Fig 1.
Characteristics of the Puf family in P. ultimum.
(A) Predicted Pumilio repeats in Puf proteins. (B, C) Transcript levels of the Puf genes measured using RNA-seq (Left) and/or qRT-PCR (Right) (B), and sexual development of P. ultimum (C) when mycelia were inoculated in V8 liquid medium for 24, 36, 48, or 96 h. Cultures were stained with the lactophenol-trypan blue for 30 s before microscope observation. “IO” and “MO” indicate the oogonia with immature oospore and mature thick-walled oospore, respectively. Bar, 20 μm.
Fig 2.
CRISPR/Cas9-mediated PuM90 gene knockout and complementation.
(A) Schematic diagram of homology-directed repair-mediated modification of the target gene. Top: an ‘all-in-one’ plasmid (pYF515) harboring both Cas9 and sgRNA cassettes was co-transformed with a plasmid (pBS-SK II+) containing homologous donor DNA hph with PuM90 flanking sequences. Locations of the primers used to screen the HRR mutants and Sanger sequencing traces of junction regions confirming that the PuM90 ORF was precisely replaced. Bottom: analysis of genomic DNA from the wild-type (WT), empty-vector control line (EV), and PuM90-knockout mutants (ΔPuM90-1/2/3) using the primers shown at the top and actin primers as a positive control. (B) Schematic representation of the ΔPuM90 mutant complementation strategy and the plasmids used for second transformation in Pythium. Top: PuM90-m with two black triangles indicates PuM90 modified with two sgRNA targeting sequences. Locations of the primers used to screen the complementation mutants and Sanger sequencing traces of junction regions confirming that the PuM90 ORF was precisely complemented. Bottom: analysis of genomic DNA from the wild-type (WT), complemented transformants (ΔPuM90-C1/2), and empty control line of ΔPuM90 (ΔPuM90-EV) using the primers shown at the top and actin primers as a positive control.
Fig 3.
PuM90-knockout disrupts oospore formation.
(A) Morphology of oogonia and oospores generated by WT, EV, PuM90-knockout transformants (ΔPuM90-1/2/3), complemented transformants (ΔPuM90-C1/2), and the empty control line of ΔPuM90 (ΔPuM90-EV). Oospores were generated on 14-day-old V8 solid medium (first row), 7-day-old V8 liquid medium (second row), or infected root tissue at 72 hpi (third row) and stained with lactophenol-trypan blue. (B–D) Statistical analysis of oogonium number from 14-day-old cultures on V8 solid medium (B), oospore diameter (C), and thickness of the oospore wall (D) from 7-day-old cultures grown in V8 liquid medium. Bar, 20 μm. Asterisks indicate significant differences comparing with WT at P < 0.05 (*) or P < 0.01 (**).
Fig 4.
PuM90-knockout transformants produce abnormal oospores.
(A) Oospores from 2-, 4-, and 7-day-old cultures stained with lactophenol-trypan blue were observed under a light microscope. Bar, 20 μm. (B) Oospores from 7-day-old cultures were observed with a transmission electron microscope (TEM). The area between the two yellow arrows represents the oospore wall. Bar, 2 μm. (C) Oospores from 7-day-old cultures were observed with a light microscope after staining with 1000 ppm Congo red for 24 h. Bar, 20 μm.
Fig 5.
Mutations of key amino acids in the TRM of the Puf domain compromise PuM90 function.
(A) Protein structure of the Puf RNA-binding domain in PuM90 was predicted using SWISS-MODEL (https://swissmodel.expasy.org/). Individual amino acids predicted to interact with RNA are colored in red and mutated to AAA in ΔPuM90-CM. The predicted RNA bases targeted by each Pumilio repeat are shown. (B) Morphology of oogonia and oospores from the WT, PuM90-complemented transformants (ΔPuM90-C1), and key amino acid mutation transformants (ΔPuM90-CM1/2/3). Oospores were generated on 14-day-old V8 solid medium (top) or in 7-day-old V8 liquid medium (bottom) cultures. (C–E) Statistical analysis of oogonium number from 14-day-old cultures on V8 solid medium (C), thickness of oospore wall (D), and oospore diameter (E) from 7-day-old cultures on V8 liquid medium. Bar, 20 μm. Asterisks indicate significant differences comparing with WT at P < 0.05 (*) or P < 0.01 (**).
Fig 6.
PuM90 binds to the 3′-UTR of PuFLP and represses PuFLP expression.
(A) The number of differentially regulated genes between the PuM90 knockout mutant ΔPuM90-1 and WT at 24 h and 96 h. Three genes (PYU1_T006554, PYU1_T013662, and PYU1_T003505) were upregulated in the mutants compared to the WT at 96 h and also contained the UGUA[A/U/C]AUA motif in their 3′-UTRs. (B) Transcript levels of the three candidate genes measured through RNA-seq and qRT-PCR. (C) Sanger sequencing trace of the 30-nt PuFLP-3′BS used for the electrophoretic mobility shift assay (EMSA) assay. The UGUACAUA core binding motif is indicated with a black box. (D) EMSA results showing that PuM90-RBD bound to PuFLP-3′BS, while PuM90-RBDAAA or GST did not bind to PuFLP-3′BS, and PuM90-RBD did not bind to PuFLP-3′BSmu. (E) Microscale thermophoresis (MST) results showing that PuM90-RBD bound to PuFLP-3′BS (Kd = 2.66 μM, green trace), while PuM90-RBDAAA did not bind to PuFLP 3′BS (Kd = 0 μM, red trace), and PuM90-RBD did not bind to PuFLP-3′BSmu (Kd = 0 μM, blue trace).
Fig 7.
PuFLP overexpression affects oospore development.
(A) PuFLP transcript and protein levels measured via qRT-PCR and Western blotting, respectively. Total protein was extracted from each strain for SDS-PAGE and immunoblotting was performed with GFP antibodies. Relative transcript levels were calculated using the WT as a reference. The experiments were repeated independently three times. (B) Morphology of oogonia and oospores generated in 14-day-old cultures of WT, an empty-vector control (OE-PuFLP-EV), and PuFLP overexpression transformants (OE-PuFLP-1/2/3). (C) Statistical analysis of oogonium numbers generated in 14-day-old cultures. Bar, 20 μm. Asterisks indicate significant differences comparing with WT at P < 0.05 (*) or P < 0.01 (**).
Fig 8.
Replacement of the UGUACAUA motif in PuFLP-3′BS with ACACACAC (PuFLP3′BSmu) affects oospore development.
(A) Morphology of oogonia and oospores generated in 14-day-old cultures of the WT, PuM90-complemented transformant ΔPuFLP-C, and PuFLP-3′BS mutation transformant PuFLP3′BSmu. (B) Statistical analysis of oogonium number generated in 14-day-old cultures. Bar, 20 μm. (C) Relative transcript levels of PuFLP were analyzed by qRT-PCR, using the WT results as a reference. The experiments were repeated independently three times. Asterisks (**) indicate significant differences comparing with WT at P < 0.01.
Fig 9.
A proposed model for PuM90 function.
PuM90 protein could specifically bind to a UGUACAUA motif in the 3′-UTR of PuFLP mRNA, as the post-transcriptional way to repress PuFLP mRNA level to facilitate oospore formation. PuFLP is not regulated by PuM90 in PuM90 deletion mutants resulting in increased transcript levels of PuFLP. Exorbitant PuFLP mRNA leads to abnormal oospore. Thus, the RNA-binding protein PuM90 acts as a novel oospore formation regulator to promote the oomycete development.