Identification of Defense-related Proteins in the Root Necrotic Mutant rn1 in Soybean

The root necrotic mutant rn1 in soybean is caused presumably by insertion of the endogenous CACTA-element, Tgm9. It has been shown that excision of Tgm9 from the W4 locus resulted in a high frequency of mutation that negatively regulates the cell death process in rn1 mutants. Tgm9 in T322 genotype, generated from an Asgrow line, is highly active and we have recently demonstrated that the element transposes to all soybean chromosomes with a preference for genic regions. The rn1 mutant was identified from Tgm9-induced mutant population and multiple mutations mapped to rn1 locus and are allelic.  Earlier it was shown that the mutant gains tolerance to Phytophthora sojae, the oomycete root pathogen. In this study we investigated the proteomes of necrotic and healthy roots identified from segregants of soybean plants heterozygous for Rn1. Proteins from roots of 5 to 7 day-old seedlings exhibiting either root necrosis or no root necrosis were isolated in the presence of a cocktail of phosphatase inhibitors. Protein samples were prepared individually from three biological replications for both (i) necrotic and (ii) non-necrotic roots and labeled with six iTRAQ reagents with molecular weights varying from 126 to 131. Six labeled protein samples (two treatments X three replications) were mixed prior to conducting LC-ESI-MS/MS. The proteins that accumulated in necrotic roots include several types of pathogenesis related proteins, leucine-rich repeat containing protein, trypsin and protease inhibitors, enzymes involved in antimicrobial compound biosynthesis, and glutathione S-transferases. Classification of phosphoproteins based on biological processes revealed that the identified phosphoproteins in necrotic roots are involved in signal transduction; whereas, in healthy roots are involved in carbohydrate metabolic (80%) and transport (20%) processes.