Identification and functional characterization of core effectors from cyst nematodes through comparative genomics

Abstract

Plant parasitic nematodes (PPNs), are one of the most important constraints in agricultural production causing substantial losses in quantity and quality of food. The economically most important PPNs are the sedentary endoparasitic root-knot and cyst nematodes. Heterodera sacchari, a sedentary endoparasitic cyst nematode, is an important parasite of sugarcane and rice crops and is an emerging threat to intensive upland rice cultivation especially in West Africa. Like other species of cyst nematodes, pre-parasitic infective juveniles (J2s) of H. sacchari penetrate the host roots and induce a metabolically active syncytial feeding cell system in the stele. To gain more detailed insights into the H. sacchari-rice interaction, we established an in vitro culture system for H. sacchari on Nipponbare rice in pluronic gel. We confirmed that unlike other cyst nematodes, H. sacchari reproduces by parthenogenesis and males are rare except under high stress conditions. At 25 days post inoculation (dpi), developing females start to produce eggs inside the body. At around 40 dpi, the cuticle of the females started to tan, and the females became darker as the life cycle progressed. We found that H. sacchari can successfully complete its life cycle in 7-9 weeks at 25 °C on Nipponbare rice in pluronic gel. Results of hatching assays showed that hatching of juveniles from eggs contained in cysts was stimulated by 3mM Zinc Chloride but not by rice root exudates. The established in vitro culture system can be efficiently used to collect post-infective stages of nematode and host tissue at different time points for various studies and also to screen different rice cultivars for nematode resistance/susceptibility. Using in vitro material, we generated transcriptome data from two different life stages (pre-parasitic J2s and 15 days post-infection) using Illumina MiSeq next-generation sequencing with a total of 17,086,132 paired-end 2x 250 bp reads.<br /> A multi-gene phylogenetic analysis using Core Eukaryotic Gene Mapping Approach (CEGMA) genes from a range of nematodes showed that H. sacchari and the cereal cyst nematode H. avenae (both monocot parasites) evolved from a common dicot-parasitic ancestor. Comparisons of putative effector and non-effector genes from H. sacchari to their closest homologues in potato cyst nematode G. rostochiensis showed that effector genes are more divergent than non-effector genes. We found some effectors with conserved expression profile and likely function (e.g. cellulase, chorismate mutase, 19C07). Transcripts encoding genes similar to the SPRYSEC family of effectors were also present in H. sacchari dataset, suggesting conservation of this effector family across cyst nematodes. The transcriptome of H. sacchari contains seven transcripts that encode proteins similar to CLE effectors. The CLE like peptides encoded by H. sacchari are more similar to rice CLEs than any other plant or nematode CLEs. We also demonstrated that exogenous application of H. sacchari CLE peptide induced a short root phenotype in rice while peptides from the dicot parasite H. glycines did not produce any phenotype. These results provide insights in effector evolution that co-occurred with the transition from a dicot-parasitic to a monocot-parasitic lifestyle.