Identification of CEP peptides encoded by the tobacco (Nicotiana tabacum) genome and characterization of their roles in osmotic and salt stress responses.

Members of the CEP (C-terminally Encoded Peptide) gene family have been shown to be involved in various developmental processes and stress responses in plants. In order to understand the roles of CEP peptides in stress response, a comprehensive bioinformatics approach was employed to identify NtCEP genes in tobacco (Nicotiana tabacum L.) and to analyze their potential roles in stress responses. Totally 21 NtCEP proteins were identified and categorized into two subgroups based on their CEP domains. Expression changes of the NtCEP genes in response to various abiotic stresses were analyzed via qRT-PCR and the results showed that a number of NtCEPs were significant up-regulated under drought, salinity, or temperature stress conditions. Furthermore, application of synthesized peptides derived from NtCEP5, NtCEP13, NtCEP14, and NtCEP17 enhanced plant tolerance to different salt stress treatments. NtCEP5, NtCEP9 and NtCEP14, and NtCEP17 peptides were able to promote osmotic tolerance of tobacco plants. The results from this study suggest that NtCEP peptides may serve as important signaling molecules in tobacco's response to abiotic stresses.

Improving a Quantitative Trait in Rice by Multigene Editing with CRISPR-Cas9.

CRISPR-Cas9 system is one sequence-specific nuclease (SSN) that has several advantages over zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), such as multiplex genome editing. With multiplex genome editing, CRISPR-Cas9 becomes a preferred SSN to edit many quantitative trait loci (QTL) simultaneously for trait improvement in major crops. A multiplexed CRISPR system is also important for deletion of a large fragment within a chromosome, analysis of the function of gene families, exon exchange, gene activation, and repression. Therefore, assembly of several single guide RNAs (sgRNAs) into one binary vector is the main step in multigene editing by CRISPR-Cas9. Different vector construction methods have been practiced including Golden Gate assembly. This chapter provides a detailed protocol for the construction of a T-DNA binary vector for expressing Cas9 and three sgRNAs for simultaneous targeting of three QTL genes for improving seed trait in rice.

Multiplex QTL editing of grain-related genes improves yield in elite rice varieties.

KEY MESSAGE: Significant yield increase has been achieved by simultaneous introduction of three trait-related QTLs in three rice varieties with multiplex editing by CRISPR-Cas9. Using traditional breeding approaches to develop new elite rice varieties with high yield and superior quality is challenging. It usually requires introduction of multiple trait-related quantitative trait loci (QTLs) into an elite background through multiple rounds of crossing and selection. CRISPR-Cas9-based multiplex editing of QTLs represents a new breeding strategy that is straightforward and cost effective. To test this approach, we simultaneously targeted three yield-related QTLs for editing in three elite rice varieties, namely J809, L237 and CNXJ. The chosen yield-related QTL genes are OsGS3, OsGW2 and OsGn1a, which have been identified to negatively regulate the grain size, width and weight, and number, respectively. Our approach rapidly generated all seven combinations of single, double and triple mutants for the target genes in elite backgrounds. Detailed analysis of these mutants revealed differential contributions of QTL mutations to yield performance such as grain length, width, number and 1000-grain weight. Overall, the contributions are additive, resulting in 68 and 30% yield per panicle increase in triple mutants of J809 and L237, respectively. Our data hence demonstrates a promising genome editing approach for rapid breeding of QTLs in elite crop varieties.