Construction of a genomewide RNAi mutant library in rice

Abstract

SummaryLong hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification-mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down-regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross-silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs. Our results indicate that RNAi mutant library is a high-efficient approach for genomewide gene identification in plants.

Genome sequence of the model rice variety KitaakeX

“…japonica ), which originated at the northern limit of rice cultivation in Hokkaido, Japan [3], has emerged as a model for rice research [4] because it is extremely early flowering, easy to propagate, and short in stature [5]. Kitaake has been used to establish multiple mutant populations, including an RNAi mutant collection [6], T-DNA insertion collections [4], [7], and a whole-genome sequenced mutant population of KitaakeX, a Kitaake variety carrying the Xa21 immune receptor gene (formerly called X.Kitaake) [8, 9]. Kitaake has been used to explore diverse aspects of rice biology, including flowering time [10], disease resistance [11], [12], [13], small RNA biology [14], and the CRISPR-Cas9 and TALEN technologies [15], [16].…”

Vacuum and Co-cultivation Agroinfiltration of (Germinated) Seeds Results in Tobacco Rattle Virus (TRV) Mediated Whole-Plant Virus-Induced Gene Silencing (VIGS) in Wheat and Maize

“…Hence, massive numbers of new genes have been identified, and functional analysis is required to decipher related biological traits and explore potential applications of these genes. A widely used approach in functional analysis is to generate mutations using chemical or physical agents (Kodym and Afza, 2003; Belfield et al, 2012; Hanafy and Mohamed, 2014; Serrat et al, 2014; Dhaliwal et al, 2015; Li et al, 2016; Zhang et al, 2016), T-DNA insertion (Weigel et al, 2000; Alonso et al, 2003), RNA interference (RNAi) (Kusaba, 2004; Mahmood-ur-Rahman et al, 2008; Wang et al, 2008, 2013), or genome editing (Shan et al, 2013; Wang et al, 2014; Kumar and Jain, 2015). However, those techniques often generate large numbers of mutants, and require tedious screening or genetic transformation procedures.…”

RNA Silencing in Plants: Mechanisms, Technologies and Applications in Horticultural Crops

“…However, mutants of such genes could be recovered for gene function analysis by incomplete gene knockdown with RNA silencing technologies. This is demonstrated in rice where transformation with hpRNA libraries results in the recovery of essential gene mutants [77]. iii) RNA silencing technologies allow for tissue-specific silencing of a gene using a tissue-specifically expressed transgene, whereas genetic mutation result in gene knock-out in all tissues.…”

“…A number of cloning vectors have therefore been developed to facilitate the preparation of hpRNA constructs, such as pHannibal and pHellsgate vectors [74, 75]. In addition, a method based on rolling-circle DNA replication by ϕ29 polymerase has been established to prepare genomewide hpRNA libraries [76, 77]. …”