Advances in the identification of novel factors required in soybean nodulation, a process critical to sustainable agriculture and food security

Nodulation is a process of organogenesis that results from a symbiotic relationship between legume plants and soil-dwelling, nitrogen-fixing bacteria, called rhizobia. The rhizobia are housed in newly formed structures on the host roots, called nodules. Within nodules, the rhizobia fix atmospheric N2 into useable forms of nitrogen for the plant. This process is highly important to agriculture, as nitrogen is critical for plant growth and development and is typically the main component of fertilizers. Although fertilizers are effective, they are expensive and often pollute, making biological alternatives, such as legume nodulation, attractive for use in agriculture. Nodulation is regulated by the auto regulation of nodulation (AON) pathway, which enables the host plant to balance its needs between nitrogen acquisition and energy expenditure. Current research is elucidating the nodule development and AON signalling networks. Recent technological advances, such as RNA-sequencing, are revolutionizing the discovery of genes that are critical to nodulation. The discovery of such genes not only enhances our knowledge of the nodulation signalling network, but may help to underpin future work to isolate superior legume crops via modern breeding and engineering practices. Here, recent advances using the cutting-edge technique of RNA sequencing to identify new nodulation genes in soybean are discussed

Molecular analysis of lipoxygenases associated with nodule development in soybean

We utilized transcriptional profiling to identify genes associated with nodule development in soybean. Many of the candidate genes were predicted to be involved in processes such as defense, metabolism, transcriptional regulation, oxidation, or iron storage. Here, we describe the detailed characterization of one specific class of genes that encode the enzyme lipoxygenase (LOX). The LOX9 and LOX10 genes identified by microarray analysis represent novel soybean LOXs expressed in developing nodules. LOX expression during nodulation was relatively complex, with at least eight different LOX genes expressed in soybean nodules. Histochemical analyses utilizing LOX9 promoter::beta-glucuronidase (GUS) fusion constructs in transgenic soybean hairy roots suggest that this gene is involved in the growth and development of specific cells within the root and nodules. In soybean roots, LOX9 was expressed specifically in the developing phloem. In nodules, the expression of LOX9 was correlated with the development of cells in the vasculature and lenticels. The use of RNAi in transgenic hairy roots reduced LOX expression by approximately 95%. Despite this significant reduction in LOX expression, there was no detectable effect on the development of roots or nodules. Our findings are discussed with respect to the potential function of LOXs in nodulation

Exploring the source of TYLCV resistance in Nicotiana benthamiana

Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most devastating pathogens of tomato, worldwide. It is vectored by the globally prevalent whitefly, Bemisia tabaci, and is asymptomatic in a wide range of plant species that act as a virus reservoir. The most successful crop protection for tomato in the field has been from resistance genes, of which five loci have been introgressed fromwild relatives. Of these, the Ty-1/Ty-3 locus, which encodes an RNA-dependent RNA polymerase 3 (RDR3), has been the most effective. Nevertheless, several TYLCV strains that break this resistance are beginning to emerge, increasing the need for new sources of resistance. Here we use segregation analysis and CRISPR-mediated gene dysfunctionalisation to dissect the differential response of two isolates of Nicotiana benthamiana to TYLCV infection. Our study indicates the presence of a novel non-RDR3, but yet to be identified, TYLCV resistance gene in a wild accession of N. benthamiana. This gene has the potential to be incorporated into tomatoes

Are the current gRNA ranking prediction algorithms useful for genome editing in plants?

Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules

Dynamic movement of the Golgi unit and its glycosylation enzyme zones

Harada A., Kunii M., Kurokawa K., et al. Dynamic movement of the Golgi unit and its glycosylation enzyme zones. Nature Communications 15, 4514 (2024); https://doi.org/10.1038/S41467-024-48901-1.Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small ‘Golgi units’ that have 1-3 μm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call ‘zones’. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis