Running head: Rice miRNAs in Immunity against the Blast ... · OsmiR156b/h resulted in increased tillers but reduced plant height and panicle size (Xie et al., 2006). These literatures

1

Running head: Rice miRNAs in Immunity against the Blast Disease 1

Corresponding author: Wen-Ming Wang 2

Address: Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China 3

Tel: 86-28-86290949 4

E-mail: [email protected] 5

Research Area: Signaling and Response 6

Plant Physiology Preview. Published on December 13, 2013, as DOI:10.1104/pp.113.230052

Copyright 2013 by the American Society of Plant Biologists

www.plantphysiol.orgon February 7, 2019 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

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Multiple Rice miRNAs Are Involved in Immunity against the Blast 7

Fungus Magnaporthe oryzae1 8

9

Yan Li2, Yuan-Gen Lu2, Yi Shi2, Liang Wu, Yong-Ju Xu, Fu Huang, Xiao-Yi 10

Guo, Yong Zhang, Jing Fan, Ji-Qun Zhao, Hong-Yu Zhang, Pei-Zhou Xu, 11

Jian-Min Zhou, Xian-Jun Wu, Ping-Rong Wang and Wen-Ming Wang* 12

Rice Research Institute and College of Agronomy, Sichuan Agricultural University, 13

Chengdu 611130, China (Y.L., Y.-G.L., Y.S., Y.-J.X., F.-H., X.-Y.G., Y.Z., J.F., J.-Q.Z., 14

H.-Y.Z., P.-Z.X., X.-J.W., P.-R.W., W.-M.W.); National Key Facility for Crop Resources 15

and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural 16

Sciences, Beijing 100081, China (L.W.); Institute of Genetics and Developmental Biology, 17

Chinese Academy of Sciences, Beijing 100101, China (J.-M.Z.)；Rice and Sorghum 18

Institute, Sichuan Academy of Agricultural Sciences, Deyang 618000, China (X.-Y.G.) 19

20

One-sentence summary 21

Deep sequencing small RNA libraries from susceptible and resistant lines identified 22

multiple miRNAs differentially responsive to the infection of the blast fungus Magnaporthe 23

oryzae and ectopic expression of either miR160a or miR398b confers elevated resistance to 24

the blast disease. 25



3

1This work was supported by National Natural Science Foundation of China (grant number 26

31101406 to Y.L.), Sichuan Agricultural University Start-up Fund to W.-M.W., and by the 27

Special Fund for Agro-scientific Research in the Public Interest (201203014 to F.H.). 28

2These authors contributed equally to the paper. 29

* Address correspondence to [email protected] 30



4

ABSTRACT 31

MicroRNAs (miRNAs) are indispensable regulators for development and defense in 32

eukaryotes. However, the miRNA species have not been explored for rice immunity against 33

the blast fungus Magnaporthe oryzae, the most devastating fungal pathogen in rice 34

production worldwide. Here, by deep sequencing small RNA (sRNA) libraries from 35

susceptible and resistant lines at normal conditions and upon M. orzyzae infection, we 36

identified a group of known rice miRNAs that were differentially expressed upon M. 37

oryzae infection. They were further classified into three classes based on their expression 38

patterns in the susceptible line LTH (Lijiangxin Tuan Hegu, Oryza sativa L. japonica) and 39

in the resistant line IRBLkm-Ts that contains a single resistance gene locus Pikm within 40

LTH background. RNA-blotting assay on nine of them confirmed sequencing results. 41

Real-time reverse transcription (RT) PCR assay showed the expressions of a part of target 42

genes were negatively correlated with the expressions of miRNAs. Moreover, transgenic 43

rice plants over-expressing miR160a and miR398b displayed enhanced resistance to M. 44

oryzae as demonstrated by decreased fungal growth, increased H2O2 accumulation at the 45

infection site and up-regulated expression of defense-related genes. Taken together, our 46

data indicate that miRNAs are involved in rice immunity against M. oryzae and 47

over-expression of miR160a or miR398b can enhance rice resistance to the disease. 48



5

In plant-pathogen co-evolution, plants mount two-layered immune system to protect 49

themselves from being damaged by pathogenic microbes. In the first layer of immunity, 50

plants detect conserved molecular features of microbes, termed pathogen-associated 51

molecular patterns (PAMPs) to trigger immunity (PTI) that is efficient to prevent a large 52

number of potential pathogenic microbes from invasion (Zipfel and Felix, 2005). 53

Successful pathogens deliver effectors into plant cells to suppress PTI and establish 54

parasitism (Boller and He, 2009; Dou and Zhou, 2012). In turn, the second layer of plant 55

immunity, called effector-triggered immunity (ETI), is initiated upon the recognition of 56

effectors by the cognate intracellular immune receptors, i.e. nucleotide-binding-site 57

leucine-rich repeat (NBS-LRR) type proteins. ETI is usually concomitant with 58

hypersensitive response (HR), a programmed cell death at the infection site to inhibit the 59

diffusion of the invading pathogen (Alfano and Collmer, 2004; Chisholm et al., 2006; Jones 60

and Dangl, 2006). In plant-fungal interactions, PTI and ETI equip plant pre- and 61

post-invasive resistance, respectively. Increasing evidence demonstrates that small RNAs 62

are involved in both PTI and ETI signaling and act as key fine-tuning regulators 63

(Padmanabhan et al., 2009; Katiyar-Agarwal and Jin, 2010). Small RNAs are short 64

non-coding RNAs which guide gene silencing either by regulating chromatin 65

methyl-modification or by mRNA degradation and translational repression (Baulcombe, 66

2004; Seo et al., 2013). They are classified into microRNAs (miRNAs) and small 67

interfering RNAs (siRNAs) based on their biogenesis and origin. Both miRNAs and 68

siRNAs are involved in regulation of diverse biological processes including development 69

and responses to biotic and abiotic stresses (Katiyar-Agarwal and Jin, 2010; Chen, 2012; 70

Khraiwesh et al., 2012). In Arabidopsis, a set of miRNAs were identified to be responsive 71

to the PAMP molecule flg22 or to the infection of non-pathogenic strain P. syringae 72

DC3000 (hrcC-) (Fahlgren et al., 2007; Li et al., 2010; Zhang et al., 2011). The first 73

identified PTI-related small RNA is miR393 that can be induced by the PAMP molecule 74

flg22 and positively contributes to PTI by suppressing auxin signaling via silencing auxin 75

receptors (Navarro et al., 2006). In addition, miR160a, miR398b and miR773 are involved 76

in regulation of callose deposition and thus act in PTI signaling (Li et al., 2010). Some 77

miRNAs also exhibit differential induced responses to the infection of pathogenic and 78



6

avirulent strains of P. syringae DC3000 and thus are involved in ETI signaling (Zhang et 79

al., 2011). More recently, some miRNAs are found to guide the cleavage of NBS-LRR type 80

disease resistance (R) genes in Solanaceae and Leguminosae species, indicating that these 81

miRNAs are key regulators in ETI (Zhai et al., 2011; Li et al., 2012; Shivaprasad et al., 82

2012). 83

The study of miRNAs in rice has been progressed from computational prediction to 84

experimental identification and functional characterization (Reinhart et al., 2002; Rhoades 85

et al., 2002; Jones-Rhoades and Bartel, 2004; Liu et al., 2005). The first set of rice miRNAs 86

experimentally identified was reported in 2004 (Wang et al., 2004). Later on, more 87

miRNAs either conservative or novel in rice were identified from rice shoot, root, 88

inflorescence, panicle, calli, developing grains, and immature seeds (Liu et al., 2005; 89

Sunkar et al., 2005; Luo et al., 2006; Zhu et al., 2008; Xue et al., 2009). By exposing rice 90

seedlings to drought or salt stress and through high throughput sequencing, Sunkar et al. 91

(2008) identified 23 new miRNAs and 40 candidates (Sunkar et al., 2008). By subject to 92

drought stress from tillering to inflorescence formation stages, 30 miRNAs were identified 93

to be differentially expressed under drought conditions (Zhou et al., 2010). By comparing 94

samples from normal conditions and exposed to oxidative stress, Li et al. (2011) identified 95

seven miRNA families that are differentially responsive to oxidative stress and discovered 96

32 new rice miRNAs (Li et al., 2011). By analyzing 62 small RNA libraries that represent 97

several tissues from control plants and those subjected to different environmental stress 98

treatments, Jeong et al. (2011) re-evaluated ~400 annotated miRNAs and found 76 new 99

miRNAs that are responsive to water stress, nutrient stress, or temperature stress (Jeong et 100

al., 2011). In addition, because miRNA relies on DCL1 for maturation, rice loss of function 101

mutation in OsDCL1 is lethal at seedling stage (Liu et al., 2005), implying that miRNA is 102

essential for development. MiR166 was ectopically expressed in mutants with mutations in 103

SHOOTLESS2 (SHL2), SHL4/SHOOT ORGANIZATION2 (SHO2), and SHO1 that are the 104

orthologues of Arabidopsis RNA-dependent RNA polymerase 6, ARGONAUTE 7, and 105

Dicer-like 4, respectively, leading to the silencing of the miR166 target genes OSHB1 and 106

OSHB2, two HD-ZIPIII genes (Nagasaki et al., 2007). Members of miR156 families target 107

the SQUAMOSA (SQUA) promoter-binding-like (SPL) genes and over-expression of 108



7

OsmiR156b/h resulted in increased tillers but reduced plant height and panicle size (Xie et 109

al., 2006). These literatures demonstrate that miRNAs play indispensable roles for 110

development and abiotic stress responses in rice. However, it remains elusive to identify the 111

miRNA species involved in rice immunity against the blast fungus M. oryzae. 112

The rice-rice blast fungus plant-pathogen system has become a model in the study of 113

plant-fungal interactions because rice is one of the most important staple food for the most 114

population of the world and rice blast is the most devastating disease for rice production 115

(Liu et al., 2013). The blast fungus M. oryzae can infect rice plants at any developmental 116

stages. After attaching onto the surface of a rice cell, conidium germinates to form a germ 117

tube at about 2 hours post inoculation (hpi), and then the germ tube differentiates an 118

appressorium at 2-20 hpi, which guides the fungus to penetrate into the underneath 119

epidermal cell at 24-30 hpi (Ribot et al., 2008). After entrance into the host cell, the fungus 120

develops bulbous infectious hyphae without visible damages to the host at first. With the 121

development of colonization, the fungus destroys the infected cells at 2-5 dpi and spreads 122

the disease by sporulation (Ribot et al., 2008). Both PTI and ETI are involved in rice 123

immunity against the blast fungus (Liu et al., 2013). Chitin, a conserved fungal-derived 124

PAMP, can be recognized by rice pattern-recognition receptors CEBiP (chitin 125

oligosaccharide elicitor-binding protein) to trigger innate immunity (Kaku et al., 2006; 126

Kishimoto et al., 2010; Shimizu et al., 2010). Knockdown of CEBiP in rice cell lines results 127

in obvious suppression of chitin-triggered oxidative burst (Kaku et al., 2006). The 128

PTI-related genes, such as OsKS4 and OsNAC4, can be induced by chitin in wild type 129

plants, but suppressed in susceptible transgenic lines (Park et al., 2012). M. oryzae delivers 130

a batch of effectors into rice cells through biotrophic interfacial complex (BIC) or invasive 131

hyphae to enhance its virulence (Mosquera et al., 2009; Khang et al., 2010; Mentlak et al., 132

