All of the NLS tests were carried out in the P. sojae reference isolate P6497 (race 2). Cultures were routinely grown and maintained in cleared V8 medium at 25°C in the dark. P. sojae transformants were incubated in 12-well plates containing liquid V8 media supplemented with 50 μg/ml G418 (Geneticin) for 2–3 days before examination by confocal microscopy.

Phytophthora protein sequences and IDs were obtained from FungiDB (Stajich et al., 2012). Their corresponding accession numbers in the GenBank database are listed in Table S1. Ribosomal protein and core histone sequences of Arabidopsis, yeast and human were obtained from the GenBank database: Arabidopsis L27a (accession number, NP_177217), H3.1 (NP_201339), H4, (NP_190179); human H3.k (P68431), H4.j (AAA52652), L27a (NP_000981); Saccharomyces cerevisiae L28 (NP_011412), H3 (or Hht2p, CAY82162); H4 (or Hhf2p, NP_014368). Protein sequence alignments were carried out using Clustal Omega (Sievers et al., 2011). Additional sequence information for the proteins PHYSO_357835, PHYSO_480605, PHYSO_251824, PHYSO_561151, and PHYSO_533817 can be found in the Data Sheet 1 (Supplemental Sequences).

All the primers used in this study are listed in Table S2 in the Supplementary Material. All the fusion protein reporter constructs in which the NLS was fused to the N-terminus of GFP or 2XGFP were based on the plasmid backbone pYF2-2XGFP (Fang and Tyler, 2016). To test reporters with the NLS fused to the C-terminus of GFP, a new plasmid backbone pYF3-2XGFP was generated by inserting an eGFP fragment with attached C-terminal multiple cloning sites and stop codon (Bsr GI-Hpa I-Bsp EI-Mlu I-TAA) into the Afl II and Apa I sites of the backbone pYF2-GFP. To clone NLS candidates efficiently and economically, NLSs derived from exogenous genes smaller than ~80 bp were created by oligo annealing and inserted into the Spe I and Sac II sites of pYF2-2XGFP, or the Bsr GI and Bsp EI sites of pYF3-2XGFP. PCR products from P. sojae genes were inserted into the Stu I site of pYF2-2XGFP or the Hpa I site of pYF3-2XGFP by blunt ligation. The P. sojae H2B nuclear marker fusions (H2B-GFP and H2B-mCherry) and the P. capsici fibrillarin nucleolar marker fusion (FIB-mCherry) were created using the pGFPN or pMCherryN plasmids (Ah-Fong and Judelson, 2011). Point mutations and deletions of DNA sequences were made through QuikChange Lightning Multi Site-Directed Mutagenesis (Agilent Technologies).

To express 2XGFP fusions in mammalian cells, genes encoding 2XGFP and its SV40 NLS and M9NLS fusions were PCR-amplified from P. sojae expression plasmids using primers pYF2_GW_F and GFP_stop_GW_R, and integrated into pcDNA 3.2/V5-DEST by Gateway™ cloning (Thermo Scientific). Integration of 2XGFP into pcDNA 3.2/V5-DEST also generated a plasmid backbone pcDNA 3.2-2XGFP, in which a preserved Eco RV site originating from the pYF2 backbone was used for insertion of PY-NLS amplicons by blunt ligation.

PCR-amplification was conducted using Phusion® High-Fidelity DNA Polymerase (NEB). Standard molecular techniques were performed as described by Sambrook and Russell (2001) or according to instructions from kit manufacturers.

Polyethylene glycol (PEG)-mediated protoplast transformations were conducted by an improved protocol as described previously (Fang and Tyler, 2016). In general, P. sojae protoplasts were isolated by enzyme digestion using 0.5% Lysing Enzymes from Trichoderma harzianum (Sigma L1412) and 0.5% CELLULYSIN® Cellulase (Calbiochem 219466) in 0.4 M mannitol, 20 mM KCl, 20 mM MES, pH 5.7, 10 mM CaCl₂. One milliliter of protoplasts at a density of 2 × 10⁶–2 × 10⁷/ml and 20–30 μg DNA were used for single plasmid transformations. For co-transformation experiments, 20–30 μg of the plasmid carrying the NPT II selectable marker gene was used, together with an equimolar ratio of any other DNAs included. Protoplasts were regenerated overnight in regeneration media (pea broth containing 0.5 M mannitol). Regenerated protoplasts were collected and grown as colonies in solid regeneration media containing 50 μg/ml G418 (Geneticin) for 2 days. G418-resistant colonies were transferred to 12-well plates containing liquid V8 media supplemented with 50 μg/ml G418 and incubated for 2–3 days at 25°C before confocal microscopy observation. Under these conditions, P. sojae transformant colonies typically continue to express genes introduced on exogenous DNA for 1–3 weeks, even if the genes are not integrated into the chromosomes. Here we did not test whether the exogenous DNA had integrated in the chromosomes; thus we refer to the mycelial colonies recovered by our procedure as transient transformants.

