Resistance genes mediate differential resistance to pine defensive substances α-Pinene and H₂O₂ in Bursaphelenchus xylophilus with different levels of virulence

Abstract

The pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, is the pathogen of pine wilt disease (PWD) which can devastate forests. PWN can be of high or low severity and the mechanisms underlying the differences in virulence are unclear. Therefore, it is necessary to study the relationship between differentiation of PWN severity and its resistance to the main defensive substances of pine species (i.e., α-pinene and H₂O₂). The feeding rate and fecundity of PWN was examined at different levels of virulence under conditions of α-pinene and H₂O₂ stress. Moreover, the expression patterns of the main resistance genes of PWN with different virulence were determined under conditions of α-pinene and H₂O₂ stress. The feeding rate and fecundity of the high virulence strain AMA3 were higher than those of the low virulence strain YW4. The expression levels of the autophagy gene BxATG5, cytochrome P450 gene BxCYP33D3, and glutathione S-transferase genes BxGST1 and BxGST3 in AMA3 increased significantly upon exposure to α-pinene for 2 h, while these genes showed smaller degrees of upregulation in YW4. Under conditions of H₂O₂ stress, the expression levels of BxATG5, catalase genes Bxy-ctl-1and Bxy-ctl-2, and the 2-cysteine peroxiredoxin gene BxPrx in AMA3 were higher than those in YW4. These findings suggest that high virulence PWN has greater resistance to pine defensive substances α-pinene and H₂O₂ than low virulence PWN, and resistance genes mediate the differential resistance of PWN strains. This study will contribute to the clarification of the mechanism underlying virulence differentiation of PWN and will advance understanding of the pathogenic mechanism of PWD.

Keywords

Pine wood nematode / Virulence / Defensive substances / Autophagy gene / Detoxification gene / Antioxidant gene

References

[1] Aikawa T, Kikuchi T. Estimation of virulence of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) based on its reproductive ability. Nematology, 2007, 9(3): 371-377.

[2] Baker CJ, Orlandi EW. Active oxygen in plant pathogenesis. Annu Rev Phytopathol, 1995, 33(1): 299-321.

[3] Boya R, Reggiori R, Codogno P. Emerging regulation and functions of autophagy. Nat Cell Biol, 2013, 15(7): 713-720.

[4] Cheng HR, Lin MS, Li WQ, Fang ZD. Pine wilt disease on Pinus thunbergii in Nanjing. For Pest Dis, 1983, 4: 1-5. (in Chinese)

[5] Ding XL, Ye JR, Lin SX, Wu XQ, Li DW, Nian B. Deciphering the molecular variations of pine wood nematode Bursaphelenchus xylophilus with different virulence. PLoS ONE, 2016 11 5 e0156040

[6] Du YY, Wang PC, Chen J, Song CP. Comprehensive functional analysis of the catalase gene family in Arabidopsis thaliana. J Integr Plant Biol, 2008, 50(10): 1318-1326.

[7] Eo J, Takemoto S, Otobe K. Is there a relationship between the intrinsic rate of propagation and in-vitro migration and virulence of the pinewood nematode, Bursaphelenchus xylophilus. Eur J Plant Pathol, 2011, 130: 231-237.

[8] Futai K. Pine wood nematode, Bursaphelenchus xylophilus. Annu Rev Phytopathol, 2013, 51(51): 61-83.

[9] He LX, Ji J, Qiu XW, Zhang LP. Occurrence and control measures of pine wood nematode around the world. J For Eng, 2014, 28(3): 8-13. (in Chinese)

[10] He LX, Wu XQ, Yu LZ, Ji J, Ye JR. The difference of H₂O₂ and oxidative enzyme in the interaction of different resistance pines and Bursaphelenchus xylophilus. J Nanjing For Univ (Nat Sci Edn), 2010, 34(6): 13-17.

[11] Huang L, Wang P, Tian MQ, Zhu LH, Ye JR. Major sperm protein BxMSP10 is required for reproduction and egg hatching in Bursaphelenchus xylophilus. Exp Parasitol, 2019, 197: 51-56.