2012). Some effectors are recognized by intracellular receptors, termed resistance (R) 133

proteins, to trigger race-specific resistance which contributes to the post-invasive resistance. 134

In the past decades, twenty-one rice blast R genes have been characterized by molecular 135

cloning, 19 of which encode NBS-LRR proteins, while Pi-d2 and Pi21 encode a 136

receptor-like kinase (RLK) protein and a Proline-rich protein, respectively (Fukuoka et al., 137

2009; Liu et al., 2010; Liu et al., 2013). Pikm locus contains two NBS-LRR genes, 138



8

Pikm1-TS and Pikm2-TS, both are required to recognize the cognate effector Avr-Pikm to 139

trigger effective resistance to the blast disease (Li et al., 2007; Yoshida et al., 2009). The 140

synergistic function of the two adjacent genes confers Pikm-specific resistance strongly 141

against infection of the rice blast fungus (Ashikawa et al., 2008). Pi2 and Piz-t are alleles 142

showing high similarity to Pi9 and they confer broad-spectrum resistance against different 143

sets of M. oryzae isolates (Qu et al., 2006; Zhou et al., 2006). However, up to date, limited 144

literatures imply that small RNAs may be involved in rice immunity against the blast 145

fungus M. oryzae. A recent report assayed the accumulation of miRNAs in rice treated by 146

elicitors extracted from blast fungal mycelia (Campo et al., 2013). A new rice miRNA, 147

osa-miR7695, is identified to be involved in rice resistance by negatively regulating the 148

transcript of OsNramp6 (Natural resistance-associated macrophage protein 6) (Campo et al., 149

2013). Currently, miRBase lists over 700 mature miRNA sequences cloned or predicted in 150

rice (http://www.mirbase.org) (Kozomara and Griffiths-Jones, 2011). Nevertheless, whether 151

and which of the known miRNAs are involved in rice immunity against the blast fungus 152

remain to be identified. 153

To obtain the miRNAs involved in rice immunity against the blast fungus, we 154

performed a systemic screen by comparing the abundance of miRNAs in the susceptible 155

line LTH and the resistance line IRBLkm-Ts that carries the Pikm locus. By deep 156

sequencing six small RNA libraries from samples of LTH and IRBLkm-Ts collected at 0, 157

12, and 24 hpi of M. oryzae, we identified candidate miRNAs that may be involved in rice 158

immunity because of their differential responses to the infection of the blast fungus. 159

Overexpression of miR160a and miR398b in transgenic rice plants enhanced resistance to 160

the blast fungus. Taken together, our data demonstrate that it is feasible to engineering rice 161

resistance by identification and ectopic expression of individual miRNA that is involved in 162

immunity against the blast fungus. 163

164

RESULTS 165

Identification of the Monogenic Resistant Line with Strong Defense Responses 166

To identify miRNAs most possibly involved in rice immunity against the blast disease, we 167

decided to choose an accession harboring an R gene that enables strong defense responses. 168



9

Previous study indicates that R genes at two loci render broad-spectrum resistance to the 169

blast disease in rice production (Zhou et al., 2006; Li et al., 2007; Ashikawa et al., 2008). 170

We thus chose rice lines containing R genes at these loci, i.e. IRBLkm-Ts and IRBLz5-CA, 171

in our investigation, and focused on one of them for further investigation. IRBLkm-Ts and 172

IRBLz5-CA are monogenic lines containing the resistance gene Pikm and Pi2 that were 173

introduced into LTH by backcrossing, respectively (Tsumematsu H, 2000). LTH is a 174

japonica variety highly susceptible to over 1300 regional isolates of M. oryzae worldwide 175

and no major R gene is ever identified in it (Lin et al., 2001). To evaluate Pikm- and 176

Pi2-mediated defense responses, three-leaf-old seedlings were inoculated with conidial 177

mixture of 15 M. oryzae strains collected from our rice blast disease nursery in Ya’an, 178

Sichuan province, China. Disease phenotypes were recorded at 10 dpi. Compared to LTH, 179

both IRBLkm-Ts and IRBLz5-CA displayed obvious resistant phenotypes (Fig. 1A). Then 180

by quantitative RT-PCR (qRT-PCR), we examined the expression of five defense-related 181

genes, including OsPR1, OsPR10, and the PTI-related genes OsPBZ1, OsKS4 and OsNAC4 182

(Park et al., 2012; Yamaguchi et al., 2013). The transcripts of all the five genes were 183

significantly increased in IRBLkm-Ts and IRBLz5-CA in comparison with those in LTH 184

(Fig. 1B-F). Moreover, OsRP1, OsPR10, OsPBZ1, and OsKS4 were induced earlier and to 185

greater amplitude in IRBLkm-Ts than in IRBLz5-CA (Fig. 1B-E). These data suggest that 186

Pikm renders defense responses faster and stronger than that does Pi2 against M. oryzae. 187

Because the production of H2O2 in infected leaf cells is a signal that precedes 188

hypersensitive responses (HR) that often culminates the defense responses in ETI (Levine 189

et al., 1994), we examined H2O2 accumulation and fungal growth at 2 and 10 dpi by 190

3,3’-diaminobenzidine and trypan-blue staining, respectively. As anticipated, in contrast to 191

no or trace amount of H2O2 accumulation in leaf cells of LTH, we observed high levels of 192

H2O2 accumulation around the appressoria at 2 dpi in the leaf cells of both IRBLkm-Ts and 193

IRBLz5-CA with stronger in IRBLkm-Ts (Fig. 1G). At 10 dpi, we observed massive aerial 194

mycelia with a great number of conidiophores on leaves of LTH, though H2O2 was also 195

accumulated in some cells (Fig. 1G, Supplemental Fig. S1A). In contrast, little hyphae and 196

no conidiophores were observed on leaves of IRBLkm-Ts and IRBLz5-CA, and high levels 197

of H2O2 were accumulated in the cells attacked by the appressoria (Fig. 1 G, Supplemental 198



10

Fig. S1B-C). 199

Taken together, these data suggest that the defense responses in IRBLkm-Ts are 200

stronger than that in IRBLz5-CA. Therefore, we used IRBLkm-Ts in the subsequent 201

screening for miRNAs involved in defense against M. oryzae. 202

203

Deep-Sequencing Analysis on Small RNA Libraries from M. oryzae-Free and Infected 204

Rice Leaves 205

To obtain miRNAs involved in rice defense against M. oryzae, three-week-old seedlings of 206

LTH and IRBLkm-Ts were inoculated with spore mixture of M. oryzae. Infected leaves 207

were collected at 12 and 24 hpi with leaves before inoculation as control. Six small RNA 208

libraries were constructed with the small RNA extracted from the collected leaves and 209

subjected to high-throughput sequencing. In total, 3,791,618, 1,277,195 and 2,559,590 210

reads for LTH and 2,723,456, 1,867,650 and 2,000,003 reads for IRBLkm-Ts from samples 211

collected at 0, 12 and 24 hpi matched to rice genome, respectively (Supplemental Table S1). 212

These reads represent 1,952,485, 765,215 and 1,379,760 unique sRNA sequences for LTH; 213

and 1,686,864, 1,127,034, and 1,139,492 unique sRNA sequences for IRBLkm-Ts at the 214

three time points, respectively (Supplemental Table S1). The size of most small RNAs is 21 215

and 24 nucleotides (nt), while the size of most known miRNAs is 21 nt (Mi et al., 2008; Wu 216

et al., 2009; Zhao et al., 2010). Consistently, small RNAs in our libraries were 217

predominantly 21 and 24 nt (Fig. 2A), while miRNAs were 21 nt (Fig. 2B), indicating that 218

our data is highly reliable. There were 50%-85% of small RNAs matched to rice genome in 219

the six libraries (Supplemental Table S1, Fig. 2C). Five to twenty-five percent of reads in 220

the libraries of inoculated samples (collected at 12 and 24 hpi) matched to M. oryzae 221

genome, while only about 1% reads in the libraries constructed from the control samples 222

matched to M. oryzae genome (Supplemental Table S1, Fig. 2C). These data suggest that 223

the small RNAs identified were of rice and M. oryzae origins. 224

However, there were 20%-30% reads that could not match to the rice or M. oryzae 225

genome in each library (Supplemental Table S1), which is presumably due to the DNA 226

sequence polymorphisms between the rice cultivars / M. oryzae isolates used and the 227

reference genomes. 228



11

Then the rice small RNA sequences were classified into different categories based on 229

their match to the rice genome (Supplemental Table S1, Fig. 2D). The largest category of 230

the small RNAs was heterochromatic siRNAs (hc-siRNAs), followed by small RNAs 231

matched to protein-coding genes (Fig. 2D). Over 20 thousand reads from each library that 232

accounted for 10%-20% matched to rice mitochondria and chloroplast genome 233

(Supplemental Table S1, Fig. 2D). There were 2%-3% of reads matched to nat-siRNAs but 234

no ta-siRNAs (Fig. 2D). Reads matched to rice miRNAs accounted for 5%-10%, which is 235

consistent with a previous report (Fig. 2D) (Wu et al., 2009). Compared with control 236

sample at 0 dpi, miRNA reads were decreased significantly at 12 hpi in LTH, whereas 237

increased at 24 hpi, suggesting that the blast fungus suppresses miRNA biogenesis in 238

compatible rice-blast fungus interaction during pre-penetration stage (Supplemental Table 239

S1, Fig. 2D). In contrast, the miRNA reads were increased in IRBLkm-Ts along with the 240

infection of M. oryzae (Supplemental Table S1, Fig. 2D), implying that miRNAs might 241

play roles in rice immunity against M. oryzae. Because of the large volume of total 242

sequence data and our primary focus in this study on learning about how the known 243

miRNAs were involved in rice immunity, novel miRNAs due to M. oryzae infection or the 244

M. oryzae-derived miRNAs and their biological implications will be the focus of future 245

research. 246

247

Candidate MicroRNAs Involved in Rice -M. oryzae Interaction 248

The miRNAs involved in immunity must display differential expression upon M. oryzae 249

infection. According to this speculation, we compared the miRNA accumulation patterns 250

between the libraries from LTH and IRBLkm-Ts upon M. oryzae infection, and the 251

miRNAs with reads changing over-2-fold in any two of the six libraries were selected for 252

further investigation (Supplemental Table S2). Accordingly, these miRNAs were classified 253

into 39 patterns by analysis with Short Time-series Expression Miner (STEM) software 254

(Ernst and Bar-Joseph, 2006) based on their change trends, of which 19 patterns showed 255

significant changes in the accumulation of miRNAs upon M. oryzae infection 256

(Supplemental Table S3). We further classified the miRNAs of the 19 patterns into 3 257

classes based on their change upon M. oryzae infection: 1) no obvious change in LTH, but 258



12

significantly increased in IRBLkm-Ts (i.e. patterns #2, #11, #22) or reduced in LTH, but no 259

obvious change or increased in IRBLkm-Ts (i.e. #7); 2) no significant change in LTH, but 260

decreased greatly in IRBLkm-Ts (i.e. #21) or increased in LTH, but no obvious change or 261

decreased in IRBLkm-Ts (i.e. #08, #16, #18, #25, #30, #32, #40, #42 and #45); 3) 262

increased in both LTH and IRBLkm-Ts upon M. oryzae infection, including the patterns 263

