Genetic analysis implicates a molecular chaperone complex in regulating epigenetic silencing of methylated genomic regions.

DNA cytosine methylation confers stable epigenetic silencing in plants and many animals. However, the mechanisms underlying DNA methylation-mediated genomic silencing are not fully understood. We conducted a forward genetic screen for cellular factors required for the silencing of a heavily methylated p35S:NPTII transgene in the Arabidopsis thaliana rdm1-1 mutant background, which led to the identification of a Hsp20 family protein, RDS1 (rdm1-1 suppressor 1). Loss-of-function mutations in RDS1 released the silencing of the p35S::NPTII transgene in rdm1-1 mutant plants, without changing the DNA methylation state of the transgene. Protein interaction analyses suggest that RDS1 exists in a protein complex consisting of the methyl-DNA binding domain proteins MBD5 and MBD6, two other Hsp20 family proteins, RDS2 and IDM3, a Hsp40/DNAJ family protein, and a Hsp70 family protein. Like rds1 mutations, mutations in RDS2, MBD5, or MBD6 release the silencing of the transgene in the rdm1 mutant background. Our results suggest that Hsp20, Hsp40, and Hsp70 proteins may form a complex that is recruited to some genomic regions with DNA methylation by methyl-DNA binding proteins to regulate the state of silencing of these regions.

CRISPR/Cas9-Based Genome Editing Toolbox for Arabidopsis thaliana.

CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways can be exploited to knock out or replace genes. Also, cytidine or adenine base editors (CBEs or ABEs) fused to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system. Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing in other plant species such as rice.

A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis.

The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4⁻10%) and T2 (32.5⁻46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis, especially in multiplex applications.

CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation.

Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.

A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi.

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an antiviral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus can inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homolog, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signaling components have been coopted to play multiple functions in plants.

Efficient Generation of diRNAs Requires Components in the Posttranscriptional Gene Silencing Pathway.

It has been reported that double-stranded break (DSB)-induced small RNAs (diRNAs) are generated via the RNA-directed DNA methylation pathway and function in DSB repair in Arabidposis. However, important questions remain regarding the biogenesis and function of diRNAs. Here, we used CRISPR/Cas9- or TALEN-triggered DSBs to characterize diRNAs in Arabidopsis and rice. We found that 21-nt diRNAs were generated from a 35S promoter::GU-US reporter transgene targeted by CRISPR/Cas9. Unexpectedly, Pol II transcription of the transgene was required for efficient diRNA production and the level of diRNA accumulation correlated with the expression level of the transgene. diRNAs were not detected from CRISPR/Cas9- or TALEN-induced DSBs within the examined endogenous genes in Arabidopsis or rice. We also found that DCL4 and RDR6 that are known to be involved in posttranscriptional gene silencing were required to generate diRNAs. Our results suggest that DSBs are necessary but not sufficient for efficient diRNA generation and a high level of diRNAs is not necessary for DSB repair.

Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis.

The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.

The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.

Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.

Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis.

The transition from dormancy to germination in seeds is a key physiological process during the lifecycle of plants. Abscisic acid (ABA) is the sole plant hormone known to maintain seed dormancy; it acts through a gene expression network involving the transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3). However, whether other phytohormone pathways function in the maintenance of seed dormancy in response to environmental and internal signals remains an important question. Here, we show that the plant growth hormone auxin, which acts as a versatile trigger in many developmental processes, also plays a critical role in seed dormancy in Arabidopsis. We show that disruptions in auxin signaling in MIR160-overexpressing plants, auxin receptor mutants, or auxin biosynthesis mutants dramatically release seed dormancy, whereas increases in auxin signaling or biosynthesis greatly enhance seed dormancy. Auxin action in seed dormancy requires the ABA signaling pathway (and vice versa), indicating that the roles of auxin and ABA in seed dormancy are interdependent. Furthermore, we show that auxin acts upstream of the major regulator of seed dormancy, ABI3, by recruiting the auxin response factors AUXIN RESPONSE FACTOR 10 and AUXIN RESPONSE FACTOR 16 to control the expression of ABI3 during seed germination. Our study, thus, uncovers a previously unrecognized regulatory factor of seed dormancy and a coordinating network of auxin and ABA signaling in this important process.

Molecular basis of age-dependent vernalization in Cardamine flexuosa.

Plants flower in response to many varied cues, such as temperature, photoperiod, and age. The floral transition of Cardamine flexuosa, a herbaceous biennial-to-perennial plant, requires exposure to cold temperature, a treatment known as vernalization. C. flexuosa younger than 5 weeks old are not fully responsive to cold treatment. We demonstrate that the levels of two age-regulated microRNAs, miR156 and miR172, regulate the timing of sensitivity in response to vernalization. Age and vernalization pathways coordinately regulate flowering through modulating the expression of CfSOC1, a flower-promoting MADS-box gene. The related annual Arabidopsis thaliana, which has both vernalization and age pathways, does not possess an age-dependent vernalization response. Thus, the recruitment of age cue in response to environmental signals contributes to the evolution of life cycle in plants.

[Molecular cloning and prokaryotic expression of phenylalanine ammonia-lyase gene FdPAL from Fagopyrum dibotrys].

OBJECTIVE: To clone and characterize the DNA and cDNA sequences of phenylalanine ammonia-lyase gene (PAL) from Fagopyrum dibotrys, and investigate the biological activity of the obtained PAL. METHOD: Using homology cloning and RT-PCR techniques, the DNA and full-length cDNA sequences of PAL gene were amplified from F. dibotrys. The obtained sequences were analyzed by bioinformatics software. The ORF of PAL gene was cloned into expression vector pET-30b(+) and transformed into Escherichia coli BL21 (DE3) for expression the recombined protein. The catalytic activity of the recombined protein was determined by Spectrophotometer and thin layer chromatography (TLC) methods. RESULT: The DNA sequence of PAL gene (designated as FdPAL, GenBank accession number: HM628904) was 2 583 bp in size, of which consisted two extrons and a single intron, and the full-length cDNA of FdPAL was 2 169 bp in size, which contained an ORF. The deduced protein of FdPAL contained 722 amino acids with calculated molecular weight (MW) of 78.31 kDa and an isoelectric point (pI) of 5.94. The SDS-PAGE results showed that the molecular weight of recombinant FdPAL protein was 75.37 kDa, which is consistent with the predictions. After 4 hours of induction, the enzymatic specific activity of FdPAL reached the summit, up to 4 386 nmol x g(-1) x min(-1). The reaction products were also identified by TLC, using L-Phe and trans-cinnamic acid as the internal standard. CONCLUSION: The PAL gene (both DNA sequence and full-length cDNA sequence) was cloned from F. dibotrys, and it has the same classic characters as other PALs in plants. The recombinant FdPAL was efficiently expressed in E. coli and had the activity for catalyzing the conversion from L-phenylalanine to cinnamic acid.