Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.
Oomycetes are a class of eukaryotic microorganisms with life cycles and growth habits similar to filamentous fungi, but are not true fungi. Although sexual reproduction, which produce oospores, is an essential stage of life cycle, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. In this study, we developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium ultimum, a notorious oomycete pathogen that is responsible for a variety of diseases in a broad range of plant species. We further identified the Puf family RNA-binding protein PuM90 and the flavodoxin-like protein PuFLP as major functional factors involved in P. ultimum oospore formation. We proposed a new model that PuM90 acts as a stage-specific post-transcriptional regulator by specifically binding to the 3′-UTR of PuFLP and then repressing PuFLP mRNA level. This study describes new technologies and data that will help to elucidate sexual reproduction and post-transcriptional regulation in oomycetes.
Citation: Feng H, Wan C, Zhang Z, Chen H, Li Z, Jiang H, et al. (2021) Specific interaction of an RNA-binding protein with the 3′-UTR of its target mRNA is critical to oomycete sexual reproduction. PLoS Pathog 17(10): e1010001. https://doi.org/10.1371/journal.ppat.1010001
Editor: Howard S. Judelson, University of California, Riverside, UNITED STATES
Received: July 9, 2021; Accepted: October 3, 2021; Published: October 14, 2021
Copyright: © 2021 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files. The RNA-seq data have been deposited in the National Center for Biotechnology Information (NCBI) database [BioProject ID: PRJNA540115 (Run IDs: SRR15049129-SRR15049146)].
Funding: This work was supported by grants to W. Y. from the National Natural Science Foundation of China (NSFC; 31972250 and 31772140), grants to Y. W. from the NSFC (31721004) and the China Agriculture Research System (CARS-004-PS14), and grants to H. F. from the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX20_0594). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Oomycetes are a class of eukaryotic microorganisms with life cycles and growth habits similar to filamentous fungi, but are not true fungi . Unlike fungi, oomycetes produce oospores during sexual reproduction [2–4]. In both homothallic and heterothallic oomycete species, fertilization results in thick-walled zygotes called oospores, which can be produced within infected plant tissue and released into the soil as the plant tissue degrades. Oospores are highly resistant to environmental influences and can persist in the soil for several years . The sexual cycle enhances fitness through formation of recombinant genotypes that may be more pathogenic or resistant to fungicides than their parents . Consequently, understanding the biological processes and molecular mechanisms that regulate sexual reproduction is essential for precise and efficient prevention of plant disease outbreaks associated with these pathogens.
One of the most destructive oomycete pathogens , Pythium ultimum, displays a wide host range and has been recorded on hundreds of plant species worldwide, causing damping-off and root rot that lead to huge yield losses . P. ultimum is homothallic, meaning that a single isolate can mate with itself to complete the sexual stage; however, outcrossing has also been reported . P. ultimum includes two varieties: P. ultimum var. ultimum (the most common and pathogenic group) and P. ultimum var. sporangiferum (a rare and less pathogenic group); the former scarcely produces zoospores and almost solely produces oospores . Therefore, sexual reproduction is likely more important in the life cycle of P. ultimum var. ultimum (hereinafter referred as P. ultimum).
To date, only a few genes involved in the sexual reproduction of oomycetes have been identified. In Pythium oligandrum, small tyrosine rich (PoStr) proteins play a key role in oospore formation. Oospores of PoStr family-silenced mutants displayed major ultrastructural changes and were sensitive to degradative enzyme treatment . In Phytophthora sojae, knockout mutants of PsYPK1, a serine/threonine protein kinase-encoding gene, lost the ability to produce oospores ; and silencing mutants of PsGK5, one of the genes coding G-protein-coupled receptors with a phosphatidylinositol phosphate kinase domain, was also defective in oospore formation . In Phytophthora infestans, silencing of a loricrin-like protein-encoding gene significantly affected oospore formation . Despite the identification of the regulators above, the regulatory mechanisms of oospore formation and sexual reproduction process in oomycetes remain largely unknown.
The release of genome sequences has opened the door for elucidation of P. ultimum biology at the molecular level . However, barriers for functional genomics remain, and one of the greatest is genetic manipulation technologies. Although protoplast electroporation and Agrobacterium tumefaciens-mediated transformation were proved to be available in P. ultimum [15,16], few studies have reported using these methods. In recent years, the CRISPR/Cas system has been applied to oomycetes such as Phytophthora sojae , Phytophthora palmivora , Phytophthora capsici , Phytophthora infestans , Aphanomyces invadans , and Peronophythora litchii , greatly promoting functional genomics research. Although polyethylene glycol (PEG)-mediated protoplast transformation has been reported in a few Pythium species , CRISPR/Cas system-mediated genetic manipulation methods, such as gene knockout and in situ complementation, have not yet been reported in any Pythium species.
Regulation of gene expression at the post-transcriptional level, including RNA stability, translation, and localization, is important for the growth and development of eukaryotic organisms . RNA-binding proteins are a major group of regulators that directly or indirectly participate in these processes . Among them, Puf proteins, named for Drosophila Pumilio (PUM) and Caenorhabditis elegans fem-3 binding factor (FBF), share a Puf RNA-binding domain and are present in various eukaryotic species. Puf proteins are involved in diverse biological processes, including embryonic development , nervous system function and development , stem cell and germ cell maintenance , rRNA processing and ribosome biogenesis , and chemotactic cell movement . They commonly repress level of targeted mRNAs, although a few reports have suggested roles in stabilization of target transcripts . For example, in humans, Pum1 acts as a negative regulator of LGP2, which is a master activator of innate immunity genes . In Saccharomyces cerevisiae, Puf3 protein specifically promotes the degradation of COX17 mRNA, and Puf5 (Mpt5) protein negatively regulates synthesis of HO endonuclease by binding to the 3’-UTR of HO mRNA . Yeast Puf6p binds to the Ash1p 3′-UTR and downregulates level of Ash1p mRNA . PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum . Generally, classical Puf proteins have eight Pumilio repeats organized into a crescent-shaped structure that bind to the 3′-UTRs of mRNAs. Each Pumilio repeat contains a tripartite recognition motif (TRM) and generally recognizes a single base of the target RNA via stacking interactions and hydrogen bonding . Hence, upon identification of the TRMs in each Pumilio repeat, it is possible to predict the specific RNA bases to which a Puf protein binds.