#27, #29, #39, #46 and #47. Theoretically, the first two classes of miRNAs should play 264

positive and negative roles in rice resistance, respectively; while the third class may be 265

involved in regulation of basal responses to the M. oryzae infection because of their 266

up-regulation in both susceptible and resistant lines. 267

Next, the miRNAs belonging to the classes mentioned above with more than 100 reads 268

in one of the six libraries were selected for further analysis (Supplemental Table S2). In 269

total, 33 miRNAs were selected with the pattern number listed in the bracket following 270

their name in Table 1. The expressions of nine miRNAs were analyzed by sRNA-blotting to 271

verify the deep sequencing results, including three positive regulators (i.e. miR160a, 272

miR164a and miR168a), three negative regulators (i.e. miR396, miR827 and miR1871), and 273

three basal responsive regulators (i.e. miR169a, miR172a, and miR398b) (Fig. 3A). The 274

sRNA-blotting analysis was basically consistent with the sequencing results. As shown in 275

Fig. 3A, the accumulation of miR160a and miR164a were significantly induced to higher 276

levels upon M. oryzae infection in IRBLkm-Ts. MiR172a and miR398b were increased in 277

both LTH and IRBLkm-Ts, whereas miR1871, miR396d and miR827a were obviously 278

increased in LTH. 279

280

Expression Pattern of The miRNA Target Genes upon M. oryzae Infection 281

Because the regulatory roles of miRNAs in gene expression is to cleave target mRNAs or 282

repress translation, we firstly examined the transcripts of some target genes which were 283

previously validated by degradome assay using qRT-PCR (Supplemental Table S4) (Wu et 284

al., 2009; Li et al., 2010; Zhou et al., 2010). As our anticipation, the expressions of certain 285

targets of miR160, miR164, miR827, miR172 and miR398 exhibited negative correlation to 286

the abundance of miRNAs (Fig. 3B-G), which is consistent with one of the miRNAs’ roles 287

to guide degradation of their target genes. Among the tested target genes, Os04g43910 288



13

encodes putative auxin response factor 16 (ARF16) and Os07g46990 encodes superoxide 289

dismutase 2 (SOD2); and they were previously confirmed as the target of miR160 and 290

miR398 by degradome cDNA library assay, respectively (Wu et al., 2009). Os04g48410 291

encoding a copper chaperone for SOD was identified as another target of miR398 (Li et al., 292

2010). In addition, Os12g41680, Os04g48390, and Os05g03040 were confirmed as the 293

targets of miR164, miR827 and miR172 by degradome sequencing, respectively (Wu et al., 294

2009; Li et al., 2010; Zhou et al., 2010). The negative correlation between the abundant of 295

miRNA and its target indicates that they are truly target of the miRNAs and thus may be 296

involved in regulation of rice immunity against M. oryzae. Nevertheless, nine of the 297

confirmed or predicted target genes exhibited partially negative correlation with the 298

miRNA abundance (Supplemental Fig. S3 and Supplemental Table S4), thus whether they 299

are targets of the miRNAs regulated via translational repression needs further experimental 300

verification. 301

302

Enhanced Defense Responses to M. oryzae in The Transgenic Rice Lines 303

Over-Expressing miR160a and miR398b 304

Next, we took transgenic approach to verify representatives of the miRNAs involved in 305

defense responses to M. oryzae. Kasalath is an indica accession that possesses high 306

re-generation rate during tissue culture and is sensitive to most M. oryzae strains (Li et al., 307

2007), thus suitable for employment in transgenic analysis. MiR160a represents positive 308

regulators of resistance; its expression was not much changed in LTH but increased 309

approximately 4-fold upon M. oryzae infection in IRBLkm-Ts (Table 1 and Fig. 3A). Two 310

independent transgenic lines with high miR160a expression were identified by miRNA 311

real-time RT-PCR and RNA-blot analyses in comparison with the control line (Fig. 4A,B). 312

Consistently, the expression of its target genes Os02g41800 and Os04g43910, both of 313

which encode putative auxin response factor 16 (ARF16), were reduced to less than 10% of 314

that in the control line (Fig. 4C). The transcript of Os04g59430, another target of miR160 315

encoding a B3 DNA binding domain containing protein, was also significantly suppressed 316

though not as much as that of Os02g41800 and Os04g43910 (Fig. 4C). However, the 317

expression of Os10g33940 and Os06g47150, both of which also encode putative ARF16, 318



14

were up-regulated to levels higher than that in the control line (Supplemental Fig. S4), the 319

simplest explanation is that miR160a may repress their translation or they are under 320

feedback regulation by their products. Next, we examined the transgenic lines by 321

inoculating with mixed M. oryzae strains, and disease phenotypes were recorded at 10 dpi. 322

The number of lesions in the transgenic lines over-expressing miR160a was obviously less 323

than that in the control line (Fig. 4D). In addition, the disease lesions were mainly scored as 324

type 1 - 2 in the miR160a over-expression line #4, which was obviously less severe than the 325

type 3 - 4 in the control line (Fig. 4D). These observations suggested that the resistant level 326

was higher in the transgenic lines. Consistent with the resistant phenotype, the spore 327

number and relative fungal mass in the infected leaves, indicated as the ratio of the M. 328

oryzae Pot2 gene versus the rice ACTIN1 (Berruyer et al., 2006; Park et al., 2012), were 329

significantly reduced in the transgenic lines than that in the control line (Fig. 4E,F). 330

MiR398b represents regulators acting in basal responses because its expression is 331

increased in both LTH and IRBLkm-Ts upon M. oryzae infection. The abundant of 332

miR398b increased approximately 8-fold at 24 hpi in LTH and increased in IRBLkm-Ts 333

over 5-, 8-fold at 12, 24 hpi, respectively (Fig. 3A). We also identified two transgenic lines 334

in which the accumulation of miR398b was significantly increased (Fig. 5A,B). 335

Consistently, its confirmed target genes Os03g22810, Os04g48410, Os07g46990 and 336

predicted target gene Os11g09780 were all suppressed greatly in the two miR398b 337

transgenic lines (Fig. 5C) (Wu et al., 2009; Li et al., 2010; Zhou et al., 2010). Then the 338

miR398b transgenic lines were examined by inoculation with mixed M. oryzae strains and 339

disease phenotypes were recorded at 10 dpi. The number of lesions in the transgenic lines 340

over-expressing miR398b was obviously less than that in the control line (Fig. 5D). In 341

addition, the disease lesions were obviously reduced to type 2 and type 3 in the miR398b 342

over-expression lines #9 and #10, respectively, which were less severe compared to type 3 - 343

4 in the control line (Fig. 5D). These observations suggested that the resistant level was 344

increased in the transgenic lines. Consistent with the resistant phenotype, the spore number 345

and relative fungal biomass in the infected leaves were significantly reduced in transgenic 346

lines than that in the control line (Fig. 5E,F). 347

Resistant phenotypes are usually accompanied with up-regulation of defense-related 348



15

genes, thus we examined the transcription of some defense-related genes in one line 349

over-expressing miR160a or miR398b, respectively. As our anticipation, both miR160a and 350

miR398b transgenic lines displayed expression of OsPR1 and OsPR10 significantly higher 351

than that of the control line and the expression level in the miR398-overecpression line was 352

higher than that in the miR160a-over-expression line (Fig. 6A,B). In addition, the 353

expression of PTI-related genes OsKS4 and OsNAC4 was significantly enhanced in the 354

miR398b transgenic line, but was not obviously affected in the miR160a transgenic line 355

(Fig. 6C,D). Furthermore, the transcripts of both OsKS4 and OsNAC4 were significantly 356

enhanced at 1 hour after treated by flg22 and chitin in the miR398b-overexpression line, but 357

were not affected in the miR160a-overexpression line (Fig. 6E,F), suggesting that miR398b 358

is most likely involved in PTI-signaling to regulate defense responses, while miR160a may 359

be in ETI-signaling. 360

To visualize how the transgenic lines over-expressing miR160a and miR398b mount 361

resistance on cellular level, the virulent M. oryzae strain Zhong-8-10-14 expressing 362

enhanced GFP (eGFP) was inoculated on leaf sheath. Then the pathogenesis was observed 363

under Laser Scanning Confocal Microscope (LSCM). The spores were germinated and 364

formed appressoria on sheath of control plants at 12 hpi; and invasive hyphae were formed 365

at 24 hpi and proliferated widely at 36 hpi (Fig. 7A). In contrast, the spores did not 366

germinate and appressoria were not generated until 24 hpi in transgenic plants 367

over-expressing miR160a and 36 hpi over-expressing miR398b, respectively (Fig. 7B-C and 368

F). Furthermore, invasive hyphae were hardly observed on both transgenic plants until 36 369

hpi (Fig. 7B-C and G). 370

Then, the accumulation of H2O2 and fungal growth were examined at 2 and 10 dpi. 371

Microscopic analysis showed that at 2 dpi, H2O2 was accumulated around the 372

appressorium in the leaf cells of miR160a and miR398b over-expression transgenic lines, 373

whereas no or little H2O2 was accumulated in leaf cells in control line (Fig. 7D). At 10 dpi, 374

we observed the well-grown aerial hyphae and massive production of conidiophores in 375

control line, although H2O2 was also accumulated in the cells from where the aerial 376

hyphae were generated (Fig. 7E, Supplemental Fig. S5A). Conversely, in the miR160a and 377

miR398b over-expression lines, we observed high levels of H2O2 accumulation in the leaf 378



16

cells beneath the appressoria and few aerial hyphae but no conidiophores were observed 379

(Fig. 7E and Supplemental Fig. S5B-C). These observations indicated that the fungus 380

failed to colonize the leaves. 381

Taken together, the results from transgenic analysis indicate that both miR160a and 382

miR398b contribute to suppression of fungal infection and may act differentially in rice 383

immunity against the blast fungus. 384

385

DISCUSSION 386

Host miRNAs play pivotal roles in plant-microbe interactions. Identification of host 387

miRNAs that are differentially induced by pathogen infection is the first step to investigate 388

their functions in host immunity against the pathogen. Previously, a set of miRNAs were 389

identified to be involved in plant immunity against bacterial and viral pathogens (Navarro 390

et al., 2006; Du et al., 2011; Radwan et al., 2011; Seo et al., 2013). Recently, host miRNAs 391

are also found to be responsive to the infection of a increasing list of filamentous pathogens, 392

such as Cronartium quercuum (Lu et al., 2007), Erysiphe graminis (Xin et al., 2010), 393

Fusarium virguliforme (Radwan et al., 2011), Phytophthora sojae (Guo et al., 2011), and 394

Verticillium dahliae (Yin et al., 2012; Yang et al., 2013). In the present study, small RNA 395

accumulation in rice seedlings of susceptible and resistant lines upon M. oryzae infection 396

was systemically assayed by deep sequencing. The changes in abundance of nine miRNAs 397

were verified by RNA blotting analyses, suggesting that the deep sequencing data are 398

reliable. Totally, we found representatives from 25 known rice miRNA families that were 399

presumably involved in innate immunity against the infection of the blast fungus M. oryzae 400

(Table 1). There seems more known miRNAs that negatively regulate rice immunity than 401

those do positively. Members of four miRNA families, i.e. miR160a/b/c/d/f, miR164a/b/f, 402

miR167a/b/c, and miR168a, were reduced or not much changed in expression in the 403

susceptible line, but significantly increased in the resistant line upon M. oryzae infection, 404

thus they may positively regulate rice immunity against the fungus. In contrast, members 405

from sixteen families were up-regulated in expression in the susceptible line but 406

down-regulated or did not exhibit significant changes in the resistant line after M. oryzae 407

infection, thus they may negatively regulate rice immunity. Members from ten miRNA 408



