Phytoene Desaturase Research Articles

Virus-Induced Gene Silencing Simultaneously Exploits ‘Attract and Kill’ Traits in Plants and Insects to Manage Huanglongbing

Abstract The vector-borne disease Huanglongbing (HLB) causes severe economic losses to citrus production worldwide with no available cure. Herein, we applied virus-induced gene silencing technology to engineer citrus that preferentially attracted and specifically killed Diaphorina citri, the vector associated with HLB. We engineered the infectious citrus tristeza virus (CTV-T36) clone to carry three truncated genes. The triple construct (CTV-tAwd-tWnt-tPDS) produces small interfering RNAs (siRNAs) against phytoene desaturase, PDS, to yield a phenotype with visual, olfactory, and gustatory cues that preferentially attracted D. citri. In addition, siRNAs targeted two genes related to flight in D. citri, abnormal wing disc (DcAwd) and wingless (DcWnt), that caused wing malformations and decreased survival in psyllids that fed on plants inoculated with the engineered virus. During two successive generations, D. citri reared on CTV-tAwd-tWnt-tPDS-inoculated plants exhibited higher mortality across life stages as well as reduced fecundity and fertility as compared with those reared on non-infected plants or CTV-wt-inoculated plants. Furthermore, CTV-tAwd-tWnt-tPDS-inoculated plants shortened the lifespan of D. citri by more than 20 days. Morphological abnormalities were noted in those adults that did successfully emerge on plants inoculated with CTV-tAwd-tWnt-tPDS, including cocked wings with a bowl-shaped depression and/or a convex shape. Phloem sap from CTV-tAwd-tWnt-tPDS-inoculated plants decreased the survival of D. citri adults, confirming that siRNAs were present in the sap of these plants. Collectively, we provide proof of concept for a novel variant of the attract-and-kill method where the cultivated crop is potentially transformed into a hyper-attractive population and transmission sink for a phytopathogen vector.

Improving the Genome Editing Efficiency of CRISPR/Cas9 in Melon and Watermelon

CRISPR/Cas9 is a powerful genome editing tool for trait improvement in various crops; however, enhancing mutation efficiency using CRISPR/Cas9 in watermelon and melon remains challenging. We designed four CRISPR systems with different sgRNA expression cassettes to target the phytoene desaturase (PDS) gene in melon. The constructed vectors were delivered to host plants using Agrobacterium-mediated transformation. Phenotypic and genotypic analyses of the edited melon seedlings revealed that the CRISPR systems with tRNA and Csy4 spacers driven by the Pol II-type promoter significantly improved mutation efficiency, reaching 25.20% and 42.82%, respectively. Notably, 78.95% of the mutations generated by the Csy4 system involved large-fragment deletions (LDs) between the two target sites. In watermelon, the Csy4 system achieved a PDS editing efficiency of 41.48%, with 71.43% of the edited seedlings showing LD between the two target sites. Sequencing analysis indicated that the edited melon seedlings exhibited heterozygous, three-allele mutation and chimeric events; the edited watermelon seedlings included 2/14 homozygous mutations. Compared to the commonly used Pol III promoter, using the Pol II promoter to drive sgRNA expression cassettes containing Csy4 showed the best improvement in CRISPR/Cas9 editing efficiency in melon; this system was also effective in watermelon.

Exploring an economic and highly efficient genetic transformation and genome-editing system for radish through developmental regulators and visible reporter.

Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes-mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red-violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots-to-shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2-ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2-ipt combination. Then, Wus2-ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene-edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome-editing genetic improvement of other vegetable species.

Combining systems and synthetic biology for in vivo enzymology

Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax, and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.

Efficient gene editing of a model fern species through gametophyte-based transformation.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species through gametophytes due to the complex characteristics of fern genomes, genetics, and physiology. Here, we established a protocol for gametophyte-based screening of single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas9-mediated gene knockout in a model fern species, Ceratopteris richardii. We utilized the C. richardii ACTIN promoter to drive sgRNA expression and the enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes, such as Nucleotidase/phosphatase 1 (CrSAL1) and Phytoene Desaturase (CrPDS), which resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly (P<0.05) reduced stomatal conductance (gs), leaf transpiration rate (E), guard cell length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, and E as well as most of the stomatal traits and ABA-induced ROS production in guard cells compared to the wild-type (WT) plants. Taken together, our optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery, and agricultural applications.