In the genome of P. ultimum, four genes encode Puf proteins, but their biological functions remain uninvestigated. In this study, we successfully developed a CRISPR/Cas9 system-mediated gene knockout and in situ complementation in P. ultimum. We characterized PuPuf1 (named PuM90), which exhibited increased expression levels specifically during the stage of oospore development in P. ultimum. We revealed that PuM90 is required for P. ultimum oospore formation and further identified a flavodoxin-like protein (designated PuFLP) regulated by PuM90 through specific binding to the PuFLP 3′-UTR. Our findings reveal the mechanism through which the RNA-binding protein PuM90 acts as a major regulator of P. ultimum oospore formation by repressing PuFLP mRNA level. This study represents the first report of both a CRISPR/Cas9-mediated gene editing system in Pythium and a post-transcriptional regulatory mechanism for oomycete sexual reproduction.
PuM90 is specifically transcribed during P. ultimum oospore formation
Through Hidden Markov Model (HMM) and Basic Local Alignment Search Tool (BLAST) searches, we identified four genes (designated PuPuf1–PuPuf4) encoding Puf family RNA-binding proteins in P. ultimum (S1 Table), in which four to eight Pumilio repeats were predicted (Pfam ID: PF00806; Fig 1A). Each P. ultimum Puf protein exhibited a 1:1 orthologous relationship with those identified in other oomycetes (S1 Fig). Similar to its orthologs, PuPuf1 contained eight predicted Pumilio repeats (Fig 1A and S1 Table). PuPuf1 was designated PuM90 due to the given name of its ortholog PiM90 in Phytophthora infestans [36,37]. Based on RNA sequencing analysis (RNA-seq) of P. ultimum mycelia cultured in V8 liquid medium for 24, 36, 48, or 96 h, we found that the transcript level of PuM90 was strongly increased at 96 h (16.0-fold compared to 24 h; Fig 1B). The transcription pattern of PuM90 was confirmed through a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay (Fig 1B). According to our observations, immature oospores appeared at 48 h and mature thick-walled oospores appeared at 96 h (Fig 1C); therefore, the formation of mature oospores coincides with the increase in PuM90 transcript levels.
(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.
PuM90 gene knockout and in situ complementation in P. ultimum
Using a CRISPR/Cas9-mediated gene replacement (with the hph gene encoding hygromycin B phosphotransferase) strategy for PuM90 gene knockout (Fig 2A), we obtained 100 primary transformants and identified 12 candidate mutants through screening based on genomic DNA (gDNA) PCR and sequencing. Three of these candidate mutants were randomly selected for further purification using the single mycelial fragment isolation method. Six single mycelial lines were isolated from each transformant, yielding 18 pure lines in total. Using gDNA PCR with sequencing for confirmation, all pure lines showed homogeneous sequence profiles (Fig 2A), indicating that they carried the same PuM90 knockout on both alleles of the diploid genome.
(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.
An in situ gene complementation assay was conducted according to a previously described protocol for Phytophthora sojae . After culturing PuM90 knockout mutants for at least 10 generations on antibiotic-free plates, the mutants could no longer grow on G418 antibiotic plates. To avoid persistent cleavage of the newly transformed PuM90 gene from the previous RNA template, the open reading frame (ORF) sequence of PuM90 with two mutated single guide RNA (sgRNA) sites (PuM90-m) was exploited as donor DNA in the complementation system. This mutated donor DNA vector, along with the Cas9-expressing vector and new RNA templates targeting the hph gene, was co-transformed into the P. ultimum knockout line (ΔPuM90-1 in this study) that had lost G418 resistance. After screening based on gDNA PCR and sequencing, 10 independent PuM90-m complemented strains from 86 primary transformants were identified (Fig 2B and S3 Table).
In this study, we present the results obtained from three representative PuM90 knockout mutants (ΔPuM90-1/2/3) and two representative PuM90 complemented strains (ΔPuM90-C1/2). The wild-type strain (WT) and two representative empty-vector lines (EV, in which the PuM90 knockout was not successful, for comparison with the WT; and ΔPuM90-EV, in which PuM90-m was not successfully complemented, for comparison with ΔPuM90-1) were included as controls (Fig 2).
PuM90 knockout disrupts oospore formation in P. ultimum
After 14 days of growth on V8 agar medium, all three tested PuM90 knockout mutants (ΔPuM90-1/2/3) and ΔPuM90-EV generated abnormal oospores exhibiting thinner oospore walls (termed abnormal-1 type) or empty oogonia rather than normal oospores (termed abnormal-2 type), while few abnormal oospores were found in the WT, EV, and ΔPuM90-C1/2 lines (Fig 3A and 3B). In addition, no significant differences in the total number of oogonia were observed (Fig 3B), indicating that PuM90 might be associated with oospore development rather than oogonia development. With ΔPuM90-1/2/3 and ΔPuM90-EV, abnormal oospores were observed not only in the culture medium but also in infected soybean hypocotyl tissue (Fig 3A). In both cases, we found that abnormal-1 type oospores were larger than normal oospores. The average oospore diameters were 22.9–23.9 μm in ΔPuM90-1/2/3 and ΔPuM90-EV, which were significantly larger than 14.9–15.9 μm in the WT, EV, and ΔPuM90-C1/2 cultures (Fig 3A and 3C).
(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 (**).
