Differential expression of candidate salivary effector proteins in field collections of Hessian fly, Mayetiola destructor.

Evidence is emerging that some proteins secreted by gall-forming parasites of plants act as effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs) that are the putative effectors responsible for the physiological changes elicited in susceptible seedling wheat by Hessian fly, Mayetiola destructor (Say), larvae have been documented. However, how the genes encoding these candidate effectors might respond under field conditions is unknown. The goal of this study was to use microarray analysis to investigate variation in SSGP transcript abundance amongst field collections from different geographical regions (southeastern USA, central USA, and the Middle East). Results revealed significant variation in SSGP transcript abundance amongst the field collections studied. The field collections separated into three distinct groups that corresponded to the wheat classes grown in the different geographical regions as well as to recently described Hessian fly populations. These data support previous reports correlating Hessian fly population structure with micropopulation differences owing to agro-ecosystem parameters such as cultivation of regionally adapted wheat varieties, deployment of resistance genes and variation in climatic conditions.

Biological and molecular characterization of Hessian fly (Diptera: Cecidomyiidae) from Israel.

Samples of a dipteran pest of wheat were tested to confirm identity, describe local populations and suggest the use of deploying resistance (R) genes in wheat cultivars for control of Mayetiola destructor, Hessian fly (HF). Morphological evaluation of adults and a free-choice oviposition preference test documenting that females overwhelmingly preferred to oviposit on wheat instead of barley supported they were HF. Using the cytochrome c oxidase subunit I (coxI), the Barcoding Region, nine haplotypes were revealed. Two were found only in the Israeli collections and averaged 3% sequence divergence compared to the other seven haplotypes found in the United States, Israel and Syria. In evaluations of virulence, the Israeli HF in culture was virulent to 11 of the 19 (R) genes tested, and complementation analysis documented that, for four of the R genes tested, the Israeli HF shared loci for virulence with HF from the United States. Levels of HF infestation at seven Israeli fields were at least at the 5-8% level, which historically has indicated a significant yield loss. Microsatellite genotyping of the five HF collections from Israel revealed mixed populations in Israel that are distinctly separate from the single population in Syria.

Cloning and characterization of mariner-like elements in the soybean aphid, Aphis glycines Matsumura.

Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is currently the most important insect pest of soybean (Glycine max (L.) Merr.) in the United States and causes significant economic damage worldwide, but little is known about the aphid at the molecular level. Mariner-like transposable elements (MLEs) are ubiquitous within the genomes of arthropods and various other invertebrates. In this study, we report the cloning of MLEs from the soybean aphid genome using degenerate PCR primers designed to amplify conserved regions of mariner transposases. Two of the ten sequenced clones (designated as Agmar1 and Agmar2) contained partial but continuous open reading frames, which shared high levels of homology at the protein level with other mariner transposases from insects and other taxa. Phylogenetic analysis revealed Agmar1 to group within the irritans subfamily of MLEs and Agmar2 within the mellifera subfamily. Southern blot analysis and quantitative PCR analysis indicated a low copy number for Agmar1-like elements within the soybean aphid genome. These results suggest the presence of at least two different putative mariner-like transposases encoded by the soybean aphid genome. Both Agmar1 and Agmar2 could play influential roles in the architecture of the soybean aphid genome. Transposable elements are also thought to potentially mediate resistance in insects through changes in gene amplification and mutations in coding sequences. Finally, Agmar1 and Agmar2 may represent useful genetic tools and provide insights on A. glycines adaptation.

Assessment of structural variation and molecular mapping of insertion sites of Desmar-like elements in the Hessian fly genome.

