Discovery, cloning and heterologous expression of secreted potato proteins reveal erroneous pre-mRNA splicing in Aspergillus oryzae.
A novel transposon assisted signal trapping (TAST) technology, developed to specifically select only the secreted proteins, was used to discover novel extracellular plant proteins from Solarium tuberosum infected with Phytophthora infestans. Analysis of 384 hits provided 191 P. infestans and S. tuberosum sequences of secreted proteins, with an approx. 2/3 of these originating from potato. Subsequent screening for interesting genes was carried out using bioinformatics. A selected variety of the discovered sequences are presented, including a novel S. tuberosum xyloglucan endotransglucosylase (StXTH), which was cloned and subjected to detailed heterologous expression studies in Aspergillus oryzae. RT-PCR analysis of mRNA from A. oryzae StXTH1 transformants revealed that parts of the mRNA pool had been incorrectly processed, and only weak and inconsistent indications of active protein could be detected. A high AT content of StXTH1 and the occurrence of A. oryzae intron donor, acceptor, and branch point recognition sites resulted in erroneous intron interpretation (cryptic introns) of parts of the mRNA coding sequence. This may explain the difficulties generally experienced in expressing plant genes in filamentous fungi.
Hamann
Thomas
T
Molecular Biotechnology, Novozymes A/S, Smørmosevej 25 1B1, DK-2880 Bagsvaerd, Denmark.
Lange
Lene
L
D001236:Aspergillus oryzae / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods*; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D012326:RNA Splicing / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D011198:Solanum tuberosum / Q000502:physiology*
D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins