Telomere-targeted retrotransposons in the rice blast fungus Magnaporthe oryzae: agents of telomere instability

Abstract

The fungus Magnaporthe oryzae is a serious pathogen of rice and other grasses. Telomeric restriction fragments in Magnaporthe isolates that infect perennial ryegrass (prg) are hotspots for genomic rearrangement and undergo frequent, spontaneous alterations during fungal culture. The telomeres of rice-infecting isolates are very stable by comparison. Sequencing of chromosome ends from a number of prg-infecting isolates revealed two related non-LTR retrotransposons (M. oryzae Telomeric Retrotransposons or MoTeRs) inserted in the telomere repeats. This contrasts with rice pathogen telomeres that are uninterrupted by other sequences. Genetic evidence indicates that the MoTeR elements are responsible for the observed instability. MoTeRs represent a new family of telomere-targeted transposons whose members are found exclusively in fungi.

Figure 1

Changes in telomeric restriction fragments following two cycles of plant infection. Spores were harvested from leaf lesions, and DNA was extracted from single-spore cultures. DNAs were digested with PstI, fractionated by electrophoresis, and electroblotted to membranes. The immobilized DNAs were then hybridized with a ³²P-labeled telomere probe and, finally, exposed to phosphorimage screens. (A) Telomere changes in the rice pathogen 70-15. (B) Telomere changes in LpKY97-1A. The numbers on the right of each phosphorimage represent molecular sizes in kilobases. The white arrowhead marks LpKYTEL2, a telomeric fragment that undergoes rearrangement very rarely.

Figure 2

Organization of telomeric DNA in three prg-infecting strains of M. oryzae. The three strains analyzed were LpKY97-1A, FH, and CHW. Telomeres were cloned either as PstI to blunt end or ApaI to blunt end restriction fragments, and their organizations were determined by restriction mapping and sequencing. Panels A\x{2013}K show the organization of 11 cloned telomeric fragments. Telomeric sequence ([TTAGGG]_n) is represented by concatenated circles and the variant telomere tract (consensus sequence: [TTTGGG]₈[TTCGGG]₂) is depicted by concatenated, closed circles. Repeats inserted in the telomere array are shown as boxes, and regions within the boxes having the same shading represent sequences that are identical (or nearly identical). The numbers within the boxes represent the length of each element. Truncated repeats are indicated by using dotted lines to mark the truncation boundary. Chromosome unique DNA is illustrated with a line, and the different line styles indicate that they have different sequences. The sequences of short telomere tracts (and telomere-like tracts) at the junctions of adjacent insertions are listed below the relevant junctions. Positions of PstI and ApaI restriction sites are shown. Boxes and lines are drawn to scale with the exception of the circles that represent the telomeres and telomere-like sequences.

Figure 3

Comparison of homologous chromosome ends in strains 70-15 and FH. Panels A\x{2013}D show alignments of four pairs of chromosome ends. Telomere and telomere-like sequences are represented with circles, and the telomere-embedded repeats are depicted as boxes as described in the legend to Figure 2. The TLH regions in the 70-15 subtelomeres are highlighted with dark gray background shading. For cosmid clones that did not contain the terminal telomere repeat array, the direction of the telomere is indicated. Transposon insertions and other repeats in the subtelomere regions are represented by colored boxes with arrows or arrowheads indicating their orientations. Features that are described in the text are labeled. Regions of alignment between the 70-15 chromosome and the FH homolog are connected by light gray shading. Sequences linked to telomere 11 of FH align with two sequences that are well separated in the 70-15 genome, and so two alignments are shown. Sequences that were present near the FH telomeres but absent from the 70-15 genome are drawn as dotted lines, with different line styles representing different sequences. The star-shaped area of shading between telomere 11 homologs represents an inversion that contains a transposon insertion, which in turn caused a deletion in the recipient chromosome. All features are drawn to scale with the exception of the circles that represent the telomeres and telomere-like sequences.

Figure 4

Organization of MoTER1 and MoTER2. (A) Overall structures showing positions of relevant features. Both elements are drawn to scale. Coding regions are depicted by a dark-gray arrow. Blocks of tandem repeats are shown as medium-gray boxes. The sequences and copy numbers of the individual repeat units are listed in Table S6. Light-gray shading connecting the termini of MoTER1 and MoTER2 shows regions of significant sequence identity between the two elements. The positions of the REL-endo domains and the probes used in this study are shown. (B) Alignment of the MoTeR1 REL-endo domain with those found in other retrotransposons. The names of the retroelements are listed on the left. Arrows indicate the positions of the CCHC and REL domains. Asterisks indicate the characteristic AD..D residues. Values in parentheses indicate the numbers of amino acid residues between the highly conserved motifs.

Figure 5

Genomic distribution of MoTER elements. PstI-digested DNA samples (∼200 ng/lane) were fractionated by electrophoresis and electroblotted to membranes. The immobilized DNAs were then hybridized with a MoTER1 probe (A). After exposure to a phosphorimage screen, the probe was stripped off and the DNAs were reprobed with a MoTER2-specific fragment (B). Finally, the DNA samples were hybridized with the telomere probe (C). Asterisks highlight MoTeR-containing fragments that did not hybridize to the telomere probe. Isolate names are listed above the relevant lanes, and molecular sizes are shown on the left. The white arrowhead marks a highly degenerate internal MoTeR sequence.

Figure 6

Segregation of telomere instability in a genetic cross. DNA was extracted from single-spore cultures of each progeny isolate (G1). These isolates were subcultured for four additional generations, new single-spore cultures (G5) were established, and DNA was extracted from the resulting cultures. The genomic DNA samples were digested with PstI, fractionated by electrophoresis, and electroblotted to a membrane. The membrane was probed sequentially with ³²P-labeled telomere and MoTeR1 fragments. The left and right lanes contain the DNAs from the parental strains. DNA samples from the G1 and G5 subcultures of an individual progeny isolate were loaded in adjacent lanes to allow comparison of their telomeric restriction fragment profiles. Molecular sizes are shown on the left. The white arrowhead marks 2539TEL10, a MoTeR2-containing telomere that was highly stable. (A) Phosphorimage of the TTAGGG-probed gel. Boxes outline fragments that segregated among the progeny but that were not present in either parent. (B) The same membrane used in A was stripped and reprobed with a fragment from the MoTeR1 RT. Asterisks mark hybridizing fragments that were present in the G0 culture but absent in the G5. Open circles show fragments that appeared in the G5 cultures.

Figure 7

Proposed transposition mechanism for the MoTeR elements. (A) Nicking of the telomere repeat top strand by the MoTeR RT. The nick site is identified with an asterisk (note that the specific nick position is not known). (B) Annealing between the 3′ end on the top strand of the nicked telomeric DNA and the CCCTAA sequences predicted to occur at, or near, the 3′ end of a MoTeR transcript. (C) First-strand synthesis of MoTeR cDNA and nicking of the bottom telomeric DNA strand. The model shows a 3′ overhang with four nucleotides, but the length and type of overhang may be different (note that a 5′ overhang would produce a target-site deletion, as opposed to a duplication). (D) Annealing between the 3′ end released by nicking the bottom strand and the 3′ end of the newly synthesized MoTeR cDNA, followed by second-strand synthesis with concomitant RNA strand displacement. (E) Ligation of remaining nicks would result in a MoTeR element inserted precisely in the telomere repeats.