo, Xanthomonas oryzae pv. Oryzae; hpi, hours post infection; ta-siRNAs,

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trans-acting siRNAs; ROS, Reactive Oxygen Species; LRR, Leucine-Rich Repeat;

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GA, gibberellin; BR, Brassinosteroid. 1

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Abstract

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Endogenous small RNAs are newly identified players in plant immune responses, yet

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their roles in rice (Oryza sativa) responding to pathogens are still less understood,

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especially for pathogens that can cause severe yield losses. We examined the small

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RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of

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Xanthomonas oryzae pv. oryzae (Xoo) virulent strain PXO99, the causal agent of rice

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bacterial blight disease. Dynamic expression changes of some miRNAs and

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trans-acting siRNAs were identified, together with a few novel miRNA targets,

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including an RLK gene targeted by osa-miR159a.1. Coordinated expression changes

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were observed among some small RNAs in response to Xoo infection, with small

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RNAs exhibiting the same expression pattern tended to regulate genes in the same or

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related signaling pathways, including auxin and GA signaling pathways, nutrition and

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defense related pathways. These findings reveal the dynamic and complex roles of

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small RNAs in rice-Xoo interactions, and identified new targets for regulating plant

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responses to Xoo.

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Keywords

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Small RNA, Rice bacterial blight disease, osa-miR159

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1. Introduction Small RNAs are important regulators that modulate gene expression at both

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transcriptional and posttranscriptional levels. MicroRNAs (miRNAs) and small

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interfering RNAs (siRNAs), which differ in their biogenesis and functions, are two

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major categories of plant endogenous small RNAs (Axtell, 2013; Li et al., 2014a; Li

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and Zhou, 2013; Wu, 2013). Plant miRNAs are approximately 21-nucleotides (nt) in

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length and are produced from hairpin-shaped RNA precursors; whereas plant siRNAs

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are usually 21- to 24-nt long and generated from double-stranded RNA molecules.

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According to their origins, plant siRNAs can be divided into several categories,

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including non-miRNA hairpin-derived small RNAs, heterochromatic siRNAs,

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trans-acting siRNAs (ta-siRNAs), phased siRNAs, natural antisense siRNAs, and

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millions of unclassified small RNAs (Axtell, 2013).

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Plants have evolved a complicated and efficient immune system to respond to

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bacterial infections, in which small RNAs are indispensable key players (Boccara et

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al., 2014; Katiyar-Agarwal and Jin, 2010; Li et al., 2012; Liu et al., 2014; Pumplin

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and Voinnet, 2013; Ruiz-Ferrer and Voinnet, 2009; Seo et al., 2013). In Arabidopsis

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(Arabidopsis thaliana), bacterial infection can induce the expressions of ath-miR393,

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ath-miR160, and ath-miR167 (Fahlgren et al., 2007; Navarro et al., 2006), and repress

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the expression of ath-miR398 (Jagadeeswaran et al., 2009), in accompany with the

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opposite expression changes of their target genes. Osa-miR160 and osa-miR398 are

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also important for rice (Oryza sativa) immunity against the blast fungus (Li et al.,

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2014b). Bacterial infection in Arabidopsis also induces the expression of two other

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types of endogenous small RNAs, the natural antisense siRNAs and the long siRNAs

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(Katiyar-Agarwal et al., 2006; Katiyar-Agarwal et al., 2007). Recently, a novel

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category of siRNAs, the phased siRNAs that derived from host disease resistance

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genes, was identified in multiple plant species with important functions in

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plant-bacteria interactions (Fei et al., 2013; Kallman et al., 2013; Shivaprasad et al.,

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2012; Zhai et al., 2011). Intriguingly, bacteria could directly inject effector proteins

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into Arabidopsis cells to repress the biogenesis, stability and activity of the host

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miRNAs (Navarro et al., 2008; Qiao et al., 2013; Weiberg et al., 2013). Xanthomonas oryzae pv. oryzae (Xoo) is the causal pathogen of rice bacterial

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blight, one of the most destructive bacterial diseases with severe yield losses in

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irrigated rice (Gnanamanickam et al., 1999). Previous studies on rice responding to

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Xoo infections were predominantly focused on protein-coding genes (Bart et al.,

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2010; Deng et al., 2012; Ding et al., 2012; Fitzgerald et al., 2005; Gomi et al., 2010;

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Jiang et al., 2013; Lee et al., 2009; Li et al., 2006; Shen et al., 2010; Sun et al., 2004;

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Tao et al., 2009; Yuan et al., 2010), whereas the roles of rice small RNAs in this

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process are still poorly understood. To investigate the functions of small RNAs in

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rice-Xoo interactions, we profiled the expression patterns of small RNAs in the leaves

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of Xoo-infected and mock-inoculated rice using high-throughput sequencing

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technology. We found that different groups of rice small RNAs were dynamically

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regulated at specific time points during the first 24 hours post Xoo infection.

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Especially, some coordinately expressed miRNAs and ta-siRNAs involved in the

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regulation of genes in the same or related signaling pathways. We also identified and

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validated that a RLK gene could be directly targeted by osa-miR159a.1 after Xoo

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infection.

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2. Results

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2.1 Expression profiles of rice small RNAs after Xoo infection

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To explore the roles of small RNAs in the rice-bacteria interactions, we

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systematically examined the expression profiles of rice small RNAs during the first 24

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hours after infection with Xoo PXO99, the causal pathogen of bacterial blight on rice

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leaves. RNA samples were collected from the pathogen-infected and mock leaves

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(treated by water) at 2, 6, 12, and 24 hours post infection (hpi), respectively. Small

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RNAs were isolated from each sample and sequenced by the Illumina sequencing

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technology. After removing adaptors and low quality reads, a total of 91,685,066 4

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reads with lengths of 18- to 30-nt were obtained (Fig. S1A), representing

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approximately 10 million high quality reads from each library. Consistent with previous reports in rice (Du et al., 2011; Jeong et al., 2011;

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Rodrigues et al., 2013; Song et al., 2012a; Song et al., 2012b; Wei et al., 2011; Zhai et

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al., 2013), 24-nt small RNAs were the most abundant population in both the total

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reads and the non-redundant sequences, and the 21- and 22-nt small RNAs were the

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second largest populations (Fig. S1B). The expression abundance of small RNAs

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varied a lot within the population, with ~77% of the non-redundant sequences in each

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library were sequenced only once, whereas ~3% of the non-redundant sequences,

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which accounted for ~70% of the total reads, were sequenced 10 or more times (Fig.

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1A and 1B).

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Genomic comparison revealed that ~90% of the total small RNAs had perfect

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matches on the rice genome (Fig. S1A). To further classify these mapped small

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RNAs, we compared their mapping locations with the rice genomic annotations.

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About 20% sequenced small RNAs were known rice miRNAs, and the rest were

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mainly derived from rRNAs, repeat regions, and the sense or antisense strand of

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protein coding genes (Fig. 1C). In concert with previous findings, 89% of small RNAs

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derived from miRNA genes were 21- or 22-nt in length, whereas 73% repeat-region

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derived small RNAs were 24-nt long (Fig. 1D).

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2.2 Dynamic expression alterations of rice small RNAs in response to Xoo

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infection

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To identify small RNAs that may involve in Xoo infection response, we

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compared the small RNA expression profiles of Xoo-infected rice with those of the

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controls. To ensure the reliability of the comparison results, only small RNAs with 10

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or more reads in at least one library were included in the analysis. About 10-14% of

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small RNAs were differentially regulated at different time points after Xoo infection

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(13.8% at 2 hpi, 13.6% at 6 hpi, 10.0% at 12 hpi, and 10.7% at 24 hpi, respectively) 5

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(Fig. 2A). Notably, the amount of small RNAs induced by Xoo was comparable

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among the four time points, whereas the amount of the repressed small RNAs was

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gradually decreased, especially at 12 hpi and 24 hpi (Fig. 2A). To further examine the dynamic expression changes of small RNAs, we focused

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on the 9586 highly expressed small RNAs with abundance ≥50 reads in all eight

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libraries. Among them, 1030, 810, 477, and 717 small RNAs were induced or

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repressed by Xoo infection at 2 hpi, 6 hpi, 12 hpi, and 24 hpi, respectively (Fig. 2B).

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In addition, about 4% of the differentially expressed small RNAs were repressed at 2

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hpi, but consistently induced at the later time points (Fig. 2B).

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2.3 MiRNAs and ta-siRNAs exhibited variable expression patterns in response to

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Xoo infection

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To investigate how small RNAs modulate plant immune system in response to

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Xoo infection, we first focused on the expression changes of miRNAs and trans-acting

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siRNAs (ta-siRNAs), which have been demonstrated to play critical roles in plant

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immune responses (Katiyar-Agarwal and Jin, 2010; Li et al., 2014b; Pumplin and

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Voinnet, 2013; Ruiz-Ferrer and Voinnet, 2009; Seo et al., 2013). Taken together, 509

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out of the 592 known rice miRNA precursors (miRBase release 20) and all known

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ta-siRNAs derived from the three known rice TAS genes were detected in our

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samples. In addition to the known miRNAs, we also found that a few miRNA

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isoforms and miRNA*s had higher or comparable read abundance than their

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corresponding miRNAs in our data sets (Table S1), indicating that they may have

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more important functions than their corresponding miRNAs under our examined

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conditions. For example, the expression of osa-miR397b was almost undetectable in

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both the control and Xoo infected samples, whereas six of its isoforms with shorten or

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shifted sequences had thousands of reads in most samples (Fig. S2). Comparing with

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the public AGO protein association data of rice small RNAs, osa-miR393b-3p, the

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pairing sequence of osa-miR393b, also had higher AGO association than

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osa-miR393b (Table S2). We next analyzed the expression changes of abundant small RNAs (with 40 or

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more reads in at least one library) mapped to miRNA and ta-siRNA precursors. Using

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1.5-fold expression change at one or more examined time points, 48 mature miRNAs,

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miRNA isoforms or miRNA*s, and ta-siRNAs were identified as differentially

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expressed between the Xoo-infected rice and controls (Table S3). These differentially

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expressed small RNAs were further classified into four classes based on their

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temporal expression dynamics (Fig. 3A). The expressions of small RNAs in cluster I,

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including osa-miR160, osa-miR167, osa-miR827, osa-miR393, osa-miR393b-3p and

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TAS3b (D6+), were rapidly induced by Xoo infection at 2 hpi, but the induction was

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gradually tapered off at the sequential time points, and completely repressed at 24 hpi

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(Fig. 3A). Such expression differences were also confirmed by quantitative RT-PCR

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and northern-blot hybridization (Fig. 3B and 3C), indicating the fidelity of the

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sequencing results. Small RNAs in cluster II, mainly comprised of miRNA397,

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miR397* and their isoforms, as well as miR1432 and its isoforms, were specifically

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induced at 6 hpi, but without obvious expression changes at other time points (Fig.

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3A). The expression profile of small RNAs in cluster III was in contrast to those in

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cluster I, which were repressed by at 2 hpi but consistently induced at 6, 12, and 24

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hpi (Fig. 3A).

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miR166a and miR398b, were almost repressed at all examined time points (Fig. 3A).

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It is worth to note that miR398b and miR398b* showed different expression patterns

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(Fig. 3A), indicating that they might play different roles in the Xoo resistant response.

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The IV cluster of small RNAs, including miR159a and its isoform,

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2.4 MiRNAs and ta-siRNAs with same expression patterns tend to function

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synergistically on the same or related signaling pathways

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Using previously reported plant miRNA target prediction criteria (Allen et al.,

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2005; Zhao et al., 2012), we identified 95 putative targets (Table S4) for the 7

ACCEPTED MANUSCRIPT differentially expressed miRNAs and ta-siRNAs. Most of the predicted targets were

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homologous to the known targets of Arabidopsis miRNAs and ta-siRNAs. The

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predicted targets of miR160 and TAS3b were validated by 5′-RNA ligase-mediated

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(RLM)-RACE experiments. Direct cleavage on the mRNAs of OsARF18

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(Os06g47150) by osa-miR160 and on the mRNAs of OsARF15 (Os05g48870) by rice

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TAS3b (D6+) were detected (Fig. 4), which were consistent with previous reports (Li

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et al., 2010; Wu et al., 2009; Zhou et al., 2010). Similarly, we also confirmed the

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miRNA::target relationships between osa-miR393b and auxin receptors (OsTIR1,

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Os05g05800 and OsAFB2, Os04g32460) (Xia et al., 2012; Zhou and Wang, 2013), as

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well as for osa-miR398b and a gene encoding Copper/zinc superoxide dismutase

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(OsCSD1, Os03g11960) (Fig. 4). Functional analysis for these target genes revealed

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that they were enriched in biological processes related to plant immune system, such

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as stimulus responses, regulation of cell death, and several signaling pathways (Fig.

