Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa

Abstract

We report the isolation of 15 Neurospora crassa mutants defective in "quelling" or transgene-induced gene silencing. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. We show that when qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene.

Figure 2

RNase protection experiments for sense al-1 RNA transcripts in strain 6xw. (A) RPA was performed on total RNA extracted from wild-type untransformed strain illuminated for 20 min with light (lane 1), dark-grown transformant 6xw (lane 2), 6xw illuminated for 20 min (lane 3), dark-grown wild type (lane 4). Arrows indicate the sizes of the protected fragments obtained from the unspliced endogenous al-1 mRNA (206 nt), from the mature endogenous al-1 mRNA (133 nt), and from the transgenic al-1 sense RNA (96 nt). Undigested al-1 RNA probe (266 nt) is shown in lane 5. (B) Map of endogenous and potential transgenic sense transcripts. The pALC4 plasmid, which contains a single intron, was used to generate an RNA probe capable of hybridizing to sense al-1 mRNA. This RNA probe is indicated as a solid black line. The diagonal portion of the RNA probe represents plasmid sequences not present in the N. crassa al-1 mRNA. Shaded boxes represent sense RNA from the endogenous native al-1 RNA gene (Upper) and a putative sense RNA from the al-1 transgene (Lower).

Figure 6

RNase protection experiments for transgenic sense al-1 RNA transcripts in mutant strains. RPA was performed, using an RNA probe as described in Fig. 2B, on total RNA extracted from dark-grown mutants representative of each group (M10, M30, M20, M17) (lanes 1–4), from dark-grown transformant 6xw (lane 5), and from an untransformed strain illuminated for 20 min (lane 6). Undigested al-1 RNA probe is shown in lane 7. Arrows indicate the size of the protected fragments obtained from the unspliced endogenous al-1 sense RNA (206 nt), the mature endogenous al-1 mRNA (133 nt), and the transgenic al-1 sense RNAs (96 nt). Undigested probe (lane 7) of 266 nt is indicated by an arrow. Sizes of the protected fragments are as in Fig. 2B.

Figure 1

Southern hybridization analysis of the stable albino transformant strains. (A) Schematic representation of al-1 endogenous locus and of plasmid pX16 used in transformation experiments. (B) SmaI/HindIII-digested genomic DNA extracted from wild-type untransformed strain (lane 1), strain 6xw (lane 2), and strain 5xw (lane 3) was hybridized with an al-1 probe able to detect both endogenous and transgenic al-1 copies. The 3.1-kb band corresponding to the endogenous al-1 gene and the 5.5-KB band corresponding to the tandem repeated insertions are indicated by arrows.

Figure 3

Southern hybridization analysis of the mutants. SmaI/HindIII-digested genomic DNA extracted from five representative mutants of group 1 (M37, M40, M7, M10, M11), mutant strains of group 2 (M30, M34, M35, M43), the quelled strain (6xw), and a wild-type untransformed strain (WT). The DNA digestions were blotted and probed with a DNA fragment able to detect both endogenous and transgenic al-1 copies. The 3.1-kb band corresponding to the endogenous al-1 gene and the 5.5-kb band corresponding to the plasmid arranged in tandem repeats are indicated.

Figure 4

Analysis of al-1 gene expression in mutant strains. Total RNA extracted from mycelia, illuminated for 20 min, of eight representative mutants (M7, M30, M37, M40, M41, M43, M10, M11), strain 6xw, and wild type. After blotting, RNAs were hybridized with an al-1 probe. Control hybridization was performed with the photoregulated al-2 gene.

Figure 5

Expression of qa-2 transgenes in qde mutants. (A) Histograms indicate the relative dry weight of mycelia from qde mutants grown in different media as indicated. Three representative mutants (M7, M10, M17) are compared with the 6xw and wild-type strains. (B) Northern blot analysis of qa-2 gene expression in qde mutants and strain 6xw. Total RNA extracted from mycelia grown in presence of 10 mM quinic acid as inducer of qa-2 expression was hybridized with a qa-2 probe. A sod-1 probe was used as normalization control.