To explore the effects of PuM90 knockout on oospore formation, oospore morphology was investigated when mycelia were inoculated in V8 liquid medium for 2, 4, and 7 days. No difference was apparent in the 2-day-old oogonia of different strains. However, about half of the 4-day-old oogonia of the PuM90 knockout mutants showed decreased amounts of cytoplasm in oogonia compared with the WT and EV. Moreover, the cytoplasm of 7-day-old oogonia of the PuM90 knockout mutants was completely void and empty, whereas the majority of oogonia of the WT and EV at the same stage contained mature thick-walled oospores (Fig 4A). In ΔPuM90-1/2/3 and ΔPuM90-EV, we observed that the thickness of the oospore wall was 0.29–0.36 μm, which was reduced by almost 10 fold compared to the WT, EV, and ΔPuM90-C1/2 (2.01–2.28 μm) (Fig 3D). The difference in oospore wall thickness was confirmed through observations using a transmission electron microscope (TEM) (Fig 4B). The thickness of abnormal-1 type oospores was significantly less in ΔPuM90-1/2/3 and ΔPuM90-EV, and therefore the oogonia were more easily dyed when exposed to 1000 ppm Congo Red solution for 24 h (Fig 4C).
(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.
Compared to the WT, EV, and ΔPuM90-C1/2, ΔPuM90-1/2/3 and ΔPuM90-EV showed no significant differences in mycelial growth rate on either nutrient-rich V8 medium (S2A and S2B Fig) or nutrient-poor Plich medium (S2A and S2C Fig), indicating that PuM90 knockout had no effect on vegetative growth of P. ultimum. All hypocotyls of etiolated soybean seedlings inoculated with mycelia of ΔPuM90-1/2/3 or ΔPuM90-EV developed disease symptoms, with lesion sizes (S2A Fig) and relative biomass of P. ultimum (S2D Fig) comparable to the WT, EV, and ΔPuM90-C1/2 at 24 h after infection. These results suggest that PuM90 may function specifically in oospore formation in P. ultimum.
Mutations of key amino acids in the TRM of the Puf domain compromise PuM90 function
PuM90 protein contains a typical Puf RNA-binding domain which consists of eight Pumilio repeats and was predicted to organize into a typical crescent-shaped structure (Figs 1A and 5A). According to a sequence alignment of the Pumilio repeats and reports in yeast , we predicted the key amino acids in the TRM that may contact specific targeted RNA bases (Fig 5A). Using an in situ complementation strategy, we generated P. ultimum mutants that had all 24 amino acids (3 amino acids per repeat × 8 repeats) in PuM90 substituted with alanine (ΔPuM90-CM1/2/3) (Fig 5A and S3 Fig). Similar to ΔPuM90-1/2/3, almost all oospores generated by ΔPuM90-CM1/2/3 were abnormal, including types abnormal-1 and abnormal-2, whereas ΔPuM90-C1 generated normal oospores matching those of the WT (Fig 5B–5E), revealing that mutations of all 24 key amino acids in eight Pumilio repeat compromise the biological functions of PuM90.
(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 (**).
The Puf domain of PuM90 binds to the 3′-UTR of candidate target mRNAs
Interaction of the TRM in the Pumilio repeat with specific RNA bases can result in degradation or instability of the targeted transcripts [40–42]. According to the reported RNA-binding specificity of the TRM , we predicted that PuM90 might bind to 3′-UTRs containing a UGUA[A/U/C]AUA motif (Fig 5A). A total of 117 P. ultimum genes contained this motif in the predicted 3’ UTR region (within 100 nt after the stop codon). We performed RNA-seq when mycelia were cultured in V8 liquid medium for 24 h and 96 h, and found that there were more genes differentially expressed at the 96 h (986 genes) compared to the 24 h (80 genes) between ΔPuM90-1 and WT (S2 Table). Among the 363 genes that were significantly upregulated (compared to WT) at 96 h, we identified three candidate PuM90 target genes containing a UGUA[A/U/C]AUA motif in the 3′-UTR (PYU1_T006554, PYU1_T013662, and PYU1_T003505; Fig 6A and 6B). qRT-PCR analysis confirmed upregulation of the three candidate genes at 96 h (Fig 6B). The PYU1_T013662-encoded protein contains a flavodoxin-2 domain (Pfam ID: PF02525), and therefore was named Flavodoxin-Like Protein (PuFLP).
(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).
Using the electrophoretic mobility shift assay (EMSA), we found that the RNA-binding domain of PuM90 (PuM90-RBD) was sufficient for binding to the selected 30-nt-long 3′-UTR binding sites (3′BSs; upstream 11 nt + UGUANAUA motif + downstream 11 nt) of all three candidate genes (Fig 6C–6E and S4A and S4B Fig). When all of the key amino acids in the TRM of the Pumilio repeat were mutated, the peptide PuM90-RBDAAA exhibited no detectable binding to the 3′BSs (Fig 6D and 6E and S4A and S4B Fig). PuFLP-3′BS formed a weak RNA–protein complex only at a high concentration of PuM90-RBDAAA (Fig 6D and S5A Fig). These data suggest that PuM90-RBD directly binds to the predicted 3′-UTR binding site of three candidate targets and that key amino acids in the TRM of PuM90-RBD were required for this interaction.
Identification of the flavodoxin-like protein PuFLP as a major PuM90 target
To determine the biological functions of the candidate targets, gene overexpression assays were conducted using the constitutively active Ham34 promoter. We obtained no transformants exhibiting overexpression of PYU1_T006554, and we therefore deleted PYU1_T006554. Compared with the WT and corresponding empty-vector control, no significant phenotypic difference was observed when PYU1_T006554 was knocked out (S6 Fig) or PYU1_T003505 was overexpressed (S7 Fig; assuming that the GFP tag was not affecting the function of the protein); however, overexpression of PuFLP affected oospore formation.