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5B).

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Intriguingly, we found that miRNAs and ta-siRNAs with similar expression

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patterns tend to regulate genes involved in the same or related signaling pathways

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(Table S4). For example, sixty percent (12 of 20) of small RNAs in cluster I targeted

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genes involved in the auxin signaling pathway, including ARFs and TIRs (Fig. 4,

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Table 1 and S4). Furthermore, we found that the expression patterns of these target

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genes were anti-correlated with the expression patterns of miRNAs and ta-siRNAs

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(Fig. 3A and 5A). For example, contrary to the expression pattern of osa-miR160

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(Fig. 3A and 3B), the mRNA levels of its two targets, OsARF18 and OsARF16

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(Os10g33940), were consistently repressed at 2, 6, and 12 hpi, but significantly

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increased at 24 hpi in Xoo-infected rice (Fig. 5A). Similar anti-correlated expression

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patterns were also observed for osa-miR167, osa-miR827, TAS3b and their targets

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(Fig. 5A). In addition to auxin responsive genes, small RNAs in cluster I also targeted

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several transporter genes, such as OsMATE1, OsSPX-MFS1, OsSPX-MFS2, OsST1,

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and OsST2 (Table 1), which might function synergistically with the auxin pathway to

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repress pathogen growth in rice. Similarly, osa-miR398b in cluster IV targeted genes involved in reactive oxygen

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species (ROS) pathway. The predicted targets of osa-miR398b were OsCSD1,

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OsCSD2, and OsCOX5b.1 (Table S4), which are orthologs of ath-miR398 targets in

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Arabidopsis (Beauclair et al., 2010; Dugas and Bartel, 2008; Jones and Takemoto,

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2004; Li et al., 2010; Sunkar et al., 2006; Yamasaki et al., 2007). Here we confirmed

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that OsCSD1 was targeted by osa-miR398b. Except for OsCSD1, several OsCSD

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genes have been reported to be targeted by miR398b (Li et al., 2014b). As

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antioxidants, CSDs can protect plants from pathogen induced ROS damage (Heller

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and Tudzynski, 2011; Mittler et al., 2004).

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2.5 MiR159 directly targeted GAMYB and a Leucine-Rich Repeat (LRR) protein

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Osa-miR159a.1 was another Xoo repressed miRNA. Consistent with previous

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reports (Tsuji et al., 2006; Wu et al., 2009), the target relationships between

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Osa-miR159a.1 and GAMYB transcription factor (OsGAMYB1, Os01g59660) were

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detected in our samples (Fig. 3A and 4). GAMYB is an important transcriptional

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factor for gibberellin (GA) signaling pathway which regulate rice development and

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immunity (Bari and Jones, 2009; Yang et al., 2013). When the expression of

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osa-miR159a.1 was decreased at 24 hpi (Fig. 3A), the expression of OsGAMYB1 was

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increased at the same time point (Fig. 5A).

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Intriguingly, we predicted and confirmed that OsLRR-RLK2 (Os12g10740),

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which encodes a protein with leucine-rich repeat and a receptor-like kinase domain,

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was an authentic target of osa-miR159a.1 in rice (Fig. 4). Furthermore, we showed

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that the expression of OsLRR-RLK2 was induced at 24 hpi when osa-miR159a.1 was

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repressed at the same time point (Fig. 3A and 5A), demonstrating the effective

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regulation of OsLRR-RLK2 expression by osa-miR159a.1 under Xoo infection. LRR 9

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domain-containing proteins are central players in the plant immune system (Dangl

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and Jones, 2001; Dodds and Rathjen, 2010; Jaillais et al., 2011; Jones and Takemoto,

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2004; Kobe and Kajava, 2001). Thus, osa-miR159a.1 may directly regulate rice

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pathogen responses through targeting OsLRR-RLK2.

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3. Discussion

Bacterial blight, a serious disease of rice, is caused by Xoo infection and results

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in severe yield losses. As small RNAs have been proven to play crucial roles in

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diverse physiological and pathological processes in plants, understanding the roles of

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rice small RNAs in bacterial blight disease progress will shed light on the cultivation

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of rice cultivars with resistance to Xoo infection. In this study, to illuminate the

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functions of small RNAs responding to Xoo infection at early stages, we

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systematically examined the sequential expression profiles of rice small RNAs after

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Xoo infection by high-throughput sequencing. We observed the dynamic expression

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changes of small RNAs at four time points after Xoo infection, identified 48 miRNAs,

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miRNA isoforms, and ta-siRNAs that were differentially expressed in Xoo-infected

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rice, and also found the synergetic functions of miRNA in gene regulatory network.

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A previous study has shown that the progression of bacterial blight in rice can be

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dissected into multiple stages with distinct symptoms (Adhikari et al., 1994),

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reflecting the dynamic responses of rice after Xoo infection. Here, we have for the

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first time provided the expression profiles of small RNAs in rice leaves within the

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first 24 hours after Xoo infection, which will serve as a resource for the identification

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of functions small RNAs involving in rice pathogen responses. Majority of the small

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RNAs with differential expression between the control and Xoo infected rice

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exhibited varied abundance at different post infection time points, in addition, small

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RNAs regulating the same or related signaling pathways tended to have similar

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expression patterns, indicating their active functions in regulating pathogen responses

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(Fig. 6). For example, small RNAs in cluster I predominantly (60%) regulate genes

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ACCEPTED MANUSCRIPT involved in the auxin pathway (Fig. 3, 4 and 5; Table 1). Auxin has been reported to

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play key roles in plant-pathogen interactions through modulating the pathogen growth

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(Bari and Jones, 2009; Ding et al., 2008; Domingo et al., 2009; Kazan and Manners,

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2009; Navarro et al., 2006; Robert-Seilaniantz et al., 2011; Truman et al., 2010; Wang

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et al., 2007), and ARFs could function as either activators or repressors in certain cell

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types or environments (Guilfoyle and Hagen, 2007). Moreover, Transport Inhibitor

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Response 1 (TIR1) family of auxin receptors was directly regulated by miR393 that

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also plays important roles in plant-pathogen interactions (Navarro et al., 2006).

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Intriguingly, osa-miR393b-3p, the pairing sequence of osa-miR393b in cluster I

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was also rapidly induced by Xoo infection at 2 hpi (Fig. 3A). In Arabidopsis,

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ath-miR393b-3p is associated with AGO2 and targets three membrane trafficking

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genes, including MEMBRIN 12 (MEMB12) which is a negative regulator of

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Pathogenesis-Related 1 (PR1) secretion (Pumplin and Voinnet, 2013; Zhang et al.,

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2011). Although the sequences of miR393b-3p are different in rice and Arabidopsis,

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osa-miR393b-3p was also predicted to target OsMEMB12 (Table S4). The

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co-evolutionary target relationship of miR393b-3p and MEMB12 indicated that

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miR393b-3p might be important to plant disease resistance (Fig. 6).

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Unlike miRNAs in cluster I, most miRNAs in cluster II were mainly induced at 6

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hpi (Fig. 3A) and have been reported to be involved in abiotic stresses (Khraiwesh et

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al., 2012; Gielen et al., 2012; Guleria et al., 2011). For example, dramatic expression

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changes of miR397 and miR408 had been observed under drought and metal stresses

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(Gielen et al., 2012; Khraiwesh et al., 2012). In addition, osa-miR397 regulates the

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Brassinosteroid (BR) signaling pathway by targeting OsLAC (Zhang et al., 2013). As

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BR has been found to modulate plant immunity (Albrecht et al., 2012; Belkhadir et al.,

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2012; Nakashita et al., 2003; Wang, 2012), the induced expression of miR397 under

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Xoo infection indicates that miR397 might play important roles in plant disease

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responses (Fig. 6).

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ACCEPTED MANUSCRIPT Besides Xoo induced miRNAs, we also identified several miRNAs with

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repressed expression after Xoo infection, such as miR159a.1 and miR398b (Fig. 3A).

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Among them, miR159a.1 was confirmed to regulate one LRR protein kinase,

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OsLRR-RLK2 (Fig. 4 and 5A). It is well known that LRR protein kinases are essential

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for plant disease response (Dodds and Rathjen, 2010; Jaillais et al., 2011).

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Osa-miR159a.1 may be directly involved in rice pathogen responses through

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regulating OsLRR-RLK2 (Fig. 6). Osa-miR398b is another Xoo repressed miRNA that

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was reported to negatively regulate responses against bacterial pathogens

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(Jagadeeswaran et al., 2009; Li et al., 2010). However, osa-miR398b was increased by

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blast fungus and is a positively regulator for fungal pathogens in rice (Li et al., 2014b).

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Thus, osa-miR398b may regulate rice innate immunity in the contrary manners for

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bacterial and fungal pathogens. Several OsCSD genes involved in ROS pathway were

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reported to be targeted by miR398b except for OsCSD1 (Fig. 4) (Li et al., 2014b).

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Different from miR398b, miR398* in cluster III increased by Xoo infection was

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predicted to target OsRAPTOR1 (Fig. 3A and Table S4). The RAPTOR proteins are

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binding partners of the target of rapamycin (TOR) kinase, which is important for cell

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growth and nutrient responses (Dobrenel et al., 2011; Dobrenel et al., 2013). Thus,

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miR398b and miR398* may regulate rice pathogen responses in different manners.

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In addition, we also identified reversed expression changes between osa-miR827

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and its target genes, OsSPX-MFS1 and OsSPX-MFS2 (Lin et al., 2010), after Xoo

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infection. The OsSPX-MFS1 and OsSPX-MFS2 proteins belong to Major Facilitator

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Superfamily (MFS) secondary transporter family and contain a domain homologous

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to the sugar transporter domain (pfam00083 domain). Plenty of studies have shown

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that the MFS transporters directly involve in the host-pathogen interactions in

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Arabidopsis and Maize (Pao et al., 1998; Marger and Saier, 1993; Saier et al., 1999;

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Sa-Correia and Tenreiro, 2002; Simmons et al., 2003; Remy et al., 2013). Moreover,

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another group of sugar transporters, the SWEET family protein, has been shown to

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involve in the intercellular exchange and to provide nutrition for pathogens in rice

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ACCEPTED MANUSCRIPT (Chen et al., 2010). Although little is known about the roles of OsSPX-MFS1 and

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OsSPX-MFS2 in pathogen response in rice, the expression patterns of these genes

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revealed in our study indicated that they may function like the SWEET sugar

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transporters to involve in the nutrition support regulation of Xoo. It will be interesting

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to generate further functional analysis on these proteins to illuminate the overlapping

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networks between nutrition and pathogen infections.

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In conclusion, the present study provides the first small RNA expression analysis

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at the early stages of bacterial blight disease progress in rice. In contrast to the

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control, the small RNAs changes in compatible interactions between Xoo and rice

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revealed that some small RNAs clustered together to modulate the phytohormone and

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defense-related pathways. The expression analysis of small RNAs’ target genes at

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different time points provided clues for their roles in responding to Xoo infection in

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rice leaves. The findings on the Xoo-responsive small RNAs and their target genes

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might expand our understanding of the mechanism of rice-Xoo interactions, which

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provides a new strategy to generate disease-resistant plants.

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4. Materials and methods

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4.1 Sample collection, RNA isolation, and small RNA sequencing Japonica rice varieties (Oryza sativa, Nipponbare) were grown in the

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experimental fields in Beijing. The leaves of 40-day-old rice seedling were infected

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by Xoo PXO99 and mock-inoculated with water. Parts of the leaves, which were

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below the infected locations 1 cm in length, were collected at 2, 6, 12, and 24 hours

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post infection (hpi), respectively. The samples were collected from multiple plants

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and were pooled together to avoid the individual genotype differences. Total RNAs of

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each sample were isolated using Trizol reagent (Invitrogen), and small RNAs were

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collected and sequenced by the Illumina’s sequencing technology. The equal amount

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of small RNAs was used for library construction.

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4.2 Bioinformatics analysis of the deep sequencing data sets The adapter sequences were trimmed. Sequences with low phred quality scores

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or shorter than 18 nucleotides were removed. The total number of reads for each data

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set was normalized to 10 million. Total sequences of the eight data sets were aligned

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to the rice genomic sequences using the BLASTN (Altschul et al., 1990) program.

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The rice genomic sequences were downloaded from Rice Genome Annotation Project

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(RGAP) version 6.1. We also aligned these sequences to the rice miRNA precursors

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downloaded from the miRBase database release 20 (Kozomara and Griffiths-Jones,

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2011) and to the three rice TAS3 genes using the BLASTN program. Rice gene

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annotations were downloaded from the RGAP version 6.1. The transposons and

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repeat regions in rice genome were identified by RepeatMasker software. Target gene

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prediction for miRNAs and ta-siRNA was performed as previously described (Zhao et

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al., 2012).

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The statistical analyses and the data plotting were carried out in R. Expression

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comparison of small RNAs was performed at each time point separately. Small RNAs

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with at least 10 reads in both conditions were included in the expression comparison

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analysis. At least 1.5 fold expression change was used as the criterion to select

385

differentially expressed small RNAs. GOEAST (Zheng and Wang, 2008) was used to

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perform the Gene Ontology enrichment analysis. The NCBI Conserved Domain

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Database (Marchler-Bauer et al., 2013) was used to annotate the domains of proteins.

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4.3 Small RNA northern-blot hybridization Small RNA northern-blot hybridization was carried out as previously described

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(Zhao et al., 2012), using 20 µg of total RNAs in each experiment. Probes, which

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were labeled by [γ-32P] ATP, were the complementary sequences of the corresponding

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small RNAs. The sequences of all probes used in this study are listed in

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Supplementary Table S5.

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4.4 Quantitative RT-PCR Expression levels of small RNAs were analyzed by Quantitative RT-PCR

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experiments using All-in-One™ miRNA qRT-PCR Reagent Kits (GeneCopoeia). In

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the quantitative RT-PCR experiments of small RNAs, 1 µg of total RNAs treated with

400

DNase I (New England Biolabs) was used in each reaction with U6 snRNA as the

401

internal control. In the quantitative RT-PCR experiments for mRNAs, 1 µg of total

402

RNAs treated with DNase I (New England Biolabs) was synthesized to cDNA by

403

M-MuLV (New England Biolabs) using poly (dT) oligonucleotides with the mRNA of

404

OsNAB (Os06g11170) as the reference gene. SYBR® Green PCR Master Mix

405

(Applied Biosystems) was used in all quantitative RT-PCR experiments. The relative

406

expression fold changes of miRNAs, ta-siRNAs, and genes were calculated using the

407

2 δ-δ Ct method (Livak and Schmittgen, 2001). Primers used in all the quantitative

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RT-PCR experiments were listed in Supplementary Table S6.

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409 4.5 5′′-RLM-RACE

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The 5′-RLM-RACE experiments were carried out using the FirstChoice

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RLM-RACE Kit (Ambion) following the previously reported procedures (Zhao et al.,

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2012). All primers for specific target genes used in this study are listed in

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Supplementary Table S7.

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4.6 Accession numbers

All sequencing data generated in this study are available in the NCBI Gene

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Acknowledgements

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Expression Omnibus (GEO) database under the accession number GSE58385.

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This work was supported by the National Natural Science Foundation of China

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(grant No. 31371318 to M. W.), the National Basic Research Program of China (grant

15

ACCEPTED MANUSCRIPT 423

No. 2011CB100703 to Y.-T. J.), and the State Key Laboratory of Plant Genomics

424

(grant No. SKLPG2011B0105 to X.-J. W.).

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Sunkar R., Kapoor A., Zhu J.K., 2006. Posttranscriptional induction of two Cu/Zn

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Tao, Z., Liu, H., Qiu, D., Zhou, Y., Li, X., Xu, C., Wang, S., 2009. A pair of allelic

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WRKY genes play opposite roles in rice-bacteria interactions. Plant Physiol. 151,

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Truman W.M., Bennett M.H., Turnbull C.G., Grant M.R., 2010. Arabidopsis auxin

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mutants are compromised in systemic acquired resistance and exhibit aberrant

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accumulation of various indolic compounds. Plant Physiol. 152, 1562-1573.

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GAMYB controls different sets of genes and is differentially regulated by microRNA

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Wang D., Pajerowska-Mukhtar K., Culler A.H., Dong X., 2007. Salicylic acid inhibits

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Wei, L.Q., Yan, L.F., Wang, T., 2011. Deep sequencing on genome-wide scale

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pollen of Oryza sativa. Genome Biol. 12, R53.

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Weiberg, A., Wang, M., Lin, F.M., Zhao, H., Zhang, Z., Kaloshian, I., Huang, H.D.,

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interference pathways. Science 342, 118-123.

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ACCEPTED MANUSCRIPT Wu, G., 2013. Plant microRNAs and development. Journal of genetics and genomics

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Wu, L., Zhang, Q., Zhou, H., Ni, F., Wu, X., Qi, Y., 2009. Rice MicroRNA effector

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complexes and targets. Plant Cell 21, 3421-3435.

699

Xia, K., Wang, R., Ou, X., Fang, Z., Tian, C., Duan, J., Wang, Y., Zhang, M., 2012.

700

OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more

701

tillers, early flowering and less tolerance to salt and drought in rice. PLoS One 7,

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e30039.

703

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Chem. 282, 16369-16378.

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Xanthomonas oryzae overcomes rice defenses by regulating host copper

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717

Cao X., Yu B., Timmermans M., Yang B., Chen X., Meyers BC., 2013. Plant

718

MicroRNAs Display Differential 3' Truncation and Tailing Modifications That Are

719

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721

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ACCEPTED MANUSCRIPT miRNA393( *)-mediated silencing of a Golgi-localized SNARE gene, MEMB12. Mol.

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Cell 42, 356-366.

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Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size

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731

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738

sequencing reveals endogenous small RNA targets in rice. Front Biol. 5, 67-90.

741 742 743 744 745 746

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723

747 748 749 750 27

ACCEPTED MANUSCRIPT

Figure legends

752

Fig. 1. Profiling of rice small RNAs after Xoo infection.

753

A: Abundance distribution of total sequenced reads in the eight libraries. B:

754

Distribution of reads with different abundance among non-redundant sequences. C:

755

Classification of total sequenced small RNAs with perfect matches on the rice

756

genome. D: Length distributions of small RNAs derived from different genomic

757

regions. nt, nucleotide.

RI PT

751

758

Fig. 2. Expression changes of the rice small RNAs after Xoo infection.

760

A: Scatter plots of small RNA read abundance in the Xoo-infected rice (Xoo) and the

761

controls (CK) at the four time points. Only small RNAs with 10 or more reads in at

762

least one library were included in these plots. B: Heatmap of small RNA expression

763

changes after Xoo infection by hierarchical clustering analysis. Only small RNAs with

764

at least 50 reads in each of the eight libraries were included in the analysis.

M AN U

SC

759

TE D

765

Fig. 3. Clustered expression of miRNAs and ta-siRNAs.

767

A: Clustering of expression changes of miRNAs and ta-siRNAs after Xoo-infection. B:

768

Quantitative RT-PCR analysis of differentially expressed miRNAs and ta-siRNAs in

769

Xoo-infected rice and the controls. Error bars indicate the standard deviation of three

770

replicates. C: Northern-blot hybridization analysis of differentially expressed

771

miRNAs in Xoo-infected rice and the controls.

AC C

772

EP

766

773

Fig. 4. Validations of selected target genes of miRNAs and ta-siRNA by

774

5′-RLM-RACE analysis of the cleavage sites.

775 776

Fig. 5. Expressions and functional analysis of selected genes targeted by

777

differentially expressed miRNAs and ta-siRNA after Xoo infection.

28

ACCEPTED MANUSCRIPT A: Quantitative RT-PCR analysis was performed on selected target genes after Xoo

779

infection. Error bars represent the standard deviation of three replicates. Expression

780

changes of the miRNAs and ta-siRNA are indicated by the heatmaps (also shown in

781

Figure 3A). B: Unbiased Gene Ontology enrichment analysis of target genes of the

782

differentially expressed miRNAs and ta-siRNAs in Xoo-infected rice.

RI PT

778

783

Fig. 6. Model of small RNA mediated pathways in response to Xoo infection.

785

The up-regulated miRNAs are shown in red, down-regulated miRNAs in green. The

786

solid lines indicate validated miRNA-target pairs and dashed lines indicate putative

787

miRNA-target pairs.

SC

784

M AN U

788 789 790 791

795 796 797 798 799 800 801

EP

794

AC C

793

TE D

792

802 803 804 805 806 807 29

ACCEPTED MANUSCRIPT

Supplementary data

809

Fig. S1. Statistics of the sequencing and mapping results and size distributions of the

810

rice small RNAs.

811

Fig. S2. Small RNAs mapped to the osa-miR397b precursor sequence.

812

Table S1. Reads of highly expressed miRNA isoforms and miRNA*s in our data sets.

813

Table S2. Reads of miRNAs and miRNA*s in the OsAGO1 associated small RNA

814

data sets.

815

Table S3. Differentially expressed miRNAs, miRNA*s, isoforms, and ta-siRNAs.

816

Table S4. Predicted target genes of the differentially expressed miRNAs and

817

ta-siRNAs in Xoo-infected rice, and the alignments between small RNAs and their

818

targeting sites.

819

Table S5. Probes used for the small RNA northern-blot hybridizations.

820

Table S6. Primers used for the quantitative RT-PCR experiments.

821

Table S7. Primers used for the 5′-RLM-RACE experiments.

AC C

EP

TE D

M AN U

SC

RI PT

808

30

ACCEPTED MANUSCRIPT

Tables Table 1. Putative target genes of miRNAs and ta-siRNAs in cluster I. Gene name

Locus ID

osa-miR160

OsARF18

Os06g47150

Auxin response factor 18

OsARF10

Os04g43910

Auxin response factor 10

OsARF8

Os02g41800

Auxin response factor 8

OsARF13

Os04g59430

Auxin response factor 13

OsARF16

Os10g33940

Auxin response factor 16

OsARF25

Os12g41950

Auxin response factor 25

OsARF17

Os06g46410

Auxin response factor 17

OsNBS-LRR1

Os07g29820

NBS-LRR disease resistance protein

OsARF2

Os01g48060

Auxin response factor 2

OsARF15

Os05g48870

Auxin response factor 15

OsARF3

Os01g54990

Auxin response factor 3

OsARF4

Os01g70270

Auxin response factor 4

OsARF14

Os05g43920

Auxin response factor 14

OsFBX32

Os01g69940

F-box domain containing protein 32

OsTIR1

Os05g05800

F-box domain and LRR containing protein

OsAFB2

Os04g32460

F-box domain and LRR containing protein

OsLRR-RI

Os03g09070

LRR domain containing protein

OsLRR-RLK1

Os03g51440

LRR receptor-like protein kinase

OsMEMB12

Os03g45260

Vesicle transport v-SNARE protein

osa-miR390

OsLRR-EXS

Os02g10100

LRR receptor protein kinase EXS precursor

osa-miR159a.2

OsAT-GTL1

Os03g02240

GT-2-like 1 transcription factor

OsAP2L1

Os03g60430

APETALA2-like transcription factor

OsAP2L2

Os05g03040

APETALA2-like transcription factor

OsAP2L3

Os07g13170

APETALA2-like transcription factor

OsAP2L4

Os04g55560

APETALA2-like transcription factor

OsAP2L5

Os06g43220

APETALA2-like transcription factor

OsMATE1

Os06g49310

MATE efflux family protein, transporter

OsSPX-MFS1

Os04g48390

SPX and major facilitator superfamily domain

osa-miR393b-3p

AC C

osa-miR172

TE D

osa-miR393

Auxin response factor 6

Auxin response factor 12

EP

osa-miR394

Os02g06910 Os04g57610

SC

TAS3 (D6+)

