Disruption of the Subtilase Gene, albin1 , in Ophiostoma piliferum

Abstract

Wood sapstaining fungi produce multiple proteases that break down wood protein. Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B. Hoffman and C. Breuil, Curr. Genet. 41: [168][169][170][171][172][173][174][175] 2002). In this work we examined the role of the subtilase Albin1 from Ophiostoma piliferum. Reamplification of cDNA ends PCR was used to obtain the albin1 gene sequence. The encoded subtilase is probably extracellular and involved in nutrient acquisition. This gene was disrupted with an Agrobacterium tumefaciens-mediated transformation system. Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood. Thus, albin1 plays an important role in O. piliferum's ability to acquire nitrogen from wood proteins.Sapstaining fungi infect wood and produce a dark blue to black discoloration that lowers the value of the wood. These fungi also are of concern in some importing countries, as they may be pathogenic. The forest products industry spends millions of dollars annually on fungicides to control sapstaining fungi, but these chemicals do not have the duration of effectiveness desired and present significant environmental concerns. Biological control agents, such as Cartapip-97, an albino strain of Ophiostoma piliferum, have been examined as an alternative method for preventing sapstain (4, 47). However, these agents may grow slowly and have not been shown to be reliable under field conditions. To develop new strategies for controlling sapstain and to improve the available biological control agents, we need to better understand how sapstaining fungi and biological control agents grow on wood and modify wood color.Sapstaining fungi cannot degrade structural components in wood. Instead, they utilize nonstructural components, including triglycerides, fatty acids, and proteins (14,32,36,41,48). Nitrogen levels in trees are low, and easily assimilated nitrogen sources, such as ammonia, are insufficient to support fungal growth (21,28). Trees store most of their nitrogen in an organic form as proteins and amino acids (25,39). To extract nitrogen from these protein sources and sustain their growth on wood, sapstaining fungi produce several extracellular proteases.Subtilases, a type of serine protease, are the dominant extracellular proteases produced by the sapstaining fungus Ophiostoma floccosum (1). Subtilase genes are common in sapstaining fungal species, and three major groups of fungal subtilases have been identified (16). One group contains intracellular subtilases thought to play a variety of housekeeping roles (22,27,33). The other two groups contain extracellular fungal subtilases, several of which are involved in pathogenesis (5,22,27,40,43).In this study we focused on the commercially important sapstaining fungus O. piliferum, which was used to develop the albino biological control agent Cartapip-97. This sp...

Agrobacterium-Mediated Transformation of Non-Plant Organisms

“…Agrobacteriummediated transformation is not restricted to eukaryotes as Agrobacterium is also able to transform the gram positive bacterium Streptomyces lividans. (Tsuji et al, 2003) Colletotrichum trifolii (Takahara et al, 2004) Coniothyrium minitans (Rogers et al, 2004;Li et al, 2005) Cryphonectria parasitica (Park and Kim, 2004) Fusarium circinatum (Covert et al, 2001) Fusarium culmorum (Michielse et al, 2005) Fusarium oxysporum (Mullins et al, 2001;Takken et al, 2004;Khang et al, 2005 (Michielse et al, 2005) Ophiostoma piceae (Tanguay and Breuil, 2003) Ophiostoma piliferum (Hoffman and Breuil, 2004) Paecilomyces fumosoroseus (Michielse et al, 2005) Paracoccidioides brasiliensis (Leal et al, 2004) Penicillium chrysogenum (Sun et al, 2002) Saccharomyces cerevisiae (Bundock et al, 1995;Piers et al, 1996;Risseeuw et al, 1996;Bundock et al, 2002) Trichoderma asperellum (Michielse et al, 2005) Trichoderma atroviride (Zeilinger, 2004) Pardo et al, 2002 ) …”