Three representative lines (OE-PuFLP-1/2/3), which showed 9-, 10- and 19-fold increases of PuFLP transcript levels (compared to the WT and empty-vector control OE-PuFLP-EV), were selected for phenotypic analysis (Fig 7A). PuFLP protein levels in OE-PuFLP-1/2/3 were measured by Western blot (Fig 7A). Similar to PuM90 knockout mutants, OE-PuFLP-3 generated abnormal oospores of types abnormal-1 and abnormal-2 and almost no normal oospores. Likely due to their lower overexpression levels, OE-PuFLP-1 and OE-PuFLP-2 generated fewer abnormal oospores (abnormal-1: 13% and 33%; abnormal-2: 17% and 13%) than OE-PuFLP-3, but significantly more than the WT and OE-PuFLP-EV (Fig 7B and 7C). In addition, the transcript level of PuFLP was increased at 36 h and 48 h and then repressed at 96 h when PuM90 was most strongly induced (S8 Fig). These results indicate that PuFLP in P. ultimum may play a negative regulatory role during the late stage of oospore formation.
(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 (**).
Mutation of the 3′-UTR binding site of PuFLP affects oospore development
Microscale thermophoresis (MST) experiments revealed that PuM90-RBD bound to PuFLP-3′BS with Kd = 2.66 μM (Fig 6E). Replacement of the UGUACAUA motif in PuFLP-3′BS with ACACACAC (PuFLP-3′BSmu) abolished its binding to PuM90-RBD (Fig 6D and 6E and S5B Fig), indicating that the UGUACAUA motif of the PuFLP 3′-UTR was essential for PuM90 binding. Using an in situ complementation strategy (S9 Fig), we generated P. ultimum mutants in which the UGUACAUA motif in PuFLP-3′BS was substituted with ACACACAC (PuFLP3′BSmu) and used the PuFLP-complemented transformants (ΔPuFLP-C) as control. Similar to the phenotypes of PuM90 knockout mutants, PuFLP3′BSmu generated large amounts of abnormal oospores including types abnormal-1 (21%) and abnormal-2 (13%), which were significantly higher levels than in the WT (abnormal-1: 4%; abnormal-2: 3%), whereas ΔPuFLP-C generated normal oospores consistent with the WT (Fig 8A and 8B). In PuFLP3′BSmu, transcript levels of PuFLP were 5-fold higher than in the WT and ΔPuFLP-C (Fig 8C). These results indicate that when the UGUACAUA motif in its 3′-UTR was mutated, PuM90 lost the ability to negatively regulate PuFLP, resulting in increased PuFLP transcript levels that affected oospore formation.
(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.
Sexual reproduction is a key process in the pathogenic life cycle of oomycetes, especially those in which asexual reproduction does not occur . However, due to the lack of genome editing technologies and functional genomic studies on oomycetes, little is known about the molecular mechanisms of sexual reproduction in oomycetes. Previous transcriptional and functional studies have indicated that M90 is involved in sexual reproduction in the oomycete Peronophythora litchii; however, the mechanism through which this RNA-binding protein regulates the biological process remains unknown . In this study, we provide new insights into the functional mechanisms of PuM90 in the regulation of oospore formation in P. ultimum. The results demonstrated that at the level of post-transcriptional regulation, a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA is critical for sexual reproduction in P. ultimum.
Among the few molecular genetic strategies which have been employed to investigate the biology of Pythium, gene silencing via RNA interference (RNAi) is the most commonly applied, e.g., in Pythium oligandrum and Pythium guiyangense [23,44]. Despite these successes, application of the RNAi strategy has several drawbacks. For example, the degree of gene silencing is unpredictable, and some of the transformants may be unstable [17,45]. In 2016, the CRISPR/Cas9 system was successfully applied to Phytophthora sojae for the first time , and subsequently to other Phytophthora species [18,22]; however, this method had not yet been applied to Pythium. In this study, we adapted the CRISPR/Cas9-mediated genome editing tool to P. ultimum to delineate the biological functions and functional mechanisms of PuM90 (Fig 2). This initial report of gene knockout and in situ complementation in P. ultimum indicates that this method is broadly applicable to Pythium species.
Puf proteins are highly conserved in terms of protein structure, especially that of the RNA-binding domain, which typically contains eight Pumilio repeats . Despite Puf proteins being ubiquitous among eukaryotes, their functions differ widely among organisms, including roles in stem cell maintenance, development, ribosome biogenesis, and human diseases . Our data indicate that PuM90 expression was elevated during the oospore formation stage (Fig 1), and knockout of PuM90 resulted in severe impairment of oospore formation, which was recovered with the reintroduction of PuM90 into the mutant (Figs 3 and 4).
Puf proteins generally bind to the 3′-UTR of single-stranded RNA targets in a sequence-specific manner, destabilizing the target RNA . For example, in C. elegans, a cytoplasmic Puf RNA-binding protein (called fem-3 binding factor or FBF) binds specifically to the regulatory region of the fem-3 3′-UTR, repressing fem-3 expression and mediating the sperm/oocyte transition . It is reasonable to speculate that PuM90 may bind to the 3′-UTR of its target mRNAs. Based on bioinformatics-based prediction of the 3′-UTR binding motif, RNA-seq for identification of differentially expressed genes, and EMSA for exploration of protein–RNA interactions, we found that PuM90 binds to the PuFLP 3′-UTR to repress PuFLP mRNA level (Fig 6). PuFLP possesses a domain with a flavodoxin-like fold. Flavodoxins are small soluble electron transfer proteins that occur widely among bacteria and some eukaryotes such as oomycetes and metazoans. Flavodoxins participate in various metabolic pathways; in some bacteria, they have been identified as essential proteins and promising therapeutic targets for fighting bacterial infections, including that of Helicobacter pylori, the most prevalent human gastric pathogen . In addition to overexpression (Fig 7), knockout of PuFLP also results in abnormal oospore development of P. ultimum, indicating that the PuFLP pathway may represent a promising target for fungicide development to control diseases caused by oomycete pathogens.
