Modulations in gene expression and mapping of genes associated with cyst nematode infection of soybean

Infection of the soybean root by the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) induces a well-documented, yet poorly understood, response by the host plant. The plant response, involving the differentiation of a feeding structure, or "syncytium," facilitates the feeding and reproduction of the nematode to the detriment of the host. We used a genetic system involving a single dominant soybean gene conferring susceptibility to an inbred nematode strain, VL1, to characterize the nematode-host interaction in susceptible line PI 89008. The restriction fragment length polymorphism marker pB053, shown to map to a major SCN resistance locus, cosegregates with resistance among F2 progeny from the PI 89008 x PI 88287 cross. Cytological examination of the infection process confirmed that syncytium development in this genetic system is similar to that reported by others who used noninbred nematode lines. Our study of infected root tissue in the susceptible line PI 89008 revealed a number of genes enhanced in expression. Among these are catalase, cyclin, elongation factor 1alpha, beta-1,3-endoglucanase, hydroxy-methylglutaryl coenzyme A reductase, heat shock protein 70, late embryonic abundant protein 14, and formylglycinamidine ribonucleotide synthase, all of which we have genetically positioned on the public linkage map of soybean. Formylglycinamidine ribonucleotide synthase was found to be tightly linked with a major quantitative trait locus for SCN resistance. Our observations are consistent with the hypothesis proposed by others that feeding site development involves the dramatic modulation of gene expression relative to surrounding root cells.     

Identification of soybean microRNAs involved in soybean cyst nematode infection by deep sequencing

Soybean cyst nematode (SCN), Heterodera glycines, is the most devastating pathogen of soybean worldwide. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are known to play important role in plant stress response. However, there are few reports profiling the miRNA expression patterns during pathogen stress. We sequenced four small RNA libraries from two soybean cultivar (Hairbin xiaoheidou, SCN race 3 resistant, Liaodou 10, SCN race 3 susceptible) that grown under un-inoculated and SCN-inoculated soil. Small RNAs were mapped to soybean genome sequence, 364 known soybean miRNA genes were identified in total. In addition, 21 potential miRNA candidates were identified. Comparative analysis of miRNA profiling indicated 101 miRNAs belong to 40 families were SCN-responsive. We also found 20 miRNAs with different express pattern even between two cultivars of the same species. These findings suggest that miRNA paly important role in soybean response to SCN and have important implications for further identification of miRNAs under pathogen stress.     

Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles

With the availability of microarray technology, the expression profiles of thousands of genes can be monitored simultaneously to help determine the mechanisms of these biological processes. We conducted Affymetrix GeneChip microarray analyses of the Arabidopsis-cyst nematode interaction and employed a statistical procedure to analyze the resultant data, which allowed us to identify significant gene expression changes. Quantitative real-time RT-PCR assays were used to confirm the microarray analyses. The results of the expression profiling revealed 128 genes with altered steady-state mRNA levels following infection by the sugar beet cyst nematode (Heterodera schachtii; BCN), in contrast to only 12 genes that had altered expression following infection by the soybean cyst nematode (H. glycines; SCN). The expression of these 12 genes also changed following infection by BCN, i.e. we did not identify any genes regulated exclusively by SCN. The identification of 116 genes whose expression changes during successful cyst nematode parasitism by BCN suggests a potential involvement of these genes in the infection events starting with successful syncytium induction. Further characterization of these genes will permit the formulation of testable hypotheses to explain successful cyst nematode parasitism.     

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence‐related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full‐length cDNAs of GmSAMT1 from a SCN‐resistant soybean line and from a SCN‐susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli‐expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μm . To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN‐susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.     

Relationship of cyst nematode gene frequencies to soybean resistance

Summary Soybean (S, Glycine max (L.) Merr.) lines with relatively few cysts of soybean cyst nematode (CN, Heterodera glycines Ichinohe) populations are usually called CN-resistant. The phenotype of number of cysts per plant is of the CN-S (Cyst Nematode-Soybean) association and determined by the interactions of genes for avirulence-resistance. The acronym alins was proposed for these alleles for incompatibility, with xalin representing the interaction X of one microsymbiont malin with its host h-alin. These alins are dominant in the gene-for-gene model but may be mostly recessive with CN-S. Definitive genetic studies have been hindered by the heterogeneity of sexually reproducing CN populations and lack of the appropriate genetic models. Loegering's abstract interorganismal genetic model was modified so that one model represented all four possible interactions of dominant-recessive alins for an incompatible phenotype. This involved redefining the Boolean algebra symbol 1 to represent both the alins AND their frequencies. The model was used to derive the relationship: {ie893-01} where the expectation E of cysts (of any CN-S combination, as proportion of number of cysts on a check cultivar) is proportional to the product of CN genotypic frequencies expressed as functions of m-alin frequencies. Each m-alin is at a different locus, i.e., {ie893-02}. The number of terms multiplied for each CN-S is equal to the number of alins in the S line (or F₂ plant). There are too many unknowns in the equation to solve for any of them. The relationship does explain the continuous distributions of phenotypes that were nearly always observed. Basic genetic principles were used to concurrently derive the models and to obtain discontinuous distributions of numbers of cyst phenotypes in segregating generations due to one recessive alin in a CN-susceptible soybean line.Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 9739     

