Genome editing of a rice CDP-DAG synthase confers multipathogen resistance.

The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes1. Here we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis2. Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In rice, PtdIns(4,5)P2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor3. By using targeted genome editing, we obtained an allele of RBL1, named RBL1Δ12, which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops.

Characterization and acceleration of genome shuffling and ploidy reduction in synthetic allopolyploids by genome sequencing and editing.

Polyploidy and the subsequent ploidy reduction and genome shuffling are the major driving forces of genome evolution. Here, we revealed short-term allopolyploid genome evolution by sequencing a synthetic intergeneric hybrid (Raphanobrassica, RRCC). In this allotetraploid, the genome deletion was quick, while rearrangement was slow. The core and high-frequency genes tended to be retained while the specific and low-frequency genes tended to be deleted in the hybrid. The large-fragment deletions were enriched in the heterochromatin region and probably derived from chromosome breaks. The intergeneric translocations were primarily of short fragments dependent on homoeology, indicating a gene conversion origin. To accelerate genome shuffling, we developed an efficient genome editing platform for Raphanobrassica. By editing Fanconi Anemia Complementation Group M (FANCM) genes, homoeologous recombination, chromosome deletion and secondary meiosis with additional ploidy reduction were accelerated. FANCM was shown to be a checkpoint of meiosis and controller of ploidy stability. By simultaneously editing FLIP genes, gene conversion was precisely introduced, and mosaic genes were produced around the target site. This intergeneric hybrid and genome editing platform not only provides models that facilitate experimental evolution research by speeding up genome shuffling and conversion but also accelerates plant breeding by enhancing intergeneric genetic exchange and creating new genes.

Comparative transcriptomic profiling of the two-stage response of rice to Xanthomonas oryzae pv. oryzicola interaction with two different pathogenic strains.

BACKGROUND: Two-tiered plant immune responses involve cross-talk among defense-responsive (DR) genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered immunity (ETI) and effector-triggered susceptibility (ETS). Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an important bacterial disease that causes serious threats to rice yield and quality. Transcriptomic profiling provides an effective approach for the comprehensive and large-scale detection of DR genes that participate in the interactions between rice and Xoc. RESULTS: In this study, we used RNA-seq to analyze the differentially expressed genes (DEGs) in susceptible rice after inoculation with two naturally pathogenic Xoc strains, a hypervirulent strain, HGA4, and a relatively hypovirulent strain, RS105. First, bacterial growth curve and biomass quantification revealed that differential growth occurred beginning at 1 day post inoculation (dpi) and became more significant at 3 dpi. Additionally, we analyzed the DEGs at 12 h and 3 days post inoculation with two strains, representing the DR genes involved in the PTI and ETI/ETS responses, respectively. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the common DEGs, which included 4380 upregulated and 4019 downregulated genes and 930 upregulated and 1383 downregulated genes identified for the two strains at 12 h post inoculation (hpi) and 3 dpi, respectively. Compared to those at 12 hpi, at 3 dpi the number of common DEGs decreased, while the degree of differential expression was intensified. In addition, more disease-related GO pathways were enriched, and more transcription activator-like effector (TALE) putative target genes were upregulated in plants inoculated with HGA4 than in those inoculated with RS105 at 3 dpi. Then, four DRs were randomly selected for the BLS resistance assay. We found that CDP3.10, LOC_Os11g03820, and OsDSR2 positively regulated rice resistance to Xoc, while OsSPX3 negatively regulated rice resistance. CONCLUSIONS: By using an enrichment method for RNA-seq, we identified a group of DEGs related to the two stages of response to the Xoc strain, which included four functionally identified DR genes.

OsGELP77, a QTL for broad-spectrum disease resistance and yield in rice, encodes a GDSL-type lipase.

Lipids and lipid metabolites have essential roles in plant-pathogen interactions. GDSL-type lipases are involved in lipid metabolism modulating lipid homeostasis. Some plant GDSLs modulate lipid metabolism altering hormone signal transduction to regulate host-defence immunity. Here, we functionally characterized a rice lipase, OsGELP77, promoting both immunity and yield. OsGELP77 expression was induced by pathogen infection and jasmonic acid (JA) treatment. Overexpression of OsGELP77 enhanced rice resistance to both bacterial and fungal pathogens, while loss-of-function of osgelp77 showed susceptibility. OsGELP77 localizes to endoplasmic reticulum and is a functional lipase hydrolysing universal lipid substrates. Lipidomics analyses demonstrate that OsGELP77 is crucial for lipid metabolism and lipid-derived JA homeostasis. Genetic analyses confirm that OsGELP77-modulated resistance depends on JA signal transduction. Moreover, population genetic analyses indicate that OsGELP77 expression level is positively correlated with rice resistance against pathogens. Three haplotypes were classified based on nucleotide polymorphisms in the OsGELP77 promoter where OsGELP77Hap3 is an elite haplotype. Three OsGELP77 haplotypes are differentially distributed in wild and cultivated rice, while OsGELP77Hap3 has been broadly pyramided for hybrid rice development. Furthermore, quantitative trait locus (QTL) mapping and resistance evaluation of the constructed near-isogenic line validated OsGELP77, a QTL for broad-spectrum disease resistance. In addition, OsGELP77-modulated lipid metabolism promotes JA accumulation facilitating grain yield. Notably, the hub defence regulator OsWRKY45 acts upstream of OsGELP77 by initiating the JA-dependent signalling to trigger immunity. Together, OsGELP77, a QTL contributing to immunity and yield, is a candidate for breeding broad-spectrum resistant and high-yielding rice.