17

families were up-regulated in expression in both the susceptible and resistant lines, thus 409

they may act in fundamental rice responses to M. oryzae infection (Table 1 and Fig. 3). 410

Three miRNA families had members differentially responsive to M. oryzae infection, 411

including miR167, miR169, and miR444. While miR167a/b/c might act as positive regulator 412

for rice immunity, miR167d/e/f/g/h/i/j act as negative regulator. MiR169a/b/c might act in 413

basal responses to M. oryzae infection, whereas miR169f/g/h/i/j/k/l/m might negatively 414

function in rice immunity against the blast fungus. Similarly, miR444b.1/c.1 might function 415

in basal responses to M. oryzae infection, whereas miR444b.2/c.2 might negatively regulate 416

rice immunity against the blast fungus (Table 1 and Fig. 3). Thus the identified known 417

miRNAs involved in rice immunity provides a good starting point to further pursue how 418

rice resistance to the blast fungus is regulated through diverse aspects. 419

In the present investigation, we detected that miR160a was up-regulated, while its 420

target gene Os04g43910 (ARF16) was down-regulated in the resistant line, but both 421

miRNA and its target were not much changed in the susceptible line upon M. oryzae 422

infection, implying that miR160a may positively regulate rice immunity by silencing IAA 423

signaling (Table 1, and Fig. 3). Then we obtained transgenic rice lines over-expressing 424

miR160a that exhibited enhanced resistance to the blast disease. RNA blotting and 425

qRT-PCR analyses verified that miR160a was highly expressed in contrast to the 426

significantly suppressed expression of its three target genes (i.e. Os02g41800, Os04g43910 427

and Os04g59430) (Fig. 4). Nevertheless, the expression of Os10g33940 and Os06g47150, 428

two targets of miR160a, were significantly up-regulated in the transgenic lines 429

over-expressing miR160a (Supplemental Fig. S4). One explanation is that miR160a may 430

repress their translation. This possibility could be easily confirmed if their antibody were 431

available. Alternatively, these two target genes of miR160a may be under feedback 432

regulation by their products as the case for the targets of miR172 in Ariabidopsis (Schwab 433

et al., 2005). It seems that miR160a may act in post-invasive defense responses, because the 434

expression of the PTI-related genes OsKS4 and OsNAC4 were not induced in the 435

miR160a-overexpression line upon M. oryzae infection or after PAMP (flg22 and chitin) 436

treatment (Fig. 6). However, miR160a acts in regulation of PTI and PAMP-triggered 437

defense responses in Arabidopsis (Li et al., 2010). Therefore, the functions of miR160a 438



18

seem to be conserved with diversification in plant innate immunity between monocots and 439

dicots. 440

Our data also revealed that the expression of miR398b was induced upon M. oryzae 441

infection in both the susceptible and resistant lines, while its target genes Os07g46990 442

(SOD2) and Os04g48410 (encoding a copper chaperone for SOD) were down-regulated 443

(Table 1, Fig. 3). By transgenic approach, we identified two transgenic lines 444

over-expressing miR398b. Both transgenic lines exhibited resistance to the blast fungus as 445

revealed by weaker disease phenotypes, less fungal mass, up-regulated expression of 446

defense-related genes, and stronger H2O2 production (Fig. 5, 6, 7 and Supplemental Fig. 447

S5). The two PTI-related genes OsKS4 and OsNAC4 as well as OsPR1 and OsPR10 were 448

significantly induced in the miR398b-overexpression transgenic line upon M. oryzae 449

infection and after PAMP treatments, indicating that miR398b might act in positively 450

regulation of both pre- and post-invasive defense responses against the infection of the blast 451

fungus. The cellular responses of the miR398b overexpression line to an eGFP-tagged 452

strain showed that both conidial germination and appressorium formation were largely 453

delayed, thus substantiated the roles of miR398b in positive regulation of pre-invasive 454

defense (Fig. 7). However, from literatures, miR398b seems to negatively regulate PTI and 455

ETI responses against bacterial pathogen, because the accumulation of miR398b is slightly 456

down-regulated after flg22 treatment in Arabidopsis (Li et al., 2010). The abundance of 457

miR398b can also be down-regulated by the infection of the incompatible bacteria Pst 458

DC3000 (avrRpm1 and/or avrRpt2), but not the compatible strain Pst DC3000 459

(Jagadeeswaran et al., 2009). Moreover, transgenic lines overexpressing miR398b leads to 460

repression of flg22- and hrcC--induced callose deposition and hyper-sensitivity to the 461

virulent strain DC3000 and the avirulent strain DC3000 hrcC- (Li et al., 2010). Thus, 462

miR398b may act contrarily between monocots and dicots in the regulation of innate 463

immunity. 464

The findings that miR160a and miR398b positively regulate rice immunity against M. 465

oryzae raise interesting questions concerning the potential roles of their target genes in 466

resistance. Auxin is regarded as a negative regulator for plant resistance (Navarro et al., 467

2006; Fu et al., 2011). ARF proteins are key factors in auxin signal pathway. They bind to 468



19

auxin-responsive elements (AuxREs) in promoters to regulate transcription of primary 469

auxin-response genes (Hagen and Guilfoyle, 2002). In Arabidopsis, miR393 specifically 470

targets TIR/AFB transcripts to silence auxin signaling pathway and enhance plant innate 471

immunity against bacterial pathogen (Navarro et al., 2006). Overexpression of miR160a 472

suppresses the expression of ARF16 and enhances flg22- and nonpathogenic bacteria 473

DC3000 (hrcC-)-induced callose deposition (Li et al., 2010). Rice gene GH3-2 encodes an 474

indole-3-acetic acid (IAA)-amido synthetase, which inactivates IAA signaling by 475

catalyzing the formation of an IAA-amino acid conjugation to suppress pathogen-induced 476

IAA accumulation, thus contribute to broad-spectrum resistance against both bacterial and 477

fungal pathogens in rice (Fu et al., 2011). In this study, we demonstrated that miR160a also 478

targets putative ARF16 in rice and contributes to resistance against M. oryzae, suggesting 479

the conservative role of miR160 in plant innate immunity by silencing auxin pathway genes 480

in both monocots and dicots. 481

SODs are a group of metalloenzymes converting superoxide anion (O2−) into hydrogen 482

peroxide and oxygen molecules to protect plant from damages caused by free radical 483

species (Beyer et al., 1991). Overexpressing tobacco Mn-SOD in alfalfa (Medicago sativa 484

L.) enhanced plant tolerance to freezing stress, suggesting a protective role in minimizing 485

oxygen free radical production after freezing stress (McKersie et al., 1993). 486

Down-regulation of miR398 by oxidative stresses leads to higher transcription of CSD1 and 487

CSD2, and enhances oxidative stress tolerance in Arabidopsis (Sunkar et al., 2006). 488

Conversely, miR398 level is up-regulated by sucrose, resulting in decreased CSD1 and 489

CSD2 mRNA and protein accumulation (Dugas and Bartel, 2008). Here, we showed that 490

miR398b accumulation is significantly increased at 24 hpi in the susceptible line LTH and 491

increased at 12 hpi in the resistant line IRBLkm-Ts upon M. oryzae infection (Table 1 and 492

Fig. 3). Three genes, i.e. Os03g22810, Os07g46990 and Os04g48410 that encode SOD, 493

SOD2 and copper chaperone for SOD, respectively, were confirmed as the targets of 494

miR398b by degradome sequencing or RNA ligase-mediated 5’ rapid amplification of 495

cDNA ends (RLM 5’-RACE) (Wu et al., 2009; Li et al., 2010; Zhou et al., 2010). 496

Os11g09780 was also predicted as a target of miR398b and encodes a hypothetical protein 497

(Wu et al., 2009). However, we were not successful in confirming whether the mRNA of 498



20

Os11g09780 was cleaved at the predicted target site via RLM 5’-RACE. It is highly 499

feasible that the cleaved mRNA of Os11g09780 is degraded quickly after its cleavage or 500

difficult to be detected because the target site is close to the 3’ end of the cDNA. Consistent 501

with this speculation, Os11g09780 was not detected in previous degradome analyses (Wu et 502

al., 2009; Li et al., 2010; Zhou et al., 2010). Alternatively, Os11g09780 may not be truly the 503

target of miR398b. Nevertheless, consistent with the up-regulated miR398b levels, its target 504

genes were down-regulated, and the transgenic plants overexpressing miR398b displays 505

significantly lower SOD/SOD2 transcripts and higher resistance to M. oryzae, indicating 506

that miR398b may contribute to ROS production by suppressing SOD level in cells to 507

counter-attack the invading blast fungus. It is possible that miR398b guides the degradation 508

of SOD transcripts to keep dynamic balance of superoxide anion and hydrogen peroxide 509

metabolism during M. oryzae infection. Thus future work will focus on functional 510

investigation of the target genes of miRNAs and functional identification of the 511

components of the regulation network. 512

Thus, by deep-sequencing of small RNA libraries constructed from samples of 513

susceptible and resistant lines, we identified a set of known miRNAs that acts either 514

positively or negatively in regulation of rice immunity against the blast fungus. By 515

transgenic approach, we further demonstrated that ectopic expression of a single miRNA is 516

able to establish rice resistance to the blast disease. 517

518

MATERIALS AND METHODS 519

Plant Materials and Growth Conditions 520

Rice susceptible line LTH and its resistant monogenic lines IRBLkm-Ts and IRBLz5-CA 521

were grown in a growth room maintained at 26℃ and 70% relative humidity with a 14/10 522

h of day/night light. The indica accession Kasalath (Oryza sativa ssp indica) was used for 523

transgenic analysis. 524

525

Library Construction, Sequencing and Bioinformatics Analysis of Small RNAs 526

Small RNA library construction and Illumina sequencing were performed as described (Mi 527



21

et al., 2008). The small RNA reads with length over 16 were mapped to the O. sativa 528

nuclear, chloroplast, and mitochondrial genomes (http://rice.plantbiology.msu.edu/, version 529

6.0) and M. oryzae genomes (http://www.broadinstitute.org). The perfect genome-matched 530

sRNAs were analyzed following a previous report (Wu et al., 2009). The normalized 531

abundance of small RNAs were calculated as reads per million. 532

533

Small RNA Gel-Blot Analysis 534

RNA-blot analyses for small RNAs from total extracts were performed as previously 535

described (Qi et al., 2005; Mi et al., 2008). Three-leaf-old plants spraying-inoculated with 5 536

×105 spores/mL was used for RNA extraction at 0, 12, 24, 48, and 72 hpi, respectively. 537

MiRNA probes were end labeled with γ-32P-ATP by T4 polynucleotide kinase (NEB). 538

Probes used for RNA-blot were listed in Supplemental Table S4. 539

540

Construction of miRNA Overexpression Transgenic Lines 541

To make over-expression constructs, genomic sequences from 330 bp upstream to 242 bp 542

downstream of miR160a and from 289 bp upstream to 306 bp downstream of miR398b 543

were amplified from Nipponbare (NPB) genomic DNA. PCR products were cloned into the 544

binary vector 35S-pCAMBIA1300 at KpnI/SalI sites. Primers used for making constructs 545

were listed in Supplemental Table S5. Constructs were transformed into Kasalath via 546