Although PuFLP3′BSmu generated abnormal oospores (Fig 8), the proportion was lower than those of the PuM90 knockout mutants (Fig 3). Sequences flanking the core consensus motif in the 3′-UTR were important for binding . Replacement of the UGUACAUA motif in PuFLP-3′BS with ACACACAC (PuFLP-3′BSmu) inhibited but did not fully prevent its binding to PuM90-RBD (Fig 6D and 6E and S5B Fig). Therefore, the flanking regions of the PuFLP-3′BS may play a role in binding of the PuM90-RBD. In addition, each Puf protein generally binds to a large set of mRNAs encoding functionally and cytotopically related proteins . In yeast, Puf1p- and Puf2p-associated mRNAs disproportionately encode membrane-associated proteins, while Puf3p specifically binds to mRNAs encoding mitochondrial proteins . In the present study, the 100-nt region after the stop codon was considered the potential 3′-UTR sequence (Fig 6A); however, some genes may be regulated by a 3′-UTR longer than 100 nt. Some studies have shown that Puf proteins can bind to coding sequences (CDSs) or 5′-UTRs. In Drosophila motoneurons, Pum can bind directly to mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para) . In Cryptococcus neoformans, Pum1, an ortholog of both S. cerevisiae Puf3p and Drosophila melanogaster Pumilio, binds exclusively to the consensus-binding element UGUACAUA in the 5′-UTR of its own mRNA to participate in the regulation of hyphal morphogenesis . Thus, PuM90 may employ other affinity mechanisms for binding to other target genes, perhaps via other cis elements in the CDSs or 5′-UTRs.
A typical Puf protein has a C-terminal RNA-binding domain comprised of eight tandem Pumilio repeats along with N- and C-terminal flanking regions. The eight Pumilio repeats are arranged to form an arch, with each repeat composed of three α-helices. The second helix of each repeat contains a TRM, where the residue at position 2 stacks with the cognate base and residues at positions 1 and 5 directly interact with RNA bases through polar interactions. We identified the residues at positions 1, 2, and 5 of the second helix and mutated the corresponding amino acids in all eight repeats. This mutation disrupted RNA binding and the mutants generated abnormal oospores, indicating that PuM90 is a typical Puf protein, and that its ability to specifically bind to target RNA is responsible for its role (Fig 5). However, mutations of 24 amino acids may cause the collapse of the protein structure, resulting in disrupted RNA binding and generating abnormal oospores, thus further researches are needed to ensure structural stability of the protein. By contrast, the N-terminal and central regions differ among Puf proteins. Puf proteins usually collaborate with protein partners to regulate the expression of target mRNA(s). Puf proteins from Drosophila and C. elegans must interact with other regulatory proteins, such as Nanos and Brat, to function . Three unique domains in the N-terminus of Drosophila Pumilio possess repressive activity and can function autonomously . Although there were no predicted functional domains, we speculate that the N-terminal and central regions of PuM90 might play important roles in cofactor recruitment or possess repressive activity; further investigation of these roles is needed.
In phylogenetic analysis, the Puf proteins exhibited a high conservation in oomycetes (S1 Fig). Although the M90 proteins in some oomycete plant pathogens have been revealed to have similar protein domain structure, and/or transcription patterns and biological functions during sexual development [36,37,43], further researches are still needed to learn whether the PuM90-mediated functional mechanisms, including the sequence-specific interaction with the 3′-UTR of target genes and the network of regulated genes, are conserved. In addition to M90, the other Puf members (Puf2–Puf4) showed diverse domain structures and transcription patterns (Fig 1B and S1 Table), thus may possess different functions and mechanisms.
In conclusion, we propose a new model of the role of the Puf RNA-binding protein PuM90 in the sexual development of P. ultimum (Fig 9). PuM90 acts as a stage-specific post-transcriptional regulator by specifically binding to the 3′-UTR of PuFLP and then repressing PuFLP mRNA level. We identified the flavodoxin-like protein PuFLP as a major functional factor involved in oomycete sexual reproduction. We developed a CRISPR/Cas9-mediated method for gene knockout and in situ complementation in Pythium, providing a robust platform for further molecular studies in Pythium. This study describes new technologies and data that will help to elucidate sexual reproduction and post-transcriptional regulation in oomycetes.
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.
Materials and methods
Source and culturing of P. ultimum
The Pythium ultimum var. ultimum strain F18-6 used as the WT strain was isolated from soybean field soil in Shandong Province, China . All strains employed in this study were routinely grown on 10% V8 agar medium at 25°C in the dark. For growth rate analysis, the strains were cultured on V8 medium and Plich medium at 25°C in the dark. Plich medium is a minimal medium with low nutritional content containing 0.5 g KH2PO4, 0.25 g MgSO4∙7H2O, 1 g asparagine, 1 mg thiamine, 0.5 g yeast extract, 10 mg β-sitosterol, 25 g glucose, and 15 g agar per liter [58,59]. The diameter of each colony was measured at 24 hours post inoculation (hpi) on medium, and the average diameter was determined from two measurements taken at right angles to each other. The experiments were repeated three times in triplicate for each assay. Results were compared by t-test in Excel.
RNA-seq sampling and sequencing
RNA-seq samples were collected from mycelia cultured on 10% V8 liquid medium at 25°C in the dark. For the WT strain, samples were collected at 24, 36, 48, and 96 hpi, and designated 24h, 36h, 48h, and 96h, respectively. Among PuM90 knockout mutants, ΔPuM90-1 was selected, and samples were collected at 24 and 96 hpi, when PuM90 expression had not and had been induced, respectively. Three independent biological replicates were conducted for each treatment. RNA was extracted using the EZNA Total RNA Kit I (Omega Bio-tek, Norcross, GA, USA). RNA-seq was conducted by BGI Genomics Co. (Shenzhen, China) using the BGISEQ-500 system with 100-bp paired-end reads. The filtered clean reads from RNA-seq have been deposited in the National Center for Biotechnology Information (NCBI) database [BioProject ID: PRJNA540115 (Run IDs: SRR15049129-SRR15049146)].
