Hunter S. Beard

Identification of molecular markers in soybean comparing RFLP, RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.

Profiling local gene expression changes associated with Eimeria maxima and Eimeria acervulina using cDNA microarray

Eimeria parasites show preferential sites of invasion in the avian intestine and produce a species-specific host immune response. Two economically important species, Eimeria acervulina and Eimeria maxima, preferentially invade and develop in the avian duodenum and jejunum/ileum, respectively. To investigate local host immune responses induced by parasite infection, global transcriptional changes in intestinal intraepithelial lymphocytes (IELs) induced by oral inoculation of chickens with E. acervulina or E. maxima were monitored using cDNA microarrays containing 400 unique chicken genes. Multiple gene transcripts were significantly up- or down-regulated following primary or secondary infection with E. acervulina or E. maxima. In general, infection by either parasite resulted in the expression changes of more genes following primary infection than following secondary infection, and E. acervulina caused more changes than did E. maxima. Although different regions of the small intestine were infected, similar changes in the levels of several cytokine mRNAs were observed in both Eimeria species following primary infection. Also identified was a set of transcripts whose expression was commonly enhanced or repressed in intestinal IELs of chickens infected with either parasite. Microarray analysis of chicken genes induced or repressed following Eimeria infection offers a powerful tool to enhance our understanding of host–parasite interactions leading to protective immunity.

Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions

Our laboratory has been working toward increasing our understanding of the genetic control of cold hardiness in blueberry (Vaccinium section Cyanococcus) to ultimately use this information to develop more cold hardy cultivars for the industry. Here, we report using cDNA microarrays to monitor changes in gene expression at multiple times during cold acclimation under field and cold room conditions. Microarrays contained over 2,500 cDNA inserts, approximately half of which had been picked and single-pass sequenced from each of two cDNA libraries that were constructed from cold acclimated floral buds and non-acclimated floral buds of the fairly cold hardy cv. Bluecrop (Vaccinium corymbosum L.). Two biological samples were examined at each time point. Microarray data were analyzed statistically using t tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). Interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Many of the genes induced only under cold room conditions could be divided into three major types: (1) genes associated with stress tolerance; (2) those that encode glycolytic and TCA cycle enzymes, and (3) those associated with protein synthesis machinery. A few of the genes induced only under field conditions appear to be related to light stress. Possible explanations for these differences are discussed in physiological context. Although many similarities exist in how plants respond during cold acclimation in the cold room and in the field environment, there are major differences suggesting caution should be taken in interpreting results based only on artificial, cold room conditions.

Molecular cloning and expression of two cDNAs encoding asparagine synthetase in soybean

Two cDNA clones (SAS1 and SAS2) encoding different isoforms of asparagine synthetase (AS; EC 6.3.5.4) were isolated. Their DNA sequences were determined and compared. The amino-terminal residues of the predicted SAS1 and SAS2 proteins were identical to those of the glutamine binding domain of AS from pea, asparagus, Arabidopsis and human, suggesting that SAS1 and SAS2 cDNAs encode the glutamine-dependent form of AS. The open reading frames of SAS1 and SAS2 encode a protein of 579 and 581 amino acids with predicted molecular weights of 65 182 and 65 608 Da respectively. Similarity of the deduced amino acid sequences of SAS1 and SAS2 with other known AS sequences were 92% and 93% for pea AS1; 91% and 96% for pea AS2; 88% and 91% for asparagus; 88% and 90.5% for Arabidopsis; 70.5% and 72.5% for E. coli asnB and 61% and 63% for man. A plasmid, pSAS2E, was constructed to express the soybean AS protein in Escherichia coli. Complementation experiments revealed that the soybean AS protein was functional in E. coli. Southern blot analysis indicated that the soybean AS is part of a small gene family. AS transcript was expressed in all tissues examined, but higher levels were seen in stem and root of light-grown tissue and leaves of dark-treated tissue.

Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots

BackgroundExtensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction.ResultsHere we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of composite plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control).ConclusionsKnowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots, resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes.

A physical map of a gene-dense region in soybean linkage group A2 near the black seed coat and Rhg 4 loci

Abstract Soybean (Glycine max L. Merrill) linkage group A2 contains a major resistance gene to the soybean cyst nematode (Heterodera glycines Ichinohe) at the Rhg4 locus near a gene encoding aspartokinase homoserine dehydrogenase (AK-HSDH) and also near the I locus affecting seed coat color. To identify clones related to this region of the genome, we used a PCR assay using primers designed from a gene encoding AK-HSDH to screen approximately 40,000 clones from a bacterial artificial chromosome (BAC) library constructed from genomic DNA of the susceptible cv. Williams 82. The identified BACs were screened with a second PCR assay using primers designed from DNA sequence associated with the I locus to confirm the location of the BACs. Only BAC Gm_ISb001_056_G02 (56G2) was positive for both assays. BAC 56G2 contains several genes previously associated with stress or defense response including genes with high sequence similarity to those encoding chalcone synthase, glucosyl-transferase, a heat-shock transcription factor, a membrane-associated salt-inducible protein, adenosyl homocysteinase, a protein kinase, and a G10-like protein. The map contributes to our understanding of the organization of the soybean genome and to the completion of a physical map of the soybean genome. In addition, the genes identified provide landmarks to identify BAC clones near the Rhg4 locus in resistant soybean genomic libraries and provide a foundation for comparison of soybean cyst nematode resistant and -susceptible DNA sequences in this region.

