Characterization of the Gene Encoding S-adenosyl-L-methionine (AdoMet) Synthetase in Penicillium chrysogenum; Role in Secondary Metabolism and Penicillin Production

The filamentous fungus Penicillium chrysogenum (recently reidentified as Penicillium rubens) is used in the industrial production of the b-lactam antibiotic penicillin. There are several mechanisms regulating the production of this antibiotic, acting both at the genetic and epigenetic levels, the latter including the modification of chromatin by methyltransferases. S-adenosyl-L-methionine (AdoMet) is the main donor of methyl groups for methyltransferases. In addition, it also acts as a donor of aminopropyl groups during the biosynthesis of polyamines. AdoMet is synthesized from L-methionine and ATP by AdoMet-synthetase. In silico analysis of the P. chrysogenum genome revealed the presence of a single gene (Pc16g04380) encoding a putative protein with high similarity to well-known AdoMet-synthetases. Due to the essential nature of this gene, functional analysis was carried out using RNAi-mediated silencing techniques. Knock-down transformants exhibited a decrease in AdoMet, S-adenosyl-L-homocysteine (AdoHcy), spermidine and benzylpenicillin levels, whereas they accumulated a yellow-orange pigment in submerged cultures. On the other hand, overexpression led to reduced levels of benzylpenicillin, thereby suggesting that the AdoMet synthetase, in addition to participate in primary metabolism, also controls secondary metabolism in P. chrysogenum.

Unmarked gene editing in Clavibacter michiganensis using CRISPR/Cas9 and 5-fluorocytosine counterselection

Plant pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis (Cm), which couples the expression of cas9 and sgRNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the Cm rpsL gene to generate a streptomycin resistant mutant, and complete gene knockout, in which the deletion of the Cm celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 100 μg/ml 5-fluorocytosine (5-FC), in which an unmarked katA mutant lacking the transformation vector was recovered. Compared to conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient as it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into Cm and other Clavibacter species.

Uridine Treatment of the First Known Case of SLC25A36 Deficiency

SLC25A36 is a pyrimidine nucleotide carrier playing an important role in maintaining mitochondrial biogenesis. Deficiencies in SLC25A36 in mouse embryonic stem cells have been associated with mtDNA depletion as well as mitochondrial dysfunction. In human beings, diseases triggered by SLC25A36 mutations have not been described yet. We report the first known case of SLC25A36 deficiency in a 12-year-old patient with hypothyroidism, hyperinsulinism, hyperammonemia, chronical obstipation, short stature, along with language and general developmental delay. Whole exome analysis identified the homozygous mutation c.803dupT, p.Ser269llefs*35 in the SLC25A36 gene. Functional analysis of mutant SLC25A36 protein in proteoliposomes showed a virtually abolished transport activity. Immunoblotting results suggest that the mutant SLC25A36 protein in the patient undergoes fast degradation. Supplementation with oral uridine led to an improvement of thyroid function and obstipation, increase of growth and developmental progress. Our findings suggest an important role of SLC25A36 in hormonal regulations and oral uridine as a safe and effective treatment.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

Background Agrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function. Results In this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean. Conclusion A rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

Plant regeneration via somatic embryogenesis in diploid cultivated cotton (Gossypium arboreum L.)

The cultivated diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function compared to the allotetraploid cotton species through genetic transformation, the reliable and efficient method for high frequency somatic embryogenesis and plant regeneration in G. arboreum is urgent need to be established. Callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB (MS salts and B5 vitamins) medium with 0.09 µM 2,4-D and 2.32 µM KT. The embryogenic callus was induced on MS5 medium from the suspended cultures of several cycles of alternate liquid-solid culture, which was critical step for somatic embryogenesis. The liquid medium of MS4 was supplemented with 0.1g/L NaCl, 0.1g/L KCl and 0.1g/L CuSO4. The solid medium of MS5 for embryogenic callus effective induction was supplemented with 37.59 mM KNO3 + 62.47 µM NH4NO3 and 2.46 µM IBA + 0.93 µM KT or 0.045 µM 2,4-D + 2.46 µM IBA + 0.465 µM KT. During callus growing on different media, callus was effectively selected for subculture or treatment according to cell morphology to induce embryogenic callus and somatic embryos. Somatic embryo maturation and germination were better on MS5 medium with maltose or glucose + maltose than the single glucose. The regenerated plantlets with well-developed roots were directly transferred to soil or grafted onto the germinated cotton plantlets. The feasible process of plant regeneration via somatic embryogenesis in diploid cultivated species was established and needed to be improved and optimized for the gene functional analysis and gene editing in the diploid cotton species.

Toward comprehensive functional analysis of gene lists weighted by gene essentiality scores

ABSTRACTGene functional enrichment analysis represents one of the most popular bioinformatics methods for annotating the pathways and function categories of a given gene list. Current algorithms for enrichment computation such as Fisher’s exact test and hypergeometric test totally depend on the category count numbers of the gene list and one gene set. In this case, whatever the genes are, they were treated equally. However, actually genes show different scores in their essentiality in a gene list and in a gene set. It is thus hypothesized that the essentiality scores could be important and should be considered in gene functional analysis. For this purpose, here we proposed WEAT (https://www.cuilab.cn/weat/), a weighted gene set enrichment algorithm and online tool by weighting genes using essentiality scores. We confirmed the usefulness of WEAT using two case studies, the functional analysis of one aging-related gene list and one gene list involved in Lung Squamous Cell Carcinoma (LUSC). Finally, we believe that the WEAT method and tool could provide more possibilities for further exploring the functions of given gene lists.

Comprehensive analysis of AHL gene family and their expression under drought stress and ABA treatment in Populus trichocarpa

The AT-hook motif nuclear-localized (AHL) family is a plant transcription factor family, which plays an important role in growth and development and stress responses. We identified and analyzed 37 AHL genes in poplar (Populus trichocarpa). Phylogenetic analysis classified the PtrAHL members into three subfamilies based on their conserved domain. All PtrAHL paralogous pairs evolved under purifying selection. The promoter analysis revealed the presence of stress-related and phytohormone-related cis-elements of the PtrAHL genes. Our analysis of the tissue-specific expression pattern of PtrAHL genes indicated their significance in tissue and organ development. Network-based prediction suggested that PtrAHL genes may interact with histone deacetylases (HDAC) and participate in the development of organs, such as roots. Drought negatively impacts plant growth and development. ABA is produced under osmotic stress condition, and it takes an important part in the stress response and tolerance of plants. Real-time quantitative PCR (qRT-PCR) showed that PtrAHL genes were induced by drought stress and ABA treatment. These insights into the expression of PtrAHL genes under stress provide a basis for PtrAHL gene functional analysis. Our study will help develop new breeding strategies to improve drought tolerance in poplar.

A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility.

A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor C19ORF57/MIDAP

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with 3 cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse, compared to a new HSF2BP knock-out mouse showed that it behaves as a hypomorphic allele. HSF2BP-S167L females show reduced fertility with small litter sizes. To obtain mechanistic insights, we identified C19ORF57/MIDAP as a strong interactor and stabilizer of HSF2BP by forming a higher-order macromolecular structure involving BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L mutation showed a strongly decreased expression of both MIDAP and HSF2BP at the recombination nodules. Although HSF2BP-S167L does not affect heterodimerization between HSF2BP and MIDAP, it promotes a lower expression of both proteins and a less proficient activity in replacing RPA by the recombinases RAD51/DMC1, thus leading to a lower frequency of cross-overs. Our results provide insights into the molecular mechanism of two novel actors of meiosis underlying non-syndromic ovarian insufficiency.SummaryFelipe-Medina et al. describe a missense variant in the meiotic gene HSF2BP in a consanguineous family with Premature Ovarian Insufficiency, and characterize it as an hypormorphic allele, that in vivo impairs its dimerization with a novel meiotic actor, MIDAP/ C19ORF57, and affect recombination at double-strand DNA breaks.