HEK293 cells were maintained in DMEM media (Mediatech) supplemented with 10% fetal bovine serum (Tissue Culture Biologicals), penicillin-streptomycin solution (1 IU/ml penicillin and 100 ug/ml streptomycin, Mediatech). Transfection was done according to the manufacturer's protocol. Briefly, HEK293 cells were plated on a glass coverslip in a 24-well plate (Greiner Bio-One) at a density sufficient for 70% confluency at the time of transfection. Cells were transfected with 500 ng DNA and 1 μl of Lipofectamine 2000 (Invitrogen) per well for 48 h. For immunocytochemistry, the transfected cells were fixed with 3.7% (w/v) paraformaldehyde in PBS for 15 min at RT. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at RT and incubated with 1% bovine serum albumin in PBS for 1 h at RT. Then, the cells were incubated with rabbit anti-GFP antibody conjugated with AlexaFluor 488 (Invitrogen) overnight at 4°C. Cells on the coverslip were mounted on a glass slide using ProLong Gold antifade with DAPI (Invitrogen) and dried overnight at RT in darkness.

Laser scanning confocal microscopy (Zeiss LSM 780 NLO) was used to monitor the subcellular localization of fluorescent protein fusions in P. sojae and human cell transformants. Clumps of 2–3 days old transformed P. sojae mycelia grown in liquid V8 media were removed with a toothpick, washed, and maintained in modified Plich media (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 dissolved in 1 l water) before observation. For DAPI staining, mycelia samples were pre-treated with Phosphate-buffered saline (PBS, pH 7.4) containing 0.2 μg/ml DAPI for 25 min in dark, and washed twice with PBS according to Hardham (2001). Images were captured using a 63X oil objective with excitation/emission settings (in nm) of 405/410–490 for DAPI, 488/504–550 for GFP, and 561/605–650 for mCherry. For each sample, at least three independent P. sojae transformants were examined. Images were adjusted using the microscope's built-in Zen 2012 software (Blue and/or Black edition according to different purposes). Images were cropped, and the tonal range was increased by adjusting highlights and shadows without altering the color balance.

To quantitate the ratio of nuclear to cytoplasmic localization, nuclear and cytoplasmic intensities were collected by manually defining the nuclear and cytoplasmic regions in each image as described in Hunter et al. (2014). The mean intensity values of the defined regions were generated by the Zen 2012 (Blue edition) “measure” tool, which ignores any adjustments of tonal range. In most cases, nuclei were identified by the morphology of the GFP-stained region (a large, uniformly stained, irregular ovoid region, often with an unstained nucleolus), and were not confirmed by co-expression of H2B-mCherry. Where nuclei could not be identified due to very poor nuclear localization, H2B-mCherry was co-expressed to verify the nuclei. The nuclear to cytoplasmic fluorescence ratio was calculated using the means (± standard error) of the log₂ transformed ratios from 30 pairs of adjacent nuclear and cytoplasmic regions, chosen at random from each of three independent P. sojae transformants. This procedure produced the log₂ (nuclear to cytoplasmic ratio) or LNC used to characterize the nuclear localization activity of most constructs in this study. Significance was tested with two-sample, unpaired t-tests of the LNC-values performed by the software GraphPad Prism 7, and a false discovery rate (FDR)-adjusted p′-value (Benjamini and Hochberg, 1995) of 0.001–0.05, as noted in each figure legend.

To assess the activity of NLSs, we implemented a classic in vivo NLS assay, in which a candidate NLS was fused to GFP or to two fused GFP moieties (2XGFP) and then transiently expressed in individual P. sojae transformants. The fusion of two GFP molecules created a protein (~55 kDa) larger than the NPC threshold for passive diffusion. Subcellular localization of the proteins was visualized by live-cell imaging in at least three individual transformant colonies per construct, using confocal laser scanning microscopy. To assist in verifying the nuclear localization of a protein, the commonly used nuclear dye DAPI (4′, 6-diamidino-2-phenylindole) was initially employed, because it had been reported to label the nuclei of living Hardham, 2001; Zhang et al., 2012 or fixed (Gamboa-Mélendez et al., 2013) Phytophthora tissues. However, when living P. sojae hyphae were stained with DAPI, the NLS-fused GFPs were extensively distributed into the cytoplasm (Figure S1A). This contrasted with the clear nuclear localization observed in hyphae that were not stained with DAPI (Figure S1A). In particular, when using a 2XGFP reporter, fused to a strong synthetic NLS, PsNLS (Fang and Tyler, 2016), we noticed that regions of hyphae with poor DAPI staining exhibited strong GFP nuclear localization while regions of hyphae well-stained with DAPI showed poor GFP nuclear localization (Figure S1B). This suggested that DAPI staining caused mis-localization of nuclear-localized proteins to the cytoplasm. Indeed, a time-lapse experiment tracking a hypha during DAPI staining showed that after 18 min staining, the nuclei disintegrated and the nuclear-localized PsNLS-2XGFP was released into the cytoplasm (Figure S1C and Supplemental Video 1).