OsARF6 OsARF12

M AN U

osa-miR167

Gene annotation

RI PT

ID

osa-miR827

OsSPX-MFS2

containing protein, transporter Os02g45520

SPX and major facilitator superfamily domain containing protein, transporter

osa-miR395

OsST1

Os03g09930

Sulfate transporter

OsST2

Os03g09940

Sulfate transporter

ACCEPTED MANUSCRIPT

A

B

40% 20% 0%

80% 60% 40% 20% 0%

No. of reads per sequence

C

D 80%

SC

60%

% of non-redundant sequences

80%

CK-2hpi CK-6hpi CK-12hpi CK-24hpi Xoo-2hpi Xoo-6hpi Xoo-12hpi Xoo-24hpi

RI PT

100%

CK-2hpi CK-6hpi CK-12hpi CK-24hpi Xoo-2hpi Xoo-6hpi Xoo-12hpi Xoo-24hpi

M AN U

% of total reads

100%

No. of reads per sequence

miRNA

Gene

Gene antisense

Repeat

rRNA

Unclassified

2%

TE D

60%

miRNA

6%

14%

Repeat rRNA

6%

AC C

21%

20%

Gene antisense

EP

20% 31%

40%

Gene

tRNA

Unclassified

0

80% 60% 40% 20% 0 20

22

24

26 28

20

22

24

26

20

28

Length of sequence

(nt)

22

24

26

28

RI PT

ACCEPTED MANUSCRIPT

A

SC

2000

M AN U

No. of reads (Xoo)

4000

2000

0

AC C

2 hpi 6 hpi

12 hpi

24 hpi

6 hpi

12 hpi

24 hpi

2000 4000 0 No. of reads (CK)

EP

0

B

2 hpi

TE D

No. of reads (Xoo)

0 4000

2000 4000 No. of reads (CK) -3 0 3 Log2 fold change (Xoo/CK)

ACCEPTED MANUSCRIPT

A

B -1.5 0 1.5 Log2 fold change (Xoo/CK)

2 1

Ⅲ

Ⅳ 2 hpi

6 hpi 12 hpi 24 hpi

osaosa- TAS3b osamiR160a miR167 (D6+) miR827

SC

0

Relative expression (24 hpi)

TE D AC C

EP

Ⅱ

CK Xoo

RI PT

Relative expression (2 hpi)

3

M AN U

Ⅰ

osa-miR394 osa-miR160e osa-miR160a osa-miR160f osa-miR159a.2 osa-miR390 osa-miR827 osa-miR395b osa-miR393a osa-miR393b-3p osa-miR393b-isoform1 TAS3b (D6+) osa-miR167a osa-miR167d-isoform1 TAS3a (D6+) osa-miR166g osa-miR166k osa-miR172a osa-miR162a osa-miR168a osa-miR397a-isoform5 osa-miR397a-isoform1 osa-miR397a osa-miR397a-isoform2 osa-miR397a-isoform3 osa-miR397a-isoform4 osa-miR408-5p-isoform1 osa-miR408-5p osa-miR408-5p-isoform2 osa-miR169a osa-miR164a osa-miR1432-isoform4 osa-miR1432 osa-miR1432-isoform1 osa-miR1432-isoform2 osa-miR1432-isoform3 osa-miR397b* osa-miR530 osa-miR820a-isoform2 osa-miR820a osa-miR820a-isoform1 osa-miR398b* osa-miR398b*-isoform2 osa-miR398b*-isoform1 osa-miR159a.1 osa-miR159a.1-isoform1 osa-miR166a osa-miR398b

1.5

CK Xoo

1 0.5 0

osa- TAS3b miR160a (D6+)

C 12 hpi 24 hpi 2 hpi 6 hpi CK Xoo CK Xoo CK Xoo CK Xoo osa-miR393 osa-miR827 5S rRNA

7/7

2216 ACGGACCGAGGGACATACGGAC 2195 ||||||||||||||||||||o| osa-miR160 1 TGCCTGGCTCCCTGTATGCC-G 21 1/9

SC

OsARF18

RI PT

ACCEPTED MANUSCRIPT

1/9 4/9

4210 AAGAACTGGAACGTTCTGGAAA 4189 ||||||||||||||||||o||| TAS3b(D6+) 1 TTCTTGACCTTGCAAGAC-TTT 21

M AN U

OsARF15

6/6

OsTIR1

5744 AGGTTTCCCTAGCGTAAC-AG 5725 ||||||||||||||||||o|| osa-miR393b 1 TCCAAAGGGATCGCATTGATC 21 1/11

8/11

2244 AGGTTTCCCTAGCGTAAC-AG 2225 ||||||||||||||||||o|| osa-miR393b 1 TCCAAAGGGATCGCATTGATC 21

TE D

OsAFB2

1/11

9/10 1/10

OsGAMYB1

3279 AGAACCTCACTTCCCTCGAGGT 3258 |o|||||o|||||||||||||| osa-miR159a.1 1 T-TTGGATTGAAGGGAGCTCTG 21 8/8

AC C

EP

OsLRR-RLK2 3053 AAACTTAACTTCCCTC-TGAT 3034 ||||o|||||||||||oo||| osa-miR159a.1 1 TTTGGATTGAAGGGAGCTCTG 21

OsCSD1

2/9

1/9

2/9

2/9

1788 TGATACA-AAGGGTCCAGCGGGTGTACAGAAAGTAGTAG 1751 ||||||||||||||||||o|| osa-miR398b 1 TGTGTTCTCAGGTCGCCCCTG 21

Osa-miR827

OsARF15

TAS3b(D6+) 6 12 24 hpi

2 1 0 1 0

OsGAMYB1

Osa-miR159a.1

2

6 12 24 hpi

TE D

Relative expression

6 12 24 hpi

OsARF2

OsLRR-RLK2

Relative Relative expression expression

6 12 24 hpi

2 OsSPX-MFS2

B

0 2 3 2 1 0 2

Relative Relative expression expression

OsSPX-MFS1

OsARF25

2

RI PT

OsARF16

Relative expression

Relative expression

Osa-miR160

Osa-miR167

SC

OsARF18

CK Xoo

4

M AN U

Relative expression

A

Relative expression

ACCEPTED MANUSCRIPT

1 0

2 hpi

1 0 2 hpi 1 0

2 hpi

3 2 1 0 24 hpi 2 1 0

24 hpi

Enrichment FDR (log10 scale) -6

EP

-8

AC C

-10

-4

-2

0 cellular response to oxygen-containing compound cellular response to chemical stimulus positive regulation of programmed cell death positive regulation of cell death cellular response to organic substance signal transduction cellular response to stimulus cell communication cellular response to carbohydrate stimulus gibberellic acid mediated signaling pathway sugar mediated signaling pathway positive regulation of abscisic acid mediated signaling pathway cellular response to superoxide response to molecule of bacterial origin response to biotic stimulus response to other organism

ACCEPTED MANUSCRIPT

Transporters

RI PT

(SPX-MFSs, STs)

Xoo

SC

miR395

(ARFs, TIR1)

Basal defense

miR397

miR398

miR159

TE D EP

Auxin pathway

miR393b-3p

MEMB12

AC C

miR160, miR167, miR393, miR390, TAS3 (D6+)

M AN U

miR827

PR1 secretory pathway

BR pathway

ROS pathway

(LACs)

(CSDs)

Disease response

ROS levels

LRR-RLK2

GA pathway (GAMYB)

Disease response

ACCEPTED MANUSCRIPT A

CK-2hpi CK-6hpi CK-12hpi CK-24hpi Xoo-2hpi Xoo-6hpi Xoo-12hpi Xoo-24hpi

13,223,792 9,359,058 10,600,905 9,459,069 9,704,567 12,750,187 12,176,523 14,410,965

11,953,532 (0.90) 8,401,969 (0.90) 9,627,901 (0.91) 8,735,312 (0.92) 8,293,538 (0.85) 11,571,927 (0.91) 10,631,522 (0.87) 13,062,994 (0.91)

No. of nonredundant sequences

No. of non-redundant sequences with perfect matches to the rice genome

2,417,998 1,757,965 2,362,990 2,117,206 2,274,736 2,669,014 2,536,280 3,008,435

1,728,245 (0.71) 1,295,006 (0.74) 1,805,264 (0.76) 1,697,695 (0.80) 1,449,849 (0.64) 2,007,775 (0.75) 1,718,435 (0.68) 2,246,700 (0.75)

SC

2,000,000

CK-2hpi CK-6hpi CK-12hpi

M AN U

1,600,000

1,200,000

800,000

400,000

0 18

19

TE D

No. of nonredundant sequences

B

No. of reads with perfect matches to the rice genome

No. of reads generated

RI PT

Library

20

21

22

23

24

25

26

CK-24hpi Xoo-2hpi Xoo-6hpi Xoo-12hpi Xoo-24hpi

27

28

29

30

Length of small RNAs (nt) CK-2hpi CK-6hpi

EP

4,000,000

3,000,000

CK-12hpi CK-24hpi Xoo-2hpi

AC C

No. of total reads

5,000,000

Xoo-6hpi Xoo-12hpi

2,000,000

Xoo-24hpi

1,000,000

0 18

19

20

21

22

23

24

25

26

27

28

29

30

Length of small RNAs (nt)

Fig. S1. Statistics of the sequencing and mapping results and size distributions of the rice small RNAs. A, Number of total reads, nonredundant sequences, mapped reads, mapped nonredundant sequences in the eight data sets. B, Size distribution of nonredundant sequences and total reads in the eight data sets.

EP

TE D

M AN U

SC

AGGGAAGGCATTATTGAGTGCAGCGTTGATGAACCTGCCGGCCGGCTAAATTAATTAGCAAGAAAGTCTGAAACTGGCTCAAAGGTTCACCAGCACTGCACCCAATCACGCCTTTGCT ..........TTATTGAGTGCAGCGTTGAT........................................................................................ ..........TTATTGAGTGCAGCGTTGATG....................................................................................... ...........TATTGAGTGCAGCGTTGATGA...................................................................................... ...........TATTGAGTGCAGCGTTGATGAA..................................................................................... ...........TATTGAGTGCAGCGTTGATGAAC.................................................................................... ...........TATTGAGTGCAGCGTTGATGAACC................................................................................... ............ATTGAGTGCAGCGTTGAT........................................................................................ ............ATTGAGTGCAGCGTTGATG....................................................................................... ............ATTGAGTGCAGCGTTGATGA...................................................................................... ............ATTGAGTGCAGCGTTGATGAA..................................................................................... ............ATTGAGTGCAGCGTTGATGAAC.................................................................................... ............ATTGAGTGCAGCGTTGATGAACC................................................................................... ............ATTGAGTGCAGCGTTGATGAACCT.................................................................................. .............TTGAGTGCAGCGTTGATG....................................................................................... .............TTGAGTGCAGCGTTGATGA...................................................................................... .............TTGAGTGCAGCGTTGATGAA..................................................................................... .............TTGAGTGCAGCGTTGATGAAC.................................................................................... .............TTGAGTGCAGCGTTGATGAACC................................................................................... .............TTGAGTGCAGCGTTGATGAACCT.................................................................................. ..................................................................................AGGTTCACCAGCACTGCACCC............... ....................................................................................GTTCACCAGCACTGCACCCAATC........... .....................................................................................TTCACCAGCACTGCACCCAAT............ .....................................................................................TTCACCAGCACTGCACCCAATC........... .....................................................................................TTCACCAGCACTGCACCCAATCA.......... ......................................................................................TCACCAGCACTGCACCCA.............. ......................................................................................TCACCAGCACTGCACCCAA............. ......................................................................................TCACCAGCACTGCACCCAAT............ ......................................................................................TCACCAGCACTGCACCCAATC........... ........................................................................................ACCAGCACTGCACCCAAT............ ........................................................................................ACCAGCACTGCACCCAATC........... ........................................................................................ACCAGCACTGCACCCAATCA.......... ........................................................................................ACCAGCACTGCACCCAATCACGCC...... .........................................................................................CCAGCACTGCACCCAATCACGCCT.....