Genome-wide profiling of rice Double-stranded RNA-Binding Protein 1-associated RNAs by targeted RNA editing.

RNA-binding proteins (RBPs) play essential roles in regulating gene expression. However, the RNA ligands of RBPs are poorly understood in plants, not least due to the lack of efficient tools for genome-wide identification of RBP-bound RNAs. An RBP-fused adenosine deaminase acting on RNA (ADAR) can edit RBP-bound RNAs, which allows efficient identification of RNA ligands of RBPs in vivo. Here, we report the RNA editing activities of the ADAR deaminase domain (ADARdd) in plants. Protoplast experiments indicated that RBP-ADARdd fusions efficiently edited adenosines within 41 nucleotides (nt) of their binding sites. We then engineered ADARdd to profile the RNA ligands of rice (Oryza sativa) Double-stranded RNA-Binding Protein 1 (OsDRB1). Overexpressing the OsDRB1-ADARdd fusion protein in rice introduced thousands of A-to-G and T-to-C RNA‒DNA variants (RDVs). We developed a stringent bioinformatic approach to identify A-to-I RNA edits from RDVs, which removed 99.7% to 100% of background single-nucleotide variants in RNA-seq data. This pipeline identified a total of 1,798 high-confidence RNA editing (HiCE) sites, which marked 799 transcripts as OsDRB1-binding RNAs, from the leaf and root samples of OsDRB1-ADARdd-overexpressing plants. These HiCE sites were predominantly located in repetitive elements, 3'-UTRs, and introns. Small RNA sequencing also identified 191 A-to-I RNA edits in miRNAs and other sRNAs, confirming that OsDRB1 is involved in sRNA biogenesis or function. Our study presents a valuable tool for genome-wide profiling of RNA ligands of RBPs in plants and provides a global view of OsDRB1-binding RNAs.

A secreted fungal subtilase interferes with rice immunity via degradation of SUPPRESSOR OF G2 ALLELE OF skp1.

Serine protease subtilase, found widely in both eukaryotes and prokaryotes, participates in various biological processes. However, how fungal subtilase regulates plant immunity is a major concern. Here, we identified a secreted fungal subtilase, UvPr1a, from the rice false smut (RFS) fungus Ustilaginoidea virens. We characterized UvPr1a as a virulence effector localized to the plant cytoplasm that inhibits plant cell death induced by Bax. Heterologous expression of UvPr1a in rice (Oryza sativa) enhanced plant susceptibility to rice pathogens. UvPr1a interacted with the important rice protein SUPPRESSOR OF G2 ALLELE OF skp1 (OsSGT1), a positive regulator of innate immunity against multiple rice pathogens, degrading OsSGT1 in a protease activity-dependent manner. Furthermore, host-induced gene silencing of UvPr1a compromised disease resistance of rice plants. Our work reveals a previously uncharacterized fungal virulence strategy in which a fungal pathogen secretes a subtilase to interfere with rice immunity through degradation of OsSGT1, thereby promoting infection. These genetic resources provide tools for introducing RFS resistance and further our understanding of plant-pathogen interactions.

A novel type of Brassica napus with higher stearic acid in seeds developed through genome editing of BnaSAD2 family.

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.

Fine-tuning OsCPK18/OsCPK4 activity via genome editing of phosphorylation motif improves rice yield and immunity.

Plants have evolved complex signalling networks to regulate growth and defence responses under an ever-changing environment. However, the molecular mechanisms underlying the growth-defence tradeoff are largely unclear. We previously reported that rice CALCIUM-DEPENDENT PROTEIN KINASE 18 (OsCPK18) and MITOGEN-ACTIVATED PROTEIN KINASE 5 (OsMPK5) mutually phosphorylate each other and that OsCPK18 phosphorylates and positively regulates OsMPK5 to suppress rice immunity. In this study, we found that OsCPK18 and its paralog OsCPK4 positively regulate plant height and yield-related traits. Further analysis reveals that OsCPK18 and OsMPK5 synergistically regulate defence-related genes but differentially regulate development-related genes. In vitro and in vivo kinase assays demonstrated that OsMPK5 phosphorylates C-terminal threonine (T505) and serine (S512) residues of OsCPK18 and OsCPK4, respectively. The kinase activity of OsCPK18T505D , in which T505 was replaced by aspartic acid to mimic T505 phosphorylation, displayed less calcium sensitivity than that of wild-type OsCPK18. Interestingly, editing the MAPK phosphorylation motif in OsCPK18 and its paralog OsCPK4, which deprives OsMPK5-mediated phosphorylation but retains calcium-dependent activation of kinase activity, simultaneously increases rice yields and immunity. This editing event also changed the last seven amino acid residues of OsCPK18 and attenuated its binding with OsMPK5. This study presents a new regulatory circuit that fine tunes the growth-defence tradeoff by modulating OsCPK18/4 activity and suggests that CRISPR/Cas9-mediated engineering phosphorylation pathways could simultaneously improve crop yield and immunity.

A secreted fungal effector suppresses rice immunity through host histone hypoacetylation.

Histone acetylation is a critical epigenetic modification that regulates plant immunity. Fungal pathogens secrete effectors that modulate host immunity and facilitate infection, but whether fungal pathogens have evolved effectors that directly target plant histone acetylation remains unknown. Here, we identified a secreted protein, UvSec117, from the rice false smut fungus, Ustilaginoidea virens, as a key effector that can target the rice histone deacetylase OsHDA701 and negatively regulates rice broad-spectrum resistance against rice pathogens. UvSec117 disrupts host immunity by recruiting OsHDA701 to the nucleus and enhancing OsHDA701-modulated deacetylation, thereby reducing histone H3K9 acetylation levels in rice plants and interfering with defense gene activation. Host-induced gene silencing of UvSec117 promotes rice resistance to U. virens, thus providing an alternative way for developing rice false smut-resistant plants. This is the first direct evidence demonstrating that a fungal effector targets a histone deacetylase to suppress plant immunity. Our data provided insight into a counter-defense mechanism in a plant pathogen that inactivates host defense responses at the epigenetic level.

A versatile nanoluciferase toolkit and optimized in-gel detection method for protein analysis in plants.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01210-7.

A CRISPR/LbCas12a-based method for highly efficient multiplex gene editing in Physcomitrella patens.

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non-vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co-delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss-of-function mutants of multiple genes from different gene families.

Genome editing with the CRISPR-Cas system: an art, ethics and global regulatory perspective.

Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR-Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR-Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR-Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre-clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR-Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR-Cas-modified organisms/products.

Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus.

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.

Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co-expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA-gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen-activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR-edited mutants. The true knock-out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR-induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45-86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene-free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.

Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development.

Multilocular silique is a desirable agricultural trait with great potential for the development of high-yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two subgenomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T0 with different single-guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild-type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.

Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells.

CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct. Here, we report a strategy to quickly assembly multiple sgRNAs in one construct using a polycistronic-tRNA-gRNA (PTG) strategy. Taking advantage of the endogenous tRNA processing system in mammalian cells, we efficiently express multiple sgRNAs driven using only one Pol III promoter. Using an all-in-one construct carrying PTG, we disrupt the deacetylase domain in multiple histone deacetylases (HDACs) in human cells simultaneously. We demonstrate that multiple HDAC deletions significantly affect the activation of the Wnt-signaling pathway. Thus, this method enables to efficiently target multiple genes and provide a useful tool to establish mutated cells mimicking human diseases.

Direct phosphorylation and activation of a mitogen-activated protein kinase by a calcium-dependent protein kinase in rice.

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.

Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice.

MicroRNAs constitute a large group of endogenous small RNAs of ~22 nt that emerge as vital regulators, mainly by targeting mRNAs for post-transcriptional repression. Previous studies have revealed that the miR164 family in Arabidopsis is comprised of three members which guide the cleavage of the mRNAs of five NAC genes to modulate developmental processes. However, the functions of the miR164-targeted NAC genes in crops are poorly deciphered. In this study, the conserved features of six miR164-targeted NAC genes (OMTN1-OMTN6) in rice are described, and evidence is provided that four of them confer a negative regulatory role in drought resistance. OMTN proteins have the characteristics of typical NAC transcriptional factors. The miR164 recognition sites of the OMTN genes are highly conserved in rice germplasms. Deletion of the recognition sites impaired the transactivation activity, indicating that the conserved recognition sites play a crucial role in maintaining the function of the OMTN proteins. The OMTN genes were responsive to abiotic stresses, and showed diverse spatio-temporal expression patterns in rice. Overexpression of OMTN2, OMTN3, OMTN4, and OMTN6 in rice led to negative effects on drought resistance at the reproductive stage. The expression of numerous genes related to stress response, development, and metabolism was altered in OMTN2-, OMTN3-, OMTN4-, and OMTN6-overexpressing plants. Most of the up-regulated genes in the OMTN-overexpressing plants were down-regulated by drought stress. The results suggest that the conserved miR164-targeted NAC genes may be negative regulators of drought tolerance in rice, in addition to their reported roles in development.