To identify a reliable strategy to label P. sojae nuclei, we tested other nuclear staining dyes, such as Hoechst 33342. However, P. sojae hyphae were not permeable to that dye (data not shown). We also tried to stain the nuclei of fixed hyphae using DAPI according to a protocol described by Gamboa-Mélendez et al. (2013), but redistribution of nuclear GFP into the cytoplasm was still commonly observed. Therefore, we sought to label P. sojae nuclei by expressing a nuclear-targeted fluorescent protein. P. sojae core histone H2B fused to mCherry was selected according to Ah-Fong and Judelson (2011), and this fusion showed predominant nuclear localization in P. sojae hyphae (Figure 1A). Expression of H2B-mCherry appeared to cause some toxicity to P. sojae, because reduced numbers of transiently transformed P. sojae colonies were obtained with this construct. Similar results were observed with a P. sojae histone H1 fusion protein, and even worse with histone H3 and H4 fusions (data not shown). Thus, we settled on H2B-mCherry as a nuclear marker for co-expression with NLS-GFP reporter genes.

To examine cNLS activity in P. sojae, three well-characterized monopartite cNLS sequences were individually fused to the N-terminus of 2XGFP and expressed in P. sojae transformants. To quantify the activities of different NLSs, the ratio of fluorescence intensity in nuclei compared to the cytoplasm (Nuc:Cyt) was measured within ~ 30 hyphae from at least three individual P. sojae transformants for each fusion. Due to the wide range of nuclear-cytoplasmic ratios observed, we found it convenient to express these ratios as log₂(Nuc:Cyt), or LNC. As expected, 2XGFP alone was extensively localized to the cytoplasm (LNC = 0.52; Figures 1A,D). The well-studied cNLS derived from SV40 large T antigen (SV40 NLS) produced incomplete albeit statistically significant (FDR p′ < 0.001) nuclear localization in any fusion configuration (fusion at N- or C-terminus of 2XGFP) or copy number (LNC = 1.43 − 2.35; Figures 1A,B,D). In comparison, H2B-GFP exhibited an LNC of 7.94. Another monopartite cNLS prototype, the c-Myc NLS, produced similar results (LNC = 1.55; Figures 1A,D). To characterize cNLSs in native P. sojae nuclear proteins, we also analyzed eight protein fragments that contained monopartite cNLS motifs predicted by PSORT II (PSORT, 1997). As summarized in Table S3, we observed that none of the predicted monopartite cNLSs were sufficient for nuclear localization of GFP reporters.

To examine the nuclear localization activities of bipartite cNLSs, the classic bipartite cNLS of nucleoplasmin (NPL) was assayed in P. sojae. This sequence produced strong nuclear localization (LNC = 5.33; Figures 1A,D). Small unstained regions in the centers of nuclei were validated as nucleoli by co-expression of the nucleolar marker, fibrillarin (from Phytophthora capsici; Genbank accession No. KY452016) fused to mCherry (Figure 1C). To explore the activity of bipartite cNLSs further, four protein segments that were predicted to contain bipartite cNLS sequences (by PSORT II) were identified from nuclear-localized P. sojae proteins, and assayed in P. sojae transformants. Of these four, two (PsH3_116−136 and PHYSO_561151_505−521) were incapable of producing strong localization of GFP reporters into P. sojae nuclei (Table S3). On the other hand, two regions carrying predicted bipartite cNLSs showed strong activity, namely one at the C-terminus of PHYSO_561151 and one at the N-terminus of PsL28. At the C-terminus of PHYSO_561151, the functional residues proved to be residues 504–520, displaced one position from the inactive predicted sequence at 505–521 (LNC = 4.68). At the N-terminus of PsL28, the functional residues appeared to be residues 7–27 (LNC = 3.59) (Figures 1E,F). Sequence comparisons of the functional bipartite cNLS sequences in PsL28 and PHYSO_561151 revealed the presence of additional arginine or lysine residues in the first of the two basic amino acid clusters that comprise the bipartite motif, compared to the canonical consensus developed from mammalian and yeast proteins (highlighted in red in Figure 1E). Mutation of these additional basic amino acids from the PsL28 or PHYSO_561151 bipartite NLSs showed that these residues were essential for the activity of these NLSs (Figures 1F,G). The human NPL bipartite cNLS however lacked any additional positive residues in the first basic cluster, but was functional. This cNLS has four consecutive lysines in the second cluster and two additional lysines between the two clusters; one or both of these features may make this cNLS functional in P. sojae. Although PSORT II did not accurately predict the functional bipartite NLSs in PsH3 and PsL28, both NLStradamus and cNLS Mapper identified broader regions that encompassed those extended NLSs (Tables S3, S4). Both programs also correctly predicted that PsH3_116−136 would not be functional.

