Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin

Min Hye Jeong, Jung A. Kim, Seogchan Kang, Eu Ddeum Choi, Youngmin Kim, Yerim Lee, Mi Jin Jeon, Nan Hee Yu, Ae Ran Park, Jin Cheol Kim, Soonok Kim, Sook Young Park

Research output: Contribution to journalArticlepeer-review

Abstract

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

Original languageEnglish (US)
Pages (from-to)491-497
Number of pages7
JournalMycobiology
Volume49
Issue number5
DOIs
StatePublished - 2021

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Infectious Diseases

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