The project started a week after the beginning of the first COVID-19 lockdown in the United Kingdom, in April 2020. The focus was therefore given to the in silico approaches that were not requiring access to the lab. The first work performed was to analyse the lifestage-specific trans-kingdom RNAseq timecourse generated. This timecourse not only provided a reference dataset to the community but also was used to identify co-expression clusters enriched in effector-encoding genes. Meanwhile, several hundred Heterodera schachtii’s gland cells were isolated by collaborators from the Iowa State University and an RNAseq library was generated from the gland cells RNAs extracted. We conducted the gland cells RNAseq analysis and used the gland cell-specific expression dataset obtained to refine our list of candidate regulators, focusing on those presenting a high level of expression in the gland cells.
Once back in the laboratory, an RNAi-mediated silencing of the four most interesting candidates were done before performing a comparative RNAseq analysis in order to identify regulated genes. This allowed the identification of a SUbventral Gland Regulator, coined the SUGR, that was further analysed. The SUGR regulates a set of 297 genes, among them a large set (45%) of gene encoding for secreted proteins, including known and candidate subventral gland effectors. Further analysis were conducted to confirm this hypothesis, among them an in situ hybridization on regulated known and candidate subventral gland effectors in order to localize their expression. The majority of the selected genes were found specifically expressed in the subventral glands, or in both the subventral and dorsal glands. To go further in the analysis of the SUGR and assess its impact on Heterodera schachtii’s pathogenicity, a root penetration assay was performed where SUGR-silenced nematodes were used to infect mustard roots and compared to the appropriate control. This showed the silencing of the SUGR impairs the ability of the nematode to penetrate the plant root and further confirms the SUGR is critically important at the early stage of infection, when the J2s secrete subventral effectors to digest the plant cell wall and enter the plant root.
As it is known that root-derived molecules attract J2s, a similar signal might also activate the SUGR-encoding gene expression, consistent with its early expression during the plant penetration stage. H. schachtii’s juveniles were soaked in mustard roots filtrate before being flash-frozen in liquid nitrogen. SUGR-encoding gene expression was measured by real time qPCR in the root-filtrate and water-soaked nematodes used as a control. This showed the SUGR-encoding gene was activated by the mustard root filtrates, suggesting one or several molecules were detected by the nematode as an pathogenicity activation signal.
Additional analysis performed on the root filtrates showed the activation signal is heat resistant, resistant to proteolysis and has a maximum molecular weight of 3kDa. Biochemical characterization of this root extract may allow the identification of the molecule that activates H. schachtii pathogenicity. Interestingly, soaking the juveniles into root extracts from non-host plants has opposite effect on the SUGR-encoding gene, triggering a repression of its expression, implying H. schachtii can distinguish between host and non-host and responds accordingly.
The identification of a subventral gland regulator and its characterization offers two different possibilities for control strategies. As the genes directly regulated by the SUGR are involved into the plant penetration process, preventing the SUGR from binding to its target genes would directly impact the nematode’s pathogenicity. Alternatively, identifying plant-derived molecules able to repress the SUGR-encoding gene expression would similarly prevent the nematode to efficiently penetrate the root. Being plant-derived, their usage would not be impacted by the current and future EU regulations on the usage of synthetic pesticides as pest control strategies.