Although a number of PY-NLS sequences have been characterized in human and yeast proteins, few of them have been reported in other organisms including oomycetes. To examine the activity of PY-NLS sequences in oomycetes, four well-characterized human and yeast PY-NLSs (two basic, bPY-NLS; two hydrophobic, hPY-NLS) were fused to the N-terminus of 2XGFP and their localization was examined in P. sojae transformants (Figure 2A). None of these PY-NLSs produced strong nuclear localization of the 2XGFP reporter (Figures 2B,C). Only the bPY-NLS in hnRNP M produced significantly more nuclear localization than 2XGFP alone (FDR p′ < 0.001) (Figures 2B,C). We also tested a synthetic peptide, M9M, that is a chimera of the hnRNP A1 (M9) hPY-NLS and the hnRNP M bPY-NLS (Figure 2A). This peptide has high affinity to the Kapβ2 PY-NLS binding site in human and is usually used as a Kapβ2-specific inhibitor (Cansizoglu et al., 2007). In P. sojae this peptide produced significantly more nuclear localization than the four PY-NLS prototypes (FDR p′ < 0.001), although the localization was still weaker than that produced by the SV40 NLS (Figures 2B,C).

Given that some prototypical PY-NLSs produced weak nuclear localization in P. sojae, we considered the possibility that a fully functional PY-NLS in P. sojae may require additional elements, as was the case for cNLSs. To search for functional PY-NLS sequences in P. sojae nuclear-localized proteins, we began by scanning the P. sojae proteome using the human/yeast PY-NLS consensus sequences (the “PL” rule was allowed for bPY-NLS, Figure 3A). Then we filtered for structural disorder and overall positive charge in the NLS. This two step scan revealed 25 proteins containing candidate hPY-NLSs and 200 containing candidate bPY-NLSs. Twelve candidates annotated as RNA-processing proteins or transcription factors were selected for further analysis.

We also extended the consensus-sequence-based search by including the “PL” option for hPY-NLSs. The rationale for this was that it was reported that the tyrosine in the C-terminal PY motif shows degeneracy in yeast (Süel et al., 2008). However, since yeast Kap104 only recognizes bPY-NLS motifs, the degeneracy of tyrosine in hPY-NLS was not clear. Thus, we reasoned that the “PL” rule for the bPY-NLSs may also apply to hPY-NLSs. By adding the “PL-rule” in the hPY-NLS search (Figure 3A), we obtained another 82 hPY-NLS candidates having C-terminal “PL” motifs. One candidate (PHYSO_480605, annotated as an mRNA maturation protein) was selected for further analysis because the putative PY-NLS was the only predicted NLS in the protein (Figure 3B). Another candidate, PHYSO_251824, had already been selected for testing because it also contained a separate conventional PY-NLS motif.

To validate the subcellular localization of the proteins, the 13 full-length proteins were tagged with GFP and expressed in P. sojae transformants. Five candidates showed strong nuclear localization in P. sojae hyphae at steady state, including PHYSO_480605 (Figure 3B and Table 1). PHYSO_561151 and PHYSO_357835 also showed nucleolar localization (Figure 3B). (Although PHYSO_561151 was initially selected as a PY-NLS candidate, its predicted PY-NLS proved to be inactive, as described below, and we subsequently determined that it contained a functional bipartite cNLS, as described above). Other candidates either could not be PCR amplified, contained incorrect intron annotations causing frame shifts, or appeared toxic when overexpressed in P. sojae transformants (data not shown).

To determine whether the predicted PY-NLSs in the nuclear-localized proteins were sufficient to mediate nuclear localization of reporter proteins, protein segments containing the motifs were fused to 2XGFP at either the N- or C-terminus, based on their positions in the native proteins. One PY-NLS-containing segment, PHYSO_357835_338−387, mediated very efficient nuclear localization (Figure 3C). Other candidates, namely PHYSO_480605₁₋₃₂ and PHYSO_251824_239−358, produced incomplete nuclear localization with some remaining cytoplasmic signals (Figure 3C). In contrast, the PY-NLS-containing segments PHYSO_561151_225−445 and PHYSO_533817_314−463 did not produce any nuclear localization (Figure 3C, Figures S2, S3). As noted above, nuclear localization of PHYSO_561151 subsequently turned out to be mediated by a bipartite cNLS at its C-terminus, while the nuclear localization of PHYSO_533817 was determined by an unidentified sequence between residues 172–314 (Figure S3).

Because four of the protein segments carrying candidate PY-NLSs showed weak or non-existent nuclear localization activity in P. sojae, it was unclear if the problem was the general reliability of the three PY-NLS prediction rules or whether the P. sojae import machinery did not efficiently utilize the predicted PY-NLS motifs. To address this question, parallel experiments were carried out in human embryonic kidney 293 cells (HEK 293) to determine the activity of the putative PY-NLSs in human cells. As shown in Figures 3C,D, the subcellular localization of the PY-NLS-containing protein segments in HEK 293 cells were well correlated with their localizations in P. sojae, except for segment PHYSO_480605₁₋₃₂ that showed much stronger nuclear localization in the human cells.

