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.

Soybean cyst nematode resistance in soybean is independent of the Rhg4 locus LRR-RLK gene.

To test the function of candidate genes in soybean for resistance to the soybean cyst nematode (SCN), a large collection of EMS-mutants from the SCN-resistant soybean cultivar "Forrest" was developed for Targeting Induced Local Lesions IN Genomes (TILLING). Additionally, due to the complexity of the soybean genome, an integrated set of genomic and genetic analysis tools was employed to complement the TILLING approach. The efficiency of this integrated set of tools was tested using a candidate soybean gene for resistance to SCN, encoding a leucine-rich repeat receptor-like kinase (LRR-RLK) that was identified by map-based cloning at the Rhg4 locus. The Rhg4 locus is one of the major quantitative trait loci controlling soybean resistance against SCN race 3 (HG type 0) in cv. Forrest, but the gene(s) sequence for resistance remains to be determined. Using TILLING, a Forrest mutant containing a nonsense mutation in the LRR domain of the candidate resistance protein was identified and confirmed; however, the SCN-resistant phenotype of the mutant was not altered. Haplotyping and EcoTILLING of recombinant inbred lines along with complementation analysis corroborated the TILLING result and ruled out the possibility of functional redundancy by a second copy of the LRR-RLK gene identified in the soybean genome. This study validates the use of TILLING, in combination with an integrated set of genomic tools, as an efficient means of testing candidate genes for SCN resistance in soybean.

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.

Arabidopsis photorespiratory serine hydroxymethyltransferase activity requires the mitochondrial accumulation of ferredoxin-dependent glutamate synthase.

The dual affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase for O(2) and CO(2) results in the net loss of fixed carbon and energy in a process termed photorespiration. The photorespiratory cycle is complex and occurs in three organelles, chloroplasts, peroxisomes, and mitochondria, which necessitates multiple steps to transport metabolic intermediates. Genetic analysis has identified a number of mutants exhibiting photorespiratory chlorosis at ambient CO(2), including several with defects in mitochondrial serine hydroxymethyltransferase (SHMT) activity. One class of mutants deficient in SHMT1 activity affects SHM1, which encodes the mitochondrial SHMT required for photorespiration. In this work, we describe a second class of SHMT1-deficient mutants defective in a distinct gene, GLU1, which encodes Ferredoxin-dependent Glutamate Synthase (Fd-GOGAT). Fd-GOGAT is a chloroplastic enzyme responsible for the reassimilation of photorespiratory ammonia as well as for primary nitrogen assimilation. We show that Fd-GOGAT is dual targeted to the mitochondria and the chloroplasts. In the mitochondria, Fd-GOGAT interacts physically with SHMT1, and this interaction is necessary for photorespiratory SHMT activity. The requirement of protein-protein interactions and complex formation for photorespiratory SHMT activity demonstrates more complicated regulation of this crucial high flux pathway than anticipated.

Functional characterization of the plastidic phosphate translocator gene family from the thermo-acidophilic red alga Galdieria sulphuraria reveals specific adaptations of primary carbon partitioning in green plants and red algae.

In chloroplasts of green plants and algae, CO(2) is assimilated into triose-phosphates (TPs); a large part of these TPs is exported to the cytosol by a TP/phosphate translocator (TPT), whereas some is stored in the plastid as starch. Plastidial phosphate translocators have evolved from transport proteins of the host endomembrane system shortly after the origin of chloroplasts by endosymbiosis. The red microalga Galdieria sulphuraria shares three conserved putative orthologous transport proteins with the distantly related seed plants and green algae. However, red algae, in contrast to green plants, store starch in their cytosol, not inside plastids. Hence, due to the lack of a plastidic starch pool, a larger share of recently assimilated CO(2) needs to be exported to the cytosol. We thus hypothesized that red algal transporters have distinct substrate specificity in comparison to their green orthologs. This hypothesis was tested by expression of the red algal genes in yeast (Saccharomyces cerevisiae) and assessment of their substrate specificities and kinetic constants. Indeed, two of the three red algal phosphate translocator candidate orthologs have clearly distinct substrate specificities when compared to their green homologs. GsTPT (for G. sulphuraria TPT) displays very narrow substrate specificity and high affinity; in contrast to green plant TPTs, 3-phosphoglyceric acid is poorly transported and thus not able to serve as a TP/3-phosphoglyceric acid redox shuttle in vivo. Apparently, the specific features of red algal primary carbon metabolism promoted the evolution of a highly efficient export system with high affinities for its substrates. The low-affinity TPT of plants maintains TP levels sufficient for starch biosynthesis inside of chloroplasts, whereas the red algal TPT is optimized for efficient export of TP from the chloroplast.

The photorespiratory Arabidopsis shm1 mutant is deficient in SHM1.

Mitochondrial serine hydroxymethyltransferase (SHMT), combined with glycine decarboxylase, catalyzes an essential sequence of the photorespiratory C2 cycle, namely, the conversion of two molecules of glycine into one molecule each of CO2, NH4+, and serine. The Arabidopsis (Arabidopsis thaliana) mutant shm (now designated shm1-1) is defective in mitochondrial SHMT activity and displays a lethal photorespiratory phenotype when grown at ambient CO2, but is virtually unaffected at elevated CO2. The Arabidopsis genome harbors seven putative SHM genes, two of which (SHM1 and SHM2) feature predicted mitochondrial targeting signals. We have mapped shm1-1 to the position of the SHM1 gene (At4g37930). The mutation is due to a G --> A transition at the 5' splice site of intron 6 of SHM1, causing aberrant splicing and a premature termination of translation. A T-DNA insertion allele of SHM1, shm1-2, and the F1 progeny of a genetic cross between shm1-1 and shm1-2 displayed the same conditional lethal phenotype as shm1-1. Expression of wild-type SHM1 under the control of either the cauliflower mosaic virus 35S or the SHM1 promoter in shm1-1 abrogated the photorespiratory phenotype of the shm mutant, whereas overexpression of SHM2 or expression of SHM1 under the control of the SHM2 promoter did not rescue the mutant phenotype. Promoter-beta-glucuronidase analyses revealed that SHM1 is predominantly expressed in leaves, whereas SHM2 is mainly transcribed in the shoot apical meristem and roots. Our findings establish SHM1 as the defective gene in the Arabidopsis shm1-1 mutant.

A bacterial artificial chromosome based physical map of the Ustilago maydis genome.

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and co-digested with HaeIII to reduce fragments to 50-750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P < or = 1 x 10(-6) to 1 x 10(-24)). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P < or = 1 x 10(-7) to 1 x 10 (-12). The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P < or = 1 x 10(-7). The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18-20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.

EST-analysis of the thermo-acidophilic red microalga Galdieria sulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts.

When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56 degrees C. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria , 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria.