Intragenic complementation at the Lotus japonicus CELLULOSE SYNTHASE-LIKE D1 locus rescues root hair defects.

Root hair cells form the primary interface of plants with the soil environment, playing key roles in nutrient uptake and plant defense. In legumes, they are typically the first cells to become infected by nitrogen-fixing soil bacteria during root nodule symbiosis. Here, we report a role for the CELLULOSE SYNTHASE-LIKE D1 (CSLD1) gene in root hair development in the legume species Lotus japonicus. CSLD1 belongs to the cellulose synthase protein family that includes cellulose synthases and cellulose synthase-like proteins, the latter thought to be involved in the biosynthesis of hemicellulose. We describe 11 Ljcsld1 mutant alleles that impose either short (Ljcsld1-1) or variable (Ljcsld1-2 to 11) root hair length phenotypes. Examination of Ljcsld1-1 and one variable-length root hair mutant, Ljcsld1-6, revealed increased root hair cell wall thickness, which in Ljcsld1-1 was significantly more pronounced and also associated with a strong defect in root nodule symbiosis. Lotus japonicus plants heterozygous for Ljcsld1-1 exhibited intermediate root hair lengths, suggesting incomplete dominance. Intragenic complementation was observed between alleles with mutations in different CSLD1 domains, suggesting CSLD1 function is modular and that the protein may operate as a homodimer or multimer during root hair development.

Lotus japonicus Nuclear Factor YA1, a nodule emergence stage-specific regulator of auxin signalling.

Organogenesis of legume root nodules begins with the nodulation factor-dependent stimulation of compatible root cells to initiate divisions, signifying an early nodule primordium formation event. This is followed by cellular differentiation, including cell expansion and vascular bundle formation, and we previously showed that Lotus japonicus NF-YA1 is essential for this process, presumably by regulating three members of the SHORT INTERNODES/STYLISH (STY) transcription factor gene family. In this study, we used combined genetics, genomics and cell biology approaches to characterize the role of STY genes during root nodule formation and to test a hypothesis that they mediate nodule development by stimulating auxin signalling. We show here that L. japonicus STYs are required for nodule emergence. This is attributed to the NF-YA1-dependent regulatory cascade, comprising STY genes and their downstream targets, YUCCA1 and YUCCA11, involved in a local auxin biosynthesis at the post-initial cell division stage. An analogous NF-YA1/STY regulatory module seems to operate in Medicago truncatula in association with the indeterminate nodule patterning. Our data define L. japonicus and M. truncatula NF-YA1 genes as important nodule emergence stage-specific regulators of auxin signalling while indicating that the inductive stage and subsequent formation of early nodule primordia are mediated through an independent mechanism(s).

Lotus japonicus NF-YA1 Plays an Essential Role During Nodule Differentiation and Targets Members of the SHI/STY Gene Family.

Legume plants engage in intimate relationships with rhizobial bacteria to form nitrogen-fixing nodules, root-derived organs that accommodate the microsymbiont. Members of the Nuclear Factor Y (NF-Y) gene family, which have undergone significant expansion and functional diversification during plant evolution, are essential for this symbiotic liaison. Acting in a partially redundant manner, NF-Y proteins were shown, previously, to regulate bacterial infection, including selection of a superior rhizobial strain, and to mediate nodule structure formation. However, the exact mechanism by which these transcriptional factors exert their symbiotic functions has remained elusive. By carrying out detailed functional analyses of Lotus japonicus mutants, we demonstrate that LjNF-YA1 becomes indispensable downstream from the initial cortical cell divisions but prior to nodule differentiation, including cell enlargement and vascular bundle formation. Three affiliates of the SHORT INTERNODES/STYLISH transcription factor gene family, called STY1, STY2, and STY3, are demonstrated to be among likely direct targets of LjNF-YA1, and our results point to their involvement in nodule formation.

De Novo Synthesis of a Conjugative System from Human Gut Metagenomic Data for Targeted Delivery of Cas9 Antimicrobials.

