Silencing of a 14-3-3 protein expression does not affect Pratylenchus brachyurus pathogenicity in axenic carrot cylinders

Introduction: The 14-3-3 protein family are highly conserved eukaryotic-specific proteins involved in several cellular and biochemical processes through interaction with phosphorylated target proteins. It has been described that 14-3-3 isoforms plays important roles in the parasitism establishment of Heterodera glycines and other economically important phytonematodes. We evaluated the effect of a specific P. brachyurus 14-3-3 silencing on its pathogenicity in carrot cylinders. Methods: P. brachyurus mixed stages were treated with dsRNA (2mg.mL-1) targeting a 14-3-3 isoform in a basic soaking solution in the presence of the neurostimulant octopamine (50mM). As a control, non-target dsRNA-GFP was applied. After incubation at 22ºC for 16h, the nematodes were washed in a 0,1% ampicillin solution, inoculated in axenic carrot cylinders (80 nematodes/cylinder), and maintained by 80 days for determination of reproductive factor (Rf). Additionally, the expression profile of the gene across the infection cycle in soybean and in the nematodes was performed by RT-qPCR. Results: No significant difference was observed in Rf values between the treatments (dsRNA-GFP and control) after 80dpi. As described in other organisms, the proteins of this family can play several roles in the host-parasite communication. An explanation is that in carrot cylinders this communication might be compromised after all the processing, and the cylinders would work just as a culture medium with no immune response. Therefore, communication would not be necessary and no longer silencing would not result in phenotypic changes. The activation of the 14-3-3 in P. brachyurus only after the contact with the soybean plants was demonstrated by RT-qPCR, corroborating this hypothesis. We also observed a high heterogeneity between the repetitions of both treatments that could be caused by the chemical differences of the cylinders. Conclusion: The carrot cylinder bioassay does not constitute an efficient method for gene function studies of potential host-parasite communication genes.