Analysis of soybean gene regulation reveals regulatory mechanisms of Rhg1-mediated resistance to soybean cyst nematode

Yield suppression due to soybean cyst nematode (SCN) infestation has been estimated to cost one billion US dollars per year. The majority of SCN-resistant soybean varieties carry the same resistance locus (Rhg1) which is a copy number polymorphism of 1-11 copies of a 31.2kb genomic region. Since Rhg1-mediated resistance is so widely used, this research project aims to identify regulatory mechanisms in soybeans by which copy number variation (CNV) at the Rhg1 locus mediates nematode resistance. We performed genome-wide analyses using high-throughput methods in three studies: The first study focused on RNA-seq profiling to identify early response genes in SCN-infected roots. We found gene expression changes vary among susceptible (Glycine max cv. Williams 82; a single copy) and resistant soybeans (G. max cv. Peking; three copies of Rhg1, G. max cv. Fayette; 11 copies and a wild relative G. soja PI 468916; non-Rhg1 resistance; a single copy). This study demonstrates the importance of the early plant responses to migrating nematodes in pathogenicity determination. The most extensive resistant transcriptome reaction was observed in PI 468916, where the resistant response was qualitatively different from that of commonly used G. max varieties, providing further evidence for the benefits of the PI 468916 as a genetic source to improve the genetic diversity of SCN resistance in soybean. In the second study, we investigated transcriptional differences between uninfected roots of three Fayette isolines carrying 8-11 copies of the Rhg1 repeat and showing different levels of resistance to SCN, using RNA-seq and Fluidigm-based qRT-PCR. A gene co-expression network suggested key genes involved in signal transduction and ethylene-mediated signaling pathway in a complex transcriptional effect of the repeat sequence. We found a positive correlation between copy number and expression levels of phytoalexin biosynthesis-related genes, which were induced throughout the early stages of SCN infection. This result suggested increased expression of these genes in soybeans harboring Rhg1 repeats could enhance Rhg1-mediated SCN resistance. The third study revealed potential regulatory mechanisms of genes in copy-number variant Fayette lines, using small RNA sequencing (sRNA-seq) and whole genome bisulfite sequencing (WGBS). We identified phased siRNAs that may regulate defense-related genes in both cis and trans, leading to complex alterations in the sRNA regulatory network in Rhg1-mediated SCN resistance. The WGBS analysis provided preliminary evidence for epigenetic control of gene expression and transposon silencing initiated by the Rhg1 repeat and mediated by sRNAs via RNA-directed DNA methylation (RdDM) process. The results suggested that manipulation of sRNAs and DNA methylation could be an alternative method for fine-tuning of gene expression to enhance SCN resistance in soybeans with CNV at the Rhg1 locus