Anwer, Muhammad Arslan: Genome-wide-association studies (GWAS) on the interaction between the cyst nematode Heterodera schachtii and Arabidopsis thaliana. - Bonn, 2018. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Muhammad Arslan Anwer}},
title = {Genome-wide-association studies (GWAS) on the interaction between the cyst nematode Heterodera schachtii and Arabidopsis thaliana},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2018,
month = oct,

note = {Plant-parasitic cyst nematodes are obligate sedentary parasites that infect the roots of a wide range of host plants. In research, the beet cyst nematode Heterodera schachtii infecting Arabidopsis thaliana is often used as a model. H. schachtii is sexually dimorphic and differentiation into male and female is strongly influenced by interactions with the environment. Under favorable conditions the unsexed infective juveniles (J2) tend to develop into females, whereas they rather form males under adverse conditions. After hatching from the egg, J2 enters the root tissue to find out a suitable infection site. Once a feeding site is selected, they become immobile and release specific proteinaceous effectors triggering the formation a syncytial nurse cell system that serves as the only nutrient supply throughout the nematode life cycle. To identify host loci underlying variation in susceptibility, we performed a genome-wide association study (GWAS) using different accessions of Arabidopsis that were exposed to H. schachtii infections. Three different susceptibility parameters were examined: average numbers of female per plant, average numbers of male per plant and their subsequent female to male sex ratio. Based on female- to -male sex ratio, we categorized all accessions into three groups: lowly susceptible, moderately susceptible and highly susceptible. GWAS revealed a novel QTL allele at chromosome four underlying potential candidates associated to sex ratio variations. Using the physical map of this locus, we selected five candidate genes from the potential QTL: GDSL-lipase (At4g18970), PPR-protein (pentatricopeptide repeat-protein; At4g18975), AtS40-3 (senescence-associated protein; At4g18980), XTH-29 (xyloglucan endotransglucosylase 29; At4g18990) and IWS-2 (interacts with SPT6; Atg19000). Finally, extreme bulks showing lowest and highest sex ratio were selected for detailed functional characterization of candidate genes. At first, we measured the expression of candidate genes among four extreme accessions showing lowest and highest sex ratio. This analysis revealed that out of the five, expression of two genes, PPR and AtS40-3 was strongly down-regulated in lowly susceptible accessions compared with highly susceptible accessions. In comparison, expression of GDSL-lipase, iWS2, and XTH29 was unchanged among all tested accessions. Together, these data suggest that changes in expression level of PPR and AtS40-3 might be correlated with variation in nematode sex ratio. AtS40-3 and PPR are divergent genes that are organized head-to-head (in opposite orientations) in a non-overlapping manner. Because of their specific regulation, we analyzed AtS40-3 and PPR for sequence variations in their common promoter region. Notably, we observed a consistent deletion in the promoter in lowly susceptible accessions, whereas no such deletions were present in highly susceptible accessions. Based on these data, we hypothesized that the deletions in the promoter region might be one of the reasons for their strong down-regulation in root tissues in lowly susceptible accessions. To further investigate this hypothesis, we performed an in silico analysis of the commonly deleted 28 bp promoter region from susceptible line Col-0 for the occurrence of important cis-acting element. Our analysis revealed the presence of some prominent transcription factor binding sequences including a typical TATA box. To understand the mechanism that regulates the differential promoter activity for AtS40-3 between lowly and highly susceptible lines, the 1500 bp promoter upstream of ATG for AtS40-3 from the low susceptibility accession Xan-1 (carrying a deletion of 28 nt) and the susceptible accession Col-0 (without deletion) was cloned and used to drive the expression of GFP. The expression of GFP was analyzed via a transient expression system in N. benthamiana through real-time qPCR. Indeed, we found a significantly reduced expression of GFP driven by the promoter cloned from the Xan-1 accession (low susceptibility to nematodes) compared with that from Col-0 (high susceptibility to nematodes). We obtained the T-DNA-inserted loss of function mutant lines of AtS40-3 and characterized these lines via nematode infection assays. Remarkably, a prominent increase in average number of males and decrease in average number of females was observed. Consequently, female-to-male sex ratio was also changed in Xan-1 compared with Col-0. Taken together, this study provides insights into genes and factors that influence the sexual variation in cyst nematode. Further exploitation of these factors may lead to development of novel resistance resources against nematode in crop plants.},
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