Screening and Functional Verification of Selectable Marker Genes for Cordyceps militaris

The selectable marker genes are necessary resistance genes for gene knockout, gene complementation, and gene overexpression in filamentous fungi. Moreover, the more sensitive the filamentous fungi are to antibiotics, the more helpful it is to screen the target transformants. In order to obtain the antibiotic (or herbicide) which can effectively inhibit the growth of Cordyceps militaris and verify the function of the corresponding resistance gene in C. militaris, the sensitivity of C. militaris to hygromycin and glufosinate ammonium was compared to determine the resistance gene that was more suitable for the screening of C. militaris transformants. The binary vector of the selectable marker gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the selectable marker gene in C. militaris was verified by the Agrobacterium tumefaciens-mediated transformation method. The results showed that C. militaris was more sensitive to glufosinate ammonium than hygromycin. The growth of C. militaris could be completely inhibited by 250 μg/mL glufosinate ammonium. The expression cassette of the glufosinate ammonium resistance gene (bar gene) was successfully constructed by DJ-PCR. The binary vector pCAMBIA0390-Bar was successfully constructed by homologous recombination. The bar gene of the vector pCAMBIA0390-Bar was successfully integrated into the C. militaris genome and could be highly expressed in the transformants of C. militaris. This study will promote the identification of C. militaris gene function and reveal the biosynthetic pathways of bioactive components in C. militaris.

Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 µg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

The identification of Tautoneura mori as the vector of mulberry crinkle leaf virus and the infectivity of infectious clones in mulberry

Mulberry crinkle leaf virus (MCLV) is a novel geminivirus identified from mulberry. The pathogenicity and the natural vector of transmission have remained unknown for MCLV. Here, the infectious clones which consisted of the complete tandem dimeric genome of MCLV in a binary vector were constructed and agro-inoculated into mulberry seedlings. The results showed that the infectious clones of MCLV were systemically infectious to mulberry, but the infected mulberry plants did not show any virus-like symptoms. The natural transmission vectors of MCLV were also identified from possible vector insects occurring on the MCLV-infected mulberry plants. The vector ability of Tautoneura mori Matsumura was identified through inoculation assay. Three of 21 (14.3%) seedlings inoculated with T. mori collected from MCLV-infected mulberry plants grown naturally were detected to be MCLV-positive 50 days post-inoculation. These MCLV-positive mulberry plants did also not show any virus-like symptoms. Collectively, it is suggested that MCLV is infectious to mulberry plants, but MCLV alone does not induce symptoms. The leafhopper T. mori was for the first time determined experimentally to be a transmission vector of MCLV.

Generation of Iron-Independent Siderophore-Producing Agaricus bisporus through the Constitutive Expression of hapX

Agaricus bisporus secretes siderophore to uptake environmental iron. Siderophore secretion in A. bisporus was enabled only in the iron-free minimal medium due to iron repression of hapX, a transcriptional activator of siderophore biosynthetic genes. Aiming to produce siderophore using conventional iron-containing complex media, we constructed a recombinant strain of A. bisporus that escapes hapX gene repression. For this, the A. bisporushapX gene was inserted next to the glyceraldehyde 3-phosphate dehydrogenase promoter (pGPD) in a binary vector, pBGgHg, for the constitutive expression of hapX. Transformants of A. bisporus were generated using the binary vector through Agrobacterium tumefaciens-mediated transformation. PCR and Northern blot analyses of the chromosomal DNA of the transformants confirmed the successful integration of pGPD-hapX at different locations with different copy numbers. The stable integration of pGPD-hapX was supported by PCR analysis of chromosomal DNA obtained from the 20 passages of the transformant. The transformants constitutively over-expressed hapX by 3- to 5-fold and sidD, a key gene in the siderophore biosynthetic pathway, by 1.5- to 4-fold in mRNA levels compared to the wild-type strain (without Fe3+), regardless of the presence of iron. Lastly, HPLC analysis of the culture supernatants grown in minimal medium with or without Fe3+ ions presented a peak corresponding to iron-chelating siderophore at a retention time of 5.12 min. The siderophore concentrations of the transformant T2 in the culture supernatant were 9.3-fold (−Fe3+) and 8-fold (+Fe3+) higher than that of the wild-type A. bisporus grown without Fe3+ ions, while no siderophore was detected in the wild-type supernatant grown with Fe3+. The results described here demonstrate the iron-independent production of siderophore by a recombinant strain of A. bisporus, suggesting a new application for mushrooms through molecular biological manipulation.

Different Transfer Functions for Binary Particle Swarm Optimization with a New Encoding Scheme for Discounted {0-1} Knapsack Problem

The discounted {0-1} knapsack problem (DKP01) is a kind of knapsack problem with group structure and discount relationships among items. It is more challenging than the classical 0-1 knapsack problem. In this paper, we study binary particle swarm optimization (PSO) algorithms with different transfer functions and a new encoding scheme for DKP01. An effective binary vector with shorter length is used to represent a solution for new binary PSO algorithms. Eight transfer functions are used to design binary PSO algorithms for DKP01. A new repair operator is developed to handle isolation solution while improving its quality. Finally, we conducted extensive experiments on four groups of 40 instances using our proposed approaches. The experience results show that the proposed algorithms outperform the previous algorithms named FirEGA and SecEGA . Overall, the proposed algorithms with a new encoding scheme represent a potential approach for solving the DKP01.

Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains—AGL1, EHA101, and GV3101—to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

Agrobacterium tumefaciens Mediated Transformation of Symbiodinium spp.

Symbiodinium spp conducts symbiosis mutualism within a wide phyletic range of marine invertebrate hosts including corals and anemones. The present study investigates the transformation of foreign genes into the free living cultured Symbiodinium spp by co-cultured Symbiodinium cells with A. tumefaciens. Ti-plasmidbased binary vector harboring the GUS and GFP genes were transformed into Symbiodinium cells by co-cultivation. GUS histochemical assay was monitored in Symbiodinium cells under light microscopy. Putative GFP in transformed Symbiodinium cells was detected by immunoassaying with antibodies against GFP protein. These results suggest that A. tumefaciens could provide efficient tools for gene transformation of Symbiodinium cells.