To more precisely define the roles of the predicted PY-NLS motifs in the nuclear localization of each of PHYSO_357835, PHYSO_480605, and PHYSO_251824, a series of truncations and mutations were made. These analyses are detailed in the next three sections.

The aforementioned experiments indicated that residues 338–387 of PHYSO_357835, containing a predicted PY-NLS sequence, were sufficient to mediate localization of the 2XGFP reporter into the P. sojae nucleus. However, examination of the PHYSO_357835 protein sequence using PSORT II revealed the presence of another candidate NLS within this segment, namely a monopartite cNLS-like sequence (370-RHKR-373) overlapping with the epitope 2 of the PY-NLS motif (Figure 4A). To test whether nuclear localization by PHYSO_357835_338−387 required the putative cNLS separately from the epitope 2 of the PY-NLS-like sequence, histidine at 371 and lysine at 372 were mutated to alanines, resulting in 370-RAAR-373 (the canonical PY-NLS consensus requires only a single basic residue at this position). Although 2XGFP-PHYSO_357835_338−387 (H371A/K372A) appeared slightly more cytoplasmic than wild type (LNC = 1.82 compared to 2.42), the reporter remained primarily nuclear, suggesting that the nuclear localization of PHYSO_357835_338−387 was not primarily dependent on this putative cNLS.

To confirm whether the predicted PY-NLS sequence within PHYSO_357835_338−387 was the only NLS that determined the nuclear localization of PHYSO_357835_338−387, amino acid substitutions were introduced at the key residues in the predicted PY-NLS sequence. As shown in Figure 4B, mutation of the PY residues (P376A/Y377A) reduced but did not eliminate nuclear localization (LNC = 1.37). However, when the cNLS mutations H371A/K372A were combined with the PY mutations, nuclear localization produced by PHYSO_357835_338−387 was abolished (LNC = 0.26; Figure 4B), suggesting that the PY motif, in combination with the cNLS, is required for the nuclear localization of PHYSO_357835_338−387. To further explore the role of the basic region (370-RHKR-373), all the four basic residues were converted to alanines. This mutation also abolished nuclear localization (LNC = −0.02), indicating that the full set of four residues of this basic region is essential for nuclear import. Together these results suggest that both the predicted PY-NLS (defined by the residues PY in combination with at least 1 of RHKR) and the predicted cNLS (defined by all four of RHKR) are required for efficient nuclear localization of this P. sojae protein.

To test the role of this cNLS-augmented PY-NLS sequence in import of the full length PHYSO_357835 protein, mutations in the basic region (R370A/H371A/K372A/R373A) alone and combination with the PY dipeptide (R370A/H371A/K372A/R373A/P376A/Y377A) were introduced into full-length GFP-tagged protein. Mutation of the basic region resulted in substantial mislocalization of the full length protein into the cytoplasm (from LNC of 5.51 to 1.33), and additional mutation of the PY motif further decreased the LNC-value (to 0.56, not significantly different than 2XGFP alone; FDR p′ = 0.7; Figure 4C). Together, these results indicate that the augmented PY-NLS sequence of PHYSO_357835 is necessary as well as sufficient for the nuclear localization of this protein. Other than PSORT II, which predicted the RHKR element of this NLS, no current software programs, including NLStradamus or cNLS Mapper, predicted the functional NLS sequences of PHYSO_357835 (Tables S3, S4).

As noted above, the predicted PY-NLS located at 1–32 within PHYSO_480605 contains terminal PL residues rather than PY residues. To test if this variant motif is required for PHYSO_480605 nuclear localization, the protein was split at position 32 and each fragment was fused to 2XGFP and expressed in P. sojae. The N-terminal 32 residues fused to 2XGFP exhibited significant nuclear localization with some visible cytoplasmic signal (LNC = 1.48, FDR p′ < 0.001; Figures 2B, 5B), while the C-terminal fragment (residues 33–754) was exclusively distributed in the cytoplasm (Figure 5B). These results indicated that the fragment containing the variant PY-NLS was necessary for the nuclear import of PHYSO_480605, but suggested that additional amino acids downstream of the motif may contribute to the strength of the nuclear targeting (LNC of the full length protein was 5.79). In fact, an expanded fragment (1–60) that includes a stretch of positively charged amino acids (36-KFKGK-40) showed significantly increased nuclear localization (from LNC = 1.48 to 2.18, FDR p′ < 0.001; Figure 5B). However, localization was still much less than the full length protein (LNC = 5.79), suggesting that additional downstream sequences might augment nuclear localization, despite being insufficient to independently direct localization. No current software programs, including PSORT II, NLStradamus or cNLS Mapper, predicted any NLS sequences in PHYSO_357835 (Tables S3, S4).