Identification and characterization of mouse Rab32 by mRNA and protein expression analysis

Rab proteins, a subfamily of the ras superfamily, are low molecular weight GTPases involved in the regulation of intracellular vesicular transport. Cloning of human RAB32 was recently described. Presently, we report the cloning and characterization of the mouse homologue of Rab32. We show that murine Rab32 exhibits a ubiquitous expression pattern, with tissue-specific variation in expression level. Three cell types with highly specialized organelles, melanocytes, platelets and mast cells, exhibit relatively high level of Rab32. We show that in murine amelanotic in vitro transformed melanocytes as well as in human amelanotic metastatic melanoma cell lines, the expression of Rab32 is markedly reduced or absent, in parallel with the loss of expression of two key enzymes for the production of melanin, tyrosinase and Tyrp1. Therefore, in both mouse and human systems, the expression of Rab32 correlates with the expression of genes involved in pigment production. However, in melanoma samples, amelanotic due to a mutation in the tyrosinase gene, the expression of Rab32 remains at levels comparable to those observed in pigmented melanoma samples. Finally, we observed co-localization of Rab32 and the melanosomal proteins, Tyrp1 and Dct, indicating an association of Rab32 with melanosomes. Based on these data, we propose the inclusion of Rab32 to the so-called melanocyte/platelet family of Rab proteins.     

Physical mapping of resistant and susceptible soybean genomes near the soybean cyst nematode resistance gene Rhg4.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the foremost pest of soybean (Glycine max L. Merr.). The rhg1 allele on linkage group (LG) G and the Rhg4 allele on LG A2 are important in conditioning resistance. Markers closely linked to the Rhg4 locus were used previously to screen a library of bacterial artificial chromosome (BAC) clones from susceptible 'Williams 82' and identified a single 150-kb BAC, Gm_ISb001_056_G02 (56G2). End-sequenced subclones positioned onto a restriction map provided landmarks for identifying the corresponding region from a BAC library from accession PI 437654 with broad resistance to SCN. Seventy-three PI 437654 BACs were assigned to contigs based upon HindIII restriction fragment profiles. Four contigs represented the PI 437654 counterpart of the 'Williams 82' BAC, with PCR assays connecting these contigs. Some of the markers on the PI 437654 contigs are separated by a greater physical distance than in the 'Williams 82' BAC and some primers amplify bands from BACs in the mid-portion of the connected PI 437654 BAC contigs that are not amplified from the 'Williams 82' BAC. These observations suggest that there is an insertion in the PI 437654 genome relative to the 'Williams 82' genome in the Rhg4 region.     

Targeted comparative genome analysis and qualitative mapping of a major partial-resistance gene to the soybean cyst nematode

A major partial-resistance locus to the soybean cyst nematode (Heterodera glycines Ichinohe; SCN) was identified on linkage group G of soybean [Glycine max (L.) Merr.] using restriction fragment length polymorphisms (RFLPs). This locus explained 51.4% (LOD=10.35) of the total phenotypic variation in disease response in soybean Plant Introduction (PI) 209332, 52.7% (LOD=15.58) in PI 90763, 40.0% (LOD=10.50) in PI 88788, and 28.1% (LOD=6.94) in Peking. Initially, the region around this major resistance locus was poorly populated with DNA markers. To increase marker density in this genomic region, first random, and later targeted, comparative mapping with RFLPs from mungbean [Vigna radiata (L.) R. Wilcz.] and common bean (Phaseolus vulgaris L.) was performed, eventually leading to one RFLP marker every 2.6 centimorgans (cM). Even with this marker density, the inability to resolve SCN disease response into discrete Mendelian categories posed a major limitation to mapping. Thus, qualitative scoring of SCN disease response was carried out in an F₅₆ recombinant inbred population derived from EvansxPI 209332 using a 30% disease index cut-off for resistance. Using the computer program JoinMap, an integrated map of the region of interest was created, placing the SCN resistance locus 4.6 cM from RFLP marker B53 and 2.8 cM from Bng30. This study demonstrates how a combination of molecularmapping strategies, including comparative genome analysis, join mapping, and qualitative scoring of a quantitative trait, potentially provide the necessary tools for high-resolution mapping around a quantitative-trait locus.     

