Identification and Characterization of Stress Responsive Genes in Soybean and Sunflower

Stress responsive genes encode proteins involved in plants’ response to abiotic and biotic stresses. Among such stress responsive proteins, proteins encoded by resistance genes (R genes) or nucleotide binding site-leucine-rich repeats (NBS-LRRs) and mitogen-activated protein kinases (MAPKs) are the major groups of proteins regulating biotic and abiotic stresses, respectively. Previous studies in Nepal’s lab at SDSU identified and characterized coiled coil (CC)-NBS-LRRs (CNLs), resistance to powdery mildew8 (RPW8)-NBS-LRRs (RNLs), NBS-LRR (NLs), and MAPK proteins in soybean. This study focuses on R and MAPK genes in the recently sequenced genome of sunflower as well as the toll-interleukin-1 receptor-like nucleotide-binding site leucine-rich repeat (TNL) R genes of soybean. This study also uses greenhouse experiments and RNA sequencing (RNA-seq) data to characterize stress responsive genes involved in interaction effects of soybean aphid (SBA) and soybean cyst nematode (SCN) interactions on soybean. Thus the major objectives of this dissertation work were to 1) explore the TNL genes in soybean and R (CNL, TNL, RNL) genes in sunflower genomes to assess how they may have evolved and their possible role in resistance against pathogens using available transcriptomic data, 2) identify and characterize MAPK genes in sunflower, and 3) characterize induced susceptibility effects of soybean-soybean aphid and interaction effects of soybean soybean aphid-soybean cyst nematode on soybean. In this dissertation, we used in silico approaches to report genome-wide identification and characterization of soybean TNL proteins as well as sunflower R and MAPK proteins. In order to achieve these objectives, numerous bioinformatics tools were utilized: hidden markov model (HMM) profilings were performed, and annotation of protein domains were conducted. Maximum Likelihood phylogenetic trees were constructed, and nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site ratios (Ka/Ks) as a proxy for selection pressure of R genes were calculated. In addition, chromosomal distribution, intron-exon architecture; synteny as well as gene expression patterns were assessed. In order to characterize stress responsive genes involved in defense responses, we used soybean aphid (Aphis glycines; SBA) and soybean cyst nematode (Heterodera glycines; SCN) to infest soybean cultivars. We conducted greenhouse experiments to characterize induced susceptibility effects of soybean-SBA interaction, and three-way interactions among soybean, SBA, and SCN. We utilized both demographic and genetic (RNA-seq) datasets to characterize the genes involved in such interactions using biotype 1, biotype 2 soybean aphids and HG type 0 SCN on soybean. FastQC, Btrim, Trimmomatic, Salmon, iDEP, MapMan tools were used to assess the quality, trim, map, assemble, visualize, pathway analysis and biological significance of RNA sequencing data to host genome. We identified an inventory of 117 of 153 regular TNL genes in soybean, and 352 NBS-encoding genes (100 CNLs, 77 TNLs, 13 RNLs, and 162 NLs), 28 MPKs and eight MKKs in sunflower through in silico analyses. R genes in soybean and sunflower formed several gene clusters suggesting their origin by tandem duplications. The selection pressure analysis revealed R genes experiencing purifying selection (Ka/Ks < 1) in both soybean and sunflower. Sunflower MAP Kinases revealed within and between clade functional divergence, and MKK3 orthologues were highly conserved across the species representing diverse taxonomic groups of the plant kingdom. Demographic data obtained from greenhouse experiments showed that induced susceptibility as initial feeding with virulent SBA (biotype 2) increased the population of subsequent avirulent SBA (biotype 1) in both susceptible and resistant cultivars. In the three-way interaction among soybean, SBA, and SCN, the number of SCN eggs was significantly greater on the susceptible cultivar and there was no effect in the resistant cultivar in the presence of SBA. The SBA population density was negatively affected by SCN populations. RNA-seq analysis in both studies have revealed differentially expressed genes (DEGs) and transcription factor (TF) binding motifs, which were enriched for various biological processes and pathways at different time points. The DEGs were common and unique in susceptible and resistant cultivars and treatments that were enriched for various biological processes and pathways. These DEGs were also functionally related to known defense mechanisms previously reported in various hostaphid and host-nematode systems. The responses to aphid biotype 1 infestation in the presence or absence of inducer population (biotype 2) at two time points (day1 and 11 post inducer infestation) revealed significant differences on the gene enrichment and regulation in SBA resistant and susceptible cultivars. For instance, enrichment analysis showed ‘response to chitin’, ‘lignin catabolic and metabolic process’, ‘asparagine metabolic process’, ‘response to chemical’ unique to treatment with no inducer population, whereas, ‘response to reactive oxygen species’, ‘photosynthesis’, ‘regulation of endopeptidase activity’ unique to treatment with inducer population. Likewise, Soybean-SBA-SCN interaction study showed enrichment of genes in ‘Plant Pathogen Interaction’ and ‘cutin, suberine, and wax biosynthesis’ pathways at 5 (days post SBA infestation) dpi; ‘isoflavonoid biosynthesis’ and ‘one carbon pool by folate’ pathways enriched at 30 dpi in SCN resistant and susceptible cultivars. Overall, the results from this study have improved the current understanding of diversity and evolution of MAPK and R genes in sunflower and soybean, as well as have first time reported a molecular characterization of induced susceptibility effects due to SBA on soybean, and soybean- SBA-SCN interactions, which has a direct implication in disease and pest management.