Identification of host genes with a role in sex determination of the plant parasitic cyst nematode, <em>Heterodera schachtii</em>

Abstract

Cyst nematodes are biotrophic parasites that infect plant roots and cause physiological and structural modifications leading to the formation of syncytial nurse cells. They are sexually dimorphic but the sexual phenotype appears only after feeding has started. Because the sex ratio varies with environmental conditions and the genotype of the host plant, it is generally assumed that both factors play a decisive role in sex determination of cyst nematodes. Under favorable conditions, more females develop, whereas mainly male nematodes develop under adverse conditions. Since the presence of females determines reproduction and thereby soil infestation levels, the sex ratio is very important in agricultural practices: resistant crop cultivars, which suppress female formation, are often used to control cyst nematodes. However, the molecular mechanisms underlying this phenomenon have remained mostly unknown. In this study, a comparative transcriptomic analysis of male- and female-associated syncytia (MAS and FAS, respectively) of cyst nematode <em>Heterodera schachtii</em> was performed at early stages of infection to identify the host factors influencing sexual development. Therefore, a method was developed to predict the sex of developing nematodes on the basis of an empirically developed growth curve. In this way, syncytia associated to future males or females could be sampled at an early developmental stage, when typical sex-related traits of the juveniles were not yet visible. The transcriptome of the nurse cells was analyzed from samples taken by laser capture microdissection from root tissue of host plant <em>Arabidopsis thaliana</em>. A novel protocol was developed in order to improve the quality of the sampled RNA. The data revealed that gene categories belonging to cell wall biosynthesis, modification and metabolism were particularly up-regulated in FAS, whereas the transcriptome of MAS showed an enrichment of gene categories related to defense and nutrient deficiency. These results were further complemented by ultrastructural analyses and gene regulation studies via promoter::reporter lines. All the data point out that changes in the cell wall, suppression of host defense and availability of nutrients are important factors promoting the development of female nematodes. The data led to the assumption that knocking out genes that are differentially up-regulated might influence sexual differentiation of the nematodes. Therefore, knockout mutants for ten candidate genes (<em>CWLP1, BGLU28, BHLH101, DIN2, MLP like protein 423, SHVL3, IRX12, LAC11, LNG1, LTPG6</em>) were used to study their importance via nematode infection assays. Three mutants (<em>ltpg6, lng1</em> and <em>irx12</em>) of genes, which are differentially up-regulated in FAS, showed a significant decrease in the number of females as compared to the control. On the other hand, the number of males increased significantly in two of them (<em>lng1 </em>and<em> irx12</em>). In conclusion, our study supports the role of the host plant on the sex determination of cyst nematodes. Manipulation of the identified host factors may provide new options for breeding resistant crop plants in future.