Neodiversification of homeologous CLAVATA1-like receptor kinase genes in soybean leads to distinct developmental outcomes.

The CLAVATA pathway that regulates stem cell numbers of the shoot apical meristem has exclusively been studied in Arabidopsis; as such insight into other species is warranted. In this study, a GmCLV1A mutant (F-S562L) with altered lateral organ development, and two mutants of GmNARK, isolated from a Forrest M2 population (EMS-mutated soybean) were studied. GmCLV1A and GmNARK encode for LRR receptor kinases, and share 92% of protein sequence. While GmNARK is critical for systemic regulation of nodulation (new organ made on the root through symbiosis), we show that GmCLV1A functions locally and has no apparent function in nodulation or root development. However, a recessive, loss-of-function mutation (S562L) in a putative S-glycosylation site of GmCLV1A causes stem nodal identity alterations as well as flower and pod abnormalities (deformed flower and pod). The mutant also exhibits a homeotic phenotype, displaying abnormal leaf development/number, vein-derived leaf emergence, and a thick, faciated stem. The mutant phenotype is also temperature-sensitive. Interestingly, a novel truncated version of GmCLV1A was identified upstream of GmCLV1A that is absent from GmNARK, but is present upstream of the GmNARK orthologues, MtSUNN and PvNARK. Taken together, our findings indicate that GmCLV1A acts on shoot architecture, whereas GmNARK, functions in controlling nodule numbers.

Characterization of a Novel Prevacuolar Compartment in Neurospora crassa.

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts.

A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens.

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US$1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.

TILLING to detect induced mutations in soybean.

BACKGROUND: Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU). RESULTS: We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template. CONCLUSION: The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.