The clean reads were aligned to the genome of P. ultimum var. ultimum (strain DAOM BR144) with TopHat v2.1.1 (ccb.jhu.edu). A total of two mismatches and gaps per read were allowed, and data were included in further analyses only if both reads in a pair were successfully mapped. Transcript abundance was indicated as fragments per kilobase of exon model per million mapped reads (FPKM). To identify differentially expressed genes, read counts for each gene model were obtained using featureCounts software (bioinf.wehi.edu.au); the fold change and adjusted P-values were calculated using DESeq2 software (www.bioconductor.org); genes with an adjusted P-value < 0.001 and fold change ≥ 2 were considered differentially expressed.
Transformation of P. ultimum
PEG-mediated protoplast transformation was conducted as described by Hua  with some modifications to introduce DNA into P. ultimum. P. ultimum was inoculated into KPYG2 medium containing 1.5 g glucose, 1.0 g yeast extract, 1.0 g peptone, 0.1 g CaCl2·2H2O, 0.02 g cholesterol, 1 g corn oil, and 109.302 g mannitol per liter (with 1.5% agar added for solid medium). After 1 to 2 days (when young hyphae had not grown to the edge of the plates), 40–50 small pieces (3 × 3 mm) from the edge of the mycelial colonies were inoculated into three 250-mL Erlenmeyer flasks containing 50 mL liquid KPYG2 and grown for 2 to 3 days at 25°C in the dark with shaking once per day. At 2–3 days old, P. ultimum mycelial mats (agar plugs need not be excluded) were harvested and pre-treated with 0.8 M mannitol for 10 min, and then digested in 20 mL enzyme solution (0.8 M mannitol; 0.5 M KCl; 0.5 M 2-(N-morpholino) ethanesulfonic acid [MES], pH 5.7; 0.5 M CaCl2; 0.15 g lysing enzymes; and 0.1 g cellulase) for 40 min at room temperature with gentle shaking. The mixture was filtered through a Falcon Nylon Mesh Cell Strainer (BD Biosciences) and protoplasts were pelleted via centrifugation at 1500 g for 4 min in a Beckman Coulter benchtop centrifuge with swing buckets. After washing with 30 mL W5 solution (5 mM KCl, 125 mM CaCl2, 154 mM NaCl, and 177 mM glucose), protoplasts were resuspended in 10 mL W5 solution and held on ice for 30 min. Protoplasts were collected through centrifugation at 1500 g for 3 min in the Beckman Coulter centrifuge and resuspended at 106 cells/mL in MMg solution (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES, pH 5.7).
DNA transformation was conducted in a 50-mL Falcon tube, wherein 1 mL protoplasts was mixed well with 40–50 μg DNA for plasmid transformation. Then, three successive aliquots, each 580 μL, of freshly made PEG solution (40% PEG 4000 v/v, 0.2 M mannitol, and 0.1 M CaCl2) were slowly pipetted into the protoplast suspension and gently mixed. After 20 min of incubation on ice, 10 mL liquid KPYG2 with 50 μg/mL ampicillin was added, and the protoplasts were regenerated at 25°C in the dark for 15–18 h. For production of stable transformants, the regenerated protoplasts were collected through centrifugation at 2000 g for 5 min in the Beckman Coulter centrifuge, and then resuspended and evenly divided into three Falcon tubes containing 50 mL liquid KPYG2 with 1% agar (42°C), 30 μg/mL G418 (AG Scientific), and 50 μg/mL ampicillin. The resuspended protoplasts were then poured into empty 90 × 15 mm Petri dishes. Mycelial colonies could be observed after incubation for 48 h at 25°C in the dark, which were then covered with liquid V8 medium containing 1.5% agar (42°C), 60 μg/mL G418, and 60 μg/mL ampicillin. Mycelial colonies were observable after 12 h of incubation at 25°C in the dark. Visible transformants were transferred to 10% V8 agar medium with the addition of 60 μg/mL G418 and propagated for 2–3 d at 25°C prior to analysis.
Mycelia of P. ultimum with high growth rates were spread onto the medium, and the mycelial colonies obtained from the regenerated protoplasts were readily interwoven. Due to the difficulty of zoospore production, we isolated single mycelia to obtain pure transformants . To that end, 5 × 5-mm hyphal plugs were induced on V8 medium plates at 25°C for 2 days. Then, 2 × 2-cm hyphal plugs taken from near the inoculation points were homogenized with 5 mL water, and 500-μL suspensions were evenly smeared on 2% water agar plates containing 50 μg/mL ampicillin and 60 μg/mL G418, which were incubated at 25°C in the dark for approximately 24 h. Individual mycelial colonies were checked under a light microscope and transferred to V8 medium plates for subsequent experiments.
Construction of gene knockout and complementation mutants
Gene deletion mutants were generated using the CRISPR-mediated gene replacement strategy . sgRNAs were designed using the web tool at http://grna.ctegd.uga.edu and listed in S3 Table. Two plasmids, including the “all-in-one” plasmid pYF515 (containing Cas9, sgRNA, and selection marker NPTII), and the donor DNA plasmid pBS-SK II+ that contained the entire hph gene, flanked by 1 kb of homology arms surrounding the PuM90 gene, were used for PEG-mediated protoplast transformation (Fig 2A). Putative transformants were screened via PCR using the primer combinations shown in Fig 2A. Primer set F1/R1 (S3 Table), which binds within the PuM90 ORF, was used to screen for deletion of PuM90 from the genomes of resistant transformants. Primer sets F2/R2 and F3/R3 were used to detect homologous recombination repair (HRR) events (S3 Table). HRR events were analyzed through Sanger sequencing to confirm that PuM90 was cleanly replaced (Fig 2A). PYU1_T006554 deletion mutants were constructed using the same strategy (S6A Fig and S3 Table).
