Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.

RNA interference (RNAi) is induced by the direct transfer of double-stranded RNAs (dsRNAs) into protoplasts prepared from Arabidopsis thaliana seedlings. In this protoplast RNAi system, we compared the efficacies of various-sized dsRNAs (between 21 and 139 nucleotides [nt]) for inducing RNAi and assessed the dsRNA-cleaving activities of Dicer-like 3 (DCL3) and 4 (DCL4). After the direct transfer of dsRNAs into protoplasts, cleaved RNA products of 21 nt were detected from long 130- or 500-nt dsRNAs by DCL4 but not from 37-nt dsRNAs. These results indicate that DCL4 preferentially cleaves long dsRNAs in protoplasts, consistent with our previous biochemical data regarding the substrate specificity of DCL4. Direct transfer of long dsRNAs of approximately 130 nt into protoplasts induces RNAi much more effectively (by approximately 60- to 400-fold) than direct transfer of short 37-nt dsRNAs. Although transfer of 21-nt dsRNAs into protoplasts induced RNAi without DCL4 activity, the induction of RNAi was less effective (by approximately 0.01-fold) compared with long dsRNAs. These results indicate that cleavage of long dsRNAs exceeding 100 nt by DCL4 into 21-nt dsRNAs is essential for efficient induction of RNAi in plant cells.

Vacuole generation occurs frequently during the enlargement of bacterial protoplasts and spheroplasts. Gram-positive Enterococcus faecalis protoplasts and gram-negative Lelliottia amnigena spheroplasts had large and small vacuoles inside the cytoplasm, respectively. Although no vacuoles were found at the early stage of cell enlargement, all enlarged cells used in the microinjection procedures had vacuoles. The plasma membrane of L. amnigena was more flexible than that of E. faecalis. In addition, E. faecalis protoplasts had unique discoidal structures as well as spherical structures in the cytoplasm. Our findings showed that the number of vacuoles increased as the L. amnigena plasma membrane expanded and that the size of vacuoles increased as the E. faecalis plasma membrane expanded, suggesting that bacterial cell enlargement involved vacuole generation. Thus, biosynthesis of the plasma and vacuolar membranes was synchronous with the bacterial cell enlargement. Differences in the plasma membrane flexibility might influence the different types of vacuole generation.

CRISPR/Cas9 system has been widely applied in many plant species to induce mutations in the genome for studying gene function and improving crops. However, to our knowledge, there is no report of CRISPR/Cas9-mediated genome editing in melon (Cucumis melo). In our study, phytoene desaturase gene of melon (CmPDS) was selected as target for the CRISPR/Cas9 system with two designed gRNAs, targeting exons 1 and 2. A construct (pHSE-CmPDS) carrying both gRNAs and the Cas9 protein was delivered by PEG-mediated transformation in protoplasts. Mutations were detected in protoplasts for both gRNAs. Subsequently, Agrobacterium-mediated transformation of cotyledonary explants was carried out, and fully albino and chimeric albino plants were successfully regenerated. A regeneration efficiency of 71% of transformed plants was achieved from cotyledonary explants, a 39% of genetic transformed plants were successful gene edited, and finally, a 42-45% of mutation rate was detected by Sanger analysis. In melon protoplasts and plants most mutations were substitutions (91%), followed by insertions (7%) and deletions (2%). We set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and feasible in melon, generating multi-allelic mutations in both genomic target sites of the CmPDS gene showing an albino phenotype easily detectable after only few weeks after Agrobacterium-mediated transformation.

It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.

Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.

Iodine deficiency represents a public health problem worldwide. To increase the amount of iodine in the diet, biofortification strategies of plants have been tried. They rely on the exogenous administration of iodine to increase its absorption and accumulation. However, iodine is not stable in plants and can be volatilized as methyl iodide through the action of specific methyltransferases encoded by the HARMLESS TO OZONE LAYER (HOL) genes. The release of methyl iodide in the atmosphere represents a threat for the environment due to its ozone depletion potential. Rice paddies are among the strongest producers of methyl iodide. Thus, the agronomic approach of iodine biofortification is not appropriate for this crop, leading to further increases of iodine emissions. In this work, we used the genome editing CRISPR/Cas9 technology to knockout the rice HOL genes and investigate their function. OsHOL1 resulted a major player in methyl iodide production, since its knockout abolished the process. Moreover, its overexpression reinforced it. Conversely, knockout of OsHOL2 did not produce effects. Our experiments helped elucidating the function of the rice HOL genes, providing tools to develop new rice varieties with reduced iodine emissions and thus more suitable for biofortification programs without further impacting on the environment.

Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

Cannabis sativa aromatic prenyltransferase 4 (CsPT4) and 1 (CsPT1) have been shown to catalyze cannabigerolic acid (CBGA) biosynthesis, a step that rate-limits the cannabinoid biosynthetic pathway; both genes are highly expressed in flowers. CsPT4 and CsPT1 promoter driven β-glucuronidase (GUS) activities were detected in leaves of cannabis seedlings, and strong CsPT4 promoter activities were associated with glandular trichomes. Hormonal regulation of cannabinoid biosynthetic genes is poorly understood. An in silico analysis of the promoters identified putative hormone responsive elements. Our work examines hormone-responsive elements in the promoters of CsPT4 and CsPT1 in the context of physiological responses of the pathway to the hormone in planta. Dual luciferase assays confirmed the regulation of promoter activities by the hormones. Further studies with salicylic acid (SA) demonstrated that SA pretreatment increased the expression of genes located downstream of the cannabinoid biosynthetic pathway. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis. The work provides information relevant to plant biology, as we present evidence demonstrating correlations between molecular mechanisms that regulate gene expression and influence plant chemotypes.

The stress hormone abscisic acid (ABA) helps plants to survive under abiotic stresses; however, its use as an agrochemical is limited by its chemical instability and expense. Here, we report the development of an in vivo screening system to isolate chemicals able to induce ABA signalling responses in rice (Oryza sativa) protoplasts. This system consists of an ABA-hypersensitive synthetic promoter containing ABRE and DRE motifs driving a luciferase reporter gene. After efficiently transfecting rice protoplasts with this construct, we screened chemicals library with a similar molecular weight and chemical structure to ABA and identified one chemical, S7, that induced ABA signalling by mediating interactions between the group I and II OsPYL receptors and certain OsPP2CAs in a yeast two-hybrid assay. In an in vitro pulldown assay, S7 was found to mediate a weak interaction between OsPYL5/8 and various OsPP2CAs. S7 treatments did not affect seedling growth or seed germination, but could reduce water loss. Rice seedlings treated with S7 exhibited transcriptome profiles that partially overlapped those treated with ABA. Taken together, we concluded that S7 is a new partial ABA agonist, which has potential use in future dissections of ABA signalling and as an agrochemical.

Magnaporthe oryzae, the causal agent of blast disease, is one of the most destructive plant pathogens, causing significant yield losses on staple crops such as rice and wheat. The fungus infects plants with a specialized cell called an appressorium, whose development is tightly regulated by MAPK signaling pathways following the activation of upstream sensors in response to environmental stimuli. Here, we show the expression of the Glycogen synthase kinase 3 (GSK3) MoGSK1 in M. oryzae is regulated by Mps1 MAP kinase, particularly under the stressed conditions. Thus, MoGSK1 is functionally characterized in this study. MoGsk1 is functionally homologues to the Saccharomyces cerevisiae GSK3 homolog MCK1. Gene replacement of MoGSK1 caused significant delay in mycelial growth, complete loss of conidiation and inability to penetrate the host surface by mycelia-formed appressorium-like structures, consequently resulting in loss of pathogenicity. However, the developmental and pathogenic defects of Δmogsk1 are recovered via the heterologous expression of Fusarium graminearum GSK3 homolog gene FGK3, whose coding products also shows the similar cytoplasmic localization as MoGsk1 does in M. oryzae. By contrast, overexpression of MoGSK1 produced deformed appressoria in M. oryzae. In summary, our results suggest that MoGsk1, as a highly conservative signal modulator, dictates growth, conidiation and pathogenicity of M. oryzae.

