pHg/pSILBAγ vector system for efficient gene silencing in homobasidiomycetes: optimization of ihpRNA - triggering in the mycorrhizal fungus Laccaria bicolor

Abstract

pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy oriented two-step PCR cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, the pHg/pSILBAγ plasmid is used for Agrobacterium-mediated transformation. The pHg/pSILBAγ system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome and the integration sites of the transgenes can be resolved by plasmid rescue. pSILBAγ construct and two other pSILBA plasmid variants (pSILBA and pSILBAα) were evaluated for their capacity to silence Laccaria nitrate reductase gene. While all pSILBA variants tested resulted in up to 65-76% of transformants with reduced growth on nitrate, pSILBAγ produced the highest number (65%) of strongly affected fungal strains. The strongly silenced phenotype was shown to correlate with T-DNA integration in transcriptionally active genomic sites. pHg/pSILBAγ was shown to produce T-DNAs with minimum CpG methylation in transgene promoter regions which assures the maximum silencing trigger production in Laccaria. Methylation of the target endogene was only slight in RNA silencing triggered with constructs carrying an intronic spacer hairpin sequence. The silencing capacity of the pHg/pSILBAγ was further tested with Laccaria inositol-1,4,5-triphosphate 5-phosphatase gene. Besides its use in silencing triggering, the herein described plasmid system can also be used for transgene expression in Laccaria. pHg/pSILBAγ silencing system is optimized for L. bicolor but it should be highly useful also for other homobasidiomycetes, group of fungi currently lacking molecular tools for RNA silencing.

Figure 1

A. Sequence and unique restriction sites of pSILBAγ for inverted repeated sequence cloning. The acceptor and donor bases of L. bicolor nitrate reductase intron are in italics. The BamHI site in brackets is not unique. The same cloning sites are unique also in pSILBA and pSILBAα plasmid variants.
B. pSILBAγ cloning vector. SacI and NotI linearize the plasmid; XbaI liberates the silencing triggering cassette.
C. pSILBA.
D. pSILBAα.
Pgpd: glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus. NR: nitrate reductase. CUT: cutinase. TtrpC: Aspergillus nidulans tryptophan synthetase terminador.

Figure 2

Two‐step cloning of the hairpin trigger in pSILBAγ and joining the plasmid with the binary vector pHg. The T‐DNA structures generated with pSILBAγ and pSILBAα are marked as pHg/pSγ and pHg/pSα respectively. SC: silencing cassette. HRC: hygromycin resistance cassette. MCS: multiple cloning site. LB: T‐DNA left border repeat of pCAMBIA1300. RB: T‐DNA right border repeat of pCAMBIA1300. hph: hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to hygromycin B and structurally related antibiotics. Amp^r: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. Kan^r: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. 35S‐3′: cauliflower mosaic virus 35S terminator.

Figure 3

A. Growth of wild‐type dikaryon and transformed Laccaria strains after 1 month in nitrate medium in microtitre plates. Growth category group A is presented as duplicate due to wider variation within this group.
B and C. Comparison of silencing efficiency between 37 randomly selected of each pHg/pSILBA/NITRLoop (pS), pHg/pSILBAα/NITRLoop (pSalpha) and pHg/pSILBAγ/NITRLoop (pSgamma) transformed strains. The growth on nitrate was compared with wild type after 2 weeks (B) and 1 month (C), and classified in three growth categories: non‐affected (N), affected (A) and strongly affected (S).

Figure 4

A and B. HpaII/MspI restriction map of gpdII promoters in pHg/pSILBAγ/NITRLoop (A) and pHg/pSILBAα/NITRLoop (B) T‐DNAs. CCGG restriction sites of HpaII (CpG methylation sensitive) and MspI (CpG methylation insensitive) within and around the promoter sequences are marked with vertical lines. Fragments from complete DNA digestion hybridizing to gpdIIP probe are indicated by arrows above the T‐DNAs. Detected fragments are marked with thicker arrows. Fragments from incomplete digestion of pHg/pSILBAα/NITRLoop with HpaII are indicated by black arrows below the T‐DNA in (B). Red arrows correspond to fragments from complete digestion of the nitrate reductase silencing trigger loop (not revealed in the Southern). The terminator elements of the T‐DNAs are not presented.
C. Southern blot hybridized to gpdIIP probe. Results of one strongly silenced pSα, one strongly silenced pSγ and wild‐type strains are shown. If no CpG methylation is present in the target DNA both HpaII and MspI restrictions should result in identical hybridization signals. H: HpaII restriction. M: MspI restriction.

Figure 5

A. Laccaria wild‐type dikaryon and six pHg/pSILBAγ/NITRLoop‐transformed strains grown for 23 days on solid medium with ammonium or nitrate as N sources. Growth categories: N, non‐affected; A, affected; S, strongly affected.
B. Growth of wild type and transformants in liquid nitrate medium after 22 days.
C. Dry weight of mycelia produced by wild type and six pHg/pSILBAγ/NITRLoop‐transformed strains in liquid nitrate medium after 22 days.
D. Reverse transcription polymerase chain reaction (RT‐PCR) expression analysis of L. bicolor nitrate reductase‐encoding gene (protein ID 254066). Total RNA from mycelia of Laccaria S238N wild type and six pHg/pSILBAγ/NITRLoop‐transformed strains was isolated and used for first‐strand cDNA synthesis. A PCR was performed with first‐strand cDNA as template and between 25 and 30 cycles of amplification with alpha tubulin (control gene, protein ID 192524) and nitrate reductase primers. The picture shows fragments amplified after 30 cycles of PCR. For details see Experimental procedures.

Figure 6

Southern blot of wild type and five pHg/pSILBAγ/NITRLoop transformants. Genomic DNA was cut with BamHI, blotted and probed with amp probe. N: non‐affected growth; and S: strongly affected growth on nitrate medium. DNA marker: λ/EcoRV/HindIII.

Figure 7

A. HpaII (CpG methylation sensitive)/MspI (CpG methylation insensitive) restriction map of the Laccaria nitrate reductase gene. The probes used for hybridization (1–3) and expected fragments from complete digestion of the DNA are represented above the gene with solid two‐headed arrows (37 bp under the detection level). The two smallest possible fragments originating from CpG‐methylated DNA digested with HpaII are represented by dashed two‐headed arrows. The 417 bp sequence used for triggering RNA silencing is presented below the gene.
B–D. CpG methylation analysis of the Laccaria nitrate reductase gene in wild type and in silenced strains. (B) NR probe 1. (C) NR probe 2. (D) NR probe 3. pSα refers to a strain transformed with pHg/pSILBAα/NITRLoop construct. pSγ refers to strain transformed with pHg/pSILBAγ/NITRLoop construct. H: HpaII restriction. M: MspI restriction. eg; main endogene signal. st: main silencing trigger signal.

Figure 8

A. Laccaria wild‐type monokaryon (wt), mutant with 5‐Pase gene interruption and 5‐Pase‐silenced strains.
B. Reverse transcription polymerase chain reaction (RT‐PCR) expression analysis of L. bicolor synaptojanin‐like 5‐Pase‐encoding gene (protein ID 306121). Total RNA from mycelia of Laccaria H82 wild type and two pHg/pSILBAγ/INOLoop transformants (I, II) was isolated and used for first‐strand cDNA synthesis. A PCR was performed with first‐strand cDNA as template and between 25 and 30 cycles of amplification with alpha tubulin (control gene, protein ID 192524) and 5‐Pase gene primers. The picture shows fragments amplified after 27 cycles. For details see Experimental procedures.