Agrobacterium-mediated transformation. Transgenic plants were preliminarily screened by 547

PCR with the forward primer 35S-F and the specific reverse primers (OsmiR160a-R-Sal1 548

for miR160a and OsmiR398b-R-Sal1 for miR398b, respectively) ( Supplemental Table S5). 549

Then, the obtained transformants were subjected to hygromycin-sensitive test as follows. 550

Leaves from 3-4 week old plants were incubated in 0.1 mM 6-BA buffer containing 30 551

mg/L hygromycin for 3-5 days at 26℃ and 70% relative humidity with a 14/10 hours of 552

day/night light. The plants whose leaves showed hygromycin-resistant were selected as 553

positive transgenic plants for further experiments. 554

555

Quantitative RT-PCR 556



22

Three-leaf-old plants were inoculated with M. oryzae by spraying spore suspension at the 557

concentration of 5 ×105 spores/mL and samples were collected at 0, 6, 12, 24, and 48 hpi. 558

For PAMP triggered responses, three-leaf-old plants were incubated in water with or 559

without 1 μM of flg22 or 1 μg/ml of chitin for 1 hour, then the treated leaves were 560

subjected to RNA extraction with Trizl reagent (Invitrogen). Reverse transcription of RNA 561

to cDNA was conducted by using the Super-Script first-strand synthesis system 562

(Invitrogen). miRNAs were elongated and reverse transcribed by using NCodeTM miRNA 563

First-strand cDNA module (Invitrogen). SYBR Green Mix (TaKaRa) was used in real-time 564

PCR to determine the abundance of mRNA. Gene expression levels were normalized by 565

using OsActin1 as an internal control. Primers used in real-time RT-PCR were list in 566

Supplemental Table S5. 567

568

Pathogen Infection and Microscopy Analysis 569

M. oryzae strains used in this study (including B9, B15, DJ-15, D35-7, E5, LJ5-4, LJ9-5, 570

LJ15-8, NC-10, NJ-10, OJ-15, U-24, WJ-10, YC-5, and YC-7) were collected from rice 571

fields in Ya’an (E103.0, N29.98, southwest of Sichuan Province, China). The M. oryzae 572

strains were grown on complete medium (CM) with agar for 2 weeks at 28°C with 12/12 h 573

day/night light for sporulation. Inoculum concentration of each strain was adjusted to 5 574

×105 spores/mL, and the same volume of spore suspension of the 15 strains were mixed for 575

spraying inoculation (Qu et al., 2006). Three-week-old seedlings (for small RNA library 576

construction) or eight-week-old plants (for resistant evaluation of transgenic lines) were 577

prepared for spraying inoculation, and disease symptom was observed at 10 dpi. The lesion 578

types were scored from 0 (resistant) to 5 (susceptible) using a standard reference scale 579

(Mackill and Bonman, 1992). The blast sporulation rate on the lesions and the fungal mass 580

in infected rice leaves were measured as described (Park et al., 2012). 581

For cellular response in rice to M. oryzae infection, the eGFP-tagged M. oryzae strain 582

Zhong-8-10-14 was inoculated on 5-cm-long leaf sheath as described (Kankanala et al., 583

2007). The inoculated epidermal layer was excised and analyzed under Laser Scanning 584

Confocal Microscope (Nikon A1, Japan) at 12, 24 and 36 hpi, respectively. 585



23

H2O2 accumulation and fungal structure were stained by 3,3’-diaminobenzidine (DAB) 586

and trypan-blue, respectively, referring to a previous report (Xiao et al., 2003). Leaf 587

sections were placed in 1 mg/mL of DAB (Sigma), and incubated at 22℃ for 8 h at 588

illumination. The DAB-stained leaves were double stained with trypan-blue and observed 589

under microscope (Zeiss imager A2, Germany). 590

591

Supplemental data 592

Supplemental Table S1. Category of small RNAs before and after M. oryzae infection. 593

Supplemental Table S2. Normalized reads of rice miRNAs before and after M. oryzae 594

infection. 595

Supplemental Table S3. miRNA profiles assayed by STEM analysis. 596

Supplemental Table S4. List of miRNA targets assayed by RT-PCR. 597

Supplemental Table S5. Primers and probes used in this study. 598

Supplemental Figure S1. Fungal growth on susceptible and resistant lines. Representative 599

leaf section from LTH (A), IRBLkm-Ts (B), and IRBLz5-CA (C), stained by 600

3,3’-diaminobenzidine and trypan blue, respectively, to show the accumulation of H2O2 601

(reddish brown) and fungal mass (blue) at 10 days past inoculation of M. oryzae. Bars, 50 602

μm. 603

Supplemental Figure S2. Patterns of miRNA expression upon M. oryzae infection. The 604

expressions of miRNAs at 0, 12, 24 hpi in LTH and IRBLkm-Ts were categorized into 39 605

patterns according to their changes before and after M. oryzae infection by Short 606

Time-series Expression Miner (STEM) analysis. The colored profiles had a statistically 607

significant number of genes assigned. The number at the top left corner of a box indicates 608

the pattern number. For each pattern, the number of miRNAs assigned to the pattern was 609

showed at the lower left corner of the box. 610

Supplemental Figure S3. Expression of miRNA target genes upon M. oryzae infection. 611

Quantitative RT-PCR analyses of mRNA levels for genes targeted by predicted positive (A), 612

negative (B), and basal responsive (C) miRNAs. Error bars indicate SD. Student’s t test was 613

used to determine the significance of difference between 0 hpi and the indicated time points 614



24

in the indicated lines. Asterisks (* and **) indicate significant difference at a P value <0.05 615

and <0.01, respectively. The experiments were repeated two times with similar results. 616

Supplemental Figure S4. Quantitative RT-PCR assay for the indicated two target genes of 617

miR160a in the indicated transgenic lines. 618

Supplemental Figure S5. Fungal growth on transgenic lines. Representative leaf sections 619

stained by 3,3’-diaminobenzidine and trypan blue, respectively, to show the accumulation 620

of H2O2 (reddish brown) and fungal mass (blue) at 10 dpi of M. oryzae on the transgenic 621

plants expressing empty vector (A), 35S:miR160a (B), and 35S:miR398b (C). Bars, 50 μm. 622

623

ACKNOWLEDGMENTS 624

We thank Dr. Cai-Lin Lei (Institute of Crop Science, Chinese Academy of Agricultural 625

Sciences) for providing the monogenic resistant lines IRBLkm-Ts and IRBLz5-CA, Mr. 626

Yang Lei for help with Laser Scanning Confocal Microscopy, Ting-Ting Wang, Jin-Long 627

Sun and Liang Li for technique supports, Professor Li-Huang Zhu for providing the 628

eGFP-tagged strain and suggestions on writing the manuscript, and Drs Xue-Wei Chen, 629

Min He, and Wei-Tao Li for advices during experiments. 630

631

REFERENCES 632

Alfano JR, Collmer A (2004) Type III secretion system effector proteins: double agents in bacterial disease 633

and plant defense. Annu Rev Phytopathol 42: 385-414 634

Ashikawa I, Hayashi N, Yamane H, Kanamori H, Wu J, Matsumoto T, Ono K, Yano M (2008) Two 635

adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific 636

rice blast resistance. Genetics 180: 2267-2276 637

Baulcombe D (2004) RNA silencing in plants. Nature 431: 356-363 638

Berruyer R, Poussier S, Kankanala P, Mosquera G, Valent B (2006) Quantitative and qualitative influence 639

of inoculation methods on in planta growth of rice blast fungus. Phytopathology 96: 346-355 640

Beyer W, Imlay J, Fridovich I (1991) Superoxide dismutases. Prog Nucleic Acid Res Mol Biol 40: 221-253 641

Boller T, He SY (2009) Innate immunity in plants: an arms race between pattern recognition receptors in 642

plants and effectors in microbial pathogens. Science 324: 742-744 643

Campo S, Peris-Peris C, Sire C, Moreno AB, Donaire L, Zytnicki M, Notredame C, Llave C, Segundo 644

BS (2013) Identification of a novel microRNA (miRNA) from rice that targets an alternatively 645

spliced transcript of the Nramp6 (Natural resistance-associated macrophage protein 6) gene involved 646

in pathogen resistance. New Phytol 199: 212-217 647

Chen X (2012) Small RNAs in development - insights from plants. Curr Opin Genet Dev 22: 361-367 648

Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the evolution of 649



25

the plant immune response. Cell 124: 803-814 650

Dou D, Zhou JM (2012) Phytopathogen effectors subverting host immunity: different foes, similar 651

battleground. Cell Host Microbe 12: 484-495 652

Du P, Wu J, Zhang J, Zhao S, Zheng H, Gao G, Wei L, Li Y (2011) Viral infection induces expression of 653

novel phased microRNAs from conserved cellular microRNA precursors. PLoS Pathog 7: e1002176 654

Dugas DV, Bartel B (2008) Sucrose induction of Arabidopsis miR398 represses two Cu/Zn superoxide 655

dismutases. Plant Mol Biol 67: 403-417 656

Ernst J, Bar-Joseph Z (2006) STEM: a tool for the analysis of short time series gene expression data. BMC 657

Bioinformatics 7: 191 658

Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Law TF, 659

Grant SR, Dangl JL, Carrington JC (2007) High-throughput sequencing of Arabidopsis 660

microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS One 2: e219 661

Fu J, Liu H, Li Y, Yu H, Li X, Xiao J, Wang S (2011) Manipulating broad-spectrum disease resistance by 662

suppressing pathogen-induced auxin accumulation in rice. Plant Physiol 155: 589-602 663

Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K, Hayashi N, Takahashi A, Hirochika H, 664

Okuno K, Yano M (2009) Loss of function of a proline-containing protein confers durable disease 665

resistance in rice. Science 325: 998-1001 666

Guo N, Ye WW, Wu XL, Shen DY, Wang YC, Xing H, Dou DL (2011) Microarray profiling reveals 667

microRNAs involving soybean resistance to Phytophthora sojae. Genome 54: 954-958 668

Hagen G, Guilfoyle T (2002) Auxin-responsive gene expression: genes, promoters and regulatory factors. 669

Plant Mol Biol 49: 373-385 670

Jagadeeswaran G, Saini A, Sunkar R (2009) Biotic and abiotic stress down-regulate miR398 expression in 671

Arabidopsis. Planta 229: 1009-1014 672

Jeong DH, Park S, Zhai J, Gurazada SG, De Paoli E, Meyers BC, Green PJ (2011) Massive analysis of 673

rice small RNAs: mechanistic implications of regulated microRNAs and variants for differential 674

target RNA cleavage. Plant Cell 23: 4185-4207 675

Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, 676

including a stress-induced miRNA. Mol Cell 14: 787-799 677

Jones JD, Dangl JL (2006) The plant immune system. Nature 444: 323-329 678

Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C, Dohmae N, Takio K, Minami E, Shibuya 679

N (2006) Plant cells recognize chitin fragments for defense signaling through a plasma membrane 680

receptor. Proc Natl Acad Sci U S A 103: 11086-11091 681

Kankanala P, Czymmek K, Valent B (2007) Roles for rice membrane dynamics and plasmodesmata during 682

biotrophic invasion by the blast fungus. Plant Cell 19: 706-724 683

Katiyar-Agarwal S, Jin H (2010) Role of small RNAs in host-microbe interactions. Annu Rev Phytopathol 684