AC C

osa-MIR397b CK_0_0_0_0_Xoo_0_1_0_0 CK_1_1_1_2_Xoo_1_1_2_2 CK_1_0_0_0_Xoo_0_1_0_0 CK_2_0_0_0_Xoo_1_0_0_1 CK_0_0_0_0_Xoo_0_1_0_0 CK_0_0_1_0_Xoo_0_0_0_0 CK_2_1_0_1_Xoo_11_2_4_4 CK_1_0_6_3_Xoo_7_2_4_4 CK_8_4_7_14_Xoo_18_7_6_9 CK_966_490_1514_1237_Xoo_944_831_1288_1513 CK_11_6_20_23_Xoo_6_13_11_21 CK_2_1_0_0_Xoo_0_2_0_0 CK_1_0_2_1_Xoo_0_2_2_1 CK_1199_669_1622_1249_Xoo_1160_1802_2003_1678 CK_3656_2081_5195_3997_Xoo_3525_4725_5074_4472 CK_2455_1484_3411_2655_Xoo_2201_2934_3039_2819 CK_2534_1817_5714_4926_Xoo_3397_2966_5164_4481 CK_1785_1028_2704_2343_Xoo_1662_1921_2396_1991 CK_5_1_4_5_Xoo_2_3_10_3 CK_1_0_1_0_Xoo_1_0_0_0 CK_0_0_0_0_Xoo_1_0_0_0 CK_24_25_35_15_Xoo_23_20_21_19 CK_2057_1277_2749_2223_Xoo_1712_1502_1701_1595 CK_0_2_4_4_Xoo_2_1_0_3 CK_0_2_0_0_Xoo_1_0_0_0 CK_12_5_9_6_Xoo_5_5_7_6 CK_8_5_6_7_Xoo_6_5_3_6 CK_1109_627_1355_1188_Xoo_962_838_914_956 CK_0_0_0_1_Xoo_0_0_0_0 CK_33_24_43_39_Xoo_40_27_32_29 CK_2_1_2_2_Xoo_4_2_2_2 CK_0_0_1_0_Xoo_0_0_0_0 CK_0_0_0_0_Xoo_1_0_0_0

RI PT

ACCEPTED MANUSCRIPT

Fig. S2. Small RNAs mapped to the osa-miR397b precursor sequence. Sequence labeled in red was the miRBase annotated mature osamiR397b, while the sequence labeled in green was the annotated osa-miR397b*. The four numbers after “CK" represent the abundance of that small RNAs in control samples at 2 hpi, 6 hpi, 12 hpi, and 24 hpi, while the four numbers after “Xoo" represent the abundance of that small RNAs in Xoo-infected samples at 2 hpi, 6 hpi, 12 hpi, and 24 hpi.

ACCEPTED MANUSCRIPT   Table S1. Reads of highly expressed miRNA isoforms and miRNA*s in our data sets. Normalized sequencing reads small RNA sequence

