BPIs have been well studied in mammals as players in the defence against bacterial pathogens, and although BPI homologs have been identified in non-vertebrates, including C. elegans, BPIs have not been previously studied in plant parasitic nematodes (Krasity et al 2011). Here we show that BPI homologs are present in the genomes of cyst nematodes. Our work on Hs-BPI indicates that it is secreted from the nematode and plays an important role in parasitism. Thus, Hs-BPI is a nematode effector.
Searching for and analysing Hs-BPI homologues in sedentary plant parasitic nematodes (cyst nematodes- and root-knot nematode-species) led us to understand more about the evolution of BPI proteins in connection to the sedentary nematodes lifestyle. The phylogenetic analysis has shown that BPI evolved differently among cyst nematode species, clustering Heterodera spp. and Globodera spp. separately. Also, it clustered monocot-specific and dicot-specific species within Heterodera spp. cluster as well, a clustering nature that may have been influenced by the host-range differences (Fig. 3). The exclusion of the fifth in Gr-BPI has disrupted one of the 2 functional domains that were typically existing in all other tested homologues (Fig. 1).
İn our work we showed that Hs-BPI sequence contains a signal peptide domain and lacks any transmembrane domains, suggesting that Hs-BPI is a secreted protein. Additionally, we localized the corresponding mRNA in the nematode esophageal gland which would support the possibility that the protein is secreted. Usually, the expression of parasitism genes is elevated either in all parasitic stages or in some cases in specific stages depending on their function (Eves-van den Akker, 2016). In our study, the expression of Hs-BPI increased during in early post infective stage, indicating that it plays a role in parasitism. Host-induced gene silencing of Hs-BPI resulted in the decrease in the total number of infecting nematodes for 3 out of 4 transgenic lines. The fourth transgenic line (3.6) showed a trend towards a reduction in infecting nematodes, but it was not significant. Variability between lines due to the random insertion of the transgene is known in Arabidopsis (Valentine et al., 2012). Nonetheless, the three other transgenic lines showed clear results, and we are confident that Hs-BPI silencing reduces nematode parasitism.
Our study has identified Hs-BPI as a novel putative effector that help the invading nematodes during the early stages of parasitism (post infective J2 and J3). Because the size of the female and size of their feeding sites, the syncytia, was not affected in the RNAi lines, we hypothesize that Hs-BPI is probably involved in the early stages of the nematode-host interaction and does not have a role in the formation or maintenance of the feeding site. Previous work in bacteria showed that bacterial BPI can bind to Lipopolysaccharide (LPS)-binding protein (LBP) (Tobias et al 1997). In plant-microbe interactions, the bacterial LPS acts as a Microbe Associated Molecular Pattern (MAMP) that triggers Pattern-triggered Immunity (PTI) in Arabidopsis (Zeidler et al., 2004). Thus, it is possible that the cyst nematode secreted BPI is inhibiting a bacterial MAMP from triggering plant basal defences. Depleting Hs-BPI in the nematodes, as shown in the RNAi experiments, lowered H. schachtii infection levels. These results correspond to the decrease in G. pallida infection levels seen after applying LPS to potatoes (Reitz et al., 2000). It is noteworthy that our study was done under axenic conditions, which exclude the effect of any bacterial LPS. It is not clear whether the nematodes use BPI protein in the early parasitism to protect themselves from bacterial infections or to modulate the host defences in response to nematode MAMPs, or both.
Lastly, we cannot exclude that Hs-BPI has preserved its function in protecting the pre-infective nematodes within the soil from positive-gram bacterial infection while evolving its function as an effector. This may explain relatively higher level of Hs-BPI expression preinfective J2 compared to its expression level at a later parasitic life stage (15 dpi).
In this study, we identified a putative new effector, which can help understanding more about the plant nematode interaction while compatible nematode-parasitism. Also, we consider that succeeding in manipulating the expression of Hs-BPI using RNAi within a host plant, such as sugar beet, can negatively affect H. schachtii parasitism in a negative manner. Such an effect can in turn be used to enhance the resistance of the host plant toward nematode infection and increasing the yield.