As noted above, residues 239–358 of PHYSO_251824 produced only weak nuclear localization (LNC = 0.90, FDR p′ < 0.002; Figures 3C, 6C). To better identify the sequences required for localization, we generated fusions containing larger protein fragments of PHYSO_251824. The entire N-terminal (1–238) and C-terminal (239–419) segments of PHYSO_251824 were expressed as fusions with 2XGFP in P. sojae transformants. PHYSO_251824₁₋₂₃₈-2XGFP showed strictly cytoplasmic distribution (LNC = −0.52), despite possessing a putative monopartite cNLS (cNLS1, 100-RKRH-103) (Figures 6A,B). In contrast, 2XGFP-PHYSO_251824_239−419 produced predominant nuclear localization (LNC = 5.30 compared to 5.38 for the full length protein). Comparison of the localization of 2XGFP-PHYSO_251824_239−419 (LNC = 5.30) and 2XGFP-PHYSO_251824_239−358 (LNC = 0.90) indicated that residues 359–419 must contribute to NLS function (Figure 6C). Examination of those residues using PSORT II revealed another predicted monopartite cNLS (363-PSKRSKP-369, cNLS2) (Figure 6A). To test whether cNLS2 was necessary for nuclear localization produced by PHYSO_251824_239−419, the basic amino acids in cNLS2 were all substituted with alanines (K365A/R366A/K368A); this mutation dramatically reduced the nuclear localization of 2XGFP-PHYSO_251824_239−419 (LNC = 0.61 vs. 5.30; Figures 6B,C). To test whether cNLS2 was sufficient for nuclear localization, residues 363–369 were fused to 2XGFP; however these residues alone were not sufficient to direct 2XGFP into the nucleus (LNC = 0.31; Figures 6B,C). Thus, cNLS2 was revealed to be a non-autonomous enhancer of nuclear localization by PHYSO_251824_239−419.

In addition to cNLS2, residues 239–419 of PHYSO_251824 contain two predicted PY-NLS motifs. PY-NLS1 is located at positions 239–288 and has a variant PY motif (PL), while PY-NLS2 is a conventional PY-NLS located 309–358 (Figure 6A). To test the contribution of PY-NLS1, residues 239–308 were deleted. The resulting segment, 2XGFP-PHYSO_251824_309−419 showed a significantly reduced localization compared to 2XGFP-PHYSO_251824_239−419(LNC = 4.00 vs. 5.30, FDR p′ < 0.001; Figure 6C). This finding suggested that PY-NLS1 may have weak NLS activity. In support of this conclusion, extension of residues 239–308 onto PHYSO_251824₁₋₂₃₈-2XGFP resulted in the reappearance of some nuclear localization (LNC = 0.46 vs. −0.36, FDR p′ < 0.001; Figures 6B,C). To address the contribution of PY-NLS2, specific amino acids were converted into alanines in the PY-NLS2. In the context of residues 239–419, substitution of the PY residues of PY-NLS2 to alanines (P347A/Y348A) partially reduced the nuclear localization (from LNC = 5.30 to 2.42, FDR p′ < 0.001), while additional substitutions in the basic epitope (R332A/R336A/R339A/P347A/Y348A) effectively eliminated the nuclear localization (LNC of 0.77, not significantly greater than 2XGFP alone; Figures 6B,C). On the other hand, the PY-NLS2 alone (309–362) was not sufficient to direct 2XGFP into the nucleus (LNC = 0.66; Figures 6B,C), suggesting that it is necessary but not sufficient for efficient nuclear localization.

Taken together, these findings indicate the nuclear localization of PHYSO_251824 is determined by the C-terminal region, in which two weak PY-NLSs and one cNLS-like enhancer sequence operate synergistically to this protein into the nucleus. No current software programs, including PSORT II, NLStradamus or cNLS Mapper, fully predicted the functional NLS sequences of PHYSO_251824 (Tables S3, S4). PSORT II predicted cNLS2, NLStradamus predicted a fragment of PY-NLS2, while cNLS Mapper predicted a fragment of PY-NLS2 in combination with cNLS2.

To examine whether P. sojae utilizes the same nuclear import sequences for transport of conserved nuclear-localized proteins, we examined ribosomal proteins and core histones. Newly synthesized ribosomal proteins are transported into the nucleus in order to assemble with rRNAs in the nucleolus (Lafontaine and Tollervey, 2001; Gerhardy et al., 2014). Histones, including H2A, H2B, H3, H4, together with the linker histone H1, are essential components of chromatin (Baake et al., 2001).

