Systematic Mutagenesis of Serine Hydroxymethyltransferase Reveals an Essential Role in Nematode Resistance.

Rhg4 is a major genetic locus that contributes to soybean cyst nematode (SCN) resistance in the Peking-type resistance of soybean (Glycine max), which also requires the rhg1 gene. By map-based cloning and functional genomic approaches, we previously showed that the Rhg4 gene encodes a predicted cytosolic serine hydroxymethyltransferase (GmSHMT08); however, the novel gain of function of GmSHMT08 in SCN resistance remains to be characterized. Using a forward genetic screen, we identified an allelic series of GmSHMT08 mutants that shed new light on the mechanistic aspects of GmSHMT08-mediated resistance. The new mutants provide compelling genetic evidence that Peking-type rhg1 resistance in cv Forrest is fully dependent on the GmSHMT08 gene and demonstrates that this resistance is mechanistically different from the PI 88788-type of resistance that only requires rhg1 We also demonstrated that rhg1-a from cv Forrest, although required, does not exert selection pressure on the nematode to shift from HG type 7, which further validates the bigenic nature of this resistance. Mapping of the identified mutations onto the SHMT structural model uncovered key residues for structural stability, ligand binding, enzyme activity, and protein interactions, suggesting that GmSHMT08 has additional functions aside from its main enzymatic role in SCN resistance. Lastly, we demonstrate the functionality of the GmSHMT08 SCN resistance gene in a transgenic soybean plant.

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.

Vittatidera zeaphila (Nematoda: Heteroderidae), a new genus and species of cyst nematode parasitic on corn (Zea mays).

A new genus and species of cyst nematode, Vittatidera zeaphila, is described from Tennessee. The new genus is superficially similar to Cactodera but is distinguished from other cyst-forming taxa in having a persistent lateral field in females and cysts, persistent vulval lips covering a circumfenestrate vulva, and subventral gland nuclei of the female contained in a separate small lobe. Infective juveniles (J2) are distinguished from all previously described Cactodera spp. by the short stylet in the second-stage juvenile (14-17 µm); J2 of Cactodera spp. have stylets at least 18 µm long. The new species also is unusual in that the females produce large egg masses. Known hosts are corn and goosegrass. DNA analysis suggests that Vittatidera forms a separate group apart from other cyst-forming genera within Heteroderinae.

Variability in Distribution and Virulence Phenotypes of Heterodera glycines in Missouri During 2005.

The soybean cyst nematode, Heterodera glycines, is a serious economic threat to soybean producers in Missouri. Periodic monitoring for the presence, population densities, and virulence phenotypes of H. glycines is essential for determining crop losses and devising management strategies implementing the use of resistant cultivars. A survey using area-frame sampling was conducted to determine the distribution and virulence phenotypes of H. glycines in Missouri during 2005. Two samples from each of 125 fields representing eight geographical regions of Missouri were collected; 243 samples were processed for extraction of cysts and eggs. In all, 49% of samples had detectable cyst nematode populations, which ranged from 138 to 85,250 eggs per 250 cm3 of soil. Race and H. glycines type tests were conducted on populations from 45 samples. Nearly 80% of the populations that were tested, irrespective of the region, were virulent on the indicator line plant introduction (PI) 88788, which is the source of resistance for most H. glycines-resistant cultivars. More than 70% of populations could reproduce on the indicator lines PI 88788, PI 209332, and PI 548316 (Cloud), indicating that soybean cultivars with resistance derived from these sources need to be carefully monitored and used only in rotation with nonhost crops and soybean cultivars with resistance from other sources. Approximately one-third of the populations, primarily in the southern regions of Missouri, could reproduce on PI 548402 (Peking), another common source of resistance. Fewer than 10% of the populations could reproduce on PI 90763, PI 437654, PI 89772, or PI 438489B, suggesting that these sources of resistance should be used in soybean breeding programs to develop H. glycines-resistant soybean cultivars.

Genetics and Adaptation of Soybean Cyst Nematode to Broad Spectrum Soybean Resistance.

The soybean cyst nematode (SCN) Heterodera glycines is a major threat to soybean production, made more challenging by the current limitations of natural resistance for managing this pathogen. The use of resistant host cultivars is effective, but, over time, results in the generation of virulent nematode populations able to robustly parasitize the resistant host. In order to understand how virulence develops in SCN, we utilized a single backcross BC1F2 strategy to mate a highly virulent inbred population (TN20), capable of reproducing on all current sources of resistance, with an avirulent one (PA3), unable to reproduce on any of the resistant soybean lines. The offspring were then investigated to determine how virulence is inherited on the main sources of SCN resistance, derived from soybean lines Peking, PI 88788, PI 90763, and the broad spectrum resistance source PI 437654. Significantly, our results suggest virulence on PI 437654 is a multigenic recessive trait that allows the nematode to reproduce on all current sources of resistance. In addition, we examined how virulence on different sources of resistance interact by placing virulent SCN populations under secondary selection, and identified a strong counter-selection between virulence on PI 88788- and PI 90763-derived resistances, while no such counter-selection existed between virulence on Peking and PI 88788 resistance sources. Our results suggest that the genes responsible for virulence on PI 88788 and PI 90763 may be different alleles at a common locus. If so, rotation of cultivars with resistance from these two sources may be an effective management protocol.

The soybean GmSNAP18 gene underlies two types of resistance to soybean cyst nematode.