For PuM90 complementation, the knockout mutant was transformed using NPTII as the selection marker. The entire gene-coding region with mutated sgRNA sites, which was inserted into two 1.0-kb fragments flanking the target gene, was used as the donor DNA (Fig 2B). The primer set F2/R2 (S3 Table) was used to screen for the deletion of hph from the genome of resistant transformants. The primer sets F3/R3 and F4/R4 (S3 Table) were used to detect HRR events. To obtain ΔPuM90-CM (S3 Fig), PuM90-RBD, comprised of three mutated amino acid residues in each of the eight repeats, was synthesized by Genewiz (Suzhou, China). The entire gene-coding region with mutated sgRNA sites was complemented into the PuM90 knockout mutant, and the resulting transformants were screen as described above (S3 Fig and S3 Table).
To obtain transformants lacking the UGUACAUA motif in PuFLP-3′BS, we attempted to replace the UGUACAUA motif with ACACACAC using gene editing in the WT strain, but failed to obtain any mutants. PuFLP deletion mutants were generated using the same CRISPR-mediated gene replacement strategy used for PuM90 knockout (S9A Fig). The entire PuFLP with a mutated UGUACAUA motif and the intact PuFLP (with mutated sgRNA sites) as a control were separately complemented into the PuFLP knockout mutant (S9B Fig). Putative transformants were screened via PCR using various primer sets and analyzed through Sanger sequencing (S9 Fig and S3 Table).
Analysis of oospore development
To monitor and quantify oospore production, strains were grown on 10% V8 agar medium at 25°C in the dark. After 14 days, the cultures were examined via microscopy (Olympus). Three random fields at 40× magnification from each plate were selected for counting of oospores. Diameter and wall thickness of oospores in three random fields were measured using microscope graticules. To allow for explicit observation of the oospores, five 5 × 5-mm hyphal plugs were cultivated in 8 mL of V8 broth in 90-mm Petri dishes for 7 days at 25°C in the dark. Oospores stained with the lactophenol-trypan blue (10 mL lactic acid, 10 mL glycerol, 10 g phenol, and 10 mg trypan blue dissolved in 10 mL distilled water) were randomly selected for examination under an inverted microscope (Zeiss) . A TEM was used to observe oogonium sections from the WT, EV, and PuM90 deletion strains. Images were obtained using an AMT camera system. To assess colonization of soybean tissues by oospores, infected epidermal cells were collected at 72 hpi and soaked in lactophenol-trypan blue, and then the infected epidermal cells were examined under an inverted microscope. Oospores from 7-day-old cultures were infiltrated with 1000 ppm Congo red (Shanghai Ryon) and imaged after 24 h to investigate the permeability of the oospore wall. All experiments were performed at least three times independently. Means and standard deviations were calculated using data from three replicates.
A virulence assay was performed after hyphal plug inoculation of the hypocotyls of etiolated soybean seedlings of the Williams cultivar, as this cultivar is compatible with P. ultimum. Soybeans grown in a greenhouse at 25°C with a 16-h/8-h light/dark cycle for 4 days were used for hypocotyl infection. Then, hyphal plugs (5 mm in diameter) were inoculated onto the hypocotyls, which were incubated at 25°C in the dark for 24 h before photography and sampling. Each strain was tested using at least five plants. Virulence was quantified through determination of the ratio of P. ultimum DNA to soybean DNA in the infected plants, as measured by qRT-PCR. All assays were repeated independently at least three times.
Genomic DNA of the P. ultimum strains was extracted using a plant DNA kit (Tiangen) from mycelium grown in V8 broth for the gDNA PCR and in infected soybeans for the biomass assay. Total RNA of P. ultimum was extracted using the EZNA Total RNA Kit I (Omega). cDNA was synthesized from 1–5 μg total RNA with the PrimeScript First Strand cDNA Synthesis Kit (TaKaRa Bio Inc.) following the manufacturer’s protocol. Quantitative PCR was performed in 20-μL reactions containing 20 ng DNA/cDNA, 0.2 mM primers for the target or reference gene, 10 μL SYBR Premix ExTaq (TaKaRa Bio Inc.), and 6.8 μL double-distilled water. PCR was performed on an ABI Prism 7500 Fast Real-Time PCR System (Applied Biosystems Inc.) under the following conditions: 95°C for 30 s; followed by 40 cycles of 95°C for 5 s and 60°C for 34 s; and finally 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. For the gene expression assay, the actin gene (PuACTA = PYU1_T009609) from P. ultimum was used as a constitutively expressed endogenous control. To quantify P. ultimum biomass in infected soybean tissue, P. ultimum PuACTA and soybean GmCYP2 (NC_016099.2) were quantified via quantitative PCR, and the biomass ratio was calculated using the 2-[Ct(PuACTA- Ct(GmCYP2)] method . S3 Table lists the primers used in this study. Means and standard deviations were calculated using data from three replicates.
Western blotting for gene overexpression analysis
To conduct PuFLP-GFP (green fluorescent protein) fusion, the coding region of the PuFLP gene was amplified using the primers listed in S3 Table and cloned into the plasmid pTOR::eGFP . Constructs carrying eGFP-tagged PuFLP and eGFP-tag (control) were transformed into P. ultimum. Approximately 150–200 mg of mycelia from stable P. ultimum transformants was ground into powder in liquid nitrogen and resuspended in 1 mL 0.01 M phosphate-buffered saline with fresh additions of 1 mM phenylmethylsulfonyl fluoride and 10 μL of protease inhibitor cocktail (Sigma, Shanghai, China). Total proteins were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membranes. Anti-GFP antibodies (Abmart Inc., Shanghai, China) were used to detect PuFLP expression (Fig 7A). PYU1_T003505 overexpression transformants were constructed using the same strategy (S7C Fig and S3 Table).