We first examined the P. sojae ribosomal protein PsL28 which is the ortholog of the yeast ribosomal protein L28 (ScL28, former name L29, Underwood and Fried, 1990; the naming system for P. sojae ribosomal proteins follows the nomenclature of S. cerevisiae; Mager et al., 1997). Protein sequence alignment revealed that the N-termini of the L28 orthologs are highly conserved among different organisms (Figure 7A). Based on functional assays, ScL28 was reported to contain two NLSs: ScL28-NLS1, located at amino acid residues 7–13, and ScL28-NLS2 at 24–30 (Underwood and Fried, 1990). The sequence corresponding to NLS1 in PsL28 showed two amino acid differences from ScL28-NLS1, while the NLS2 sequence was exactly the same in all L28 orthologs (Figure 7A). The minimal conserved region of PsL28₁₋₃₃ that contains both NLSs was expressed as a fusion with 2XGFP and showed strong nuclear localization (Figure 7A). In contrast, when each putative NLS was tested separately by fusion with 2XGFP, neither one produced nuclear localization in P. sojae transformants (Figure 7A). However, as indicated above (Figures 1E,F), the two sequences in PsL28 corresponding to NLS1 and NLS2 together constitute an extended bipartite NLS that includes a core bipartite NLS motif (residues 11–27) together with additional flanking positive residues at the N-terminus; this extended bipartite NLS is functional even though the core bipartite NLS [PsL28₁₋₃₃(K7A/R9A/K10A)] is not (Figures 1E,F). Both NLStradamus and cNLS mapper correctly predicted this extended bipartite cNLS (Tables S3, S4).

We also tested two other ribosomal proteins, S22a and L3. Segments 21–29 of P. sojae S22a (PsS22a) and 1–22 of P. sojae L3 (PsL3), contain residues that are nearly identical to sequences that produced nuclear localization in yeast (Moreland et al., 1985; Timmers et al., 1999). However, in P. sojae transformants these segments produced only cytoplasmic accumulation (Figure S4); addition of flanking sequences (PsS22a₁₋₃₄ and PsL3₁₋₃₆) had no effect on nuclear localization either. Thus, other or additional sequences appear to be required for nuclear localization of these proteins in P. sojae. PSORT II and NLStradamus incorrectly predicted PsL3₁₋₃₆ should be nuclear localized, but cNLS Mapper did not. All three predicted that PsS22a₁₋₃₄ would not be nuclear localized and PSORT II predicted the presence of a cNLS motif elsewhere in the protein (117-RRKH-120) (Table S4).

A similar strategy was carried out to determine the activities of putative NLSs in core histones H3 and H4, which are nearly identical in amino acid sequences in all eukaryotes (Figures 7B,C). The NLSs in H3 and H4 have been experimentally characterized in human and yeast (Figures 7B,C). There is only one NLS reported in each of yeast H3 (ScH3) and H4 (ScH4) (Mosammaparast et al., 2002), while more than one NLS has been found in their human counterparts (Baake et al., 2001). The NLSs in ScH3 and ScH4 somewhat overlapped with their human orthologs, suggesting a common origin.

A series of truncations were made of P. sojae H3 (PsH3) and H4 (PsH4) according to the NLS locations in the yeast and human orthologs. The resulting fragments were tested for nuclear localization as 2XGFP fusions. As expected, the full-length PsH3 and PsH4 reporter constructs produced strong nuclear localization (Figures 7B,C). However, none of the H3 fragments responsible for nuclear localization in yeast or human (residues 1–32 or residues 33–75) individually exhibited the same NLS activities in P. sojae transformants. Region 1–75 of PsH3, which encompasses both of the two NLS regions of human H3, did produce strong nuclear localization in P. sojae (Figure 7B). Expression of a P. sojae H3 segment that lacked the N-terminal domain, H3₇₆₋₁₃₆, did not produce nuclear localization (Figure 7B), indicating that no other sequences contribute to PsH3 nuclear localization. NLStradamus predicted part of this region (10–36) but not all of it. PSORT II and cNLS mapper did not predict this region (Tables S3, S4).

In the case of PsH4, residues 1–25, which produced partial NLS activity in yeast (Mosammaparast et al., 2002) and full activity in human cells (Baake et al., 2001), produced no observable nuclear localization in P. sojae (Figure 7C). A larger N-terminal GFP fusion PsH4₁₋₄₂-2XGFP produced clear nuclear localization in P. sojae with little detectable fluorescence in the cytoplasm, while residues 1–80 of PsH4 produced complete nuclear localization (Figure 7C). This result suggests that segment 26–42 of PsH4, which was identified as an NLS in human cells, contains determinants that may contribute to NLS function in P. sojae. However, PsH4₂₆₋₄₂ alone was insufficient to mediate nuclear localization of the GFP reporter, indicating that these residues could not act as an independent NLS (Figure 7C). On the other hand, PsH4₂₆₋₈₀, which spans two regions (26–42 and 43–80) with NLS activity in human, did produced some nuclear localization in P. sojae but less than 1–80 (Figure 7C), whereas 43–103 showed no activity (Figure 7C). Together, these results suggest that all three regions corresponding to NLSs in human H4, namely 1–25, 26–42, and 43–80, must work together in P. sojae to produce efficient nuclear localization. P. sojae H4 reporters that lacked the N-terminal domains (H4₈₁₋₁₀₃) produced no nuclear accumulation (Figure 7C). NLStradamus predicted the first two of these regions, but not the third. PSORT II and cNLS Mapper predicted none of them (Tables S3, S4).

Taken together, the results above suggest that compared to their yeast or human counterparts, P. sojae has a similar but stronger set of sequence requirements for translocation of conserved nuclear proteins into the nucleus.

