A phosphoketolase Mpk1 of bacterial origin is adaptively required for full virulence in the insect-pathogenic fungus Metarhizium anisopliae Zhibing Duan, Yianfang Shang, Qiang Gao, Peng Zheng and Chengshu Wang* Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Summary Pentose metabolism through the phosphoketolase pathway has been well characterized in bacteria. In this paper, we report the identification of a phospho- ketolase homologue Mpk1 in the insect-pathogenic fungus Metarhizium anisopliae. Phylogenetic analy- sis showed that fungal phosphoketolases are of bacterial origin and diverged into two superfamilies. Frequent gene loss or lack of acquisition is evident in specific fungal lineages or species. The mpk1 gene is highly expressed when grown in trehalose-rich insect haemolymph but poorly induced by insect cuticle or carbohydrate-rich plant root exudate. In addition, mpk1 gene expression and enzyme activity could be upregulated by different sugars including xylose, tre- halose, glucose or sucrose. mpk1 null mutants gen- erated by homologous recombination grew similar to the wild type of M. anisopliae on medium amended with xylose as a sole carbon source. However, insect (tobacco hornworm, Manduca sexta) bioassays showed significantly reduced virulence in Dmpk1. The results of this study suggest that the horizontally transferred Mpk1 in M. anisopliae plays an important niche adaptation role for fungal propagation in insect haemocoel. Following the carbohydrate flux from plants to plant-feeding insects and insect pathogenic fungi, a tritrophic relationship is discussed in asso- ciation with the requirement of fungal phosphoketo- lase pathway. Introduction The phosphoketolase pathway has been largely reported in lactic acid bacteria and bifidobacteria for metabolization of pentoses and hexoses (e.g. Meile et al., 2001; Post- huma et al., 2002). Until recently, a few studies have reported phosphoketolase pathway in filamentous fungi, Penicillium chrysogenum and Aspergillus nidulans (Thykaer and Nielsen, 2007; Panagiotou et al., 2007; 2008; 2009). However, the pathway has been evident not present in yeast Saccharomyces cerevisiae (Sondereg- ger et al., 2004). Like numerous bacteria, filamentous fungi including plant pathogens and many saprophytic species that inhabit soils and can effectively consume live plants or plant materials, which are abundant sources of xylose-rich hemicelluloses. However, instead of xylose or glucose, the major blood sugar accumulated in plant- feeding insects is trehalose, e.g. up to more than 100 mM in tobacco hornworm Manduca sexta (Mullins, 1985; Thompson, 1999). Insect fungi like Metarhizium anisopliae and Beauveria bassiana are being developed into promising biocontrol agents against various insect pests (de Faria and Wraight, 2007; Wang and St Leger, 2007a). Fungal biocontrol agents infect hosts by penetration of the cuticle and growth in the insect haemocoel. To improve their cost- effective control efficacy, the mechanisms of fungal pathogenesis have to be explored. Previous efforts to understand the entomopathogenicity of M. anisopliae have identified virulence genes functioning from surface adhesion (Wang and St Leger, 2007b), breaching of the cuticle (Wang and St Leger, 2007c), haemocoel osmotic pressure adaptation (Wang et al., 2008) and evasion of the insect immune responses (Wang and St Leger, 2006). A case of horizontal gene transfer (HGT) of a chymotrypsin-encoding gene from bacteria has also been reported in M. anisopliae (Screen and St Leger, 2000). In order to respond to different ecological and physiological niches, insect pathogenic fungi have evolved ways of monitoring their immediate environments and upregulat- ing genes required for survival. Our previous study identified a M. anisopliae transcript (CN808491) that was highly expressed (1.4% of total sequenced clones) in insect haemolymph (Wang et al., 2005). A BLAST search revealed that it showed high simi- larity (E = 0) to bacterial xylulose-5-phosphate/fructose- 6-phosphate phosphoketolase (XpkA, EC 4.1.2.9, EC 4.1.2.22). In this study, full sequence of the gene was Received 15 March, 2009; accepted 4 May, 2009. *For corre- spondence. E-mail cswang@sibs.ac.cn; Tel. (+86) 21 5492 4157; Fax (+86) 21 5492 4015. Environmental Microbiology (2009) 11(9), 2351–2360 doi:10.1111/j.1462-2920.2009.01961.x © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd