IC4R005-miRNA-2012-23071571

Project Title

Identification of Novel Oryza sativa miRNAs in Deep Sequencing-Based Small RNA Libraries of Rice Infected with Rice Stripe Virus

The Background of This Project

Figure 1. sRNAs that were recognized as newly produced miRNAs from known precursors and their location in the secondary structures of their precursors.

• MicroRNAs (miRNAs) are small 19–24 nt RNAs that play essential roles in eukaryotes by targeting complementary mRNAs for degradation or translational repression. In plants, primary miRNA (pri-miRNA) is first transcribed by polymerase II, and then processed by Dicer-like 1 (DCL1) into the precursor miRNA (pre-miRNA), normally of about 70–300 nucleotides (nt). The pre-miRNA is further processed into the mature miRNA:-miRNA* duplex [3,4,5]. These processes occur in the nucleus. In the next stage, the duplex is transferred into the cytoplasm and unwound. The miRNA is then assembled into and RNA-induced silencing complex (RISC) and guides the RISC to cleave or suppress the target mRNA. miRNAs in plants regulate leaf morphogenesis, the development of roots and flowers and other key processes, and are recognized as important regulators of plant development. Recent research has revealed that miRNAs also play roles in plant defense against pathogens by regulating the expression of resistance (R) genes directly or indirectly, or targeting the viral genome to impair viral replication. Hence, the miRNA pathway also plays a key role during pathogen-plant interactions. In plants, over 4600 miRNAs have been identified from over 50 species (miRBase version 18.0, http://www.mirbase.org/cgi-bin/browse.pl). Medicago truncatula, Oryza sativa and Glycine max are the three plants that have the most identified miRNAs (respectively 674, 661 and 395 miRNAs). Some miRNA families have functions that are conserved across the plant kingdom and thus their sequences are similarly conserved (e.g. miR156, miR159, miR160 and miR165). Other miRNA families are specific to particular plants, and are not found elsewhere, indicating that they have novel and specific functions.
• icating that they have novel and specific functions. With the development of next generation sequencing technologies, deep-sequencing has provided a powerful high-throughput strategy for identifying novel miRNAs. In this way, hundreds of miRNAs have been identified from Arabidopsis, Brassica rapa, rice, wheat, barley, peanuts, grapevine and other plants. In rice, Sunkar et al identified 23 new miRNAs from three small RNA (sRNA) libraries of control rice seedlings and seedlings exposed to drought or salt stress; six of the new miRNAs are conserved in monocots. Chen et al identified 24 novel microRNA families from rice embryogenic callus, some of which were suggested to function in meristem development [33]. Li et al investigated the H 2 O 2 - regulated miRNAs in rice seedlings and discovered 32 new miRNAs. Peng et al identified 43 novel miRNAs from the sRNA libraries of rice spikelets [35], while Wang et al identified 75 novel miRNAs from the developing pollen of rice.

Plant Culture & Treatment

• RSV-infected rice were prepared as described [39]. Briefly, viruliferous adult brown planthoppers (Laodelphax striatellus Fallen) (carrying the RSV-Zhejiang isolate) were transferred onto healthy rice seedlings (Oryza sativa L. japonica. cv. Nipponbare) at the three-leaf stage for virus inoculation. Control seedlings were inoculated with non-viruliferous planthoppers. After 72 h, the planthoppers were removed. Systemic infections were confirmed by RT-PCR specific for RSV Zhejiang isolate. One week after inoculation, leaves were collected from the infected and control (Mock) plants, frozen and stored at 280uC until used. Allriceplants were grown in a glasshouse at 28–30uC day/25uC night, with a 12 h day/night light cycle under well-watered conditions.

Research Findings

• One miRNA precursor can produce several mature miRNAs with different sequences. Du et al (2011) recently reported that RSV infection induced different miRNAs to be produced from a single precursor in a phased pattern [38]. Hence, it was possible that the remaining 144 sRNAs contained some miRNAs that had been produced from a known conserved precursor but did not match known mature miRNAs. To identify these miRNAs, all 144 sRNAs were aligned with the known rice miRNA precursor stem-loop sequences. 23 of the sRNA sequences were identified among 19 known miRNA precursors, and were therefore considered to be novel miRNAs newly produced from known miRNA precursors. Their locations on the precursors are shown in Figure 1 and Figure 2.

Figure 2. sRNAs that were recognized as newly produced miRNAs from known precursors and their location in the secondary structures of their precursors.

• One miRNA precursor can produce several mature miRNAs with different sequences. Du et al (2011) recently reported that RSV infection induced different miRNAs to be produced from a single precursor in a phased pattern [38]. Hence, it was possible that the remaining 144 sRNAs contained some miRNAs that had been produced from a known conserved precursor but did not match known mature miRNAs. To identify these miRNAs, all 144 sRNAs were aligned with the known rice miRNA precursor stem-loop sequences. 23 of the sRNA sequences were identified among 19 known miRNA precursors, and were therefore considered to be novel miRNAs newly produced from known miRNA precursors. Their locations on the precursors are shown in Figure 2 and Figure 3. Among these 23 sRNAs, 12 were produced from the 59 arm and 11 were produced from the 39 arm of their respective precursor. Seq114 and 115 were located at the miRNA* region of osa-miR408; Seq116 was located at the miRNA* region of osa-miR167 h; Seq117 was located at the miRNA* region of osa-miR398a; Seq118 was located at the miRNA* region of osa-miR444c. These sRNAs may be the miRNA* or miRNA* isoforms of the corresponding miRNAs. We also searched for these 23 sRNA in the control library and other reported RSV and non-infected rice sRNA libraries, and found that they existed in the libraries with different sequencing reads. Statistical analysis showed that Seq99, Seq102 and Seq107 were significantly down-expressed in RSV-infected rice, indicating that they may have a function in response to RSV infection.
• The precursor sequences and corresponding stem-loop structures were predicted for each of the remaining 121 sRNAs that did not match the deposited rice miRNAs or miRNA precursors. 7 sRNAs (Seq119–125) that have precursor sequences capable of forming a stem-loop structure with free energies ranging from 223.5 to 267.9 kcal/mol, and that have no more than 26 hit loci in the rice genome were identified, while the other 114 sRNAs had no precursor sequence that could form a stem-loop structure. Among these 7 sRNAs, Seq125 had 23 loci in the rice genome and the other sRNAs had only one hit locus each. All 7 sRNAs could also be found with 1–5517 sequencing reads in the non-infected sRNA library reported by us and in 6 rice sRNA libraries reported by Du et al. The relative high expression level, the stable precursor structures and their stable appearance in rice sRNAs libraries indicated that the 7 sRNAs might be putative novel miRNAs (pn-miRNAs).

Labs working on this Project

• State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Ministry of China Key Laboratory of Biotechnology in Plant Protection, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China
• Plant Protection College, Yunnan Agricultural University, Kunming, China
• College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

Corresponding Author

• Jianping Chen(jpchen2001@yahoo.com.cn)