RNA-Sequencing of Heterorhabditis nematodes to identify factors involved in symbiosis with Photorhabdus bacteria

Abstract

Background: Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria.

Results: A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified.

Conclusions: The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.

Keywords: Biofilm; Early-adult stage; Heterorhabditis; Immunity; Photorhabdus; RNA-sequencing; Symbiosis; Transcriptome.

Fig. 1

A The general life cycle of the entomopathogenic nematode Heterorhabditis on an insect (made with help of BioRender). B The diagrammatic representation of initial stages of the maternal transmission of the symbiont bacteria. When the IJs exit diapause, they regurgitate the symbiont bacteria and become completely bacteria free before reassociating with their symbiont bacteria later on during the development. The reassociation of symbiont bacteria results in formation of a persistent symbiont bacterial biofilm in the posterior intestinal cells on the nematode. The green colour shows bacteria, arrow indicates direction of bacterial movement, and the yellow shaded box indicates early-adult stage of nematode. C Early-adult stage of symbiotic and axenic Heterorhabditis bacteriophora used for RNA-sequencing. The image was created by overlapping DIC and fluorescence images of axenic early-adult devoid of bacterial biofilm and the bacterial biofilm in the posterior intestine of early-adult stage of symbiotic nematode

Fig. 2

Gene Ontology (GO) annotation of differentially expressed transcripts identified in RNA-seq experiment. A. up-regulated transcripts B. down-regulated transcripts. WEGO (Web Gene Ontology Annotation Plot) tool was used for visualizing, comparing and plotting GO annotation results

Fig. 3

Pathways enriched in identified transcripts. A. KEGG pathway annotations of differentially expressed transcripts. B. KEGG pathway annotations of symbiotic-nematode specific transcripts. KEGG Automatic Annotation Server (KAAS) (https://www.genome.jp/kegg/kaas/) was used for pathway annotation analyses with permission from Kanehisa Laboratories, Japan (KEGG Copyright Permission reference number 221327) [16, 17]

Fig. 4

GO annotation of symbiotic-nematode specific transcripts. WEGO (Web Gene Ontology Annotation Plot) tool was used for visualizing, comparing and plotting GO annotation results

Fig. 5

Venn diagram showing specific genes pulled out from symbiotic-nematode specific transcriptome (based on GO annotations) putatively involved in responses to bacteria, immune and defense responses

Fig. 6

qRT PCR validation of expression patterns of the various differentially expressed and unique transcripts identified in RNA-seq experiment. Most of the tested transcripts conformed to their RNA-seq expression patterns

Fig. 7

A model presenting symbiotic events and the transcriptomic responses in early-adult stage of maternal nematodes, and their interpretation in relevance to symbiosis with Photorhabdus bacteria. Colonization processes involve two-way crosstalk between host and symbiont. From the nematode host side, based on gene expression profiling, expression of immune system components which are involved in the recognition of bacteria, bacteria-derived molecules and detection of any cellular perturbations caused by bacteria are observed. It leads to activation of signalling pathways and results in an array of host immune and defence responses such as autophagy, apoptosis and production of antimicrobial proteins. These effectors and mechanisms regulate symbiont bacterial numbers and help achieve successful symbiosis. INT9L/INT9R – posterior nematode intestinal cells; RGC- rectal gland cells