Construction and optimization of the TRV-mediated VIGS system in Areca catechu embryoids

Areca catechu, a member of the palm family Arecaceae native to tropical regions, holds significant economic importance as a tropical crop. This study explores the application of virus-induced gene silencing (VIGS) in A. catechu embryogenic callus tissues, utilizing the phytoene desaturase gene (AcPDS) of A. catechu as a reporter gene. Results demonstrate that employing EHA105 strain, an Agrobacterium infection liquid concentration of OD600 = 0.5, an infection duration of 5 min, acetosyringone (AS) concentration of 21.5 mg/L, co-cultivation at 19 °C for 2 days followed by 28 °C for 3 days, and a subsequent 14-day post-co-cultivation period, the experimental group (pTRV1+pTRV2-AcPDS) exhibited significantly reduced gene expression compared to the control group CK (pTRV1+pTRV2), manifesting the anticipated photobleaching phenotype by the 21st day. RT-PCR analysis revealed that AcPDS gene expression in TRV-mediated photobleached A. catechu embryos decreased to 0.227 times that of the control group CK. This established VIGS system in A. catechu embryoids establishes an effective experimental platform for studying functional genes in A. catechu.

Discovery of Novel (5-Mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)methyl Phenyl Carbamate as a Potent Phytoene Desaturase Inhibitor through Scaffold Hopping and Linker Modification.

Phytoene desaturase (PDS) is a key rate-limiting enzyme in the carotenoid biosynthesis pathway. Although commercial PDS inhibitors have been developed for decades, it remains necessary to develop novel PDS inhibitors with higher bioactivity. In this work, we used the scaffold hopping and linker modification approaches to design and synthesize a series of compounds (7a-7o, 8a-8l, and 14a-14d). The postemergence application assay demonstrated that 8e and 7e separately showed the best herbicidal activity at 750 g a.i./ha and lower doses (187.5 g, 375g a.i./ha) without no significant toxicity to maize and wheat. The surface plasmon resonance revealed strong binding affinity between 7e and Synechococcus PDS (SynPDS). The HPLC analysis confirmed that 8e at 750 g a.i./ha caused significant phytoene accumulation in Arabidopsis seedlings. This work demonstrates the efficacy of structure-guided optimization through scaffold hopping and linker modification to design potent PDS inhibitors with enhanced bioactivity and crop safety.

Exogenous application of the apocarotenoid retinaldehyde negatively regulates auxin-mediated root growth.

Root development is essential for plant survival. The lack of carotenoid biosynthesis in the phytoene desaturase 3 (pds3) mutant results in short primary roots (PRs) and reduced lateral root formation. In this study, we showed that short-term inhibition of PDS by fluridone suppresses PR growth in wild type, but to a lesser extent in auxin mutants of Arabidopsis (Arabidopsis thaliana). Such an inhibition of PDS activity increased endogenous indole-3-acetic acid levels, promoted auxin signaling, and partially complemented the PR growth of an auxin-deficient mutant, the YUCCA 3 5 7 8 9 quadruple mutant (yucQ). The exogenous application of retinaldehyde (retinal), an apocarotenoid derived from β-carotene, complemented the fluridone-induced suppression of root growth, as well as the short roots of the pds3 mutant. Retinal also partially complemented the auxin-induced suppression of root growth. These results suggest that retinal may play a role in regulating root growth by modulating endogenous auxin levels.

An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific RNA silencing and is graft transmissible to commercial citrus varieties.

A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Carotenoid biosynthesis profiling unveils the variance of flower coloration in Tagetes erecta and enhances fruit pigmentation in tomato

Carotenoids play a pivotal role in plant. Tagetes erecta, commonly called marigold, has increasing nutritional and economic value due to its high level of carotenoids in flower. However, the functional genes in the carotenoid biosynthesis of T. erecta have not been studied. In this work, three T. erecta varieties with flowers of yellow, yellow-orange and orange color, respectively, were examined for carotenoids composition and corresponding expression profiling of biosynthetic genes at four developmental stages. The results indicated that the varieties with higher lutein content, orange-flower ‘Juwang’ and yellow-orange ‘Taishan’, exhibited significant upregulation of genes in the upstream biosynthesis pathway, especially PDS (phytoene desaturase), PSY (phytoene synthase) and ZDS (zeta-carotene desaturase), whereas downstream carotenoid cleavage genes CCD (carotenoid cleavage dioxygenase) were markedly downregulated throughout flower development in the highest lutein containing variety ‘Juwang’. Furthermore, marigold TePDS, TePSYS3 and TeZDS were isolated and transformed into tomato. Overexpression of TePDS or TeZDS resulted in the promotion of fruit ripening and accumulation of carotenoids in the transgenic lines. On the other hand, marigold TePSYS3 showed multiple effects, not only on fruit carotenogenesis but also on pigmentation patterns in vegetative tissues and plant growth. Taken together, the variations in expression profiles of the biosynthetic genes contribute to dynamic change in carotenoid levels and diversity of flower coloration in T. erecta. These functional genes of T. erecta were verified in tomato and provide targets for genetic improvement of fruit carotenoids accumulation.