[12] Hudson AL, Sotirchos IM, Davey MW. The activity and hydrogen peroxide sensitivity of the peroxiredoxins from the parasitic nematode Haemonchus contortus. Mol Biochem Parasitol, 2011, 176: 17-24.

[13] Hu LJ, Wu XQ, Li HY, Wang YC, Huang X, Wang Y, Li Y. BxCDP1 from the pine wood nematode Bursaphelenchus xylophilus is recognized as a novel molecular pattern. Mol Plant Pathol, 2020

[14] Kikuchi T, Cotton JA, Dalzell JJ, Hasegawa K, Kanzaki N, McVeigh P, Takanashi T, Tsai IJ, Assefa SA, Cock PJA, Otto TD, Hunt M, Reid AJ, Sanchez-Flores A, Tsuchihara K, Yokoi T, Larsson MC, Miwa J, Maule AG, Sahashi N, Jones JT, Berriman M. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus. PLoS Pathog, 2011 7 9 e1002219

[15] Kim IH, Kim K, Rhee SG. Induction of an antixoidant protein of Saccharomyces cerevisiae by O₂, Fe³⁺, or 2-mercaptoethanol. Proc Natl Acad Sci USA, 1989, 86: 6018-6022.

[16] Kodydkova J, Vavrova L, Kocik M, Zak A. Human catalase, its polymorphisms, regulation and changes of its activity in different diseases. Folia Biol, 2014, 60(4): 153-167.

[17] Ktistakis Walker SA, Karanasios E (2016) Assembly of early machinery for autophagy induction: novel insights from high resolution microscopy. Oncotarget 7(52): 85678–85679. https://doi.org/10.18632/oncotarget.13144

[18] Leiers BR, Kampkotter A, Grevelding CG, Link CD, Johnson TE, Henkle-Duhrsen K. A stress-responsive glutathione S-transferase confers resistance to oxidative stress in Caenorhabditis elegans. Free Radic Biol Med, 2003, 34(11): 1405-1415.

[19] Lewinsohn E, Savage TJ, Gijzen M, Croteau R. Simultaneous analysis of monoterpenes and diterpenoids of conifer oleoresin. Phytochem Anal, 1993, 4(5): 220-225.

[20] Li LL, Tan JJ, Chen FM. Bacillus pumilus strain LYMC-3 shows nematicidal activity against Bursaphelenchus xylophilus via the production of a guanidine compound. Biocontrol Sci Technol, 2018, 28(12): 1128-1139.

[21] Li M, Zheng Y, Liang HW, Zou LH, Sun JJ, Zhang YY, Qin F, Liu SZ, Wang ZZ. Molecular cloning and characterization of cat, gpx1 and Cu/Zn-sod genes in pengze crucian carp (Carassius auratus var. Pengze) and antioxidant enzyme modulation induced by hexavalent chromium in juveniles. Comput Biochem Physiol Part C Toxicol Pharmacol, 2013, 157(3): 310-321.

[22] Li YX, Meng FL, Deng X, Wang X, Feng YQ, Zhang W, Pan L, Zhang XY. Comparative transcriptome analysis of the pinewood nematode Bursaphelenchus xylophilus reveals the molecular mechanism underlying its defense response to host-derived α-pinene. Int J Mol Sci, 2019 20 4 911

[23] Li Z, Liu XX, Chu YN, Wang Y, Zhang QW, Zhou XG. Cloning and characterization of a 2-Cys peroxiredoxin in the pine wood nematode, Bursaphelenchus xylophilus, a putative genetic factor facilitating the infestation. Int J Biol Sci, 2011, 7(6): 823-836.

[24] Li Z, Zhang QW, Zhou XG. A 2-Cys peroxiredoxin in response to oxidative stress in the pine wood nematode Bursaphelenchus xylophilus. Sci Rep, 2016 6 1 27438

[25] Liu HB, Rui L, Feng YQ, Wu XQ. Molecular characterization and functional analysis of three autophagy genes, BxATG5, BxATG9, and BxATG16 Bursaphelenchus xylophilus. Int J Mol Sci, 2019 20 18 3769

[26] Liu HB, Rui L, Feng YQ, Wu XQ. Autophagy contributes to resistance to the oxidative stress induced by pine reactive oxygen species metabolism, promoting infection by Bursaphelenchus xylophilus. Pest Manage Sci, 2020

[27] Liu HB, Wu F, Wu XQ, Ye JR. Differential effects of rapamycin on Bursaphelenchus xylophilus with different virulence and differential expression of autophagy genes under stresses in nematodes. Acta Biochim Biophys Sin, 2019, 51: 254-262.

[28] Liu HB, Wu XQ, Feng YQ, Rui L. Autophagy contributes to the feeding, reproduction, and mobility of Bursaphelenchus xylophilus at low temperatures. Acta Biochim Biophys Sin, 2019, 00(00): 1-9.

[29] Liu J, Wu XQ, Ying CX, He LX, Ye JR. Study on superoxide anion of Pinus thunbergii infected by Bursaphelenchus xylophilus and B mucronatus in the earlier interaction. J Nanjing For Univ (Nat Sci Edn)., 2008, 32(6): 24-28. (in Chinese)

[30] Livak K, Schmittgen D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^–ΔΔCt method. Methods, 2001, 25: 402-408.

[31] Matsushita M, Suzuki NN, Obara K, Fujioka Y, Ohsumi Y, Inagaki F. Structure of Atg5·Atg16, a complex essential for autophagy. J Biol Chem, 2007, 282(9): 6763-6772.

[32] Miller BC, Zhao ZJ, Stephenson LM, Cadwell K, Pua HH, Lee HK, Mizushima N, Iwasaki A, He YW, Swat W, Virgin HW. The autophagy gene ATG5 plays an essential role in B lymphocyte development. Autophagy, 2008, 4(3): 309-314.

[33] Nian B. Cloning and Functional Analysis of Glutathione S-transferase Gene BxGST3 and BxGST1 in Bursaphelenchus xylophilus, 2017, Nanjing: Nanjing Forestry University.

[34] Ogura N, Nakajima Z. In vitro occurrence of dispersal fourth stage juveniles in Bursaphelenchus xylophilus co-incubated with Monochamus alternatus. Jpn J Nematol, 2002, 32(2): 53-59.

[35] Shinya R, Takeuchi Y, Ichimura K, Takemoto S, Futai K. Establishment of a set of inbred strains of the pine wood nematode, Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae), and evidence of their varying levels of virulence. Appl Entomol Zool, 2012, 47(4): 341-350.

[36] Tan JJ, Hao DJ, Pan YW, Zhai HY, Fan BQ, Ye JR, Du YB. Effects of several pine volatiles on behavior of Bursaphelenchus xylophilus. J Northeast For Univ, 2009, 12: 60-61. (in Chinese)

[37] Tiao N, Fan JT, Fang YL, Sun JH (2006) Changes in contents of host volatile terpenes under different damaged states and electroantennogram response of Monochamus alternatus Hope to these volatiles. Acta Entomol Sin 49:179–188. https://doi.org/10.16380/j.kcxb.2006.02.003 (in Chinese)

[38] Tsai IJ, Tanaka R, Kanzaki N, Akiba M, Yokoi T, Espada M, Jones JT, Kikuchi T. Transcriptional and morphological changes in the transition from mycetophagous to phytophagous phase in the plant-parasitic nematode Bursaphelenchus xylophilus. Mol Plant Pathol, 2016, 17(1): 77-83.

[39] Tsyglakova M, McDaniel D, Hodes GE. Immune mechanisms of stress susceptibility and resilience: Lessons from animal models. Front Neuroendocrinol, 2019, 54: 100771.

[40] Vellosillo T, Vicente J, Kulasekaran S, Hamberg M, Castresana C. Emerging complexity in reactive oxygen species production and signaling during the response of plants to pathogens. Plant Physiol, 2010, 154(2): 444-448.

