Cloning of sft-4 and its influence on vitality and virulence of pine wood nematode, Bursaphelenchus xylophilus

Abstract

In our previous screening of the transcriptome of the causal agent of the devastating pine wilt disease, pine wood nematode (PWN, Bursaphelenchus xylophilus), after treatment with the nematicide fomepizole, Surfeit locus gene sft-4, which encodes a regulatory factor, was found to be downregulated. In situ hybridization results showed that the sft-4 was continuously expressed from egg to adult and was especially high in the reproductive system. Here in a study of the effect of RNA interference (RNAi) of sft-4 and recombinant SFT-4 on PWN activity, treatment with sft-4 dsRNA inhibited feeding, reproduction, oviposition and egg hatching of PWN with the greatest inhibition on reproduction and oviposition, whereas recombinant SFT-4 had the opposite effect. In addition, RNAi of sft-4 changed the female–male ratio and lifespan of PWN. In bioassays of PWNs, with RNAi of sft-4 on seedlings and 2-year-old Pinus thunbergii trees, none of the treated plants developed symptoms during the monitoring period, indicating that virulence of PWNs was either significantly weakened. These results indicate that the influence of sft-4 on PWN pathogenicity may be mainly through regulating reproductive function of PWN and its lifespan.

Keywords

Black pine / Pinaceae / Bursaphelenchus xylophilus / Sft-4 / In situ hybridization / RNAi / Pathogenicity