Identification of a new major QTL associated with resistance to soybean cyst nematode (Heterodera glycines)

Resistance of soybean [Glycine max (L.) Merr.] to cyst nematode (SCN) (Heterodera glycines Ichinohe), one of the most destructive pathogens affecting soybean, involves a complex genetic system. The identification of QTLs associated with SCN resistance may contribute to the understanding of such system. The objective of this work was to identify and map QTLs for resistance to SCN Race 14 with the aid of molecular markers. BC₃F_2:3 and F_2:3 populations, both derived from an original cross between resistant cv. Hartwig and the susceptible line BR-92–31983 were screened for resistance to SCN Race 14. Four microsatellite (Satt082, Sat_001, Satt574 and Satt301) and four RAPD markers (OPAA-11₇₉₅, OPAE-08₈₃₇, OPR-07₅₄₈ and OPY-07₂₀₃₀) were identified in the BC₃F_2:3 population using the bulked segregant analysis (BSA) technique. These markers were amplified in 183 F_2:3 families and mapped to a locus that accounts for more than 40% of the resistance to SCN Race 14. Selection efficiency based on these markers was similar to that obtained with the conventional method. In the case of the microsalellite markers, which identify homozygous resistant genotypes, the efficiency was even higher. This new QTL has been mapped to the soybean linkage group D2 and, in conjunction with other QTLs already identified for SCN resistance, will certainly contribute to our understanding of the genetic basis of resistance of this important disease in soybean. Received: 12 October 1999 / Accepted: 14 April 2000     

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective medium containing 200 μg ml⁻¹ kanamycin and 500 μg ml⁻¹ carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode. Received: 13 July 1999 / Accepted: 8 August 1999     

Characterization and expression profiling of a novel cereal cyst nematode resistance gene analog in wheat

Based on the conserved regions of known resistance genes, an NBS-LRR-type CCN resistance gene analog was isolated from the CCN resistant E-10 near isogenic lines (NILs) of wheat, designated as CreZ (GenBank accession number: EU327996). It contained a complete ORF that was 2775 bp in length and encoded 924 amino acids. Sequence comparison indicated that it shared 92% nucleotide and 87% amino acid identity with those of the known CCN-resistance gene Cre3 and had similar characteristic conserved motifs to those in other established NBS-LRR disease resistance genes. The expression profiling of CreZ indicated that it was specifically expressed in the roots of resistant plants and real-time PCR analysis demonstrated that expression levels drastically increased when the plants were inoculated with cereal cyst nematodes. It could be inferred, then, that CreZ belongs to the NBS-LRR resistance gene family and is a candidate gene for potential resistance to the cereal cyst nematode. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 6, pp. 1070–1077. The text was submitted by the authors in English.     

A bacterial artificial chromosome library for soybean and identification of clones near a major cyst nematode resistance gene

 We constructed a bacterial artificial chromosome (BAC) library for soybean (Glycine max) consisting of approximately 30 000 clones with an average insert size of 120 kilobase pairs. The library was successfully screened with restriction fragment length polymorphism (RFLP) and microsatellite markers tightly linked to a major resistance gene for the cyst nematode, Heterodera glycines. Since many soybean RFLPs hybridize to duplicate loci, BACs homologous to duplicate RFLP loci were distinguished by digestion with the restriction enzyme originally used to map the RFLP, followed by a comparison of the hybridizing fragments. Linkage mapping of BAC clones identified with markers linked to the cyst nematode resistance gene demonstrated that these clones were located at the expected chromosomal positions and that there were no indications of chimeras within the genomic inserts. Received: 3 July 1997/Accepted: 26 August 1997     

Identification of QTLs associated with resistance to soybean cyst nematode races 2, 3 and 5 in soybean PI 90763

Soybean cyst nematode (SCN) is a major soybean pest throughout the soybean growing regions in the world, including the USA. Soybean PI 90763 is an important SCN resistance source. It is resistant to several SCN populations including races 2, 3 and 5. But its genetics of resistance is not well known. The objectives of this study were to: (1) confirm quantitative trait loci (QTLs) for resistance to SCN race 3 in PI 90763 and (2) identify QTLs for resistance to SCN races 2 and 5. QTLs were searched in Hamilton × PI 90763 F_2:3populations using 193 polymorphic simple sequence repeats (SSRs) covering 20 linkage groups (LGs). QTLs for resistance to SCN were identified on LGs A2, B1, E, G, J and L. The same QTL was suggested for resistance to different SCN races where their 1-LOD support intervals of QTL positions highly overlapped. The QTL on LG G was associated with resistance to races 2, 3 and 5. The QTL on LG B1 was associated with resistance to races 2 and 5. The QTL on LG J was associated with resistance to races 2 and 3. The QTLs on LGs A2 and L were associated with resistance to race 3. The QTL on LG E was associated with resistance to race 5. We conclude that LGs A2 and B1 may represent an important distinction between resistance to SCN race 3 and resistance to SCN races 2 and 5 in soybean.