[41] Vicente CSL, Ikuyo Y, Mota M, Hasegawa K. Pinewood nematode-associated bacteria contribute to oxidative stress resistance of Bursaphelenchus xylophilus. BMC Microbiol, 2013 13 1 299

[42] Vicente CSL, Yoriko I, Ryoji S, Mota M, Hasegawa K. Catalases induction in high virulence pinewood nematode Bursaphelenchus xylophilus under hydrogen peroxide-induced stress. PLoS One, 2015 10 4 e0123839

[43] Viglierchio DR, Schmitt RV. On the methodology of nematode extraction from field samples: Baermann funnel modifications. J Nematol, 1983, 15: 438-444.

[44] Wang X, Li YX, Liu ZY, Lv Q, Jia XZ, Zhang XY. Relationship between the cytochrome P450 gene of pine wood nematode and the accumulation of pine pinene. Scientia Silvae Sinicae, 2017, 53(6): 105-110. (in Chinese)

[45] Wang Y, Yamada T, Sakaue D, Suzuki K. Variations in life history parameters and their influence on rate of population increase of different pathogenic isolates of the pine wood nematode, Bursaphelenchus xylophilus. Nematology, 2005, 7: 459-467.

[46] Wang ZL, Zhang LB, Ying SH, Feng MG. Catalases play differentiated roles in the adaptation of a fungal entomopathogen to environmental stresses. Environ Microbiol, 2013, 15(2): 409-418.

[47] Wu Y, Wickham JD, Zhao L, Sun JH. CO₂ drives the pine wood nematode off its insect vector. Curr Biol, 2019, 29(13): R619-R620.

[48] Xu JY, Wang BW, Li DL. Response pattern and functional validation of pine wood nematode (Bursaphelenchus xylophilus) BxPrx. J Hum Agric Univ (Nat Sci), 2019, 45(5): 491-495. (in Chinese)

[49] Xu XL, Wu XQ, Ye JR, Huang L. Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 Genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea). Int J Mol Sci, 2015, 16(3): 5216-5234.

[50] Xu YQ, Wang JJ, Dou W, Tang PA, An FM. Identification, characterization, and expression of P450 gene encoding CYP6BQ13v2 from the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Agric Sci China, 2009, 8(10): 1210-1218. in Chinese)

[51] Yang BJ, Pan HY, Tang J, Wang YY, Wang LF. Pine wood nematode disease, 2003, Beijing: Forestry Publishing House 45 48

[52] Ye JR. Epidemic status of pine wilt disease in China and its prevention and control techniques and counter measures. Scientia Silvae Sinicae, 2019, 55(9): 1-10. (in Chinese)

[53] Yu LZ, Wu XQ, Ye JR, Zhang SN. Relationships between nitric oxide response signal and external factors during the early interaction between Pinus thunbergii and Bursaphelenchus xylophilus. Chin J Appl Ecol, 2013, 24: 646-652. (in Chinese)

[54] Yue LN, Yang YH, Wu SW, Wu YD. Cloning and mRNA expression levels of cytochrome P450 genes CYP6AE12 and CYP9A18 in the cotton bollworm, Helicoverpa armigera (Hübner). Acta Entomol Sin, 2007, 50(3): 234-240. (in Chinese)

[55] Zamocky M, Furtmuller PG, Obinger C. Evolution of catalases from bacteria to humans. Antioxid Redox Signaling, 2008, 10(9): 1527-1548.

[56] Zhao L, Zhang X, Wei Y, Zhou J, Zhang W, Qin PJ, Chinta S, Kong XB, Liu YP, Yu HY, Hu SN, Zou Z, Butcher RA, Sun JH. Ascarosides coordinate the dispersal of a plant-parasitic nematode with the metamorphosis of its vector beetle. Nat Commun, 2016, 7: 12341.

[57] Zhou LF, Chen FM, Xie LY, Pan HY, Ye JR. Genetic diversity of pine-parasitic nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus in China. For Pathol, 2017 47 4 e12334

[58] Zulak KG, Bohlmann J. Terpenoid biosynthesis and specialized vascular cells of conifer defense. J Integr Plant Biol, 2010, 52(1): 86-97.