Metagenomic sequences represent an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequences requires the identification of suitable candidate systems as starting scaffolds for de novo DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic "trash bin" for genes associated with conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut data set representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugation genes with a differential distribution in human cohorts. We synthesized de novo an entire Citrobacter spp. conjugative system of 54 kb containing at least 47 genes and assembled it into a plasmid, pCitro. We found that pCitro conjugates from Escherichia coli to Citrobacter rodentium with a 30-fold higher frequency than to E. coli, and is compatible with Citrobacter resident plasmids. Mutations in the traV and traY conjugation components of pCitro inhibited conjugation. We showed that pCitro can be repurposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease and Citrobacter-specific sgRNAs to kill C. rodentium. Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.

Phosphate-regulated expression of the SARS-CoV-2 receptor-binding domain in the diatom Phaeodactylum tricornutum for pandemic diagnostics.

The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the eukaryotic microalgae Phaeodactylum tricornutum to develop an inducible overexpression system for SARS-CoV-2 proteins. Limiting phosphate and iron in growth media induced expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the P. tricornutum HASP1 promoter in the wild-type strain and in a histidine auxotrophic strain that alleviates the requirement for antibiotic selection of expression plasmids. The RBD was purified from whole cell extracts (algae-RBD) with yield compromised by the finding that 90-95% of expressed RBD lacked the genetically encoded C-terminal 6X-histidine tag. Constructs that lacked the TEV protease site between the RBD and C-terminal 6X-histidine tag retained the tag, increasing yield. Purified algae-RBD was found to be N-linked glycosylated by treatment with endoglycosidases, was cross-reactive with anti-RBD polyclonal antibodies, and inhibited binding of recombinant RBD purified from mammalian cell lines to the human ACE2 receptor. We also show that the algae-RBD can be used in a lateral flow assay device to detect SARS-CoV-2 specific IgG antibodies from donor serum at sensitivity equivalent to assays performed with RBD made in mammalian cell lines. Our study shows that P. tricornutum is a scalable system with minimal biocontainment requirements for the inducible production of SARS-CoV-2 or other coronavirus antigens for pandemic diagnostics.

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi.

Fungi are nature's recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.

Cloning of Thalassiosira pseudonana's Mitochondrial Genome in Saccharomyces cerevisiae and Escherichia coli.

Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana's mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.

Soil transmitted helminthiasis in Kathmandu, Nepal.

A total of 200 soil samples taken from different sites and 1,504 stool samples collected from school children (n = 188) and patients (n = 1,316) visiting the health care centres in Kathmandu Valley were included in this study. Soil samples were investigated for the presence of parasitic eggs using sucrose flotation technique. Stool samples were examined by formal-ether concentration and direct smear techniques. The contamination rate of soil with parasitic eggs and larvae was found to be 28.5% (57/ 200). The overall parasitic infection rates in school children and patients were 42.5% and 2.8%, respectively. Four types of parasites were detected from soil samples, of which Ascaris was the most common. Trichuris was most common among school children whereas Ascaris in patients. Females in both study groups had higher prevalence compared with male counterparts with significantly low rate in health care centre visiting patients (p < 0.05).

Intestinal parasitosis among the elderly people in Kathmandu Valley.

Present study was carried out among the elderly people (60+ years of age) from August 2005 to July 2006 in Kathmandu Valley to assess the prevalence of intestinal parasitosis in them. Stool samples were collected from 235 elderly people (122 from government elderly home, 66 from private elderly home and 47 from the households in a rural community). The samples were examined by formal ether sedimentation and Sheather's sucrose floatation followed by Kinyoun's modified Ziehl-Neelsen staining. The overall prevalence of intestinal parasites was found to be 41.7%, out of which 30.6% had multiple parasitism. The government elderly home had significantly higher parasitic prevalence (50.8%) followed by the rural community (46.8%) and the private elderly homes (21.2%) (P<0.05). Males (43.8%) had slightly infection rate than females (40.4%) (P>0.05). There was equal infection rate with protozoa (25.8%) and helminths (27.0%). Trichuris trichiura (39.4%) and Entamoeba histolytica (19.7%) were the commonest helminth and protozoa, respectively.