The Hessian fly (Mayetiola destructor) is an agriculturally important pest of wheat. A mariner element (Desmar1) has been previously identified in the Hessian fly genome. Using Desmar1 as a probe, we isolated individual copies of Desmar-like elements from the Hessian fly genome cloned in bacterial artificial chromosomes (BACs) and studied their structural variability and flanking DNA sequences. The partial Desmar-like copies are relatively more abundant (∼64%) than full length copies (∼36%) in the Hessian fly genome. Most of the full length copies are consistently flanked by an EcoRI restriction site that occurs 32 bp from one end and 66 bp from the other end of the mariner. Using an amplified fragment length polymorphism-PCR (AFLP-PCR) based method, we identified segregating polymorphisms associated with Desmar elements in a F2 mapping population. We were able to use the segregation data to localize the chromosomal position of three Desmar elements by linkage analysis. As paternal chromosomes are eliminated in the Hessian fly during early embryogenesis, two-thirds of the AFLPs were expected to be polymorphic in the mapping population and this was observed for AFLPs anchored to full length Desmar copies but not to the partial copies. Thus, our data indicate that dead and partial Desmar-like copies are probably associated with less polymorphic regions and may represent mariner graveyards in the Hessian fly genome.

Molecular cloning and characterization of two digestive serine proteases from the Hessian fly, Mayetiola destructor.

Full-length cDNA and genomic sequences for two genes (designated mdesprot-I and mdesprot-II) encoding digestive serine proteases in Hessian fly, Mayetiola destructor, have been cloned and characterized. The deduced amino acid sequences revealed similarity with trypsin-like digestive serine proteases from other Dipterans. Both mdesprot-I and mdesprot-II encoded proteins with secretion signal peptides at the N-terminals, indicating the proteins are secreted proteases that should function as midgut digestive proteases. A cytological analysis with fluorescent in situ hybridization revealed the cytological localization of mdesprot-I and mdesprot-II on the long arm of Autosome 2. Results are discussed in the context of the efficacy of potential protease inhibitors to develop Hessian fly resistant wheat through genetic engineering approaches.

Molecular and cytological analysis of a mariner transposon from Hessian fly.

Degenerate PCR primers for conserved regions of the mariner transposase have been shown to amplify DNA sequences from the Hessian fly (Mayetiola destructor). Using one of these sequences as a hybridization probe, a clone from an M. destructor genomic library in phage lambda was recovered and sequenced. A transposable element, Desmar 1, with perfect inverted terminal repeats and an open reading frame that encodes a mariner class transposase was found. When compared to mariner sequences in the gene database, the transposase proved to be similar to that of the active mariner Mos 1 from the fruit fly (Drosophila mauritiana). In situ hybridization of the transposon DNA sequence to salivary gland polytene chromosomes revealed the general cytological locations of mariner elements. The distribution of sequences with homology to the probe was predominantly, but not exclusively, n paracentromeric regions.

Mariner transposase-like sequences from the Hessian fly, Mayetiola destructor.

Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. They can serve as vectors in gene transfer systems and as mutagenic agents for isolation of genes. Until recently their identification has been primarily limited to organisms subjected to extensive genetic or molecular study. The Hessian fly, Mayetiola destructor (Say), is an agriculturally important pest of wheat, Triticum aestivum L., in the United States and other parts of the world. We assessed the presence of mariner transposase-like sequences in M. destructor by polymerase chain reaction (PCR) assay designed to detect conserved regions of the mariner transposase gene. DNA sequence analysis of PCR products revealed sequences with similarities to putative mariner transposase gene subfamilies from Drosophila mauritiana and horn fly, Haematobia irritans. DNA gel blot analyses indicated sequences hybridizing to the mariner transposase-like PCR clones occur at a moderate to low copy number in M. destructor. Results suggest the presence of an endogenous mobile-element system in M. destructor, which might be developed into a gene transfer system or serve in mapping genes.

Response of wheatgrasses and wheat × wheatgrass hybrids to barley yellow dwarf virus.

Resistance to barley yellow dwarf virus (BYDV), manifested by low enzyme-linked immunosorbent assay (ELISA) values in plants exposed to viruliferous aphids, was identified in several wheatgrasses (Agropyron spp.). ELISA results were similar for root and leaf extracts of infested plants. No difference in reaction to BYDV was found between plants grown in the field and those in the growth chamber. Interspecific hybrids were generated using pollen from single resistant plants of Agropyron spp. to pollinate soft red winter wheat spikes. Resistance in hybrids appeared to be at the level of virus replication rather than at the level of vector inoculation. The hybrids varied in their reaction to BYDV. Expression of BYDV resistance in hybrids was influenced not only by wheat genotype and Agropyron species but, in some cases, reaction varied even among hybrids between the same wheat genotype and Agropyron plant. Implications of these results are discussed.