Improving Soybean Resistance to Cyst Nematodes and Fusaria: Near Isoline and Transgenic Analyses of the Rhg1/Rfs2 Locus and Identification of Proteins That Bind to Receptor Kinases

Author

Ali Srour, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2012

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Lightfoot, David

Abstract

Soybean is one of the most important grain legumes grown in US and worldwide, and is a major component of human and animal protein diets. Despite improvements in management practices, and the introduction of improved soybean cultivars, soil borne pathogens continue to cause tremendous yield loss in soybean production each year. Among soil borne pathogens; Soybean Cyst Nematode (SCN) or Heterodera glycines together with Sudden Death Syndrome (SDS) induced by Fusarium virguliforme are responsible for the most damages in soybean fields. The most effective way to control these two pathogens is to develop resistant cultivars. Resistance to any population (HgType) of H. glycines, requires a functional allele at rhg1/Rfs2 locus. The rhg1/Rfs2 gene encodes a receptor-like kinase (RLK) protein. By analysing near isogenic lines (NIL) segregating for rhg1/Rfs2, rhg1-like loci were found at other locations most conservedly on LG B1. While the nature of rhg1 allele was thought to be recessive, heterozygous NIL segregating at the rhg1 locus showed that the resistant allele was dominant. Rhg1 was also inferred to be multigeneic due to absence of recombination between the RLK and other 2 genes. Functional and structural analyses were conducted on the leucine rich repeat (LRR) from RLK protein encoded by GmRLK18-1 within the Rhg1/Rfs2 locus. The LRR of GmRLK18-1 showed a high binding affinity to CLE-like peptides found in both nematode secretions and plant developmental control. Crosslinking assays and native gel analysis of GmRLK18-1-LRR validated its model as a crystal homo-dimer. Larger proteins were also shown to bind the LRR domain, in far-Western analyses both methionine synthase and cyclophilin bound strongly to the LRR domain. Homology and ab-initio modeling of the LRR domain of the GmRLK18-1 was predicted as both a monomer and a homodimer containing intrinsically unstructured regions. Amino acid substitutions found among GmRLK18-1 allotypes A87V, Q115K and H274N were predicted to play crucial roles in protein function and stability. The receptor like kinase (RLK) GmRLK18-1 within the Rhg1/Rfs2 locus underlies a pleiotropic resistance to both SCN and SDS. The resistance allele was shown to be dominant in both heterozygous NILs at Rhg1/Rfs2 and transgenics (hetero- or hemi-zygous). The RLK was found to provide a partial resistance to SCN and importantly a nearly complete resistance to both root and leaf symptoms of SDS. In the presence of Rhg4, the RLK-transgenic plants developed nearly full resistance to SCN. Therefore the RLK was proven to underlie a major portion of the Rhg1/Rfs2 locus.