Generation and characterization of a depigmented variant of Chlorella pyrenoidosa for application in food products

Microalgae are promising natural resources for an array of essential nutrients and health-promoting compounds. The past decade has witnessed their increasing popularity as a value-added ingredient in the food industry. However, some of their intrinsic properties, especially their typical green color and off-flavor, may compromise consumer acceptability and remain a key challenge for food scientists. This study aimed to generate and characterize a chemically induced pigment-deficient mutant (C49) of Chlorella pyrenoidosa. In contrast to the wild type (WT), C49 could not grow photoautotrophically or mixotrophically. However, when grown heterotrophically, it exhibited a growth curve similar to that of WT. Pigment analysis verified the presence of only trace amounts of chlorophylls (0.27 ± 0.02 mg/g) and carotenoids (0.036 ± 0.003 mg/g) in the C49 which were only ∼1.7% and ∼1% of those in the WT, respectively. C49 also had higher carbohydrate (62.75% ± 2.42%) and lower protein (26.54% ± 1.00%) content than the WT (37.41% ± 2.25% and 36.21% ± 1.97%, respectively). Genetic assays revealed inactivation of the phytoene desaturase gene as a key mechanism underlying the reduced xanthophyll-synthesizing capability of C49, thus causing its failure to accumulate chlorophyll and develop chloroplasts. Transcriptomic and RT-PCR analyses indicated that the high carbohydrate content was likely due to reduced starch degradation. Furthermore, C49 demonstrated an emulsifying capacity comparable to that of WT over a wide pH range (3–8) in oil-in-water emulsions consisting of 5% cell-disrupted homogenate in deionized water and rapeseed oil (1:1, v/v). The results of this study support that depigmented Chlorella pyrenoidosa possesses promising potential for application in the food industry.

Light Intensity Enhances the Lutein Production in Chromochloris zofingiensis Mutant LUT-4.

Chromochloris zofingiensis, a unicellular green alga, is a potential source of natural carotenoids. In this study, the mutant LUT-4 was acquired from the chemical mutagenesis pool of C. zofingiensis strain. The biomass yield and lutein content of LUT-4 reached 9.23 g·L-1, and 0.209% of dry weight (DW) on Day 3, which was 49.4%, and 33% higher than that of wild-type (WT), respectively. The biomass yields of LUT-4 under 100, 300, and 500 µmol/m2/s reached 8.4 g·L-1, 7.75 g·L-1, and 6.6 g·L-1, which was 10.4%, 21%, and 29.6% lower compared with the control, respectively. Under mixotrophic conditions, the lutein yields were significantly higher than that obtained in the control. The light intensity of 300 µmol/m2/s was optimal for lutein biosynthesis and the content of lutein reached 0.294% of DW on Day 3, which was 40.7% more than that of the control. When LUT-4 was grown under 300 µmol/m2/s, a significant increase in expression of genes implicated in lutein biosynthesis, including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene epsilon cyclase (LCYe) was observed. The changes in biochemical composition, Ace-CoA, pyruvate, isopentenyl pyrophosphate (IPP), and geranylgeranyl diphosphate (GGPP) contents during lutein biosynthesis were caused by utilization of organic carbon. It was thereby concluded that 300 µmol/m2/s was the optimal culture light intensity for the mutant LUT-4 to synthesize lutein. The results would be helpful for the large-scale production of lutein.

Identification of genus Deinococcus strains by PCR detection using the gyrB gene and its extension to Bacteria domain

In radiation-resistant bacteria belonging to the genus Deinococcus, transposition events of insertion sequences (IS elements) leading to phenotypic changes from a reddish color to white were detected following exposure to gamma irradiation and hydrogen peroxide treatment. This change resulted from the integration of IS elements into the phytoene desaturase gene, a key enzyme in the carotenoid biosynthesis pathway. To facilitate species identification and distinguish among Deinococcus strains, the gyrB gene encoding the B subunit of DNA gyrase was utilized. The s gnificance of the gyrB gene is well recognized not only in genome replication through the regulation of supercoiling but also in phylogenetic analysis providing support for 16S rRNA-based identification. Its mutation rate surpasses that of the 16S rRNA gene, offering greater resolution between closely related species, particularly those exhibiting >99% similarity. In this study, phylogenetic analysis was conducted comparing the 16S rRNA and gyrB gene sequences of Deinococcus species. Species-specific and genus-specific primers targeting Deinococcus species were designed and experimentally validated for selective amplification and rapid identification of the targeted species. This approach allows for the omission of 16S rRNA sequencing in the targeted Deinococcus species. Therefore, the gyrB gene is useful for identifying bacterial species and genus-level detection from individual microbes or microbial consortia using specialized primer sets for PCR amplification.

