Comparative study on the mRNA expression of Pinus massoniana infected by Bursaphelenchus xylophilus

Abstract

Pine wilt disease (PWD) is a devastating disease affecting the growth of Pinus massoniana, often leading to withering and death. To reveal the changes involved during disease progression, we investigated the mRNA expression profile of P. massoniana infested by Bursaphelenchus xylophilus. The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene, CC-NBS-LRR resistance-like protein, and the gene encoding a putative nematode resistance protein. Increased infestation pressure (number of nematodes inoculated) caused a continuous decline in the gene expression of stem samples. An infestation of P. massoniana also resulted in a pathway enrichment of genes involved in phenylpropanoid metabolism and flavonoid biosynthesis, which in turn reduced the levels of total phenols and total flavonoids. A downregulation of auxin responsive family protein was observed in infested samples, which resulted in a suppression of plant growth. Thus, upon B. xylophilus infestation, a downregulation of genes associated with the recognition of pathogens, PWD resistance, and growth regulation was observed in P. massoniana, together with a decrease in the levels of phytoalexin-like secondary substances, all of which resulted in withering and ultimately death of P. massoniana.

Keywords

Auxin/IAA / Bursaphelenchus xylophilus / Pinus massoniana / Resistance gene / Phytoalexin

References

[1] Cai D, Kleine M, Kifle S, Harloff HJ, Sandal NN, Marcker KA, Klein-Lankhorst RM, Salentijn EMJ, Lange W, Stiekema WJ, Wyss U, Grundler FMW, Jung C. Positional cloning of a gene for nematode resistance in sugar beet. Science, 1997, 275: 832-834.

[2] Dangl JL, Jones JD. Plant pathogens and integrated defence responses to infection. Nature, 2001, 411: 826-833.

[3] Ellis J, Dodds P, Pryor T. Structure, function and evolution of plant disease resistance genes. Curr Opin Plant Biol, 2000, 3: 278-284.

[4] Fukuda K. Physiological process of the symptom development and resistance mechanism in pine wilt disease. J For Res, 1997, 2: 171-181.

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

[6] Gao L, Tu ZJ, Millett BP, Bradeen JM. Insights into organ-specific pathogen defense responses in plants: RNA-seq analysis of potato tuber-Phytophthora infestans interactions. BMC Genom, 2013, 14: 340.

[7] Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol, 2011, 29: 644-652.

[8] Hanin M, Brini F, Ebel C, Toda Y, Takeda S, Masmoudi K. Plant dehydrins and stress tolerance: versatile proteins for complex mechanisms. Plant Signal Behav, 2011, 6: 1503-1509.

[9] Hirao T, Fukatsu E, Watanabe A. Characterization of resistance to pine wood nematode infection in Pinus thunbergii using suppression subtractive hybridization. BMC Plant Biol, 2012, 12: 13.

[10] Jones JD, Dangl JL. The plant immune system. Nature, 2006, 444: 323-329.

[11] Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T. KEGG for linking genomes to life and the environment. Nucleic Acids Res, 2008, 36: 480-484.

[12] Kortekamp A. Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen. Plant Physiol Biochem, 2006, 44: 58-67.

[13] Li G, Asiegbu FO. Induction of Pinus sylvestris PsACRE, a homology of Avr9/Cf-9 rapidly elicited defense-related gene following infection with root rot fungus Heterobasidion annosum. Plant Sci, 2004, 167: 535-540.

[14] Liu Y, Wang L, Xing X, Sun L, Pan J, Kong X, Zhang M, Li D. ZmLEA3, a multifunctional group 3 LEA protein from maize (Zea mays L.), is involved in biotic and abiotic stresses. Plant Cell Physiol, 2013, 54: 944-959.

[15] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods, 2001, 25: 402-408.

[16] McLean MD, Hoover GJ, Bancroft B, Makhmoudova A, Clark SM, Welacky T, Simmonds DH, Shelp BJ. Identification of the full-length Hs1 ^pro−1 coding sequence and preliminary evaluation of soybean cyst nematode resistance in soybean transformed with Hs1 ^pro−1 cDNA. Botany, 2007, 85: 437-441.

[17] Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Med, 2008, 5: 621-628.

[18] Park S, Han K-H. An auxin-repressed gene (RpARP) from black locust (Robinia pseudoacacia) is posttranscriptionally regulated and negatively associated with shoot elongation. Tree Physiol, 2003, 23: 815-823.

[19] Petit P, Granier T, d’Estaintot BL, Manigand C, Bathany K, Schmitter JM, Lauvergeat V, Hamdi S, Gallois B. Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis. J Mol Biol, 2007, 368: 1345-1357.

[20] Tan S, Wu S. Genome wide analysis of nucleotide-binding site disease resistance genes in Brachypodium distachyon. Comp Funct Genom, 2012, 3: 418208.

[21] Tronchet M, Balague C, Kroj T, Jouanin L, Roby D. Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Mol Plant Pathol, 2010, 11: 83-92.

[22] Westermann AJ, Gorski SA, Vogel J. Dual RNA-seq of pathogen and host. Nat Rev Microbiol, 2012, 10: 618-630.

[23] Xie W, Huang A, Li H, Feng L, Zhang F, Guo W. Identification and comparative analysis of microRNAs in Pinus massoniana infected by Bursaphelenchus xylophilus. Plant Growth Regul, 2017, 83: 223-232.

[24] Xu L, Liu ZY, Zhang K, Lu Q, Liang J, Zhang XY. Characterization of the Pinus massoniana transcriptional response to Bursaphelenchus xylophilus infection using suppression subtractive hybridization. Int J Mol Sci, 2013, 14: 11356-11375.

[25] Zhao BG. Zhao BG, Futai K, Sutherland JR, Takeuchi Y. Pine wilt disease in China. Pine wilt disease, 2008, Tokyo: Springer 18 25

[26] Zheng HY, Xu M, Xu FY, Ye JR. A comparative proteomics analysis of Pinus massoniana inoculated with Bursaphelenchus xylophilus. Pak J Bot, 2015, 47: 1271-1280.