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. 2011 Jun 2:12:288.
doi: 10.1186/1471-2164-12-288.

Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae

Cristiano C Nunes  1 , Malali Gowda, Joshua Sailsbery, Minfeng Xue, Feng Chen, Douglas E Brown, Yeonyee Oh, Thomas K Mitchell, Ralph A Dean
Affiliations

Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae

Cristiano C Nunes et al. BMC Genomics. .

Abstract

Background: Emerging knowledge of the impact of small RNAs as important cellular regulators has prompted an explosion of small transcriptome sequencing projects. Although significant progress has been made towards small RNA discovery and biogenesis in higher eukaryotes and other model organisms, knowledge in simple eukaryotes such as filamentous fungi remains limited.

Results: Here, we used 454 pyrosequencing to present a detailed analysis of the small RNA transcriptome (~ 15 - 40 nucleotides in length) from mycelia and appressoria tissues of the rice blast fungal pathogen, Magnaporthe oryzae. Small RNAs mapped to numerous nuclear and mitochondrial genomic features including repetitive elements, tRNA loci, rRNAs, protein coding genes, snRNAs and intergenic regions. For most elements, small RNAs mapped primarily to the sense strand with the exception of repetitive elements to which small RNAs mapped in the sense and antisense orientation in near equal proportions. Inspection of the small RNAs revealed a preference for U and suppression of C at position 1, particularly for antisense mapping small RNAs. In the mycelia library, small RNAs of the size 18 - 23 nt were enriched for intergenic regions and repetitive elements. Small RNAs mapping to LTR retrotransposons were classified as LTR retrotransposon-siRNAs (LTR-siRNAs). Conversely, the appressoria library had a greater proportion of 28 - 35 nt small RNAs mapping to tRNA loci, and were classified as tRNA-derived RNA fragments (tRFs). LTR-siRNAs and tRFs were independently validated by 3' RACE PCR and northern blots, respectively.

Conclusions: Our findings suggest M. oryzae small RNAs differentially accumulate in vegetative and specialized-infection tissues and may play an active role in genome integrity and regulating growth and development.

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Figures

Figure 1
Figure 1
Distribution of small RNAs with a perfect match to M. oryzae chromosome III. (A) Overall distribution of small RNAs. (B) Small RNAs mapping to unique loci. (C) Small RNAs mapping to repetitive elements. Number of small RNA alignments per 5 kb of genomic sequence is shown on the Y axis with chromosome length on the X axis (vertical lines above and below chromosome line [Y = 0] represent small RNAs mapping to sense and antisense strands, respectively). Red vertical lines indicate sequences derived from mycelia and green from appressoria.
Figure 2
Figure 2
Characterization of small RNAs from mycelia and appressoria tissues with a perfect match to the genome. (A) Proportion of small RNAs aligned to different genomic features. (B) Size distribution of small RNAs from mycelia and appressoria libraries, (C) sub-features within genes and (D) tRNAs from mycelia and appressoria libraries.
Figure 3
Figure 3
Nucleotide composition of small RNAs mapped to M. oryzae genomic features. Nucleotide frequency of small RNA alignments to sense and antisense strands of (A) genes, (B) repetitive elements, (C) tRNA and (D) rRNA loci are shown on the Y axis and nucleotide position on the X axis. Position 1 of antisense strand mapped small RNAs for genes and repeats is enriched for T (U in the RNA molecule). In contrast, C is suppressed at position 1.
Figure 4
Figure 4
Small RNAs mapped to intergenic features. Small RNAs (dotted box) mapping uniquely to intergenic regions on (A) chromosome III (3202700.3209000), (B) chromosome I (1539400.1545900) and (C) unlinked DNA (9200.13100) are enriched in mycelia. ESTs and ESSs provide additional evidence for transcriptional activity within and around these intergenic regions.
Figure 5
Figure 5
LTR-siRNAs mapping to the retrotransposable element MAGGY. (A) Distribution of small RNAs (black dotted box) to a ~ 0.8 Mb region of chromosome I show two major peaks on the plus (sense) and minus (antisense) strands. Closer inspection of boxed region reveals many small RNAs (black arrows) mapping to the full length (~ 5 kb) of MAGGY LTR-retrotransposon (red arrow). (B) Detailed inspection of boxed region shows LTR-siRNAs map in roughly equal proportions to both strands of MAGGY where the y axis corresponds to small RNA mappings to sense (red) and to antisense (green) strands and the X axis represents the full length MAGGY sequence. (C) Size distribution of MAGGY-derived LTR-siRNAs reveals a peak length of 22-23 nucleotides.
Figure 6
Figure 6
MAGGY LTR-siRNA validation by 3' RACE. (A) Detailed schematic flow of small RNA (< 30 nt) isolation, polyadenylation, cDNA synthesis and specific MAGGY LTR-siRNA amplification. (B) PCR amplified MAGGY LTR-siRNAs using the 3' reverse primer and 5' MAGGY small RNA-specific primer under three different mycelia growth conditions separated on a 3% agarose gel (see Material and Methods for tissue growth treatments). L = 100 bp DNA Ladder (New England Biolabs), lanes 1 - 3 and 7 correspond to small RNA mapping to sense and lanes 4 - 6 to anti-sense strands. (C) PCR amplification of MAGGY LTR-siRNA 7 on a 15% polyacrylamide gel as described in (A) and (B) above (lanes 3, 6 and 9). For PCR controls, lanes 1, 4 and 7 were loaded with water and lanes 2, 5 and 8 with total RNA. D = Decade DNA ladder (Invitrogen).
Figure 7
Figure 7
Small RNAs mapping to MGR583. Small RNAs mapping to the repetitive element MGR583 are highly enriched in the mycelia library (bottom of dashed box). EST data suggests MGR583 and a pseudo tRNA are transcribed as a single mRNA, which is possibly silenced by small RNAs.
Figure 8
Figure 8
Logos of tRFs mapping to tRNA families. (A) Differential mapping of tRFs to the 5' and 3' halves of mature tRNAs. tRFs originating from a single tRNAHis type (see Material and Methods) mapped more abundantly to the 3' half in the mycelia library and in roughly equal frequency to both 5' and 3' halves in the appressoria library. Numbers below each letter in the Logos represent number of nucleotides from small RNAs mappings. (B) Members of tRNAPro family clustered into two types (two Logos per library) where small RNAs mapped predominantly to one type in both libraries. While tRFs mapped preferentially to the 5'half of both tRNAPro types in the mycelia library, they mapped primarily to the 3'half of a single type in the appressoria library. (C) Members of tRNALys grouped into two types. In both libraries, tRFs mapped predominantly to the 3' half and preferentially to one tRNALys type (upper Logo in each library).
Figure 9
Figure 9
Northern blot analysis of tRFs from different tissues and stress conditions. (A) Northern blot hybridization analysis of tRNAThr reveals differential accumulation of 5' and 3' tRFs across different tissues. (B) 5' derived tRFs from tRNAGly accumulate under a variety of stress conditions, but most predominantly in spores and appressoria tissues. Predominant RNA signifies RNA of high quality and similar concentration.
Figure 10
Figure 10
Possible posttranscriptional modification sites of tRFs. (A) Genomic locus of the mature tRNAArg and flanking sequence is shown in italics and underlined letters, respectively. tRFs mapping to the 3' half of tRNAArg show non-templated CCA addition (red letters). (B) Logo of genomic tRNALeu sequence (top) and corresponding tRFs Logos from appressoria (middle) and mycelia (bottom) indicate possible posttranscriptional modification sites as indicated by dotted boxes. Numbers below Logo designate nucleotide position of the mature tRNALeu.
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