CRISPR/Cas9 based genome editing of Phytoene desaturase (PDS) gene in chilli pepper (Capsicum annuum L.)

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon–intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

Discovery of Novel Pyridazine Herbicides Targeting Phytoene Desaturase with Scaffold Hopping.

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 μg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

A highly efficient Agrobacterium-mediated infectious system for Sweet potato leaf curl virus and a deltasatellite-based VIGS vector

Plant virus disease is one of the major threats to the yield and quality of sweetpotato (Ipomoea batatas [L.] Lam.), the seventh most important food crop in the world in terms of production. Sweepoviruses are a group of monopartite geminiviruses (genus Begomovirus, family Geminiviridae) that infect Ipomoea plants and cause significant damage. However, investigations on the mechanisms of sweepovirus infection are scarce due to the low efficiency of the current infectious system. In this work, we used the genomic sequence of Sweet potato leaf curl virus (SPLCV) to construct an effective infectious clone, SPLCV-1.01. We optimized the inoculation procedure in Nicotiana benthamiana by improving the Agrobacterium virulence using an induction medium. Finally, we established a highly efficient agroinoculation protocol for SPLCV on sweetpotato. Furthermore, a simple and reliable “Agro-soaking” method was developed for SPLCV on sweetpotato and Ipomoea aquatica. The “Agro-soaking” method also worked well with Tomato yellow leaf curl China virus (TYLCCNV) and Beet severe curly top virus (BSCTV) on tomato. Based on the “Agro-soaking” method and a deltasatellite, the SBG51 VIGS vector worked with the SPLCV-1.01 infectious clone and silenced two endogenous genes IbCHL (magnesium-chelatase subunit) and IbPDS (phytoene desaturase) in two weeks. Moreover, the VIGS system identified IbADK1 (adenosine kinase 1) as a positive regulator to dampen the SPLCV accumulation in sweetpotato. Our work provides a practical method for studying the interaction between sweetpotato and SPLCV and the functional studies of genes in sweetpotato.

A novel and efficient Apple latent spherical virus-based gene silencing method for functional genomic studies in Chenopodium quinoa

Quinoa (Chenopodium quinoa Willd.), with its resilience in harsh environments and excellent nutritional value, has become crucial for global food security. Despite recent progress in genomic research, the inability to perform functional studies in quinoa due to the absence of transformation techniques remains a significant obstacle. In this work, we present the development of a novel Apple Latent Spherical Virus (ALSV)-mediated virus-induced gene silencing (VIGS) protocol that will allow to perform functional genomics studies in quinoa in a fast and simple way. The method was fine-tuned using ALSV plasmids that carry partial gene sequences of phytoene desaturase (PDS) from Nicotiana benthamiana and quinoa. The developed technique involves an initial inoculation in Nicotiana plants through agroinfiltration with Agrobacterium tumefaciens cultures carrying the different viral constructs. Viral extracts were prepared using local or systemic leaves, which were then used as inoculum to infect quinoa leaves through mechanical damage. The method was successfully tested in two contrasting quinoa varieties, although some differences were observed in infection phenotype and viral susceptibility. The effect of insertion sequence size in the viral vectors was also analyzed, resulting in differences in bleaching or chlorosis phenotype and impact on plant growth. The presence of the virus in inoculated plants was confirmed, and the reduction in PDS gene expression in silenced plants was verified. Because quinoa lacks stable transformation protocols, limiting heterologous expression assays, our ALSV-based VIGS protocol is very attractive for loss-of-function gene studies.

Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet.

The ancient crop broomcorn millet (Panicum miliaceum L.) is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance. These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger, particularly in light of the uncertainty of global climate change. However, functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques. Here, we successfully performed genome editing of broomcorn millet. We identified an elite variety, Hongmi, that produces embryogenic callus and has high shoot regeneration ability in in vitro culture. We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome-editing system for Hongmi. Using these techniques, we produced herbicide-resistant transgenic plants and edited phytoene desaturase (PmPDS), which is involved in chlorophyll biosynthesis. To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research, we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome. Together, our results open the door to improving broomcorn millet using biotechnology.

Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotypeused to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.

Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene.

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both 'Carrizo' citrange and 'Duncan' grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.