Protein expression and purification
Due to the difficulty of expressing and purifying intact PuM90, the CDSs of the PuM90 RNA binding domain (PuM90-RBD) and the PuM90 RNA binding domain with three amino acid residues mutated in all eight repeats (PuM90-RBDAAA) were separately inserted into the pGEX-4T-2 vector (containing the glutathione S-transferase [GST] tag; GE Healthcare Life Science) for in vitro assays. The plasmids (GST empty vector, GST-PuM90-RBD, and GST-PuM90-RBDAAA) were transformed into E. coli strain BL21 (DE3). A 500-mL culture of E. coli BL21(DE3) cells was grown at 37°C to an optical density at 600 nm of 0.5, after which gene expression was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (Sigma) for 4 h at 28°C. After lysing of cells, the recombinant proteins were purified on glutathione Sepharose beads (Sangon Biotech, Shanghai, China) and eluted with 5 mM glutathione dissolved in Tris-buffered saline. The concentration of purified proteins was determined using a bicinchoninic acid protein assay kit (Sangon Biotech). Protein purity was assessed via SDS-PAGE.
Electrophoretic mobility shift assay
EMSA was performed as described previously . 5-Carboxy-fluorescein (FAM)-labeled probes comprised of the core motif of the 3′-UTR of target sequences were synthesized by Genewiz (Suzhou, China). Labeled RNA fragments (50 ng) were mixed and incubated with various concentrations of purified PuM90-RBD protein at 25°C for 30 min in EMSA/Gel-Shift Binding Buffer (Beyotime). The mixtures were then loaded onto a 1% agarose gel and electrophoresed for 1 h. EMSA signals (labeled RNA fragments) were detected using Alexa 488 with the VersaDoc imaging system (Bio-Rad, Philadelphia, PA, USA).
Binding of PuM90-RBD protein to the 3′-UTR of PuFLP labeled with FAM was detected through an MST assay using the Monolith NT.115 instrument (NanoTemper Technologies), according to a previously described procedure . A constant concentration (10 μM) of the labeled 3′-UTR in MST buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 0.05% Tween 20) was titrated against increasing concentrations of PuM90-RBD protein dissolved in double-distilled water. MST premium-coated capillaries (Monolith NT.115 MO-K005) were used to load the samples into the MST instrument at 25°C using high MST power and 60% light-emitting diode power. Laser on and off times were set to 30 s and 5 s, respectively. All experiments were conducted in triplicate. Data were analyzed using NanoTemper Analysis software v. 1.2.101 (NanoTemper Technologies).
S1 Fig. Phylogenetic relationships of the identified Puf proteins in oomycetes.
The phylogenetic trees were constructed using neighbor-joining method with 1,000 bootstrap replicates in MEGA 7.0 software. Bootstrap values higher than 80 are displayed.
S2 Fig. PuM90 is not involved in mycelial growth or virulence.
(A) Growth characteristics at 24 h after inoculation on V8 medium and Plich medium, and virulence on soybean hypocotyls of WT, EV, PuM90-knockout transformants (ΔPuM90-1/2/3), complemented transformants (ΔPuM90-C1/2), and the empty control line of ΔPuM90 (ΔPuM90-EV). (B, C) Growth rates on V8 medium (B) and Plich medium (C). (D) Relative P. ultimum biomass detected through qRT-PCR at 24 h after hypocotyl infection.
S3 Fig. CRISPR/Cas9-mediated mutation of key amino acids in the RNA-binding domain of PuM90.
(A) Locations of the primers used to screen for complemented transformants (top) and Sanger sequencing traces of the mutated sequence regions in all eight Pumilio repeats (bottom). (B) Analysis of genomic DNA using the primers shown in (A) and actin primers as a positive control.
S4 Fig. Results of EMSA assays for the RNA-binding domain (RBD) of PuM90 and its candidate targets.
EMSA results showing that PuM90-RBD bound to the 3′BS of PYU1-T006554 (A) and PYU1-T003505 (B), while the mutant PuM90-RBDAAA and GST did not.
S5 Fig. Key amino acids in eight Pumilio repeats of PuM90-RBD and the UGUACAUA motif in the PuFLP 3′BS are essential for binding activity.
(A) The PuM90-RBDAAA peptide nearly abolished detectable binding to PuFLP-3′BS. (B) The PuM90-RBD peptide nearly abolished detectable binding to PuFLP-3′BSmu but formed a weak RNA–protein complex at a high PuM90-RBD concentration.
S6 Fig. PYU1_T006554-knockout does not affect oospore formation.
(A) Construction and verification of three representative candidate mutants for PYU1_T006554 knockout. (B, C) Morphology (B) and number (C) of oogonia generated in 14-day-old cultures. Bar, 20 μm.
S7 Fig. PYU1_T003505 overexpression does not affect oospore formation.
(A, B) Morphology of oogonia and oospores (A) and number of oogonia (B) generated in 14-day-old cultures. Bar, 20 μm. (C) PYU1_T003505 protein levels measured through Western blotting. (D) PYU1_T003505 transcript levels measured through qRT-PCR. Asterisks (**) indicate significant differences comparing with WT at P < 0.01.
S8 Fig. Transcription analysis of PuFLP.
Transcript levels of the PuFLP genes measured using RNA-seq (top) and qRT-PCR (bottom) when P. ultimum mycelia were cultured in V8 liquid medium for 24, 36, 48, or 96 h.
S9 Fig. CRISPR/Cas9-mediated PuFLP-3′BS mutation.
(A) Construction and verification (with Sanger sequencing traces) of the PuFLP-knockout mutant (ΔPuFLP). (B) Construction and verification (with Sanger sequencing traces) of the complementation line PuFLP3′BSmu, in which the UGUACAUA motif in PuFLP-3′BS was replaced with ACACACAC. (C) Analysis of genomic DNA PCR products using the primers shown in A and B and actin primers as a positive control.
S1 Table. Puf proteins identified in Pythium ultimum and other oomycetes.
S2 Table. Differentially regulated genes when PuM90 was knocked out.
We would like to thank Dr. Haifeng Zhang, Dr. Yan Wang, Dr. Kaixuan Duan, Dr. Bo Yang, and Dr. Meixiang Zhang (Nanjing Agricultural University, Nanjing) for helpful discussions.
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