48: 225-246 685

Khang CH, Berruyer R, Giraldo MC, Kankanala P, Park SY, Czymmek K, Kang S, Valent B (2010) 686

Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell 687

movement. Plant Cell 22: 1388-1403 688

Khraiwesh B, Zhu JK, Zhu J (2012) Role of miRNAs and siRNAs in biotic and abiotic stress responses of 689

plants. Biochim Biophys Acta 1819: 137-148 690

Kishimoto K, Kouzai Y, Kaku H, Shibuya N, Minami E, Nishizawa Y (2010) Perception of the chitin 691

oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice. Plant J 692

64: 343-354 693



26

Kozomara A, Griffiths-Jones S (2011) miRBase: integrating microRNA annotation and deep-sequencing 694

data. Nucleic Acids Res 39: D152-157 695

Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant 696

hypersensitive disease resistance response. Cell 79: 583-593 697

Li F, Pignatta D, Bendix C, Brunkard JO, Cohn MM, Tung J, Sun H, Kumar P, Baker B (2012) 698

MicroRNA regulation of plant innate immune receptors. Proc Natl Acad Sci U S A 109: 1790-1795 699

Li T, Li H, Zhang YX, Liu JY (2011) Identification and analysis of seven H(2)O(2)-responsive miRNAs and 700

32 new miRNAs in the seedlings of rice (Oryza sativa L. ssp. indica). Nucleic Acids Res 39: 701

2821-2833 702

Li Y, Wang L, Jing JX, Li ZQ, Lin F, Huang LF, Pan QH (2007) The Pikm gene, conferring stable 703

resistance to isolates of Magnaporthe oryzae, was finely mapped in a crossover-cold region on rice 704

chromosome 11. Mol Breeding 20: 179-188 705

Li Y, Zhang Q, Zhang J, Wu L, Qi Y, Zhou JM (2010) Identification of microRNAs involved in 706

pathogen-associated molecular pattern-triggered plant innate immunity. Plant Physiol 152: 707

2222-2231 708

Li YF, Zheng Y, Addo-Quaye C, Zhang L, Saini A, Jagadeeswaran G, Axtell MJ, Zhang W, Sunkar R 709

(2010) Transcriptome-wide identification of microRNA targets in rice. Plant J 62: 742-759 710

Lin ZZ, Jiang WW, Wang JL, Lei CL (2001) Research and Utilization of Universally Susceptible Property 711

of Japonica Rice Variety Lijiangxintuanheigu. Scientia Agricultura Sinjca 34: 116-117 712

Liu B, Li P, Li X, Liu C, Cao S, Chu C, Cao X (2005) Loss of function of OsDCL1 affects microRNA 713

accumulation and causes developmental defects in rice. Plant Physiol 139: 296-305 714

Liu J, Wang X, Mitchell T, Hu Y, Liu X, Dai L, Wang GL (2010) Recent progress and understanding of the 715

molecular mechanisms of the rice-Magnaporthe oryzae interaction. Mol Plant Pathol 11: 419-427 716

Liu W, Liu J, Ning Y, Ding B, Wang X, Wang Z, Wang GL (2013) Recent Progress in Understanding 717

PAMP- and Effector-Triggered Immunity Against the Rice Blast Fungus Magnaporthe oryzae. Mol 718

Plant 719

Lu S, Sun YH, Amerson H, Chiang VL (2007) MicroRNAs in loblolly pine (Pinus taeda L.) and their 720

association with fusiform rust gall development. Plant J 51: 1077-1098 721

Luo YC, Zhou H, Li Y, Chen JY, Yang JH, Chen YQ, Qu LH (2006) Rice embryogenic calli express a 722

unique set of microRNAs, suggesting regulatory roles of microRNAs in plant post-embryogenic 723

development. FEBS Lett 580: 5111-5116 724

Mackill DJ, Bonman JB (1992) Inheritance of Blast Resistance in Near-Isogenic Lines of Rice. 725

Phytopathology 82: 746-749 726

McKersie BD, Chen Y, de Beus M, Bowley SR, Bowler C, Inze D, D'Halluin K, Botterman J (1993) 727

Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.). 728

Plant Physiol 103: 1155-1163 729

Mentlak TA, Kombrink A, Shinya T, Ryder LS, Otomo I, Saitoh H, Terauchi R, Nishizawa Y, Shibuya 730

N, Thomma BP, Talbot NJ (2012) Effector-mediated suppression of chitin-triggered immunity by 731

magnaporthe oryzae is necessary for rice blast disease. Plant Cell 24: 322-335 732

Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C, Chen S, Hannon GJ, Qi Y 733

(2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal 734

nucleotide. Cell 133: 116-127 735

Mosquera G, Giraldo MC, Khang CH, Coughlan S, Valent B (2009) Interaction transcriptome analysis 736

identifies Magnaporthe oryzae BAS1-4 as Biotrophy-associated secreted proteins in rice blast 737



27

disease. Plant Cell 21: 1273-1290 738

Nagasaki H, Itoh J, Hayashi K, Hibara K, Satoh-Nagasawa N, Nosaka M, Mukouhata M, Ashikari M, 739

Kitano H, Matsuoka M, Nagato Y, Sato Y (2007) The small interfering RNA production pathway 740

is required for shoot meristem initiation in rice. Proc Natl Acad Sci U S A 104: 14867-14871 741

Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O, Jones JD (2006) A plant 742

miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312: 436-439 743

Padmanabhan C, Zhang X, Jin H (2009) Host small RNAs are big contributors to plant innate immunity. 744

Curr Opin Plant Biol 12: 465-472 745

Park CH, Chen S, Shirsekar G, Zhou B, Khang CH, Songkumarn P, Afzal AJ, Ning Y, Wang R, Bellizzi 746

M, Valent B, Wang GL (2012) The Magnaporthe oryzae Effector AvrPiz-t Targets the RING E3 747

Ubiquitin Ligase APIP6 to Suppress Pathogen-Associated Molecular Pattern-Triggered Immunity in 748

Rice. Plant Cell 24: 4748-4762 749

Qi Y, Denli AM, Hannon GJ (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. 750

Mol Cell 19: 421-428 751

Qu S, Liu G, Zhou B, Bellizzi M, Zeng L, Dai L, Han B, Wang GL (2006) The broad-spectrum blast 752

resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of 753

a multigene family in rice. Genetics 172: 1901-1914 754

Radwan O, Liu Y, Clough SJ (2011) Transcriptional analysis of soybean root response to Fusarium 755

virguliforme, the causal agent of sudden death syndrome. Mol Plant Microbe Interact 24: 958-972 756

Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 757

16: 1616-1626 758

Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA 759

targets. Cell 110: 513-520 760

Ribot C, Hirsch J, Balzergue S, Tharreau D, Notteghem JL, Lebrun MH, Morel JB (2008) Susceptibility 761

of rice to the blast fungus, Magnaporthe grisea. J Plant Physiol 165: 114-124 762

Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D (2005) Specific effects of 763

microRNAs on the plant transcriptome. Dev Cell 8: 517-527 764

Seo JK, Wu J, Lii Y, Li Y, Jin H (2013) Contribution of small RNA pathway components in plant immunity. 765

Mol Plant Microbe Interact 26: 617-625 766

Shimizu T, Nakano T, Takamizawa D, Desaki Y, Ishii-Minami N, Nishizawa Y, Minami E, Okada K, 767

Yamane H, Kaku H, Shibuya N (2010) Two LysM receptor molecules, CEBiP and OsCERK1, 768

cooperatively regulate chitin elicitor signaling in rice. Plant J 64: 204-214 769

Shivaprasad PV, Chen HM, Patel K, Bond DM, Santos BA, Baulcombe DC (2012) A microRNA 770

superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs. Plant Cell 24: 771

859-874 772

Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and characterization of microRNAs from rice. Plant 773

Cell 17: 1397-1411 774

Sunkar R, Kapoor A, Zhu JK (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase 775

genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress 776

tolerance. Plant Cell 18: 2051-2065 777

Sunkar R, Zhou X, Zheng Y, Zhang W, Zhu JK (2008) Identification of novel and candidate miRNAs in 778

rice by high throughput sequencing. BMC Plant Biol 8: 25 779

Tsumematsu H YM, Ebron LA, Hayashi N, Ando I, Kato H, Imbe T, Khush GS (2000) Development of 780

monogenic lines of rice for blast resistance. Breeding science 50: 229-234 781



28

Wang JF, Zhou H, Chen YQ, Luo QJ, Qu LH (2004) Identification of 20 microRNAs from Oryza sativa. 782

Nucleic Acids Res 32: 1688-1695 783

Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y (2009) Rice MicroRNA effector complexes and targets. Plant 784

Cell 21: 3421-3435 785

Xiao S, Brown S, Patrick E, Brearley C, Turner JG (2003) Enhanced transcription of the Arabidopsis 786

disease resistance genes RPW8.1 and RPW8.2 via a salicylic acid-dependent amplification circuit is 787

required for hypersensitive cell death. Plant Cell 15: 33-45 788

Xie K, Wu C, Xiong L (2006) Genomic organization, differential expression, and interaction of 789

SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice. Plant Physiol 790

142: 280-293 791

Xin M, Wang Y, Yao Y, Xie C, Peng H, Ni Z, Sun Q (2010) Diverse set of microRNAs are responsive to 792

powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biol 10: 123 793

Xue LJ, Zhang JJ, Xue HW (2009) Characterization and expression profiles of miRNAs in rice seeds. 794

Nucleic Acids Res 37: 916-930 795

Yamaguchi K, Yamada K, Ishikawa K, Yoshimura S, Hayashi N, Uchihashi K, Ishihama N, 796

Kishi-Kaboshi M, Takahashi A, Tsuge S, Ochiai H, Tada Y, Shimamoto K, Yoshioka H, 797

Kawasaki T (2013) A Receptor-like Cytoplasmic Kinase Targeted by a Plant Pathogen Effector Is 798

Directly Phosphorylated by the Chitin Receptor and Mediates Rice Immunity. Cell Host Microbe 13: 799

347-357 800

Yang L, Jue D, Li W, Zhang R, Chen M, Yang Q (2013) Identification of MiRNA from Eggplant (Solanum 801

melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection. 802

PLoS One 8: e72840 803

Yin Z, Li Y, Han X, Shen F (2012) Genome-wide profiling of miRNAs and other small non-coding RNAs in 804

the Verticillium dahliae-inoculated cotton roots. PLoS One 7: e35765 805

Yoshida K, Saitoh H, Fujisawa S, Kanzaki H, Matsumura H, Tosa Y, Chuma I, Takano Y, Win J, 806

Kamoun S, Terauchi R (2009) Association genetics reveals three novel avirulence genes from the 807

rice blast fungal pathogen Magnaporthe oryzae. Plant Cell 21: 1573-1591 808

Zhai J, Jeong DH, De Paoli E, Park S, Rosen BD, Li Y, Gonzalez AJ, Yan Z, Kitto SL, Grusak MA, 809

Jackson SA, Stacey G, Cook DR, Green PJ, Sherrier DJ, Meyers BC (2011) MicroRNAs as 810

master regulators of the plant NB-LRR defense gene family via the production of phased, 811

trans-acting siRNAs. Genes Dev 25: 2540-2553 812

Zhang W, Gao S, Zhou X, Chellappan P, Chen Z, Zhang X, Fromuth N, Coutino G, Coffey M, Jin H 813