Ascaris suum: cDNA microarray analysis of 4th stage larvae (L4) during self-cure from the intestine

There is spontaneous cure of a large portion of Ascaris suum 4th-stage larvae (L4) from the jejunum of infected pigs between 14 and 21 days after inoculation (DAI). Those L4 that remain in the jejunum continue to develop while those that have moved to the ileum are eventually expelled from the intestines. Although increases in intestinal mucosal mast cells and changes in local host immunity are coincidental with spontaneous cure, the population of L4 that continue to develop in the jejunum may counteract host protective mechanisms by the differential production of factors related to parasitism. To this end, a cDNA library was constructed from L4 isolated from pig jejunum at 21 DAI, and 93% of 1920 original clones containing a single amplicon in the range 400-1500 bp were verified by gel electrophoresis and printed onto glass slides for microarray analysis. Fluorescent probes were prepared from total RNA isolated from: (1) 3rd stage-larvae from lung at 7 DAI, (L3); (2) L4 from jejunum at 14 DAI (L4-14-J); (3) L4 from jejunum at 21 DAI (L4-21-J); (4) L4 from ileum at 21 DAI (L4-21-I, and; (5) adults (L5). Cy3-labeled L3, L4-14-J, L4-21-I and L5 cDNA, and Cy5-labeled L4-21-J cDNA were simultaneously used to screen the printed arrays containing the L4-21-J-derived cDNA library. Several clones showed consistent differential gene expression over two separate experiments and were grouped into 3 distinct transcription patterns. The data showed that sequences from muscle actin and myosin, ribosomal protein L11, glyceraldehyde-3-phosphate dehydrogenase and the flavoprotein subunit of succinate dehydrogenase were highly expressed in L4-21-J, but not in L4-21-I; as were a collection of un-annotated genes derived from a worm body wall-hypodermis library, and a testes germinal zone tissue library. These results suggest that only actively developing A. suum L4 are destined to parasitize the host and successfully neutralize host protective responses.

Modification of the expression of two NPR1 suppressors, SNC1 and SNI1, in soybean confers partial resistance to the soybean cyst nematode, Heterodera glycines

Systemic acquired resistance (SAR) is an enhanced defence response triggered when plants detect a pathogen. The response is extended to uninfected organs to protect against future attack. NPR1 is a nuclear leucine-rich repeat protein with a key role in SAR. It binds specifically to salicylic acid, and acts as a transcriptional coregulator of SAR activators and an inhibitor of transcriptional repressors. The proteins encoded by Suppressor of NPR1, Constitutive (SNC1) and Suppressor of NPR1, Inducible (SNI1) interact with NPR1 to regulate the expression of pathogenesis-related genes. The Arabidopsis thaliana (L.) Heynh. snc1 mutant exhibits a constitutive resistance response, but in the sni1 mutant, the SNI1 protein is rendered incapable of suppressing pathogen resistance genes. To study the influence of SNC1 and SNI1 on resistance to the soybean cyst nematode (Heterodera glycines), soybean (Glycine max (L.) Merr.) roots were separately transformed with four constructs designed to: (i) overexpress GmSNC1, the soybean orthologue of AtSNC1; (ii) overexpress AtSNI1; (iii) silence GmSNC1 and (iv) silence GmSNI1. A significant reduction of the female nematode population was observed in Treatments (i) and (iv). The expression of SAR marker genes was analysed in these treatments. The unusual pattern of expression of pathogen resistance genes shows there are differences in the effect resistance genes have on soybean and A. thaliana. Although NPR1 is involved in the cross-talk between the salicylic acid, jasmonic acid and ethylene pathways, understanding the nematode resistance mechanism in plants is still imprecise. These results provide further insights into the soybean defence response.

Putative Rust Fungal Effector Proteins in Infected Bean and Soybean Leaves

The plant-pathogenic fungi Uromyces appendiculatus and Phakopsora pachyrhizi cause debilitating rust diseases on common bean and soybean. These rust fungi secrete effector proteins that allow them to infect plants, but their effector repertoires are not understood. The discovery of rust fungus effectors may eventually help guide decisions and actions that mitigate crop production loss. Therefore, we used mass spectrometry to identify thousands of proteins in infected beans and soybeans and in germinated fungal spores. The comparative analysis between the two helped differentiate a set of 24 U. appendiculatus proteins targeted for secretion that were specifically found in infected beans and a set of 34 U. appendiculatus proteins targeted for secretion that were found in germinated spores and infected beans. The proteins specific to infected beans included family 26 and family 76 glycoside hydrolases that may contribute to degrading plant cell walls. There were also several types of proteins with structural motifs that may aid in stabilizing the specialized fungal haustorium cell that interfaces the plant cell membrane during infection. There were 16 P. pachyrhizi proteins targeted for secretion that were found in infected soybeans, and many of these proteins resembled the U. appendiculatus proteins found in infected beans, which implies that these proteins are important to rust fungal pathology in general. This data set provides insight to the biochemical mechanisms that rust fungi use to overcome plant immune systems and to parasitize cells.

Manipulation of two α‐endo‐β‐1,4‐glucanase genes, AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots

Plant endo-β-1,4-glucanases (EGases) include cell wall-modifying enzymes that are involved in nematode-induced growth of syncytia (feeding structures) in nematode-infected roots. EGases in the α- and β-subfamilies contain signal peptides and are secreted, whereas those in the γ-subfamily have a membrane-anchoring domain and are not secreted. The Arabidopsis α-EGase At1g48930, designated as AtCel6, is known to be down-regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α-EGase, AtCel2, is up-regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ-EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α-EGases are likely to be more useful than membrane-bound γ-EGases in the development of an SCN-resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis-H. schachtii interaction as a model for the soybean-H. glycines pathosystem.