Role of the methylcitrate cycle in growth, antagonism and induction of systemic defence responses in the fungal biocontrol agent Trichoderma atroviride Mukesh K. Dubey, 1 Anders Broberg, 2 Dan Funck Jensen 1 and Magnus Karlsson 1 Correspondence Mukesh K. Dubey mukesh.dubey@slu.se Received 19 June 2013 Accepted 2 October 2013 1 Uppsala BioCenter, Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Box 7026, 75007 Uppsala, Sweden 2 Uppsala BioCenter, Department of Chemistry, Swedish University of Agricultural Sciences, Box 7015, 75007 Uppsala, Sweden Methylisocitrate lyase (MCL), a signature enzyme of the methylcitrate cycle, which cleaves methylisocitrate to pyruvate and succinate, is required for propionate metabolism, for secondary metabolite production and for virulence in bacteria and fungi. Here we investigate the role of the methylcitrate cycle by generating an mcl deletion mutant in the fungal biocontrol agent Trichoderma atroviride. Gene expression analysis shows that a basal expression of mcl is observed in all growth conditions tested. Phenotypic analysis of an mcl deletion mutant suggests the requirement of MCL in propionate resistance, growth, conidial pigmentation and germination, and abiotic stress tolerance. A plate confrontation assay did not show a difference between the WT and the Dmcl strain in antagonism towards Botrytis cinerea. However, the Dmcl strain displays reduced antagonism towards B. cinerea based on a secretion assay. Furthermore, an in vitro root colonization assay shows that the Dmcl strain had reduced ability to colonize Arabidopsis thaliana roots, which results in reduced induction of systemic resistance towards B. cinerea. These data show that MCL is important not only for growth and development in T. atroviride but also in antagonism, root colonization and induction of defence responses in plants. INTRODUCTION The methylcitrate cycle is a carbon anaplerotic pathway where propionyl-CoA is converted to pyruvate (Textor et al., 1997). Methylcitrate synthase (MCS), methylcitrate dehydrogenase (MCD) and methylisocitrate lyase (MCL) are the signature enzymes of the pathway. MCS catalyses condensation of propionyl-CoA and oxaloacetate to methyl- citrate (Horswill & Escalante-Semerena, 1999), MCD catalyses methylcitrate to methylisocitrate, and MCL completes the cycle by cleaving methylisocitrate to pyruvate and succinate (Brock et al., 2001). Succinate generated by the pathway can go to the citric acid cycle, while pyruvate can be used directly for energy and biomass production (Brock et al., 2001). Propionyl-CoA used in the methyl- citrate cycle derives either from direct activation of propionate, an abundant carbon compound in soil, or from the degradation of odd-chain fatty acids and amino acids such as isoleucine, valine and methionine (Zhang et al., 2004). Besides being carbon anaplerotic, the methylcitrate cycle also detoxifies propionate, which otherwise can exert toxic effects by inhibiting several enzymes of primary metabolic pathways (Brock & Buckel, 2004; Limenitakis et al., 2013). The role of the methylcitrate cycle in propionate metabol- ism, as well as in growth, conidiation and virulence, has been demonstrated in bacterial and fungal systems (Brock, 2005; Lee et al., 2009; Mun ˜oz-Elı´as & McKinney, 2005; Upton & McKinney, 2007). In Aspergillus nidulans and Aspergillus fumigatus, disruption of the methylcitrate cycle gene leads to accumulation of propionyl-CoA, and con- sequently growth retardation, impaired conidiation and reduced polyketide secondary metabolite synthesis (Brock, 2005; Maerker et al., 2005; Zhang et al., 2004; Zhang & Keller, 2004). The importance of the methylcitrate cycle in virulence has been reported in As. fumigatus, where disruption leads to attenuated virulence in wax moth Abbreviations: ATMT, Agrobacterium tumefaciens-mediated transformation; C t , threshold cycle; CTAB, hexadecyltrimethylammonium bromide; ICL, isocitrate lyase; MCD, methylcitrate dehydrogenase; MCL, methylisocitrate lyase; MCS, methylcitrate synthase; NAG, N-acetylglucosamine; qPCR, quantitative PCR; RT-PCR, reverse transcriptase-PCR; WGA, wheatgerm agglutinin. One supplementary table and three supplementary figures are available with the online version of this paper. Microbiology (2013), 159, 2492–2500 DOI 10.1099/mic.0.070466-0 2492 070466 G 2013 SGM Printed in Great Britain