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MicroRNAs are a class of endogenous small RNA molecules (20-24 nucleotides) that have pivotal roles in regulating gene expression mostly at posttranscriptional levels in plants. Plant microRNAs have been implicated in the regulation of diverse biological processes including growth and stress responses. However, the information about microRNAs in regulating abiotic stress responses in rice is limited. We optimized a one-tube stem-loop reverse transcription quantitative PCR (ST-RT qPCR) for high-throughput expression profiling analysis of microRNAs in rice under normal and stress conditions. The optimized ST-RT qPCR method was as accurate as small RNA gel blotting and was more convenient and time-saving than other methods in quantifying microRNAs. With this method, 41 rice microRNAs were quantified for their relative expression levels after drought, salt, cold, and abscisic acid (ABA) treatments. Thirty-two microRNAs showed induced or suppressed expression after stress or ABA treatment. Further analysis suggested that stress-responsive cis-elements were enriched in the promoters of stress-responsive microRNA genes. The expressions of five and seven microRNAs were significantly affected in the rice plant with defects in stress tolerance regulatory genes OsSKIPa and OsbZIP23, respectively. Some of the predicted target genes of these microRNAs were also related to abiotic stresses. We conclude that ST-RT qPCR is an efficient and reliable method for expression profiling of microRNAs and a significant portion of rice microRNAs participate in abiotic stress response and regulation. [ABSTRACT FROM AUTHOR]

DNA test is broadly used in diagnosis of crop disease and identification of genetic modified organism (GMO) in agriculture. However, rapid, low-cost, user-friendly, and field-deployable DNA test method is still limit. Recently, the RNA programmable nuclease of CRISPR/Cas is engineered as a new nucleic acid detection platform, but their application in plant remains to explore. In this study, we evaluated the Cas12a-based DNA detection for crop disease diagnosis and GMO test. A total of 14 crRNAs were designed to target two Magnaporthe oryzae genes and a synthetic Cry1C gene which encodes Bacillus thuringiensis δ-endotoxin and has been used to develop transgenic rice cultivar (Bt-rice) in China. Using a fluorescent reporter, targeted genes were easily detected by LbCas12a after recombinase polymerase amplification (RPA) for all crRNAs, despite that the signal strength varied 2–3-folds between different crRNAs. We further combined the filter paper-based DNA extraction and lateral flow assay (LFA) with RPA-Cas12a for DNA detection. This optimized Cas12a diagnostics method is carried at body temperature and does not require extra instrument except filter paper and LFA strip. Our data show that rice blast pathogen and Bt-rice were efficiently identified from leaf disc samples using this optimized DNA test method with highly active crRNAs. Moreover, LbCas12a exhibited variable nuclease activities on different targets; therefore, highly active crRNA is critical for successful DNA test using Cas12a and LFA. Owing to its simplicity, efficiency, and low-cost, DNA test using CRISPR/Cas12a would be easily applied in field for crop disease diagnosis and GMO administration. [ABSTRACT FROM AUTHOR]

Key message: Modifications of multiple copies of the BnaSAD2 gene family with genomic editing technology result in higher stearic acid content in the seed of polyploidy rapeseed. Solid fats from vegetable oils are widely used in food processing industry. Accumulating data showed that stearic acid is more favorite as the major composite among the saturate fatty acids in solid fats in considerations of its effects on human health. Rapeseed is the third largest oil crop worldwide, and has potential to be manipulated to produce higher saturated fatty acids as raw materials of solid fats. Toward that end, we identified four SAD2 gene family members in B. napus genome and established spatiotemporal expression pattern of the BnaSAD2 members. Genomic editing technology was applied to mutate all the copies of BnaSAD2 in this allopolyploid species and mutants at multiple alleles were generated and characterized to understand the effect of each BnaSAD2 member on blocking desaturation of stearic acid. Mutations occurred at BnaSAD2.A3 resulted in more dramatic changes of fatty acid profile than ones on BnaSAD2.C3, BnaSAD2.A5 and BnaSAD2.C4. The content of stearic acid in mutant seeds with single locus increased dramatically with a range of 3.1–8.2%. Furthermore, combination of different mutated alleles of BnaSAD2 resulted in more dramatic changes in fatty acid profiles and the double mutant at BnaSAD2.A3 and BnaSAD2.C3 showed the most dramatic phenotypic changes compared with its single mutants and other double mutants, leading to 11.1% of stearic acid in the seeds. Our results demonstrated that the members of BnaSAD2 have differentiated in their efficacy as a Δ9-Stearoyl-ACP-Desaturase and provided valuable rapeseed germplasm for breeding high stearic rapeseed oil. [ABSTRACT FROM AUTHOR]

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics. [ABSTRACT FROM AUTHOR]

Key message: Genomic editing with CRISPR/Cas9 system can simultaneously modify multiple copies of theBnaFAD2 gene to develop novel variations in fatty acids profiles in polyploidy rapeseed. Fatty acid composition affects edible and processing quality of vegetable oil and has been one of the primary targets for genetic modification in oilseed crops including rapeseed (Brassica napus). Fatty acid desaturase 2 gene, FAD2, is a key player that affects three major fatty acids, namely oleic, linoleic and linolenic acid, in oilseed plants. Previously, we showed that there are four copies of BnaFAD2 in allotetraploid rapeseed. In this study, we further established spatiotemporal expression pattern of each copy of BnaFAD2 using published RNA-seq data. Genomic editing technology based on CRISPR/Cas9 system was used to mutate all the copies of BnaFAD2 to create novel allelic variations in oleic acid and other fatty acid levels. A number of mutants at two targeting sites were identified, and the phenotypic variation in the mutants was systematically evaluated. The oleic acid content in the seed of the mutants increased significantly with the highest exceeding 80% compared with wild type of 66.43%, while linoleic and linolenic acid contents decreased accordingly. Mutations on BnaFAD2.A5 caused more dramatic changes of fatty acid profile than the mutations on BnaFAD2.C5 alleles that were identified with gene editing technique for the first time. Moreover, combining different mutated alleles of BnaFAD2 can even broaden the variation more dramatically. It was found that effects of different mutation types at BnaFAD2 alleles on oleic levels varied, indicating a possibility to manipulate fatty acid levels by precise mutation at specific region of a gene. [ABSTRACT FROM AUTHOR]