he genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus

Background: Pine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA. Results: Serratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/ transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors. Conclusions: This study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment.

Developmental biology and cytogenetics of B. xylophilus

The pine wood nematode Bursaphelenchus xylophilus reproduces bisexually: a haploid sperm fertilizes a haploid oocyte, and the two pronuclei rearrange, move together, fuse, and begin diploid development. Early embryonic events taking place in the B. xylophilus embryo are similar to those of Caenorhabditis elegans, although the anterior-posterior axis appeares to be determined oppositely to that observed for C. elegans. Thai is, in the B. xylophilus embryo, the male pronucleus emerges at the future anterior end, whereas the female pronucleus appeares laterally. To understand the evolution of nematode developmental systems, we cloned the full length of Bx-tbb-1 (beta tubulin) from B. xylophilus cDNA and attempted to apply reverse genetics analysis to B. xylophilus. Several lengths of double stranded RNA (dsRNA) for the Bx-tbb-1 gene were synthesized by in vitro transcription, and both B. xylophilus and C. elegans were soaked in dsRNA for RNAi. Both nematodes could suck up the dsRNA, and we could detect the abnormal phenotypes caused by Bx-tbb-1 dsRNA in C. elegans, but not in B. xylophilus. We suspect that systemic RNAi might be suppressed in B. xylophilus and are attempting to establish other methods for functionally analyzing B. xylophilus genes.