Overexpression of geranyl diphosphate synthase (PmGPPS1) boosts monoterpene and diterpene production involved in the response to pine wood nematode invasion

Abstract

Outbtreaks of pine wood nematode (PWN; Bursaphelenchus xylophilus) represent a severe biotic epidemic for the P. massoniana in China. When invaded by the PWN, the resistant P. massoniana might secret abundant oleoresin terpenoid to form certain defensive fronts for survival. However, the regulatory mechanisms of this process remain unclear. Here, the geranyl diphosphate synthase (PmGPPS1) gene was identified from resistant P. massoniana. Tissue-specific expression patterns of PmGPPS1 at transcript and protein level in resistant P. massoniana were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Functional characteristics analysis of PmGPPS1 was performed on transgenic Nicotiana benthamiana by overexpression, as genetic transformation of P. massoniana is, so far, not possible. In summary, we identified and functionally characterized PmGPPS1 from the resistant P. massoniana following PWN inoculation. Tissue-specific expression patterns and localization of PmGPPS1 indicated that it may play a positive role involved in the metabolic and defensive processes of oleoresin terpenes production in response to PWN attack. Furthermore, overexpression of PmGPPS1 may enhance the production of monoterpene, among which limonene reduced the survival of PWN in vitro. In addition, PmGPPS1 upregulated the expression level of key genes involved in mevalonic acid (MVA) pathway, the methylerythritol phosphate (MEP) pathway and gibberellins (GAs) biosynthesis to boost the growth and development of tobacco through a feedback regulation mechanism. Our results offered new insights into the pivotal role of the PmGPPS1 involved in terpene-based defense mechanisms responding to the PWN invasion in resistant P. massoniana, and provided a new metabolic engineering scenario to improve monoterpene production in tobacco.

Identification and Defensive Characterization of PmCYP720B11v2 from Pinus massoniana

“…PWNs were removed from Pinus massoniana -carrying PWNs and introduced into the colony, which was cultured at 30 °C until the colony was entirely consumed by the PWNs. The Baermann funnel technique was used to further isolate the PWNs [ 8 , 59 , 60 ].…”

“…Meanwhile, the fresh stem samples were placed in FAA fixative for paraffin embedding and sectioning [ 8 , 61 ]. Nicotiana benthamiana was grown in a growth chamber at 26 °C with a 16 h light/8 h dark photoperiod and used for Agrobacterium-mediated transformation [ 8 , 60 ].…”

“…According to the Pfam database ( (accessed on 4 February 2022)) [ 62 ], the domains of the CYP720B proteins in the gymnosperm plant were labeled (Pfam: PF00067). Multiple protein sequence alignments for CYP720Bs of P. massoniana were carried out, taking advantage of MAFFT [ 63 ], and maximum-likelihood trees were built with CIPRES ( (accessed on 4 February 2022)) using the JTT model and 1000 bootstrap replicates [ 8 , 60 , 64 ].…”

“…The gene was compared to homologous sequences using online BLAST at the National Center for Biotechnology Information (NCBI) ( (accessed on 4 February 2022)). The sequence alignment was performed utilizing BioEdit7.2.5 [ 8 , 60 , 65 ].…”

“…The amplification was conducted in two steps: 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and 60 °C for 34 s. Three biological replicates were used, and each was checked three times as technical replicates for the qRT–PCR experiments. The data were analyzed using the 2 −ΔΔCt method [ 8 , 60 , 66 ].…”