(2011) Bacteria-responsive microRNAs regulate plant innate immunity by modulating plant 814

hormone networks. Plant Mol Biol 75: 93-105 815

Zhao CZ, Xia H, Frazier TP, Yao YY, Bi YP, Li AQ, Li MJ, Li CS, Zhang BH, Wang XJ (2010) Deep 816

sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.). BMC 817

Plant Biol 10: 3 818

Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, Bellizzi M, Wang GL (2006) The eight amino-acid 819

differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the 820

resistance specificity to Magnaporthe grisea. Mol Plant Microbe Interact 19: 1216-1228 821

Zhou L, Liu Y, Liu Z, Kong D, Duan M, Luo L (2010) Genome-wide identification and analysis of 822

drought-responsive microRNAs in Oryza sativa. J Exp Bot 61: 4157-4168 823

Zhou M, Gu L, Li P, Song X, Wei L, Chen Z, Cao X (2010) Degradome sequencing reveals endogenous 824

small RNA targets in rice (Oryza sativa L. ssp. indica). Frontiers in Biology 5: 67-90 825



29

Zhou M, Gu LF, Li PC, Song XW, Wei LY, Chen ZY, Cao XF (2010) Degradome sequencing reveals 826

endogenous small RNAtargets in rice (Oryza sativa L. ssp. indica). Front Biol 5: 67-90 827

Zhu QH, Spriggs A, Matthew L, Fan L, Kennedy G, Gubler F, Helliwell C (2008) A diverse set of 828

microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res 18: 1456-1465 829

Zipfel C, Felix G (2005) Plants and animals: a different taste for microbes? Curr Opin Plant Biol 8: 353-360 830



30

Figure legends 831

Figure 1. Comparison of defense responses among the susceptible line LTH and two 832

monogenic resistant lines. Three-leaf-old seedlings were inoculated with spore suspension 833

(5×105 spores/mL) of M. oryzae and disease phenotypes were recorded at 10 days post 834

inoculation (dpi). A, Representative leaf sections from the indicated lines to show the blast 835

disease phenotypes. B-F, Expressions of the indicated defense-related genes in LTH, 836

IRBLkm-Ts, and IRBLz5-CA upon M. oryzae infection. RNA was extracted at the 837

indicated time points for quantitative RT-PCR analysis. mRNA level was normalized to that 838

in untreated LTH (0 hr). Error bars indicate SD. Student’s t test was carried out to 839

determine the significance of difference between LTH and IRBLkm-Ts or IRBLz5-CA. 840

Asterisks (**) indicate significant difference at a P value <0.01. The experiments were 841

repeated two times with similar results. G, Representative leaf sections from the indicated 842

lines to show fungal growth and H2O2 accumulation at 2 dpi and 10 dpi, respectively. Note 843

that there was no or trace amount of H2O2 accumulation in LTH (arrow), but high levels 844

around the appressoria (arrowheads) at 2 dpi in the leaf cells of both IRBLkm-Ts and 845

IRBLz5-CA. Trypan blue and 3,3’-diaminobenzidine were used to stain the fungal structure 846

and H2O2 accumulation (reddish brown), respectively. Bars, 10 μm. 847

Figure 2. Profiling of small RNAs by deep sequencing of the libraries from different 848

samples. Small RNA libraries were constructed from samples of the indicated lines 849

collected at 0, 12, and 24 hours post inoculation (dpi) of M. oryzae and subjected to 850

deep-sequencing. A, Size distribution of sequenced small RNAs mapped to rice genome. B, 851

Size distribution of miRNAs mapped to miRNA precursor. C, Bar charts summarizing the 852

percentage of small RNA reads matched to rice and M. oryzae genome, respectively. D, Bar 853

charts summarizing the percentage of small RNA reads matched to different rice RNAs. 854

Figure 3. The expression patterns of miRNAs and their target genes upon M. oryzae 855

infection. Three-leaf-old seedlings were inoculated with 5× 105 spores/mL of M. oryzae and 856

total RNA was extracted from leaves of the indicated lines at the indicated time points. A, 857

RNA blotting analysis of miRNAs. Fifteen micrograms of small RNA was loaded. RNA 858

blots were hybridized with DNA oligonucleotide probes complementary to the indicated 859

miRNAs. U6 was used as loading control. Values below each section represent relative 860



31

abundance of miRNA normalized to U6. B to G, Quantitative RT-PCR analyses of mRNA 861

levels for genes targeted by miR160 (B, Os04g43910/ARF16), miR164 (C, Os12g41680), 862

miR827 (D, Os04g48390), miR172 (E, Os05g03040), and miR398 (F and G, 863

Os07g46990/SOD2 and Os04g48410). Error bars indicate SD. Student’s t test was used to 864

determine the significance of difference between 0 hpi and the indicated time points in the 865

same line. Asterisks * and ** indicate significant difference at a P value <0.05 and <0.01, 866

respectively. The experiments were repeated two times with similar results. 867

Figure 4. Overexpression of miR160a enhances rice resistance to M. oryzae. A and B, RNA 868

blotting analysis (A) and quantitative RT-PCR (qRT-PCR) assay (B) to examine the 869

accumulation of miR160a in the indicated transgenic lines expressing 35S:miR160a or 870

empty vector (EV). One microgram of total RNA was used for qRT-PCR analysis and 871

fifteen micrograms of small RNA was loaded for RNA-blot analysis. C, qRT-PCR assay for 872

the indicated three target genes of miR160a in the indicated transgenic lines. RNA was 873

extracted from T2 generation of 35S:MIR160a transgenic plants. D, Representative leaf 874

sections from the indicated transgenic lines to show the disease phenotypes. Three-leaf-old 875

seedlings were inoculated with 5×105 spores/mL of M. oryzae and the phenotype was 876

observed at 10 dpi. E and F, Sporulation and relative fungal growth on the inoculated leaves 877

of the indicated transgenic lines. Samples were taken for the assays at 10 dpi. Values were 878

the means of three replications. Error bars indicate SD. Student’s t test was carried out to 879

determine the significance of difference between Kasalath (EV) and 880

miR160a-overexpression transgenic plants. Asterisks * and ** indicate significant 881

difference at a P value <0.05 and <0.01, respectively. The experiments were repeated two 882

times with similar results. 883

Figure 5. Overexpression of miR398b enhances rice resistance to M. oryzae. A and B, RNA 884

blotting analysis (A) and qRT-PCR assay (B) to examine the accumulation of miR398b in 885

the indicated transgenic lines expressing 35S:miR398b or empty vector (EV). One 886

microgram of total RNA was used for qRT-PCR analysis and twenty micrograms of small 887

RNA was loaded for RNA-blot analysis, respectively. C, qRT-PCR assay for the indicated 888

four target genes of miR398b in the indicated transgenic lines. D, Representative leaf 889

sections from the indicated transgenic lines to show the disease phenotypes. 890



32

Three-leaves-old transgenic rice seedlings were inoculated with 5×105 spores/mL of M. 891

oryzae and the phenotype was observed at 10 dpi. E and F, Quantitative assay for 892

sporulation (E) and relative fungal growth (F) on the inoculated leaves of the indicated 893

transgenic lines. Samples were collected at 10 dpi. Values were the means of three 894

replications. Error bars indicate SD. Student’s t test was carried out to determine the 895

significance of difference between Kasalath (EV) and miR398b-overexpression transgenic 896

plants. Asterisks * and ** indicate significant difference at a P value <0.05 and <0.01, 897

respectively. The experiments were repeated two times with similar results. 898

Figure 6. MiR160a and miR398b positively and differentially regulate expression of 899

defense-related genes. Three-leaf-old seedlings were inoculated with 5 × 105 spores/mL of 900

M.oryzae and samples were collected at the indicated time points. RNA was extracted for 901

quantitative RT-PCR (qRT-PCR) analysis and mRNA level was normalized to that in 902

untreated control Kasalath (EV). A and B, qRT-PCR assay showing that the late responsive 903

defense-related genes PR1 (A) and PR10 (B) were up-regulated in the miR160a and 904

miR398b over-expression lines. C and D, qRT-PCR assay showing that the PTI-related 905

genes OsKS4 and OsNAC4 were up-regulated in the transgenic line over-expressing 906

miR398b but not in that of miR160a. E and F, qRT-PCR assay showing the expression 907

levels of PTI-related genes OsKS4 and OsNAC4 in the indicated transgenic lines after 908

incubating in water, 1 μg/ml of chitin, or 1 μM of flg22 for 1 hour, respectively. Note that 909

OsKS4 and OsNAC4 were significantly induced by both chitin and flg22 in the transgenic 910

line over-expressing miR398b but not in that of miR160a. Values are the means of three 911

replications. Error bars indicate SD. Student’s t test was carried out to determine the 912

significance of difference between Kasalath (EV) and miRNA overexpression transgenic 913

plants following M. oryzae or PAMP treatment. Asterisks * and ** indicate significant 914

difference at a P value <0.05 and <0.01, respectively. The experiments repeated two times 915

with similar results. 916

Figure 7. Cellular responses to the infection of M. oryzae. A to C, Representative Laser 917

Scanning Confocal Microscopic images to show infection of the eGFP-tagged strain 918

Zhong-8-10-14 on sheath cells of transgenic plants expressing empty vector (A), 919

35S:miR160a (B), and 35S:miR398b (C) at the indicated time points. Note that appressoria 920



33

(arrows) were formed at 12 hpi on EV plants, but delayed to 24 hpi on 921

miR160a-overexpression plants and 36 hpi on miR398b-overexpression plants. Invasive 922

hyphae (arrowheads) were formed at 24 hpi and extended to the neighbor cells at 36 hpi on 923

EV plants. Bars, 20 μm. D and E, Representative leaf sections from the indicated transgenic 924

lines stained by 3,3’-diaminobenzidine and trypan blue at 2 dpi (D) and 10 dpi (E), 925

respectively, to show the accumulation of H2O2 (reddish brown) at the infection site where 926

appressoria (arrows) were visualized and the fungal structures. Note that secondary conidia 927

(arrowheads in E) were generated on EV plants at 10 dpi. Bars, 10 μm. F and G, Statistic 928

analyses of conidial germination rate (F) and frequency of invasive hypha (G) at the 929

indicated time points observed on the indicated lines. Inoculated leaves were stained with 930

trypan blue and examined by microscopy. Data are mean±SEM from three independent 931

experiments each of which >200 conidia were evaluated. x2-test was used to test statistical 932

significance between EV and transgenic lines. Asterisks * and ** indicate significant 933

difference at a P value <0.05 and <0.01, respectively. 934



34

Table 1. List of candidate miRNAs involved in rice immunity against M. oryzaea 935

miRNA ID

Normalized Reads

LTH IRBLkm-Ts

M. oryzae M. oryzae

0 hr 12 24 hr 0 hr 12hr 24hr Positive regulator

osa-miR167a/b/c-5p (#7) 1772 439 6599 2462 7291 8440

osa-miR168a-5p (#7) 44893 11167 24255 20195 28750 33405

osa-miR164a/b/f (#11) 517 289 244 370 631 1185

osa-miR160a/b/c/d/f -5p(#22) 39 34 61 26 338 62

Negative regulator

osa-miR439a/b/c/d/e/f/g/h/i/j (#8) 132 204 74 15 27 20

osa-miR169f.1/g (#18) 1093 506 1360 3648 2699 1428

osa-miR169h/i/j/k/l/m (#18) 883 700 1821 3463 2419 1998

osa-miR169i-3p (#18) 180 147 487 734 501 210

osa-miR396e-3p (#18) 25 8 92 234 201 148

osa-miR444b.2/c.2 (#18) 214 147 539 956 947 365

osa-miR396c-5p (#21) 2609 2288 2838 771 1486 740

osa-miR535-3p(#25) 51 13 163 9 11 9

osa-miR167d/e/f/g/h/i/j -5p (#30) 792 773 36611 393 2106 2226

osa-miR827a/b (#30) 46 52 447 67 175 44

osa-miR1867 (#32) 106 366 70 68 29 35

osa-miR1871 (#32) 139 468 151 59 78 64

osa-miR2873a (#32) 90 248 59 49 23 67

osa-miR2878-5p (#32) 31 104 33 32 32 33

osa-miR396d/e 5p(#32) 39801 80382 45408 24966 23356 26470

osa-miR435 (#32) 166 394 189 69 82 68

osa-miR528-5p (#42) 60 386 129 75 102 67

osa-miR166k/l-3p (#42) 119 194 209 57 111 125

Basal-responsive regulator

osa-miR398b (#27) 60 27 318 36 114 452

osa-miR169a (#29) 379 492 1619 218 311 788

osa-miR169b/c(#29) 12521 7911 29151 11203 9541 15032

osa-miR1433 (#39) 40 28 218 235 220 304

osa-miR172a/d (#39) 140 355 642 608 1490 1592

osa-miR1861e/k/m (#39) 7 9 25 32 55 112

osa-miR393a (#46) 52 1156 341 153 131 267

osa-miR1425-5p(#47) 22 34 90 73 417 43

osa-miR1863a (#47) 260 433 1805 946 1081 932

osa-miR2871a-3p/b (#47) 25 68 237 134 159 142

osa-miR444b.1/c.1 (#47) 289 424 667 567 1174 255

aThe change trends of miRNA reads were analyzed by Short Time-series Expression Miner (STEM). Those 936

with significant changes and more than 100 reads in one of the six libraries were selected. The STEM pattern 937

numbers were listed in the bracket following their name. 938



Figure 1. Comparison of defense responses among the susceptible line LTH and two monogenicresistant lines Three leaf old seedlings were inoculated with spore suspension (5×105 spores/mL)resistant lines. Three-leaf-old seedlings were inoculated with spore suspension (5×105 spores/mL)of M. oryzae and disease phenotypes were recorded at 10 days post inoculation (dpi). A,Representative leaf sections from the indicated lines to show the blast disease phenotypes. B-F,Expressions of the indicated defense-related genes in LTH, IRBLkm-Ts, and IRBLz5-CA upon M.oryzae infection. RNA was extracted at the indicated time points for quantitative RT-PCR analysis.mRNA level was normalized to that in untreated LTH (0 hr). Error bars indicate SD. Student’s t testwas carried out to determine the significance of difference between LTH and IRBLkm-Ts orIRBLz5-CA. Asterisks (**) indicate significant difference at a P value <0.01. The experiments

t d t ti ith i il lt G R t ti l f ti f th i di t dwere repeated two times with similar results. G, Representative leaf sections from the indicatedlines to show fungal growth and H2O2 accumulation at 2 dpi and 10 dpi, respectively. Note thatthere was no or trace amount of H2O2 accumulation in LTH (arrow), but high levels around theappressoria (arrowheads) at 2 dpi in the leaf cells of both IRBLkm-Ts and IRBLz5-CA. Trypanblue and 3,3’-diaminobenzidine were used to stain the fungal structure and H2O2 accumulation(reddish brown), respectively. Bars, 10 μm.



Figure 2. Profiling of small RNAs by deep sequencing of the libraries from differentsamples. Small RNA libraries were constructed from samples of the indicated linescollected at 0, 12, and 24 hours post inoculation (dpi) ofM. oryzae and subjected to deep-

i A Si di t ib ti f d ll RNA d t i Bsequencing. A, Size distribution of sequenced small RNAs mapped to rice genome. B,Size distribution of miRNAs mapped to miRNA precursor. C, Bar charts summarizingthe percentage of small RNA reads matched to rice and M. oryzae genome, respectively.D, Bar charts summarizing the percentage of small RNA reads matched to different riceRNAs.



Figure 3. The expression patterns of miRNAs and their target genes upon M. oryzae infection.Three-leaf-old seedlings were inoculated with 5× 105 spores/mL of M oryzae and total RNAThree-leaf-old seedlings were inoculated with 5× 10 spores/mL of M. oryzae and total RNAwas extracted from leaves of the indicated lines at the indicated time points. A, RNA blottinganalysis of miRNAs. Fifteen micrograms of small RNA was loaded. RNA blots werehybridized with DNA oligonucleotide probes complementary to the indicated miRNAs. U6 wasused as loading control. Values below each section represent relative abundance of miRNAnormalized to U6. B to G, Quantitative RT-PCR analyses of mRNA levels for genes targetedby miR160 (B, Os04g43910/ARF16), miR164 (C, Os12g41680), miR827 (D, Os04g48390),miR172 (E, Os05g03040), and miR398 (F and G, Os07g46990/SOD2 and Os04g48410). Errorb i di t SD St d t’ t t t d t d t i th i ifi f diff b t 0bars indicate SD. Student’s t test was used to determine the significance of difference between 0hpi and the indicated time points in the same line. Asterisks * and ** indicate significantdifference at a P value <0.05 and <0.01, respectively. The experiments were repeated two timeswith similar results.



Figure 4. Overexpression of miR160a enhances rice resistance to M. oryzae. A and B, RNA blottinganalysis (A) and quantitative RT-PCR (qRT-PCR) assay (B) to examine the accumulation ofmiR160a in the indicated transgenic lines expressing 35S:miR160a or empty vector (EV). Onemicrogram of total RNA was used for qRT-PCR analysis and fifteen micrograms of small RNA wasloaded for RNA-blot analysis. C, qRT-PCR assay for the indicated three target genes of miR160a inthe indicated transgenic lines. RNA was extracted from T2 generation of 35S:MIR160a transgenicl t D R t ti l f ti f th i di t d t i li t h th diplants. D, Representative leaf sections from the indicated transgenic lines to show the diseasephenotypes. Three-leaf-old seedlings were inoculated with 5×105 spores/mL of M. oryzae and thephenotype was observed at 10 dpi. E and F, Sporulation and relative fungal growth on the inoculatedleaves of the indicated transgenic lines. Samples were taken for the assays at 10 dpi. Values were themeans of three replications. Error bars indicate SD. Student’s t test was carried out to determine thesignificance of difference between Kasalath (EV) and miR160a-overexpression transgenic plants.Asterisks * and ** indicate significant difference at a P value <0.05 and <0.01, respectively. Theexperiments were repeated two times with similar results.



Figure 5. Overexpression of miR398b enhances rice resistance to M. oryzae. A and B, RNA blottinganalysis (A) and qRT-PCR assay (B) to examine the accumulation of miR398b in the indicatedtransgenic lines expressing 35S:miR398b or empty vector (EV). One microgram of total RNA wasused for qRT-PCR analysis and twenty micrograms of small RNA was loaded for RNA-blot analysis,respectively C qRT-PCR assay for the indicated four target genes of miR398b in the indicatedrespectively. C, qRT-PCR assay for the indicated four target genes of miR398b in the indicatedtransgenic lines. D, Representative leaf sections from the indicated transgenic lines to show thedisease phenotypes. Three-leaves-old transgenic rice seedlings were inoculated with 5×105spores/mL of M. oryzae and the phenotype was observed at 10 dpi. E and F, Quantitative assay forsporulation (E) and relative fungal growth (F) on the inoculated leaves of the indicated transgeniclines. Samples were collected at 10 dpi. Values were the means of three replications. Error barsindicate SD. Student’s t test was carried out to determine the significance of difference betweenKasalath (EV) and miR398b-overexpression transgenic plants. Asterisks * and ** indicate significantdiff t P l <0 05 d <0 01 ti l Th i t t d t ti ithdifference at a P value <0.05 and <0.01, respectively. The experiments were repeated two times withsimilar results.





Figure 6. MiR160a and miR398b positively and differentially regulate expression ofdefense-related genes. Three-leaf-old seedlings were inoculated with 5 × 105 spores/mLof M oryzae and samples were collected at the indicated time points RNA was extractedof M.oryzae and samples were collected at the indicated time points. RNA was extractedfor quantitative RT-PCR (qRT-PCR) analysis and mRNA level was normalized to that inuntreated control Kasalath (EV). A and B, qRT-PCR assay showing that the lateresponsive defense-related genes PR1 (A) and PR10 (B) were up-regulated in the miR160aand miR398b over-expression lines. C and D, qRT-PCR assay showing that the PTI-related genes OsKS4 and OsNAC4 were up-regulated in the transgenic line over-expressing miR398b but not in that of miR160a. E and F, qRT-PCR assay showing theexpression levels of PTI-related genes OsKS4 and OsNAC4 in the indicated transgenicli ft i b ti i t 1 / l f hiti 1 M f fl 22 f 1 h ti llines after incubating in water, 1 μg/ml of chitin, or 1 μM of flg22 for 1 hour, respectively.Note that OsKS4 and OsNAC4 were significantly induced by both chitin and flg22 in thetransgenic line over-expressing miR398b but not in that of miR160a. Values are the meansof three replications. Error bars indicate SD. Student’s t test was carried out to determinethe significance of difference between Kasalath (EV) and miRNA overexpressiontransgenic plants following M. oryzae or PAMP treatment. Asterisks * and ** indicatesignificant difference at a P value <0.05 and <0.01, respectively. The experiments repeatedtwo times with similar results.





Figure 7. Cellular responses to the infection of M. oryzae. A to C, Representative Laser ScanningConfocal Microscopic images to show infection of the eGFP-tagged strain Zhong-8-10-14 on sheathcells of transgenic plants expressing empty vector (A), 35S:miR160a (B), and 35S:miR398b (C) at theindicated time points Note that appressoria (arrows) were formed at 12 hpi on EV plants but delayedindicated time points. Note that appressoria (arrows) were formed at 12 hpi on EV plants, but delayedto 24 hpi on miR160a-overexpression plants and 36 hpi on miR398b-overexpression plants. Invasivehyphae (arrowheads) were formed at 24 hpi and extended to the neighbor cells at 36 hpi on EV plants.Bars, 20 μm. D and E, Representative leaf sections from the indicated transgenic lines stained by 3,3’-diaminobenzidine and trypan blue at 2 dpi (D) and 10 dpi (E), respectively, to show the accumulationof H2O2 (reddish brown) at the infection site where appressoria (arrows) were visualized and the fungalstructures. Note that secondary conidia (arrowheads in E) were generated on EV plants at 10 dpi. Bars,10 μm. F and G, Statistic analyses of conidial germination rate (F) and frequency of invasive hypha (G)t th i di t d ti i t b d th i di t d li I l t d l t i d ith tat the indicated time points observed on the indicated lines. Inoculated leaves were stained with trypanblue and examined by microscopy. Data are mean±SEM from three independent experiments each ofwhich >200 conidia were evaluated. x2-test was used to test statistical significance between EV andtransgenic lines. Asterisks * and ** indicate significant difference at a P value <0.05 and <0.01,respectively.



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Running head: Rice miRNAs in Immunity against the Blast ... · OsmiR156b/h resulted in increased tillers but reduced plant height and panicle size (Xie et al., 2006). These literatures