Enhancing the stress tolerance virulence and relevant mechanism of an entomopathogen by metabolic engineering dihydroxynaphthalene (DHN)-melanin biosynthesis genes

• Main Authors: Min-Nan Tseng, 曾敏南
• Other Authors: Shean-Shong Tzean
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/qawp5b

博士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 102 === Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as ultraviolet radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN)-melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin express in the transformant of M. anisopliae was verified by spectrophotometric methods, LC/MS and confocal microscopy. In contrast to the wild type strain, the transformant displays a greater resistance to environmental stress and a higher virulence toward target insect host. However, the underlying mechanisms for these characteristics remain unclear; hence experiments were initiated to explore the possible mechanism through physiological and molecular approaches. Although both transformant and wild type strains could infect and share the same insect host range, the former germinated faster and produced more appressoria than the latter, both in vivo and in vitro. The transformant showed a significantly shorter median lethal time (LT50) when infecting the diamondback moth (Plutella xylostella) and the striped flea beetle (Phyllotreta striolata), than the wild type. Additionally, the transformant was more tolerant to reactive oxygen species (ROS), produced 40-fold more orthosporin and notably overexpressed the transcripts of the pathogenicity-relevant hydrolytic enzymes (chitinase, protease, and phospholipase) genes in vivo. In contrast, appressorium turgor pressure and destruxin A content were slightly decreased compared to the wild type. The transformant’s high anti-stress tolerance, its high virulence against five important insect pests (cowpea aphid Aphis craccivora, diamondback moth Pl. xylostella, striped flea beetle Ph. striolata, and silverleaf whitefly Bemisia argentifolii) and its capacity to colonize the root system are key properties for its potential bio-control field application.