References

[1]	Akiba M, Ishihara M, Sahashi N, Nakamura K, Ohira M, Toda T (2012) Virulence of Bursaphelenchus xylophilus isolated from naturally infested pine forests to five resistant families of Pinus thunbergii. Plant Dis 96(2):249–252
[2]	Armes N, Fried M (1996) Surfeit locus gene homologs are widely distributed in invertebrate genomes. Mol Cell Biol 16(10):5591–5596
[3]	Balklava Z, Pant S, Fares H, Grant BD (2007) Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic. Nat Cell Biol 9(9):1066-U35
[4]	Barbosa P, Faria JMS, Mendes MD, Dias LS, Tinoco MT, Barroso JG, Pedro LG, Figueiredo AC, Mota M (2012) Bioassays against pinewood nematode: assessment of a suitable dilution agent and screening for bioactive essential oils. Molecules 17(10):12312–12329
[5]	Belden WJ, Barlowe C (2001) Role of Erv29p in collecting soluble secretory proteins into ER-derived transport vesicles. Science 294(5546):1528–1531
[6]	Booth C (1971) Chapter II fungal culture media. Methods Microbiol 4:49–94
[7]	Chen G, Shan L, Zhou LX, Tang GY, Bi YP (2011) The comparison of different methods for isolating total RNA from peanuts. Chin Agric Sci Bull 27(1):214–218 (in Chinese)
[8]	Feng YQ, Rui L, Wang XY, Wu XQ (2022) Adaptation of pine wood nematode, Bursaphelenchus xylophilus, early in its interaction with two Pinus species that differ in resistance. J for Res 33:1391–1400
[9]	Futai K (2013) Pine wood nematode, Bursaphelenchus xylophilus. Annu Rev Phytopathol 51:61–83
[10]	Giallongo A, Yon J, Fried M (1989) Ribosomal protein L7a is encoded by a gene (surf-3) within the tightly clustered mouse surfeit locus. Mol Cell Biol 9(1):224–231
[11]	Grant B, Hirsh D (1999) Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell 10(12):4311–4326
[12]	Guo QQ, Du GC, Qi HT, Zhang YA, Yue TQ, Wang JC, Li RG (2017) A nematicidal tannin from Punica granatum L. Rind and its physiological effect on pine wood nematode (Bursaphelenchus xylophilus). Pestic Biochem Physiol 135:64–68
[13]	Howe KL, Bolt BJ, Cain S, Chan J, Chen WJ, Davis P, Done J, Down T, Gao S, Grove C, Harris TW, Kishore R, Lee R, Lomax J, Li YL, Muller HM, Nakamura C, Nuin P, Paulini M, Raciti D, Schindelman G, Stanley E, Tuli MA, Auken KV, Wang D, Wang XD, Williams G, Wright A, Yook K, Berriman M, Kersey P, Schedl T, Stein L, Sternberg PW (2016) WormBase: expanding to enable helminth genomic research. Nucleic Acids Res 44(D1):D774–D780
[14]	Howe KL, Bolt BJ, Shafie M, Kersey P, Berriman M (2017) WormBase ParaSite—comprehensive resource for helminth genomics. Mol Biochem Parasit 215:2–10
[15]	Lee SC, Lee HR, Kim DS, Kwon JH, Huh MJ, Park IK (2020) Emamectin benzoate 9.7% SL as a new formulation for a trunk-injections against pine wood nematode, Bursaphelenchus xylophilus. J for Res 31:1399–1403
[16]	Li YL, Fan CJ, Jiang XH, Tian XY, Han ZM (2021) Bursaphelenchus xylophilus: an important pathogenic factor of pine wilt disease and its relationship with Bursaphelenchus mucronatus. Plant Dis 105(10):3055–3062
[17]	Liu MJ, Hwang BS, Jin CZ, Li WJ, Park DJ, Seo ST, Kim CJ (2019) Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest Manag Sci 75(6):1585–1593
[18]	Mashkevich G, Repetto B, Glerum DM, Jin C, Tzagoloff A (1997) SHY1, the yeast homolog of the mammalian surf-1 gene encodes a mitochondrial protein required for respirati-on. J Biol Chem 272:14356–4364
[19]	Meng FL, Li YX, Wang X, Feng YQ, Liu ZK, Zhang W, Zhang XY (2019) Thumatin-Like Protein-1 gene (Bx-tlp-1) is associated with the pathogenicity of Bursaphelenchus xylophilus. Phytopathology 109(11):1949–1956
[20]	Meng FL, Li YX, Liu ZK, Feng YQ, Wang X, Zhang XY (2022) Expression of the thaumatin-like protein-1 gene (bx-tlp-1) from pine wood nematode Bursaphelenchus xylophilus affects terpene metabolism in pine trees. Phytopathology 112(4):888–897
[21]	Mitrovic S, Ben-Tekaya H, Koegler E, Gruenberg J, Hauri HP (2008) The cargo receptors Surf4, endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53, and p25 are required to maintain the architecture of ERGIC and Golgi. Mol Biol Cell 19(5):1976–1990
[22]	Qiu XW, Wu XQ, Huang L, Ye JR (2016) Influence of Bxpel1 gene silencing by dsRNA interference on the development and pathogenicity of the pine wood nematode, Bursaphelenchus xylophilus. Int J Mol Sci 17(1):125
[23]	Reeves JE, Fried M (1995) The surf-4 gene encodes a novel 30-KDa integral membrane-protein. Mol Membr Biol 12(2):201–208
[24]	Saegusa K, Sato M, Morooka N, Hara T, Sato K (2018) SFT-4/Surf4 control ER export of soluble cargo proteins and participate in ER exit site organization. J Cell Biol 217(6):2073–2085
[25]	Shanmugam G, Lee SK, Jeon J (2018) Identification of potential nematicidal compounds against the pine wood nematode Bursaphelenchus xylophilus through an in silico approach. Molecules 23(7):1828
[26]	Takai K, Suzuki T, Kawazu K (2003) Development and preventative effect against pine wilt disease of a novel liquid formulation of emamectin benzoate. Pest Manag Sci 59(3):365–370
[27]	Viglierchio DR, Schmitt RV (1983) On the methodology of nematode extraction from field samples: Baermann funnel modifications. J Nematol 15(3):438–444
[28]	Wang LS (2019) Physiological function of alcohol dehydrogenase gene in pine wood nematode. Dissertation, Qingdao University. (In Chinese)
[29]	Wang LS, Zhang TT, Pan ZS, Lin LL, Dong GQ, Wang M, Li RG (2019) The alcohol dehydrogenase with a broad range of substrate specificity regulates vitality and reproduction of the plant-parasitic nematode Bursaphelenchus xylophilus. Parasitology 146(4):497–505
[30]	Wang BW, Hao X, Xu JY, Wang BY, Ma W, Liu XF, Ma L (2020) Cytochrome P450 metabolism mediates low-temperature resistance in pine wood nematode. FEBS Open Bio 10(6):1171–1179
[31]	Wang M, Du GC, Fang JN, Wang LS, Guo QQ, Zhang TT, Li RG (2022) UGT440A1 is associated with motility, reproduction, and pathogenicity of the plant-parasitic nematode Bursaphelenchus xylophilus. Front Plant Sci 13:862594
[32]	Xu XL, Wu XQ, Ye JR, Huang L (2015) Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea). Int J Mol Sci 16(3):5216–5234
[33]	Yu LZ, Wu XQ, Ye JR, Zhang SN, Wang C (2012) NOS-like-mediated nitric oxide is involved in Pinus thunbergii response to the invasion of Bursaphelenchus xylophilus. Plant Cell Rep 31(10):1813–1821
[34]	Zhou X, Chen SN, Lu F, Guo K, Huang LL, Su X, Chen Y (2021) Nematotoxicity of a Cyt-like protein toxin from Conidiobolus obscurus (Entomophthoromycotina) on the pine wood nematode Bursaphelenchus xylophilus. Pest Manag Sci 77(2):686–692