cDNA Cloning and Expression in Ectomycorrhizae

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activ- ity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP- ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Be- cause the deduced EgICDH protein lacks an amino-terminal target- ing sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indi- cated that NADP-ICDH was preferentially accumulated in the epi- dermis and stele parenchyma of nonmycorrhizal and ectomycorrhi- zal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed.