The bacterial transposon Tn7 causes premature polyadenylation of mRNA in eukaryotic organisms: TAGKO mutagenesis in filamentous fungi

Abstract

TAGKO is a Tn7-based transposition system for genome wide mutagenesis in filamentous fungi. The effects of transposon insertion on the expression of TAGKO alleles were examined in Magnaporthe grisea and Mycosphaerella graminicola. Northern analysis showed that stable, truncated transcripts were expressed in the TAGKO mutants. Mapping of the 3'-ends of TAGKO cDNAs revealed that they all contain Tn7 end sequences, regardless of the transposon orientation. Polyadenylation signals characteristic of eukaryotic genes, preceded by stop codons in all frames, are located in both ends of the bacterial transposon. Thus, TAGKO transcripts are prematurely polyadenylated, and truncated proteins are predicted to be translated in the fungal mutants. Depending on the extent of protein truncation, TAGKO mutations in HPD4 (encoding p-hydroxyphenylpyruvate dioxygenase) resulted in tyrosine sensitivity in the two fungi. Similarly, a particular M.grisea CBS1 (encoding cystathionine beta-synthase) TAGKO cDNA failed to complement cysteine auxotrophy in a yeast CBS mutant. TAGKO, therefore, represents a useful tool for in vivo study of truncated gene products in filamentous fungi.

Figure 1

Chimeric, polyadenylated HPD4 transcripts in M.grisea and M.graminicola. (A) Two HPD4 mutant alleles, KO1 and KO2, are presented in each fungus. Block arrows represent the HPD4 coding regions. The orientation of the TAGKO transposon cassette is shown in arrows (Tn7-R, R→Tn7-L, L). P1 and P2 denote PCR-derived probes used in northern hybridization experiments. (B) Northern analysis of HPD4 gene expression. Approximately 10 µg of total RNA was used for each strain. Actin genes served as positive controls for gene expression in the two fungi. (C) Mapping of the 3′-ends of HPD4 cDNAs. TAGKO transcripts are chimeric and polyadenylated. Endogenous HPD4 sequences are shown in black, sequences derived from Tn7 transposon ends are shown in grey and stop codons in frame with the truncated HPD4 coding sequences are indicated. Numbers in parentheses represent the first (1) or second (2) stop codon identified in the respective Tn7 end.

Figure 2

Examination of transposon end sequences in the TAGKO cassette. An HPH gene was engineered between the transposon ends (Tn7-L and Tn7-R). Two possible orientations of transposon insertion into a gene (block arrow) are presented. Arrows indicate the direction of the HPH gene. Tn7-L (147 bp) or Tn7-R (140 bp) sequences that appeared in the 3′-ends of the TAGKO cDNAs (Fig. 1C) are shown below each diagram. Putative polyadenylation signals are in bold and stop codons in three different reading frames are underlined.

Figure 3

Phenotypes of HPD4 TAGKO mutants. (A) Predicted HPD4 gene products. Numbers in parentheses indicate the length of truncated HPD4 protein. Sequences derived from Tn 7 transposon ends in the TAGKO proteins are shown in grey. (B) Plate images showing the growth of M.grisea (after 5 days) and M.graminicola (after 8 days) strains, on MM with 4 mM of tyrosine. Note the limited growth in M.grisea KO2, and M.graminicola KO1 and KO2 mutants. Magnaporthe grisea KO1 mutant showed WT phenotypes.

Figure 4

Yeast complementation assays for M.grisea CBS1 TAGKO cDNAs. (A) Mutant alleles of the M.grisea CBS1 gene. Transposition orientations are indicated by arrows. Stop codons in frame with the truncated CBS1 coding sequences are indicated, and numbers in parentheses represent the second (2) or third (3) codon identified in Tn7-R. (B) Saccharomyces cerevisiae CBS mutant (Sc 6696) was transformed with different CBS expression clones (MgCBS). CBS1 cDNAs, isolated from WT and TAGKO M.grisea strains, were cloned into yeast expression vectors. Sc 6696 transformed with the indicated vector was grown on yeast peptone dextrose (YPD) medium. (C) The yeast transformant strains were grown on cysteine-depleted (-CYS) medium. Sc 6696 is a cysteine auxotroph. MgCBS WT, KO1 and KO2 rescued the yeast mutant phenotype, while MgCBS KO3 did not have any effect. (D) Predicted CBS1 gene products. Numbers in parentheses indicate the length of truncated CBS1 protein. Sequences derived from the Tn7 ends are shown in grey.