CK2h

CK6h

CK12h

CK24h

Xoo2h

Xoo6h

Xoo12h

Xoo24h

0

0

0

0

0

0

1

1

AGCGCCCAAGCGGTAGTTGTC

osa-MIR1423-isoform1

AGCGCCCAAGCGGTAGTTGTCTCC

56

22

43

32

28

123

80

108

osa-MIR1423-5p

AGGCAACTACACGTTGGGCGCTCG

6

7

7

15

7

8

17

11

osa-MIR1423-5p-isoform1

AGGCAACTACACGTTGGGCGCT

23

22

53

39

25

24

36

34

osa-MIR1423-5p-isoform2

CAACTACACGTTGGGCGCTCGA

29

43

84

67

54

37

63

62

osa-MIR1432

ATCAGGAGAGATGACACCGAC

3528

1721

4095

1983

2918

3402

3967

2214

osa-MIR1432-isoform1

ATCAGGAGAGATGACACCGACA

30649

16081

34556

17709

22930

25159

31382

18333

osa-MIR1432-isoform2

TCAGGAGAGATGACACCGAC

4197

2071

4422

2889

3857

4106

4423

2583

osa-MIR1432-isoform3

TCAGGAGAGATGACACCGACA

3923

1894

4567

2879

3705

3222

3852

2261

osa-MIR1432-isoform4

AGGAGAGATGACACCGACA

915

397

osa-MIR156j

TGACAGAAGAGAGTGAGCAC

88815

osa-MIR156j-isoform1

TTGACAGAAGAGAGTGAGCAC

66407

osa-MIR159a.1

TTTGGATTGAAGGGAGCTCTG

osa-MIR159a.1-isoform1

TTTGGATTGAAGGGAGCT

osa-MIR159a.2

TTGCATGCCCCAGGAGCTGCA

osa-MIR159a.2-isoform1

TTGCATGCCCCAGGAGCTGC

osa-MIR166m

TCGGACCAGGCTTCATTCCCT

osa-MIR166m-isoform1

TCGGACCAGGCTTCATTCCC

osa-MIR166m-isoform2

TCGGACCAGGCTTCATTCC

osa-MIR166m-isoform3

TCGGACCAGGCTTCATTC

osa-MIR167d

TGAAGCTGCCAGCATGATCTG

osa-MIR167d-isoform1

SC

RI PT

osa-MIR1423

M AN U

ID

974

487

588

757

1087

622

93309

103385

94805

126357

75435

114495

115711

78482

103621

87503

91185

75413

91171

94851

2493

1471

4899

2068

2221

2860

2187

3885

5254

4129

4646

3834

3386

2939

3288

1

4

3

5

2

60

11

5

11

26

34

36

28

409

73

27

44

54

65

93

62

56

37

61

169

233

257

274

180

161

132

192

326

438

449

513

290

278

236

339

318

444

408

336

192

209

241

255

10896

11861

18160

16064

14902

12520

14149

13886

TGAAGCTGCCAGCATGATCTGA

638124

725818

1101443

1079044

978240

685959

835603

950093

osa-MIR1850

TGGAAAGTTGGGAGATTGGGG

105

111

236

244

137

125

223

228

osa-MIR1850-isoform1

TGGAAAGTTGGGAGATTGGG

120

124

279

218

134

158

246

243

osa-MIR1850-isoform2

TGGAAAGTTGGGAGATTGG

57

49

110

108

68

57

101

110

osa-MIR1862a

ACGAGGTTGGTTTATTTTGGGACG

46

54

66

94

52

59

50

78

osa-MIR1862a-isoform1

ACGAGGTTGGTTTATTTTGGGA

48

75

82

81

43

46

48

68

osa-MIR1862a-isoform2

ACGAGGTTGGTTTATTTTGGG

90

102

120

140

98

97

78

87

osa-MIR1862a-isoform3

ACGAGGTTGGTTTATTTTGG

257

330

421

458

343

340

283

306

osa-MIR1862a-isoform4

ACGAGGTTGGTTTATTTTG

1261

1450

2328

2256

1422

1689

1433

1579

osa-MIR1862a-isoform5

ACGAGGTTGGTTTATTTT

2008

2156

2160

3119

2325

2713

1772

1914

osa-MIR1862a-isoform6

GTACGAGGTTGGTTTATTTTGGGA

83

46

98

142

82

75

80

104

osa-MIR1862a-isoform7

TTGGTTTATTTTGGGACGGAG

112

116

183

187

129

111

123

117

osa-MIR1862e

CTAGATTTGTTTATTTTGGGACGG

1363

1221

1647

2199

1764

1520

1528

1849

osa-MIR1862e-isoform1

ACTAGATTTGTTTATTTTG

1295

1539

1719

1943

1663

1787

1378

1517

osa-MIR1862e-isoform2

ACTAGATTTGTTTATTTT

1317

1299

521

1274

1296

1735

792

790

osa-MIR1863b.2

AGAGACTTGGCTGATGCATTACT

147

125

175

74

84

180

159

161

osa-MIR1863b.2-isoform1

AGAGACTTGGCTGATGCATTACTT

192

166

242

103

93

215

168

207

osa-MIR1863b.2-isoform2

TAGAGACTTGGCTGATGCATTACT

303

170

403

52

53

385

302

367

osa-MIR1868

TCACGGAAAACGAGGGAGCAGCCA

13

16

42

25

21

19

16

39

AC C

EP

TE D

2381

ACCEPTED MANUSCRIPT AAGCGTGCTCACGGAAAACGA

81

78

90

151

100

111

118

103

osa-MIR1868-isoform2

AAGCGTGCTCACGGAAAACGAG

30

32

35

55

37

38

31

31

osa-MIR1868-isoform3

AAGCGTGCTCACGGAAAACGAGGG

105

78

107

129

86

96

118

134

osa-MIR1868-isoform4

GCGTGCTCACGGAAAACGAGGGAG

65

53

76

68

47

81

70

110

osa-MIR1873

TCAACATGGTATCAGAGCTGGAAG

105

78

78

85

79

116

121

160

osa-MIR1873-isoform1

TCAACATGGTATCAGAGCTGGA

43

36

91

80

66

61

68

108

miR393b

TCCAAAGGGATCGCATTGATCT

0

0

0

0

0

0

0

0

miR393b-isoform1

CTCCAAAGGGATCGCATTGAT

15

28

34

26

148

25

16

17

miR393b-isoform2

TCCAAAGGGATCGCATTGATC

48

76

91

146

109

107

76

87

miR393b-isoform3

AGGACTCCAAAGGGATCGCATTGA

158

182

191

186

178

184

203

149

osa-MIR396b

TTCCACAGCTTTCTTGAACTG

1

2

2

7

29

3

3

5

osa-MIR396b-isoform1

TTCCACAGCTTTCTTGAACT

29

31

36

53

29

30

35

28

osa-MIR396b-isoform2

TTCCACAGCTTTCTTGAAC

144

210

308

352

308

280

184

246

osa-MIR396b-isoform3

TTCCACAGCTTTCTTGAA

1110

1526

osa-MIR396c

TTCCACAGCTTTCTTGAACTT

275

362

osa-MIR396c-isoform1

TTCCACAGCTTTCTTGAAC

144

210

osa-MIR396c-isoform2

TTCCACAGCTTTCTTGAA

1110

1526

osa-MIR396c-isoform3

TTTCCACAGCTTTCTTGA

25

31

osa-MIR396c-3p

GGTCAAGAAAGCTGTGGGAAG

3

osa-MIR396c-3p-isoform1

GTCAAGAAAGCTGTGGGAAGA

osa-MIR396c-3p-isoform2

AGAAAGCTGTGGGAAGAAATGGCA

osa-MIR397a

TCATTGAGTGCAGCGTTGATG

osa-MIR397a-isoform1

ATTGAGTGCAGCGTTGATGAA

osa-MIR397a-isoform2

TTGAGTGCAGCGTTGATG

osa-MIR397a-isoform3

TTGAGTGCAGCGTTGATGA

osa-MIR397a-isoform4 osa-MIR397a-isoform5 osa-MIR397b

TTATTGAGTGCAGCGTTGATG

osa-MIR397b-isoform1

ATTGAGTGCAGCGTTGATGAA

osa-MIR397b-isoform2

SC

RI PT

osa-MIR1868-isoform1

2290

2538

2128

1679

1939

310

613

443

464

478

321

308

352

308

280

184

246

2255

2290

2538

2128

1679

1939

47

60

50

46

31

31

1

2

5

2

11

2

3

18

12

13

47

34

43

25

16

873

896

983

1412

997

1032

962

931

44

27

79

77

75

77

53

62

966

490

1514

1237

944

831

1288

1513

1199

669

1622

1249

1160

1802

2003

1678

3656

2081

5195

3997

3525

4725

5074

4472

TTGAGTGCAGCGTTGATGAA

2455

1484

3411

2655

2201

2934

3039

2819

TTGAGTGCAGCGTTGATGAAC

2534

1817

5714

4926

3397

2966

5164

4481

1

1

1

2

1

1

2

2

966

490

1514

1237

944

831

1288

1513

TTGAGTGCAGCGTTGATG

1199

669

1622

1249

1160

1802

2003

1678

osa-MIR397b-isoform3

TTGAGTGCAGCGTTGATGA

3656

2081

5195

3997

3525

4725

5074

4472

osa-MIR397b-isoform4

TTGAGTGCAGCGTTGATGAA

2455

1484

3411

2655

2201

2934

3039

2819

TTGAGTGCAGCGTTGATGAAC

2534

1817

5714

4926

3397

2966

5164

4481

TTGAGTGCAGCGTTGATGAACC

1785

1028

2704

2343

1662

1921

2396

1991

TTCACCAGCACTGCACCCAATC

2057

1277

2749

2223

1712

1502

1701

1595

TCACCAGCACTGCACCCAATC

1109

627

1355

1188

962

838

914

956

osa-MIR397b-isoform6 osa-MIR397b-isoform7 osa-MIR397b-isoform8

TE D

EP

AC C

osa-MIR397b-isoform5

M AN U

2255

36

33

67

53

20

22

43

36

osa-MIR398b*

GGGCGAGCTGGGAACACACGG

216

29

87

37

29

260

122

223

osa-MIR398b*-isoform1

GGCGAGCTGGGAACACACGGT

269

58

173

62

37

234

161

194

osa-MIR398b*-isoform2

GCGAGCTGGGAACACACGGTG

51

9

32

10

6

63

42

80

osa-MIR408-5p

CAGGGATGAGGCAGAGCATGG

1599

1302

2878

7057

1483

3882

2372

7803

osa-MIR408-5p-isoform1

CAGGGATGAGGCAGAGCATG

714

929

1705

4436

923

2314

1192

3824

osa-MIR408-5p-isoform2

ACAGGGATGAGGCAGAGCATG

789

465

1125

1219

594

1226

827

1886

osa-MIR530-5p

TGCATTTGCACCTGCACCTA

4

3

0

11

8

5

10

4

miR398b

TGTGTTCTCAGGTCGCCCCTG

ACCEPTED MANUSCRIPT 2106

1978

2116

2298

1620

3512

1361

ACGGATGATTAAAGTTGGACACGG

220

170

108

92

131

302

220

244

osa-MIR812f-isoform1

GATGATTAAAGTTGGACACGGAAA

116

96

205

228

126

146

200

226

osa-MIR820a

TCGGCCTCGTGGATGGACCAG

54

34

60

40

29

111

112

135

osa-MIR820a-isoform1

TCGGCCTCGTGGATGGACCAGGA

249

122

408

114

122

509

599

592

osa-MIR820a-isoform2

TCGGCCTCGTGGATGGACCAGGAG

2353

1385

2153

653

558

4972

4288

4601

TGCATTTGCACCTGCACCTAC

osa-MIR812f

     

RI PT

2588

osa-MIR530-5p-isoform1

AC C

EP

TE D

M AN U

SC

 

ACCEPTED MANUSCRIPT Table S2. Reads of miRNAs and miRNA*s in the OsAGO1 associated small RNA data sets.

ID

OsAGO1a

OsAGO1b

OsAGO1c

(GSM455962a)

(GSM455963a)

(GSM455964a)

3302

2768

osa-miR156a*

0

0

0

osa-miR156b*

0

0

0

osa-miR156c*

0

0

0

osa-miR156d*

9

1

0

osa-miR156e*

0

0

osa-miR156f*

0

0

osa-miR156g*

0

0

osa-miR156h*

59

95

osa-miR156i*

0

0

osa-miR156j*

59

95

osa-miR156k

41

1

osa-miR156k*

0

0

osa-miR156l

25

osa-miR156l*

0

osa-miR167a

9935

osa-miR167a*

0

osa-miR167b*

0

osa-miR167c*

0

osa-miR167d

0

0

0

16

SC

0

16 2 0

0

0

0

0

1181

8163

0

0

0

0

0

0

1883

63899

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1731

531

6068

0

0

0

31005

8483

2546

0

0

0

osa-miR172a

15

0

51

osa-miR172a*

0

0

0

osa-miR172d*

0

0

0

osa-miR172b

0

0

0

osa-miR172c

0

0

0

osa-miR172c*

0

0

0

osa-miR393b

87

148

35

5861

1329

3388

147

0

494

0

0

0

osa-miR167g* osa-miR167h* osa-miR167j* osa-miR167e osa-miR167e* osa-miR168a

AC C

osa-miR168a*

EP

osa-miR167f*

0

TE D

osa-miR167d*

22237

RI PT

16861

M AN U

osa-miR156a

osa-miR393b* osa-miR528 osa-miR528* a

NCBI GEO accession number.

Table S3.   Differentially expressed miRNAs, miRNA*s, isoforms, and ta-siRNAs.

ACCEPTED MANUSCRIPT Normalized reads (CK)

Normalized reads (Xoo)

2 hpi

6 hpi

12 hpi

24 hpi

2 hpi

6 hpi

12 hpi

24 hpi

30

107

53

373

224

515

215

90

Sequences

miR394

TTGGCATTCTGTCCACCTCC

miR160e

TGCCTGGCTCCCTGTATGCCG

9

9

7

41

19

21

25

21

miR160a

TGCCTGGCTCCCTGTATGCCA

12

25

6

60

84

32

56

24

miR160f

TGCCTGGCTCCCTGAATGCCA

17

36

18

89

41

56

65

37

miR159a.2

TTGCATGCCCCAGGAGCTGC

11

26

34

36

28

409

73

27

miR390

AAGCTCAGGAGGGATAGCGCC

45

34

75

112

129

82

56

56

miR827

TTAGATGACCATCAGCAAACA

1962

1598

2640

3544

5013

2397

2679

1590

miR395b

TGAAGTGTTTGGGGGAACTC

31

34

47

miR393a

TCCAAAGGGATCGCATTGATC

48

76

91

miR393b-3p

TCAGTGCAATCCCTTTGGAAT

78

116

125

miR393b-isoform1

CTCCAAAGGGATCGCATTGAT

15

28

34

TAS3b (D6+)

TCTTGACCTTGTAAGACCCAA

1205

1260

2316

2463

3021

1367

1233

1563

miR167a

TGAAGCTGCCAGCATGATCTA

25527

29211

41840

54759

31025

29327

30122

29552

miR167d-isoform1

TGAAGCTGCCAGCATGATCTGA

638124

725818

1101443

1079044

978240

685959

835603

950093

TAS3a (D6+)

TTCTTGACCTTGCAAGACTTT

26

24

37

37

42

21

22

19

osa-MIR166g

TCGGACCAGGCTTCATTCCTC

1104

1206

1618

1609

1457

1176

816

1092

osa-MIR166k

TCGGACCAGGCTTCAATCCCT

573

671

899

925

836

667

569

637

miR172a

AGAATCTTGATGATGCTGCAT

24523

29784

44365

36480

39373

30396

26214

32927

miR162a

TCGATAAACCTCTGCATCCAG

1467

1878

2748

3437

2429

1932

2128

2315

miR168a

TCGCTTGGTGCAGATCGGGAC

160676

172489

408838

402703

295068

231431

359593

446833

miR397a-isoform5

TTGAGTGCAGCGTTGATGAAC

2534

1817

5714

4926

3397

2966

5164

4481

miR397a-isoform1

ATTGAGTGCAGCGTTGATGAA

966

490

1514

1237

944

831

1288

1513

miR397a

TCATTGAGTGCAGCGTTGATG

44

27

79

77

75

77

53

62

miR397a-isoform2

TTGAGTGCAGCGTTGATG

1199

669

1622

1249

1160

1802

2003

1678

miR397a-isoform3

TTGAGTGCAGCGTTGATGA

3656

2081

5195

3997

3525

4725

5074

4472

miR397a-isoform4

TTGAGTGCAGCGTTGATGAA

2455

1484

3411

2655

2201

2934

3039

2819

miR408-5p-isoform1

CAGGGATGAGGCAGAGCATG

714

929

1705

4436

923

2314

1192

3824

miR408-5p

CAGGGATGAGGCAGAGCATGG

1599

1302

2878

7057

1483

3882

2372

7803

miR408-5p-isoform2

ACAGGGATGAGGCAGAGCATG

789

465

1125

1219

594

1226

827

1886

miR169a

CAGCCAAGGATGACTTGCCGA

1056

215

504

541

868

531

686

720

miR164a

TGGAGAAGCAGGGCACGTGCA

7979

6396

11824

10248

7648

13209

11499

15199

miR1432-isoform4

AGGAGAGATGACACCGACA

106

50

39

57

62

146

109

107

76

87

183

159

118

111

128

26

148

25

16

17

SC

M AN U

TE D

EP

RI PT

Small RNA IDs

397

974

487

588

757

1087

622

1721

4095

1983

2918

3402

3967

2214

30649

16081

34556

17709

22930

25159

31382

18333

TCAGGAGAGATGACACCGAC

4197

2071

4422

2889

3857

4106

4423

2583

TCAGGAGAGATGACACCGACA

3923

1894

4567

2879

3705

3222

3852

2261

miR397b*

TTCACCAGCACTGCACCCAATC

2057

1277

2749

2223

1712

1502

1701

1595

miR530

AGGTGCAGAGGCAGATGCAAC

1043

682

686

221

462

790

1265

435

miR820a-isoform2

TCGGCCTCGTGGATGGACCAGGAG

2353

1385

2153

653

558

4972

4288

4601

miR820a

TCGGCCTCGTGGATGGACCAG

54

34

60

40

29

111

112

135

miR820a-isoform1

TCGGCCTCGTGGATGGACCAGGA

249

122

408

114

122

509

599

592

miR1432-isoform1 miR1432-isoform2 miR1432-isoform3

AC C

915 3528

miR1432

ATCAGGAGAGATGACACCGAC ATCAGGAGAGATGACACCGACA

ACCEPTED MANUSCRIPT GGGCGAGCTGGGAACACACGG

216

29

87

37

29

260

122

223

miR398b*-isoform2

GCGAGCTGGGAACACACGGTG

51

9

32

10

6

63

42

80

miR398b*-isoform1

GGCGAGCTGGGAACACACGGT

269

58

173

62

37

234

161

194

miR159a.1

TTTGGATTGAAGGGAGCTCTG

2381

2493

1471

4899

2068

2221

2860

2187

miR159a.1-isoform1

TTTGGATTGAAGGGAGCT

3885

5254

4129

4646

3834

3386

2939

3288

miR166a

TCGGACCAGGCTTCATTCCCC

16229

20528

22638

21922

17434

13420

12292

16123

miR398b

TGTGTTCTCAGGTCGCCCCTG

36

33

67

53

20

22

43

36

AC C

EP

TE D

M AN U

SC

RI PT

miR398b*

ACCEPTED MANUSCRIPT Table S4. Predicted target genes of the differentially expressed miRNAs and ta-siRNAs in Xoo-infected rice, and the alignments between small RNAs and their targeting sites.  RNA ID 

Locus ID 

Gene annotation

Alignment between miRNAs and their targets 

osa-miR160 

Os06g47150 

Auxin response

miRNA:

1 TGCCTGGCTCCCTGTATGCCA 22 ||||||||||||||||||||**

factor

gene: 2315 ACGGACCGAGGGACATACGGAC 2294 Auxin response

miRNA:

1 TGCCTGGCTCCCTGTATGCCA 22 ||||||||||||||||||||**

factor

RI PT

Os04g43910 

 

gene: 1549 ACGGACCGAGGGACATACGGAC 1528

Os02g41800 

 

Auxin response

miRNA:

1 TGCCTGGCTCCCTGTATGCCA 22 ||||||||||||||||||||**

factor

gene: 1683 ACGGACCGAGGGACATACGGAC 1662 Auxin response

miRNA:

1 TGCCTGGCTCCCTGTATGCCA 22

SC

Os04g59430 

 

||||||||||||||*|||*||*

factor

gene: 2235 ACGGACCGAGGGACTTACAGTC 2214 Auxin response factor

miRNA:

1 TGCCTGGCTCCCTGTATGCCA 22

M AN U

Os10g33940 

 

||||||||||||||||||||**

gene: 4027 ACGGACCGAGGGACATACGGAC 4006

osa-miR167 

Os12g41950 

Auxin response factor

miRNA:

3 AAGCTGCCAGCATGATCTGA 22 |||||||||||*||||||*|

gene: 5337 TTCGACGGTCGGACTAGAAT 5318

Os02g06910 

 

Auxin response

TE D

factor

Os04g57610 

 

Auxin response

miRNA:

|||||||||||*||||||

gene: 5368 TTCGACGGTCGGACTAGA 5351 miRNA:

Os06g46410 

Auxin response

gene: 5629 TGTTCGACGGTCGGACTAGATA 5608 miRNA:

EP

Os07g29820 

AC C

TAS3 (D6+) 

 

Os01g48060 

Os05g48870 

NBS-LRR disease

3 AAGCTGCCAGCATGATCTGA 22 |||||||||||*||||||*|

factor

 

1 TGAAGCTGCCAGCATGATCTA 22 **|||||||||||*|||||||*

factor

 

3 AAGCTGCCAGCATGATCT 20

gene: 5440 TTCGACGGTCGGACTAGAGT 5421 miRNA:

1 TGAAGCTGCCAGCATGATCTA 22 |||*||||*||||||||||||*

resistance protein

gene: 4724 ACTCCGACAGTCGTACTAGATA 4703 Auxin response

miRNA:

1 TCTTGACCTTGTAAGACCCAA 22 |||||||||||:||||||****

factor

gene: 4339 AGAACTGGAACGTTCTGGAACA 4318 Auxin response

miRNA:

1 TCTTGACCTTGTAAGACCCAA 22 |||||||||||:||||||****

factor

gene: 4077 AGAACTGGAACGTTCTGGAACA 4056

 

Os01g54990 

Auxin response

miRNA:

1 TCTTGACCTTGTAAGACCCAA 22 |||||||||||:|||||||***

factor

gene: 4785 AGAACTGGAACGTTCTGGGACC 4764

 

Os01g70270 

Auxin response factor

miRNA:

1 TCTTGACCTTGTAAGACCCAA 22 |||||||||||:||||||**|*

ACCEPTED MANUSCRIPT gene: 2912 AGAACTGGAACGTTCTGGCATG 2891

 

Os05g43920 

Auxin response

miRNA:

1 TCTTGACCTTGTAAGACCCAA 22 |||||||||||:|||||||***

factor

gene: 4809 AGAACTGGAACGTTCTGGGACA 4788

osa-miR393 

Os05g05800 

miRNA:

OsTIR1

1 TCCAAAGGGATCGCATTGATC 22 ||||||||||||||||||*||*

gene: 5845 AGGTTTCCCTAGCGTAAC-AGA 5825 miRNA:

OsAFB2

1 TCCAAAGGGATCGCATTGATC 22

RI PT

Os04g32460 

 

||||||||||||||||||*||*

gene: 3193 AGGTTTCCCTAGCGTAAC-AGA 3173

Os03g09070 

b-3p 

miRNA:

1 TCAGTGCAATCCCTTTGG-AA 21 |*|||||||||||||*||*|||

repeat domain

Os03g51440 

 

Leucine rich

containing protein

gene: 2081 ACTCACGTTAGGGAATCCGTTA 2059

LRR receptor-like

miRNA:

SC

osa-miR393

1 TCAGTGCAATCCCTTTGG--AAT 21 |||*||||||||*|||||**|||

protein kinase

gene: 1937 AGT-ACGTTAGG-AAACCGTTTA 1917 Dynein light chain

miRNA: 1 TCAGTGCAATCCCTTT-GGAAT 22

M AN U

Os01g37490 

 

||||||||||||||||*:||||*

type 1 domain

 

Os03g45260

containing protein

gene: 937 AGTCACGTTAGGGAAAGTCTTAT 915

MEMB12

miRNA: 1 TCAGTGCAATCCCTTTGGAAT 22 :|||*|*||*|||||*|*||:

gene: 987 GGTCTCTTTCGGGAA-CATTG 968

osa-miR390 

Os02g10100 

Leucine-rich

miRNA:

*|||||||:||||||||||||*

TE D

repeat receptor protein kinase

1 AAGCTCAGGAGGGATAGCGCC 22

gene: 4610 ATCGAGTCTTCCCTATCGCGGA 4589

EXS precursor

Os04g42050

Hypothetical

miRNA:

|||||||||||||||||*:|**

EP

protein

osa-miR827 

Os04g48390 

Membrane protein

gene: 533 TTCGAGTCCTCCCTATC-TGTC 513 miRNA: 1 TTAGATGACCATCAGCAAACA 22 ||||||||*|||||||||||**

 

AC C

(OsSPX-MFS1)

 

Os02g45520 

Os06g04600

Membrane protein

1 AAGCTCAGGAGGGATAGCGCC 22

gene: 453 AATCTACTTGTAGTCGTTTGAG 432 miRNA: 1 TTAGATGACCATCAGCAAA--CA 22 ||||||||||||||:||||**||*

(OsSPX-MFS2)

gene: 643 AATCTACTGGTAGTTGTTTAGGTG 620 Brix domain

miRNA:

1 TTAGATGACCATCAGCAAACA 22 :||||||||||||||||*|||*

containing protein

gene: 3870 GATCTACTGGTAGTCGT-TGTA 3850

 

Os08g01570

Retrotransposon

miRNA:

1 TTAGATGACCATCAGCAAACA 22 |:|:|||||||||||||||||*

protein

gene: 1554 AGTTTACTGGTAGTCGTTTGTT 1533

osa-miR159 a.1

 

Os01g59660 

MYB family transcription factor

miRNA:

1 TTTGGATTGAAGGGAGCT 19 :|||||*|||||||||||*

gene: 3377 GAACCTCACTTCCCTCGAG 3359

ACCEPTED MANUSCRIPT Os06g40330 

 

MYB family

miRNA:

1 TTTGGATTGAAGGGAGCT 19 *|||||*|||||||||||*

transcription factor

gene: 6273 TAACCTCACTTCCCTCGAG 6255

Os01g12700 

 

MYB family

miRNA:

1 TTTGGATTGAAGGGAGCT 19 *|||||||:|||||||||*

transcription factor

gene: 3094 TAACCTAATTTCCCTCGAG 3076

Os05g41166

MYB family

miRNA:

1 TTTGGATTGAAGGGAGCT 19 *|||||||:|||||||||*

transcription factor

RI PT

 

gene: 3255 TAACCTAATTTCCCTCGAG 3237

 

Os03g38210

MYB family

miRNA:

1 TTTGGATTGAAGGGAGCT 19 *||||*||||||||||||*

transcription factor

gene: 1168 TAACCGAACTTCCCTCGAG 1150

Os04g46384

MYB family

miRNA: 1 TTTGGATTGAAGGGAGCT 19

SC

 

|||||||:|||*||||||*

transcription factor

gene: 279 AAACCTAGCTTACCTCGAG 261

Os12g10740 

 

Leucine-rich

miRNA:

||||:|||||||||||*||*

 

Os10g05230

Zinc finger,

M AN U

repeat family protein

gene: 3053 AAACTTAACTTCCCTCTGAT 3034 miRNA: 1 TTTGGATTGAAGGGAGCT 19 :||||||||||||||||**

C3HC4 type

domain containing protein

 

Os01g12240

Hypothetical

osa-miR159

TE D

protein

Os03g02240 

a.2 

 

Os03g11960 

AC C

osa-miR398 

 

AT-GTL1

Expressed protein

EP

Os10g37240

Copper/zinc

gene: 654 GAACCTAACTTCCCTCGTT 636

miRNA:

1 TTTGGATTGAAGGGAGCT 19 *||||||:||||||||||*

gene: 3533 TAACCTAGCTTCCCTCGAG 3515 miRNA:

1 TTGCATGCCCCAGGAGCTGC 21 |||||||||||*||||||||*

gene: 2940 AACGTACGGGGGCCTCGACGA 2920 miRNA:

1 TTGCATGCCCCAGGAGCTGC 21 |||||||||||*|||||:||*

gene: 4295 AACGTACGGGGGCCTCGGCGG 4275 miRNA:

1 TGTGTTCTCAGGTCGCCC-C-TG 21 |||*|||*||||||||||*|*||

superoxide dismutase (CSD1)

gene: 1784 ACA-AAGGGTCCAGCGGGTGTAC 1763

Os08g44770

Copper/zinc

miRNA:

 

superoxide

Os01g42650

Os12g01922 

1 TGTGTTCTCAGGTCGCCC-C-TG 21 |||*|||*||||||*|||*|*||

dismutase (CSD2)

gene: 1917 ACA-AAGGGTCCAGTGGGCGTAC 1896

cytochrome c

miRNA:

1 TGTGTTCTCAGGTCGCCCCTG 22 ||||:|||||||||||**|*|*

oxidase subunit

osa-miR398* 

1 TTTGGATTGAAGGGAG-CT 19

5B (OsCOX5b.1)

gene: 245 ACACGAGAGTCCAGCGCCG-CG 225

WD domain and

miRNA:

HEAT domain containing protein (RAPTOR1)

1 GGGCGAGCTGGGAACACA-CGG 21 :|||:|||||*|||||||*|:|

gene: 902 TCCGTTCGACACTTGTGTAGTC 881

ACCEPTED MANUSCRIPT Osa‐miR395  Os03g09930 

Sulfate transporter

miRNA:

1 TGAAGTGTTTGGGGGAACTC 21 |||||||||||||*||||||*

gene: 3240 ACTTCACAAACCCACTTGAGG 3220

 

Os03g09940 

Sulfate transporter

miRNA:

1 TGAAGTGTTTGGGGGAACTC 21 ||||||||||||||||||||*

gene: 438 ACTTCACAAACCCCCTTGAGG 418

Bifunctional

miRNA:

|||||||:||||:|||||||*

3-phosphoadenosi

gene: 715 ACTTCACGAACCTCCTTGAGC 695

ne 5-phosphosulfate synthetase

Osa‐miR166  Os02g49670 

1 TGAAGTGTTTGGGGGAACTC 21

RI PT

Os03g53230 

Zinc knuckle

miRNA:

1 TCGGACCAGGCTTCATTCCCC 22

SC

 

||||||||||||||*||||||*

family protein

gene: 3125 AGCCTGGTCCGAAG-AAGGGGC 3105

Os03g10290 

Hypothetical protein

miRNA:

1 TCGGACCAGGCTTCATTCCCC 22 **|||||||||||||||||||*

M AN U

 

gene: 1664 TACCTGGTCCGAAGTAAGGGGA 1643

 

Os03g44835 

Expressed protein

miRNA:

1 TCGGACCAGGCTTCATTCCCC 22 |||||*|||||||||||||*|*

gene: 1366 AGCCTAGTCCGAAGTAAGGAGT 1345

 

Os04g48290 

MATE efflux family protein

miRNA: 1 TCGGACCAGGCTTCATTCCCC 22 *|||||||||||||*|||*||*

TE D

gene: 519 TGCCTGGTCCGAAGCAAGTGGC 498

Osa‐miR172  Os03g60430 

AP2 domain

miRNA:

||||||:|||||||||||||**

containing protein

 

Os05g03040 

AP2 domain

gene: 3893 TCTTAGGACTACTACGACGTCG 3872 miRNA:

 

EP

AC C

 

Os07g13170 

Os04g55560 

Os06g43220 

AP2 domain

1 AGAATCTTGATGATGCTGCAT 22 ||||||:|||||||||||||**

containing protein

 

1 AGAATCTTGATGATGCTGCAT 22

gene: 3962 TCTTAGGACTACTACGACGTCG 3941 miRNA:

1 AGAATCTTGATGATGCTGCA 21 ||||||:|||||||||||||

containing protein

gene: 3950 TCTTAGGACTACTACGACGT 3925 AP2 domain

miRNA:

1 AGAATCTTGATGATGCTGCAT 22 *|||||*|||||||||||||**

containing protein

gene: 3002 CCTTAGCACTACTACGACGTCG 2981 AP2 domain

miRNA:

1 AGAATCTTGATGATGCTGCAT 22 *|||||:|||||||||||||**

containing protein

gene: 3414 CCTTAGGACTACTACGACGTCG 3393

 

Os06g49310 

MATE efflux family protein

miRNA:

1 AGAATCTTGATGATGCTGCAT 22 *|*|||||||||||||||*||*

gene: 7705 CCCTAGAACTACTACGAC-TAC 7685

ACCEPTED MANUSCRIPT Osa‐miR397  Os05g38410 

Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 |||||||||||||||||||*

gene: 1126 AACTCACGTCGCAACTACTC 1107

 

Os05g38420 

Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 |||||||||||||||||||*

gene: 1124 AACTCACGTCGCAACTACTC 1105

 

Os01g44330 

Laccase precursor

miRNA: 1 TTGAGTGCAGCGTTGATGA 20

RI PT

||||*||||||||||||||*

gene: 998 AACTGACGTCGCAACTACTA 979

 

Os01g61160 

Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 ||||||||*||||||||||*

gene: 1381 AACTCACGGCGCAACTACTT 1362

Os01g62480 

Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20

SC

 

|||||:||*||||||||||*

gene: 1750 AACTCGCGCCGCAACTACTC 1731

 

Os01g62490 

Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20

M AN U

|||||:|||||||||||||*

gene: 1356 AACTCGCGTCGCAACTACTC 1337

 

Os01g63180 

Laccase-6 precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 |||||:|||||||||||||*

gene: 2706 AACTCGCGTCGCAACTACTA 2687

 

Os01g63190 

Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 ||||||*||||||||||||*

 

 

 

Os02g51440 

Laccase precursor

Laccase precursor

EP

 

Os01g63200 

Os03g16610 

AC C

 

TE D

gene: 3549 AACTCAGGTCGCAACTACTG 3530

Os11g01730 

Os12g15680 

Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 ||||*||||||||||||||*

gene: 1379 AACTGACGTCGCAACTACTA 1360 miRNA:

1 TTGAGTGCAGCGTTGATGA 20 *|||||*||||||||||||*

gene: 1236 CACTCAGGTCGCAACTACTA 1217 miRNA:

1 TTGAGTGCAGCGTTGATGA 20 |||||:|||||||||||*|*

gene: 1313 AACTCGCGTCGCAACTAGTC 1294 Laccase-23

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 |||||:||*||||||||||*

precursor

gene: 3594 AACTCGCGCCGCAACTACTT 3575 Laccase precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 |||||:|||||||||||||*

gene: 1513 AACTCGCGTCGCAACTACTA 1494

 

Os11g48060 

Laccase-22 precursor

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 ||*||||||||||||||||*

gene: 1941 AAGTCACGTCGCAACTACTA 1922

ACCEPTED MANUSCRIPT  

Os12g01730 

Laccase-23

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 |||||:||*||||||||||*

precursor

gene: 3482 AACTCGCGCCGCAACTACTT 3463

 

Os02g34600 

Calcium/calmoduli

miRNA:

|||||||||||:*||||||*

n depedent protein

 

Os01g02180 

1 TTGAGTGCAGCGTTGATGA 20

kinases

gene: 1652 AACTCACGTCGTCACTACTG 1633

Expressed protein

miRNA:

1 TTGAGTGCAGCGTTGATGA 20

RI PT

|||||||||||||||*|||* gene: 2065 AACTCACGTCGCAACGACTT 2046

 

Os04g54610 

Hypothetical

miRNA:

1 TTGAGTGCAGCGTTGATGA 20 ||*||||||||||||||*|*

protein

gene: 413 AAGTCACGTCGCAACTAATC 394

Os07g04020 

Expressed protein

miRNA: 1 TTGAGTGCAGCGTTGATGA 20

SC

 

*||||||||||:|||||:|*

gene: 74 CACTCACGTCGTAACTATTT 55

Osa‐miR408  Os02g49850 

Plastocyanin-like

miRNA:

*||||||||||||||||:|*||*

 

Os03g15340 

M AN U

domain containing protein

Plastocyanin-like

gene: 249 TACGTGACGGAGAAGGGGCTCGG 227 miRNA:

 

Os03g50140 

Plastocyanin-like

gene: 204 GACGTGACGGAGAAGGGACCCGG 182 miRNA:

TE D

 

Os06g15600 

Plastocyanin-like

gene: 373 AACGTGACGGAGAAGGGGCTCGG 351 miRNA:

Os01g03530 

protein

gene: 175 AACGTGACGGAGAAGGGGCCGC 154

Multicopper

miRNA:

EP

containing protein

gene: 2503 GACGTGACGGAGAAGGAGCCGC 2482

Nuclear

miRNA:

AC C

 

Os12g42400 

Os03g44540 

Y subunit

gene: 3014 GTCGGTTCCTACTTAACGG-TG 2994

Nuclear

miRNA:

1 TAGCCAAGAATGACTTGCCTA 22 :||||||||||||*|||||||*

transcription factor Y subunit

gene: 4053 GTCGGTTCTTACTCAACGGATG 4032

Nuclear

miRNA:

1 TAGCCAAGAATGACTTGCCTA 22 *||||||||||||*|||||||*

transcription factor

Osa‐miR164  Os02g36880 

1 CAGCCAAGGATGACTTGCCGA 22 |||||||||||||*|||||*|*

transcription factor

 

1 CTGCACTGCCTCTTCCCTGGC 22 ||||||||||||||||*:|||*

oxidase domain

Osa‐miR169  Os07g06470 

1 CTGCACTGCCTCTTCCCTGGC 22 *||||||||||||||||:|||*

domain containing

 

1 CTGCACTGCCTCTTCCCTG-GC 22 *||||||||||||||||:|*||*

domain containing protein

1 CTGCACTGCCTCTTCCCT-GGC 22 ||||||||||||||||||*|||*

domain containing protein

1 CTGCACTGCCTCTTCCCTG-GC 22

Y subunit

gene: 5295 CTCGGTTCTTACTAAACGGATG 5274

No apical

miRNA:

meristem protein

1 TGGAGAAGCAGGGCACGTGCA 22 ||||||||||||*|||||||**

gene: 1378 ACCTCTTCGTCCAGTGCACGCG 1357

ACCEPTED MANUSCRIPT  

Os06g23650 

miRNA:

No apical

1 TGGAGAAGCAGGGCACGTGCA 22 |||||||||||||||||*||**

meristem protein

gene: 1590 ACCTCTTCGTCCCGTGCTCGAG 1569

 

Os06g46270 

miRNA:

No apical

1 TGGAGAAGCAGGGCACGTGCA 22 ||||||||||||||||*|||**

meristem protein

gene: 3944 ACCTCTTCGTCCCGTGAACGAG 3923

Os12g41680 

miRNA:

No apical

1 TGGAGAAGCAGGGCACGTGCA 22 ||||||||||||||||*|||**

meristem protein

RI PT

 

gene: 5108 ACCTCTTCGTCCCGTGAACGAG 5087

 

Os04g38720 

miRNA:

No apical

1 TGGAGAAGCAGGGCACGTGCA 22 ||||||||||||*|||||||**

meristem protein

gene: 1558 ACCTCTTCGTCCAGTGCACGCG 1537 Calcium-transporti

miRNA: 1 ATCAGGAGAGATGACACCGACA 23 *||||||||||||||||*||||*

ng ATPase 9

Os03g59770 

EF hand family protein

SC

 

Os02g08018 

gene:

96 GAGTCCTCTCTACTGTGCCTGTG 74

miRNA:

1 ATCAGGAGAGATGACACCGACA 23 |||||||||||:||||||||||*

M AN U

Osa‐miR143 2 

gene: 236 TAGTCCTCTCTGCTGTGGCTGTG 214

 

Os03g59790 

EF hand family protein

miRNA:

1 ATCAGGAGAGATGACACCGACA 23 |||||||||||:||||||||||*

gene: 164 TAGTCCTCTCTGCTGTGGCTGTT 142

 

Os03g59870 

EF hand family protein

miRNA:

1 ATCAGGAGAGATGACACCGACA 23 |||||||||||:||||||||||*

TE D

gene: 149 TAGTCCTCTCTGCTGTGGCTGTG 127

Osa‐miR530  Os01g56780 

Plus-3 domain

miRNA:

|||||||:|||||||||||*|*

containing protein

 

Os02g14990 

Zinc finger,

gene: 1315 ACGTAAATGTGGACGTGGACTT 1292 miRNA:

EP

1 TGCATTTGCACCTGCACCTAC 22 ||||:|||||||||||||:*|*

C3HC4 type

domain containing

1 TGCATTTGCACCTGCACCT-A 21

gene: 709 ACGTGAACGTGGACGTGGGCGC 688

protein

Os12g02540 

AC C

 

Osa‐miR820  Os03g02010 

 

Os11g03310 

Bric-a-Brac, Tramtrack,

miRNA:

1 TGCATTTGCACCTGCACCTAC 22 ||||:|||||||||||||*|*

gene: 572 ACGTGGACGTGGACGTGGACGT 551 DNA methyltransferase

miRNA: 1 TCGGCCTCGTGGATGGACCAGGAG 25 *|:||||||||||:||||||||||*

protein

gene: 983 CGTCGGAGCACCTGCCTGGTCCTCG 959

No apical

miRNA: 1 TCGGCCTCGTGGATGGACCAGGAG 25

meristem protein

|||:||||||||||||*|*|||||* gene: 1363 AGCTGGAGCACCTACCCGTTCCTCC 1339

       

ACCEPTED MANUSCRIPT

Table S5. Probes used for the small RNA northern-blot hybridizations.

Probe sequences (5’→3’)

miR393

GATCAATGCGATCCCTTTGGA

miR827

TGTTTGCTGATGGTCATCTAA

AC C

EP

TE D

M AN U

SC

RI PT

Probe name

ACCEPTED MANUSCRIPT Primer Sequences (5’→3’)

miR160s

TGCCTGGCTCCCTGTATGCCA

miR167s

TGAAGCTGCCAGCATGATCT

TAS3as

TTCTTGACCTTGCAAGACTT

miR827s

TTAGATGACCATCAGCAAAC

U6s

TACAGATAAGATTAGCATGGCCCC

OsARF18s

TGTTACAGAATGTGAAGAGGGTG

OsARF18a

ATATACCAAATTGAGCATGCCTGG

OsARF16s

GGTAGTTGCCAGTTTTGCCTAT

OsARF16a

ACCCTCAAATGGGAAGTCAG

OsSPX-MFS2s

GACATTAGCCGTTGCAGCACTC

OsSPX-MFS2a

CGTTCTTCGGTGATTCCCTGATT

OsSPX-MFS1s

TAAATACATCACCCTCCCCAT

OsSPX-MFS1a

GATCCGAAGTTCGATCCACT CAAGGCCATTGTCAGAACAC

OsARF25a

TTTGAGGCTTCCTGGTTAGA

OsARF2s

TGTCCACTCCCCATTCAAAACG

OsARF2a

CCAAAGCCTGAGCAATGGTAGG

OsARF15s

CATTTACAGTACATCGATGA

OsARF15a

CCCATAGACGCATCAACCAA

OsGAMYB1s

CTCAAGCAGGCTGTGGGTTTT

OsGAMYB1a

AAGGGGTTGCTGCTGGAGACT

OsLRR-RLK2s

CCCTCCTGGATAACCACTTT

OsLRR-RLK2a

GCCCCGAAGCCTCAGTGTTT

AC C

EP

TE D

M AN U

OsARF25s

SC

Primer Name

RI PT

Table S6. Primers used for the quantitative RT-PCR experiments.

ACCEPTED MANUSCRIPT

OsARF18in

CTTCTTGCCATCTGATTTCT

OsARF18out

TTCTCGGGCTTGAAACATCG

OsARF15in

GCCTGCCATTTATTCACCAC

OsARF15out

CGCCTATCATTCTCGTGCTT

OsTIR1in

ATACCGATACCGCACATACT

OsTIR1out

AAGTCTATCCAACCATCAGC

OsGAMYB1in

AAGGGGTTGCTGCTGGAGAC

OsGAMYB1out

GGAATGGACCAGGATGTTGT

OsLRR-RLK2in

GGATGAAACCAATGCCAGAA

OsLRR-RLK2out

CGATAAGCGGGATGTGGTT

OsAFB2in

TTCCTCCATTTCATTGCT

OsAFB2out

ATCCCTCGCCCCAGCAGTTGT

OsCSD1in

CATCCAATTCTTGCTCCTG

OsCSD1out

GCAGTGATAACACGTCAAAAGC

SC

Primer Sequences (5’→3’)

M AN U

Primer Name

RI PT

Table S7. Primers used for the 5-RLM-RACE experiments.

AC C

EP

TE D