Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

CRISPR/Cas9‐mediated knockout of an oil palm defense‐related gene to the pathogenic fungus Ganoderma boninense

Oil palm plantation in Indonesia is significantly affected by basal stem rot disease caused by the pathogenic fungus Ganoderma boninense. Tolerant oil palm cultivars toward G. boninense have been developed through a breeding program accelerated by the implementation of the CRISPR/Cas9 technology. This study was conducted to perform a gene knockout (KO) of oil palm that confers a putative defense‐related trait toward G. boninense. A plasmid pCRISPR_EMLP containing modules, i.e., 35S‐CaMV‐promoter‐driven CRISPR/Cas9, U6‐promoter‐driven sgRNA to the target EgEMLP gene (EL695076), and hygromycin resistance gene as the selectable marker, was established for Agrobacterium‐mediated delivery into oil palm calli (OPC). The transformed OPCs were regenerated and screened in DF (de Fossard) media containing hygromycin. The working concentration of hygromycin was successfully optimized for selection at 20 ppm. Through PCR‐based selection using HYG primers, we succeeded in discerning positive transformed OPC clones. The sequenced PCR products of genomic DNA as the template amplified using EMLP1 primers showed a point mutation, causing a frameshift in the edited EgEMLP and premature stop codon. Furthermore, in silico modeling demonstrated that the mutation resulted in a change in the C‐terminal region, affecting the tertiary protein structure. Moreover, electrophoresis analysis of PCR products of cDNA as the template from transformed OPC clones showed several samples with faint or undetected bands. This indicated that the CRISPR/Cas9 module induced a mutation that could destabilize the transcribed mRNA, e.g., premature degradation. Altogether, this study has successfully implemented CRISPR/Cas9 gene editing in oil palm in a model gene that is responsible for putative defense‐related traits toward the pathogenic fungus G. boninense.

Targeted Editing of the StPDS Gene using the CRISPR/Cas9 system in Tetraploid Potato

The CRISPR/Cas9 system has been applied in a variety of plants for targeted gene editing due to its accuracy, high efficiency, and low price. It has been reported that the CRISPR/Cas9 system can lead to mutation in potato, however, the mutation patterns and genotypes of CRISPR/Cas9-induced mutants in tetraploid potato plants have not yet been disclosed in detail. In the present study, the Solanum tuberosum phytoene desaturase (StPDS) gene was selected for inducing targeted mutagenesis. The obtained StPDS knockout mutants exhibited a mutation frequency of 46.67% and clear albino phenotypes. All transgenic plants were tested for the hygromycin resistance gene and a high positive transformation frequency (96.77%) was observed in all tested resistant plants. Genotype analysis of the mutants revealed that heterozygotes accounted for 35.51%, chimeras accounted for 64.29%, and no homozygotes and biallelic mutations were detected. The patterns of the detected mutations consisted mainly of 1 to 2-nucleotide insertions and deletions, followed by 2-bp replacements. In short, the results of this study prove that the CRISPR/Cas9 system is an effective approach for cultivated potato gene engineering.

Agrobacterium-mediated transformation of the wild orchid Cattleya maxima Lindl

Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate <em>in vitro</em> growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of <em>Cattleya maxima </em>were transformed<em> </em>using the <em>Agrobacterium tumefaciens </em>method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed <em>Cattleya maxima</em> PLBs were more efficient in forming somatic embryos (60-80%) than untransformed controls (45-57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK<em> </em>plays an important role in Orchid embryogenesis.

The Sclerotinia sclerotiorum FoxE2 Gene Is Required for Apothecial Development

Sclerotinia sclerotiorum is a widely dispersed plant pathogenic fungus causing many diseases such as white mold, Sclerotinia stem rot, stalk rot, and Sclerotinia head rot on many varieties of broadleaf crops worldwide. Previous studies have shown that the Forkhead-box transcription factors (FOX TFs) play key regulatory roles in the sexual reproduction of some fungi. Ss-FoxE2 is one of four FOX TF family member genes in S. sclerotiorum. Based on ortholog function in other fungi it is hypothesized to function in S. sclerotiorum sexual reproduction. In this study, the role of Ss-FoxE2 in S. sclerotiorum was identified with a gene knock-out strategy. Following transformation and screening, strains having undergone homologous recombination in which the hygromycin resistance gene replaced the gene Ss-FoxE2 from the genomic DNA were identified. No difference in hyphae growth, number, and weight of sclerotia and no obvious change in virulence was observed among the wild type Ss-FoxE2 knock-out mutant and genetically complemented mutant; however, following induction of sclerotia for sexual development, apothecia were not formed in Ss-FoxE2 knock-out mutant. The Ss-FoxE2 gene expressed significantly higher in the apothecial stages than in other developmental stages. These results indicate that Ss-FoxE2 appears to be necessary for the regulation of sexual reproduction, but may not affect the pathogenicity and vegetative development of S. sclerotiorum significantly.

The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin

The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.

Stable Transformation of Suspension-Cultured Glycyrrhiza inflata Batalin Cells with Agrobacterium tumefaciens

A protocol for the efficient genetic transformation of licorice (Glycyrrhiza inflata Batalin) cells in suspension culture using Agrobacterium tumefaciens-mediated T-DNA delivery is described. G. inflata cells in suspension culture were infected with A. tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1303, which contains the β-glucuronidase (GUS) reporter gene and a hygromycin resistance gene (hpt II), respectively, under the transcriptional control of the CaMV35S promoter. Optimal transformation effi ciency was achieved with an A. tumefaciens suspension having an OD600 of 0.4 and a period of 24 h of co-cultivation with 3-day-old cells in a medium supplemented with 200 μM acetosyringone. The transgenic cell lines have been maintained in suspension subculture for 5 months. PCR and Southern blot analyses confirmed the stable integration of transgenes into the G. inflata genome. The introduced genes had no discernable effect on cell growth or accumulation of total licorice fl avonoids in the transgenic cell lines. This study provides the basis for the development of transgenic G. inflata cells.

Transfer, stable maintenance and expression of the mycolactone polyketide megasynthase mls genes in a recombination-impaired Mycobacterium marinum

The human pathogen Mycobacterium ulcerans produces a polyketide metabolite called mycolactone with potent immunomodulatory activity. M. ulcerans strain Agy99 has a 174 kb plasmid called pMUM001 with three large genes (mlsA1, 51 kb; mlsA2, 7.2 kb; mlsB, 43 kb) that encode type I polyketide synthases (PKS) required for the biosynthesis of mycolactone, as demonstrated by transposon mutagenesis. However, there have been no reports of transfer of the mls locus to another mycobacterium to demonstrate that these genes are sufficient for mycolactone production because in addition to their large size, the mls genes contain a high level of internal sequence repetition, such that the entire 102 kb locus is composed of only 9.5 kb of unique DNA. The combination of their large size and lack of stability during laboratory passage makes them a challenging prospect for transfer to a more rapidly growing and genetically tractable host. Here we describe the construction of two bacterial artificial chromosome Escherichia coli/Mycobacterium shuttle vectors, one based on the pMUM001 origin of replication bearing mlsB, and the other based on the mycobacteriophage L5 integrase, bearing mlsA1 and mlsA2. The combination of these two constructs permitted the two-step transfer of the entire 174 kb pMUM001 plasmid to Mycobacterium marinum, a rapidly growing non-mycolactone-producing mycobacterium that is a close genetic relative of M. ulcerans. To improve the stability of the mls locus in M. marinum, recA was inactivated by insertion of a hygromycin-resistance gene using double-crossover allelic exchange. As expected, the ΔrecA mutant displayed increased susceptibility to UV killing and a decreased frequency of homologous recombination. Southern hybridization and RT-PCR confirmed the stable transfer and expression of the mls genes in both wild-type M. marinum and the recA mutant. However, neither mycolactone nor its predicted precursor metabolites were detected in either strain. These experiments show that it is possible to successfully manipulate and stably transfer the large mls genes, but that other bacterial host factors appear to be required to facilitate mycolactone production.