Abstract
The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen responsible for pine wilt disease (PWD), a devastating forest disease with a pathogenic mechanism that remains unclear. Autophagy plays a crucial role in physiological and pathological processes in eukaryotes, but its regulatory mechanism and significance in PWN are unknown. Therefore, we cloned and characterized three autophagy genes, BxATG5, BxATG9, and BxATG16, in PWN. BxATG9 and BxATG16 were efficiently silenced through RNA interference, and we found that BxATG16 positively regulated the expression of BxATG5. Silencing BxATG9 and BxATG16 severely inhibited feeding and reproduction in PWN, indicating that autophagy is essential for these processes. We then examined the expression patterns of these three autophagy genes in PWN under the stresses of α-pinene and H2O2, the main defense substances of pine trees, and during the development of PWD using quantitative reverse transcription polymerase chain reaction. The expression levels of BxATG5, BxATG9, and BxATG16 all significantly increased after nematodes were stressed with α-pinene and H2O2 and inoculated into pine trees, suggesting that autophagy plays an important role in the defense and pathogenesis of PWN. In this study, the molecular characteristics and functions of the autophagy genes BxATG5, BxATG9, and BxATG16 in PWN were elucidated.
Highlights
The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a widespread and important quarantine pest [1], and the causative agent of pine wilt disease (PWD), a devastating forest disease that causes enormous ecological and economic losses in many parts of Europe and Asia, China [2,3,4,5]
A Blastx search of sequencing results showed that the proteins encoded by these three genes shared more than 40% homology with the autophagy proteins ATG5, ATG9, and ATG16 of some other nematodes, confirming that the three genes cloned from PWN were all autophagy genes
The results showed that the molecular weights of BxATG5, BxATG9, and BxATG16 were 29.73, 90.59, and 59.07 kDa, respectively, with Iinsto. eJ.lMecotlr. iSccip. 2o0i1n9t,s20(,p3I7)69of 5.73, 5.35, and 5.06, respectively
Summary
The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a widespread and important quarantine pest [1], and the causative agent of pine wilt disease (PWD), a devastating forest disease that causes enormous ecological and economic losses in many parts of Europe and Asia, China [2,3,4,5]. ATG9 is the only autophagy gene that encodes a transmembrane protein, which is localized in the autophagic membrane or autophagy precursor in S. cerevisiae and may regulate autophagy by altering membrane trafficking This ubiquitin-like protein may provide a membrane source for autophagic vacuole formation and is necessary for autophagosome membrane formation [26]. To investigate whether these important autophagy pathways are present in PWN, we cloned and analyzed the homologs of three autophagy genes, ATG5, ATG9, and ATG16, in PWN. To determine whether autophagy is involved in the mechanism used by PWN to overcome pine defenses and successfully infect pine trees, we analyzed the expression patterns of the three aforementioned autophagy genes in PWN under α-pinene and H2O2 stress and during the development of PWD using quantitative reverse transcription PCR (qRT-PCR). The results provide valuable information about the defense mechanisms of PWN and pathogenesis of PWD
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