What's in a worm; Uncovering polymophisms in soybean cyst nematode parasitism genes that correlate with virulence

Abstract only availableSoybean cyst nematode (SCN) is a parasitic worm that penetrates and infects soybean roots resulting in billions of dollars in crop loss annually. The most cost effective and environmentally friendly SCN management strategy is to utilize resistant soybean plants. Of particular concern, however, is that genetically diverse field populations of SCN have shown the ability to overcome current sources of resistance. Previous research has confirmed that parasitism genes expressed in esophageal gland cells encode for secretions that are involved in the process of plant parasitism and may contribute to the ability of SCN to overcome resistance. However, since the function of most of the secretions has not been identified, SCN parasitism and the mechanism by which the nematode can overcome resistance is not fully understood. The goal of this study was to use publicly available SCN inbred lines to identify polymorphisms in the molecular structure of a subset of parasitism genes and assess for correlations with virulence on resistance soybean plants. Nematode genomic DNA from 15 lines was extracted for Southern Blotting and PCR analysis of 10 SCN parasitism genes. Our results indicate that several parasitism genes belong to polymorphic gene families. Interestingly, we observed a unique banding pattern for one of the parasitism genes in a subset of inbred lines that are virulent on a particular group of SCN resistant germplasm.Plant Genomics Internship @ M

Nematode secreted proteins originating from esophageal gland cells are injected through the stylet [abstract]

Abstract only availableFaculty Mentor: Melissa G. Mitchum, Division of Plant SciencesNematode secreted proteins originating from esophageal gland cells are injected through the stylet directly into root tissues to facilitate plant parasitism. Secreted proteins are encoded by parasitism genes expressed in the nematode esophageal gland cells. The soybean cyst nematode (Heterodera glycines) Hg2DO1 gene is a candidate parasitism gene specifically expressed in the dorsal gland that encodes a 186 amino acid protein with a signal peptide. The Hg2DO1 sequence is classified as a “pioneer gene” with no homology to any other sequences present in current databases. We have determined the genomic structure of the Hg2DO1 gene and isolated both genomic DNA and corresponding cDNA sequences from the closely related nematode, Heterodera schachtii, for comparative analysis. Quantitative real-time PCR is underway to determine the developmental expression profile of the Hg2DO1 gene throughout nematode development. For functional analysis of Hg2DO1 both overexpression and RNAi constructs were generated and used to transform Arabidopsis and soybean hairy roots. These plants are being assessed for phenotypic changes and effects on parasitism. These studies are providing important insight into the mechanisms of nematode pathogenesis of plants.Nematode secreted proteins originating from esophageal gland cells are injected through the stylet directly into root tissues to facilitate plant parasitism. Secreted proteins are encoded by parasitism genes expressed in the nematode esophageal gland cells. The soybean cyst nematode (Heterodera glycines) Hg2DO1 gene is a candidate parasitism gene specifically expressed in the dorsal gland that encodes a 186 amino acid protein with a signal peptide. The Hg2DO1 sequence is classified as a “pioneer gene” with no homology to any other sequences present in current databases.  We have determined the genomic structure of the Hg2DO1 gene and isolated both genomic DNA and corresponding cDNA sequences from the closely related nematode, Heterodera schachtii, for comparative analysis. Quantitative real-time PCR is underway  to determine the developmental expression profile of the Hg2DO1 gene throughout nematode development. For functional analysis of Hg2DO1 both overexpression and RNAi constructs were generated and used to transform Arabidopsis and soybean hairy roots. These plants are being assessed for phenotypic changes and effects on parasitism. These studies are providing important insight into the mechanisms of nematode pathogenesis of plants

Functional analysis of nematode secreted proteins to devise innovative approaches for plant resistance against nematodes [abstract]

Abstract only availableApproximately 1 billion dollars in soybean yield loss is caused annually in the US by a microscopic parasite known as the soybean cyst nematode (SCN; Heterodera glycines). My research is focused on studying cyst nematode parasitism genes. Cyst nematodes secrete proteins that originate from two sets of gland cells (dorsal and subventral) through a stylet (hollow feeding tube) directly into root tissues to facilitate plant parasitism and induce a feeding cell that is necessary to sustain nematode growth and development. Stylet-secreted proteins are encoded by nematode parasitism genes. My project specifically involves a SCN parasitism gene called Hg2D01. Hg2D01 is a secreted protein with an unknown function that is specifically expressed in the dorsal gland. Hg2D01 encodes a 186 amino acid protein with a signal peptide. Hg2D01 maintains greater than 90% nucleotide and amino acid identity with the Hs2D01 gene from the closely related beet cyst nematode (Heterodera schachtii), a parasite of Arabidopsis. Through quantitative real-time PCR I have determined that Hg2D01 is expressed in all life stages throughout the 30 day life cycle of the nematode, excluding non-feeding stages such as eggs, juveniles, and adult males. The fact that Hg2D01 is only expressed in feeding life stages suggests that it likely plays an important role during nematode feeding cell formation. To test this hypothesis, we are conducting a number of studies to determine the function of Hg2D01 that include generating constructs for ectopic expression in Arabidopsis and utilizing an in-planta based RNAi approach to knock-out Hg2D01. Plants expressing dsRNA (double-stranded RNA) specifically targeting Hg2D01 have been infected with nematodes and effects on parasitism are being assessed. These studies are providing important insight into the mechanisms of nematode pathogenesis of plants and will contribute essential knowledge to our long-term goal of developing nematode-resistant crop plants.MU Monsanto Undergraduate Research Fellowshi

Characterizing the role of Glycine max NHL gene family members in plant-nematode interactions [abstract]

Abstract only availableSoybean cyst nematode (SCN; Heterodera glycines) is a microscopic parasitic roundworm of soybean that causes nearly $1 billion dollars in annual yield loss in the United States. SCN damages the plant by attaching itself to the soybean root system, where it forms a complex feeding site and drains vital nutrients from the plant. Naturally resistant soybean lines have been used as the primary strategy to manage SCN, because they have evolved a natural mechanism for resisting SCN infection. However, soybean resistance against SCN is derived from a small genetic base and repeated annual plantings of these same resistant lines has selected for populations of SCN that can reproduce on the resistant lines. Therefore, understanding the molecular mechanisms of how some soybean plants have the ability to naturally resist infection by SCN is critical for designing new strategies to improve crop plant resistance to SCN. My project focuses on soybean NDR1/HIN1-like (NHL) genes found to be expressed at higher levels specifically within SCN-induced feeding cells of resistant soybean as compared to susceptible soybean. To gain insight into the potential role of these genes in soybeans ability to resist SCN, full-length gene and cDNA sequences have been isolated using techniques known as genome walking and RACE PCR. RNAi and overexpression constructs have been generated to directly test the function of these genes in SCN resistance. To gain insight into the nematode-responsive regulation of each gene, the endogenous promoter sequences have been isolated and fused to the _-glucuronidase reporter gene for expression studies. This project will give insight into the mechanisms the soybean plant uses to defend itself against SCN infection and hopefully reveal crucial results which aid in the goal of developing SCN resistant soybean.Life Sciences Undergraduate Research Opportunity Progra

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