Two types of resistant soybean (Glycine max (L.) Merr.) sources are widely used against soybean cyst nematode (SCN, Heterodera glycines Ichinohe). These include Peking-type soybean, whose resistance requires both the rhg1-a and Rhg4 alleles, and PI 88788-type soybean, whose resistance requires only the rhg1-b allele. Multiple copy number of PI 88788-type GmSNAP18, GmAAT, and GmWI12 in one genomic segment simultaneously contribute to rhg1-b resistance. Using an integrated set of genetic and genomic approaches, we demonstrate that the rhg1-a Peking-type GmSNAP18 is sufficient for resistance to SCN in combination with Rhg4. The two SNAPs (soluble NSF attachment proteins) differ by only five amino acids. Our findings suggest that Peking-type GmSNAP18 is performing a different role in SCN resistance than PI 88788-type GmSNAP18. As such, this is an example of a pathogen resistance gene that has evolved to underlie two types of resistance, yet ensure the same function within a single plant species.

The Arabidopsis immune regulator SRFR1 dampens defences against herbivory by Spodoptera exigua and parasitism by Heterodera schachtii.

Plants have developed diverse mechanisms to fine tune defence responses to different types of enemy. Cross-regulation between signalling pathways may allow the prioritization of one response over another. Previously, we identified SUPPRESSOR OF rps4-RLD1 (SRFR1) as a negative regulator of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent effector-triggered immunity against the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4. The use of multiple stresses is a powerful tool to further define gene function. Here, we examined whether SRFR1 also impacts resistance to a herbivorous insect in leaves and to a cyst nematode in roots. Interestingly, srfr1-1 plants showed increased resistance to herbivory by the beet army worm Spodoptera exigua and to parasitism by the cyst nematode Heterodera schachtii compared with the corresponding wild-type Arabidopsis accession RLD. Using quantitative real-time PCR (qRT-PCR) to measure the transcript levels of salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathway genes, we found that enhanced resistance of srfr1-1 plants to S. exigua correlated with specific upregulation of the MYC2 branch of the JA pathway concurrent with suppression of the SA pathway. In contrast, the greater susceptibility of RLD was accompanied by simultaneously increased transcript levels of SA, JA and JA/ET signalling pathway genes. Surprisingly, mutation of either SRFR1 or EDS1 increased resistance to H. schachtii, indicating that the concurrent presence of both wild-type genes promotes susceptibility. This finding suggests a novel form of resistance in Arabidopsis to the biotrophic pathogen H. schachtii or a root-specific regulation of the SA pathway by EDS1, and places SRFR1 at an intersection between multiple defence pathways.

Plasma levels and cardiovascular gene expression of urotensin-II in human heart failure.

The peptide urotensin-II (U-II) has been described as most potent vasoconstrictor identified so far, but plasma values in humans and its role in cardiovascular pathophysiology are unknown. We investigated circulating urotensin-II and its potential role in human congestive heart failure (CHF). We enrolled control individuals (n=13; cardiac index [CI], 3.5+/-0.1 l/min/m2; pulmonary wedge pressure [PCWP], 10+/-1 mm Hg), patients with moderate (n=10; CI, 2.9+/-0.3 l/min/m2; PCWP, 14+/-2 mm Hg) and severe CHF (n=11; CI, 1.8+/-0.2 l/min/m2; PCWP, 33+/-2 mm Hg). Plasma levels of urotensin-II differed neither between controls, patients with moderate and severe CHF nor between different sites of measurement (pulmonary artery, left ventricle, coronary sinus, antecubital vein) within the single groups. Hemodynamic improvement by vasodilator therapy in severe CHF (CI, +78+/-3%; PCWP, -55+/-3%) did not affect circulating U-II over 24 h. Preprourotensin-II mRNA expression in right atria, left ventricles, mammary arteries and saphenous veins did not differ between controls with normal heart function and patients with end-stage CHF. In conclusion, urotensin-II plasma levels and its myocardial and vascular gene expression are unchanged in human CHF. Circulating urotensin-II does not respond to acute hemodynamic improvement. These findings suggest that urotensin-II does not play a major role in human CHF.

A virus-induced gene silencing method to study soybean cyst nematode parasitism in Glycine max.

BACKGROUND: Bean pod mottle virus (BPMV) based virus-induced gene silencing (VIGS) vectors have been developed and used in soybean for the functional analysis of genes involved in disease resistance to foliar pathogens. However, BPMV-VIGS protocols for studying genes involved in disease resistance or symbiotic associations with root microbes have not been developed. FINDINGS: Here we describe a BPMV-VIGS protocol suitable for reverse genetic studies in soybean roots. We use this method for analyzing soybean genes involved in resistance to soybean cyst nematode (SCN). A detailed SCN screening pipeline is described. CONCLUSIONS: The VIGS method described here provides a new tool to identify genes involved in soybean-nematode interactions. This method could be adapted to study genes associated with any root pathogenic or symbiotic associations.

A High-Throughput Automated Technique for Counting Females of Heterodera glycines using a Fluorescence-Based Imaging System.

The soybean cyst nematode (SCN), Heterodera glycines, is the most damaging pathogen of soybean. Methods to phenotype soybean varieties for resistance to SCN are currently very laborious and time consuming. Streamlining a portion of this phenotyping process could increase productivity and accuracy. Here we report an automated method to count SCN females using a fluorescence-based imaging system that is well suited to high-throughput SCN phenotyping methods used in greenhouse screening. For optimal automated imaging, females were washed from roots at 30 days post-inoculation into small Petri dishes. Using a Kodak Image Station 4000MM Pro, the Petri dishes were scanned using excitation and emission wavelengths of 470 nm and 535 nm, respectively. Fluorescent images were captured and analyzed with Carestream Molecular Imaging Software for automated counting. We demonstrate that the automated fluorescent-based imaging system is just as accurate (r(2) ≥ 0.95) and more efficient (>50% faster) than manual counting under a microscope. This method can greatly improve the consistency and turnaround of data while reducing the time and labor commitment associated with SCN female counting.