Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR/Cas9

Abstract

Phytophthora sojae is an oomycete pathogen of soybean. As a result of its economic importance, P. sojae has become a model for the study of oomycete genetics, physiology and pathology. The lack of efficient techniques for targeted mutagenesis and gene replacement have long hampered genetic studies of pathogenicity in Phytophthora species. Here, we describe a CRISPR/Cas9 system enabling rapid and efficient genome editing in P. sojae. Using the RXLR effector gene Avr4/6 as a target, we observed that, in the absence of a homologous template, the repair of Cas9-induced DNA double-strand breaks (DSBs) in P. sojae was mediated by non-homologous end-joining (NHEJ), primarily resulting in short indels. Most mutants were homozygous, presumably as a result of gene conversion triggered by Cas9-mediated cleavage of non-mutant alleles. When donor DNA was present, homology-directed repair (HDR) was observed, which resulted in the replacement of Avr4/6 with the NPT II gene. By testing the specific virulence of several NHEJ mutants and HDR-mediated gene replacements in soybean, we have validated the contribution of Avr4/6 to recognition by soybean R gene loci, Rps4 and Rps6, but also uncovered additional contributions to resistance by these two loci. Our results establish a powerful tool for the study of functional genomics in Phytophthora, which provides new avenues for better control of this pathogen.

Keywords: Avr4/6; CRISPR/Cas9; Phytophthora sojae; RXLR effector; gene replacement; genome editing; oomycetes.

Figure 1

Cas9 and guide RNA constructs for P hytophthora sojae genome editing. (A) Top: plasmid for expression of hSpCas9 fused to eGFP and a nuclear localization sequence (NLS) in P . sojae. PsNLS, a strong synthetic NLS derived from a P. sojae bZIP transcription factor; eGFP, enhanced green fluorescent protein. Bottom: P . sojae hyphae expressing PsNLS‐hSpCas9‐GFP from pYF2, counter‐stained with 4′,6‐diamidino‐2‐phenylindole (DAPI); scale bars, 10 μm. (B) Top: plasmids for expression of CRISPR constructs in P . sojae.  Cas9 expression is driven by the Ham34 promoter, on a plasmid with the selectable marker NPT II driven by the P . sojae RPL41 promoter. Transcription of single guide RNA (sgRNA) (including flanking ribozymes) is driven by the RPL41 promoter on a plasmid with an eGFP expression cassette (used as a screening marker). Bottom: double ribozyme construct for release of sgRNAs from the primary RNA polymerase II transcript.

Figure 2

Single guide RNAs (sgRNAs) for targeting of Avr4/6. (A) Two sgRNA target sites within the Avr4/6 open reading frame (ORF). Target sites of sgRNA A (sgRNA‐A) and B (sgRNA‐B) are highlighted in blue and yellow, respectively. sgRNAs A and B target Avr4/6 on the negative (−) and positive (+) DNA strand, respectively. The sgRNA‐A site overlaps with a Bst UI restriction enzyme site (CGCG) and sgRNA‐B with a Tsp 45I site (GT^G/_CAC). PAM, Protospacer Adjacent Motif (bold red). (B) In vitro cleavage assay indicating that Avr4/6 sgRNA‐B can direct Cas9 cleavage of target polymerase chain reaction (PCR) products, but sgRNA‐A cannot. The DNA template was amplified from pBS‐Avr4/6 using M13F and M13R [supplemental sequences in Appendix S1 (see Supporting Information)]. The colours of the original gel are inverted for clarity. (C) PCR and restriction enzyme analysis of P hytophthora sojae pooled transient expression transformants, indicating that only sgRNA‐B flanked by ribozymes (sgRNA‐B_R) produced amplicons resistant to restriction enzyme cleavage (arrowhead). Approximately 25%–30% of the amplicon was resistant to Tsp 45I digestion. The experiment was performed in triplicate. sgRNA‐A, ‐B, sgRNA lacking ribozymes; sgRNA‐AR, ‐BR, sgRNAs flanked byribozymes; mock, P . sojae transformants only receiving Cas9 plasmid. In (B) and (C), all gel panels placed together were from the same gel; white dividers indicate lanes not adjacent in these gels.

Figure 3

Characterization of individual non‐homologous end‐joining (NHEJ)‐mediated mutants. (A) Tsp 45I screening of Avr4/6 amplicons from six individual transformants carrying hSpCas9 and sgRNA‐B_R plasmids. Arrowhead indicates Tsp 45I‐resistant amplicons. Transformants are expected to carry a mixture of modified and non‐modified Avr4/6 genes. (B) Left: sequences of Avr4/6 mutant amplicons from single zoospore lines derived from transformants T11, T18 and T32 (T30 produced no zoospores). Target sites are highlighted in yellow and the PAM sequences are shown in bold red. Right: summary of virulence assays of Avr4/6 mutants on Rps4‐ and Rps6‐containing cultivars. V, virulent; A, avirulent; I, intermediate; I/A intermediate to avirulent; I/V intermediate to virulent.

Figure 4

Homology‐directed repair (HDR)‐mediated replacement of the Avr4/6 open reading frame (ORF) with an NPT II ORF. (A) Strategy used for gene replacement. Plasmids containing a homologous donor DNA (NPT II with Avr4/6 flanking sequences) were co‐transformed with the Avr4/6 sgRNA‐B_R and hSpCas9 constructs. (B) Three different sizes of the homology arms, 250 bp, 500 bp and 1 kb, flanking the Avr4/6 locus, were used. The NPT II gene in the hSpCas9 expression plasmid served as the control (0‐bp homologous arm, mock). Primers used to screen the HDR mutants and to validate the replaced region are shown as arrows. Primer pair F1/R1 and nested primer pairs F2/R3, F3/R2 and F2/R2 were used for HDR mutant screening. Red arrowhead indicates the CRISPR/Cas9 cleavage site (between 232 and 233 bp of Avr4/6 ORF). (C) Analysis of genomic DNA from the pooled transformants produced using the four sizes of flanking sequences, employing nested PCR. Arrowheads indicate sizes expected if HDR has occurred. ACTIN, actin control for DNA quality. (D) Screening of individual HDR transformants generated with the 1‐kb flanking sequence plasmid. The nine positive HDR mutants are highlighted in bold. (E) PCR analysis of representative zoospore‐purified lines of HDR mutants, demonstrating that T12 and T39 are homozygotes, whereas T29 is a heterozygote. DNA sizes (bp) before restriction enzyme cleavage: WT, 2936; HDR mutant, 3359; after Nco I digestion: WT, 2273 + 663; HDR mutant, 1945 + 751 + 663. The arrowhead indicates the fragment amplified from the NPT II‐replaced allele. Primer F4, Avr4/6_up500bp_PhusF (Table S1). (F) Sanger sequencing traces of junction regions confirming that the Avr4/6 ORF was cleanly replaced by the NPT II ORF in a representative zoospore‐purified clone (HDR‐T12‐1). Start and stop codons are indicated in bold red.

Figure 5

Infection phenotypes of Avr4/6 mutants. Representative photographs showing soybean seedlings inoculated on the hypocotyls with P hytophthora sojae Avr4/6 homology‐directed repair (HDR) mutants. L85‐2352 and HARO4272 contain Rps4, whereas L89‐1581 and HARO6272 contain Rps6. (A) Williams isolines. (B) Harosoy isolines. Photographs were taken at 4 days post‐inoculation.