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt+) and mating-type minus (mt-). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt+ and mt- cells, respectively. PR-IP mediates the release of protoplasts from mt- cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt- cells of the C. psl. complex. When the knockout mt- cells were mixed with wild-type mt+ cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

Alternative splicing (AS) can significantly impact the transcriptome and proteome of a eukaryotic cell. Here, using transcriptome and proteome profiling data, we analyzed AS in two life forms of the model moss Physcomitrella patens, namely protonemata and gametophores, as well as in protoplasts. We identified 12 043 genes subject to alternative splicing and analyzed the extent to which AS contributes to proteome diversity. We could distinguish a few examples that unambiguously indicated the presence of two or more splice isoforms from the same locus at the proteomic level. Our results indicate that alternative isoforms have a small effect on proteome diversity. We also revealed that mRNAs and pre-mRNAs have thousands of complementary binding sites for long non-coding RNAs (lncRNAs) that may lead to potential interactions in transcriptome. This finding points to an additional level of gene expression and AS regulation by non-coding transcripts in Physcomitrella patens. Among the differentially expressed and spliced genes we found serine/arginine-rich (SR) genes, which are known to regulate AS in cells. We found that treatment with abscisic (ABA) and methyl jasmonic acids (MeJA) led to an isoform-specific response and suggested that ABA in gametophores and MeJA in protoplasts regulate AS and the transcription of SR genes.

The upstream pseudoknots domain (UPD) of Tobacco mosaic virus (TMV) is located at the 3'-untranslated region (UTR). It plays an important role in virus replication and translation. To determine the importance of UPD and 3'-UTR, and the effects of introduced RNA elements in TMV 3'-UTR, a series of TMV mutants with internal poly(A) tract upstream of UPD was constructed for structural analysis by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). TMV(24A+UPD) and TMV(42A+UPD) formed a similar structure as that of TMV 3'-UTR, but TMV(62A+UPD) structures altered by the introduced poly(A) tract. In addition, TMV(24A+UPD) had a higher viral RNAs accumulation than TMV in N. benthamiana protoplasts, and induced lethal symptoms in the infected plants. TMV(62A+UPD) showed a drastically reduced accumulation, its coat protein was undetectable in protoplasts, and the inoculated plants remained symptomless. This study analyzed the structures of 3'-UTR of TMV and found that the longer poly(A) tract introduced upstream of UPD reduced viral RNAs accumulation and induced milder symptoms in N. benthamiana. In conclusion, different lengths of the internal poly(A) tract introduced into the TMV 3'UTR lead to structural variations that affect virus accumulation and symptom expression.

Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1-296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs.

Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR-Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.

Efficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to efficiently induce homologous recombination. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

The signal transduction of the plant hormone cytokinin is mediated by a His-to-Asp phosphorelay. The canonical cytokinin receptor consists of an extra cytoplasmic hormone binding domain named cyclase/histidine kinase associated sensory extracellular (CHASE) and cytoplasmic histidine kinase and receiver domains. In addition to classical cytokinin receptors, a different type receptor-named CHASE domain receptor serine/threonine kinase (CHARK)-is also present in rice. It contains the same ligand binding domain as other cytokinin receptors but has a predicted Ser/Thr-instead of a His-kinase domain. Bioinformatic analysis indicates that CHARK is a retrogene and a product of trans-splicing. Here, we analyzed whether CHARK can function as a bona fide cytokinin receptor. A biochemical assay demonstrated its ability to bind cytokinin. Transient expression of CHARK in protoplasts increased their response to cytokinin. Expression of CHARK in an Arabidopsis receptor double mutant complemented its growth defects and restored the ability to activate cytokinin response genes, clearly demonstrating that CHARK functions as a cytokinin receptor. We propose that the CHARK gene presents an evolutionary novelty in the cytokinin signaling system.

DNA-free genome editing was used to induce mutations in one or two branching enzyme genes (Sbe) in tetraploid potato to develop starch with an increased amylose ratio and elongated amylopectin chains. By using ribonucleoprotein (RNP) transfection of potato protoplasts, a mutation frequency up to 72% was achieved. The large variation of mutations was grouped as follows: Group 1 lines with all alleles of Sbe1 mutated, Group 2 lines with all alleles of Sbe1 as well as two to three alleles of Sbe2 mutated and Group 3 lines having all alleles of both genes mutated. Starch from lines in Group 3 was found to be essentially free of amylopectin with no detectable branching and a chain length (CL) distribution where not only the major amylopectin fraction but also the shortest amylose chains were lost. Surprisingly, the starch still formed granules in a low-ordered crystalline structure. Starch from lines of Group 2 had an increased CL with a higher proportion of intermediate-sized chains, an altered granule phenotype but a crystalline structure in the granules similar to wild-type starch. Minor changes in CL could also be detected for the Group 1 starches when studied at a higher resolution.

Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2-9.7% using the pCas9-AtU6-sgRNA vector and 14.7-20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.