The cNLSs are the best-characterized class of NLSs to date (Lange et al., 2007). The prototypical monopartite cNLS derived from the SV40 large T antigen (SV40 NLS) has been reported to direct efficient nuclear import in various organisms, including yeast, mammalian cells (Lange et al., 2007), and plants (Chang et al., 2012). However, we found that monopartite cNLSs, represented by the SV40 NLS and by the one from the c-Myc protein, produced relatively weak nuclear localization in P. sojae. It has been reported that protein context may influence the activity of NLSs, and multiple copies of a NLS can produce faster, more pronounced nuclear localization (Garcia-Bustos et al., 1991). We showed that SV40 NLS fusions at the C-terminus of GFP, and multiple copies of the NLS increased nuclear accumulation compared to a single copy at the N-terminus of GFP (Figure 1D), although some fluorescence was still visible in the cytoplasm (Figure 1B).

Based on assessment of several putative cNLSs predicted from the consensus sequences in P. sojae nuclear proteins, we found that cNLS-mediated nuclear import is conserved in P. sojae but that it exhibits some consistent differences: (1) most predicted monopartite cNLSs are not sufficient by themselves for efficient nuclear accumulation of GFP reporters. However, they may function to augment the activity of other NLSs or work collectively with other weak NLSs to accomplish efficient nuclear import of proteins. For example, the monopartite cNLS embedded inside the PY-NLS of PHYSO_357835 was essential for efficient nuclear targeting (Figure 4), while in PHYSO_251824 a C-terminal monopartite cNLS cooperated with two PY-NLSs to direct efficient nuclear localization (Figure 6). (2) With regard to bipartite cNLSs, sequence comparisons and mutational analyses of several functional bipartite cNLSs, including nucleoplasmin, PsL28, and PHYSO_561151, revealed that a functional bipartite cNLS in P. sojae typically requires additional basic amino acids in the first sub-motif, compared to the human/yeast consensus and possibly in the second also (Figures 1E,G). We did not test the contributions of non-positively charged residues in the neighborhood of the two positively charged clusters, but there is evidence that flanking residues also may be important for NLS activity (Kosugi et al., 2009; Lange et al., 2010).

None of the human and yeast PY-NLSs we tested were capable of mediating efficient nuclear entry of GFP reporters in P. sojae, though weak NLS activity was observed with the hnRNP M PY-NLS and the chimeric M9M peptide. Given that the M9M peptide is a hPY-NLS (Figure 2A), our findings suggest that, in contrast to yeast, the P. sojae nuclear transport machinery may have evolved to transport both bPY-NLS and hPY-NLS cargos.

To characterize P. sojae PY-NLSs, we analyzed five native P. sojae nuclear-localized proteins which harbored predicted PY-NLS-like motifs. Using deletion and substitution mutations, we identified PY-NLS-containing fragments from three of the proteins that could mediate different degrees of GFP translocation into the nuclei of both P. sojae and mammalian cells. In each case however, additional basic amino acids were required, either in association with one of the three PY-NLS epitopes (as in PHYSO_357835), or C-terminal to the PY-NLS motif (PHYSO_480605 and PHYSO_251824).

In yeast, Süel et al. (2008) proposed the “degeneracy rule” for the bPY-NLS in which PL is exchangeable with PY. However, yeast Kapβ2 only recognizes the basic class of PY-NLSs, so PY-motif degeneracy remains unclear for the hydrophobic class of PY-NLSs. Only a few PY-NLSs are reported to have a variant PY motif, such as the RNA-binding protein HuR which contains a PG motif instead of PY. In addition, several newly identified Kapβ2/Kap104 cargos were reported to lack a typical PY motif or even a recognizable PY-NLS (reviewed in Soniat and Chook, 2015). In our study, we found that the variant “PL”-NLS in PHYSO_480605 produced substantial nuclear localization in both P. sojae and mammalian cells, suggesting the degeneracy rule may also exist for hPY-NLS.

The PY-NLS “consensus sequence” is defined by a collection of three modular epitopes: an N-terminal hydrophobic or basic residue-enriched motif, a second R/K/H motif and the third P[Y/L] motif (Lee et al., 2006; Süel et al., 2008). Biochemical and biophysical analyses have shown that in different NLSs each epitope may contribute differently to Kapβ2 (or Kap104p) binding (Lee et al., 2006; Cansizoglu et al., 2007; Süel et al., 2008). For instance, in hnRNP M and in the yeast mRNA processing protein Hrp1, epitope 3 contributes significantly more than the other two epitopes (Cansizoglu et al., 2007; Süel et al., 2008), In contrast, in the M9NLS and Nab2p PY-NLS, epitope 3 contributes weakly to Kapβ2 (or Kap104p) binding, while strong binding is conferred by epitope 1 or by multiple positions distributed across the three epitopes (Lee et al., 2006; Süel et al., 2008). In our study, we found that mutation of PY (epitope 3) in the PHYSO_357835 PY-NLS sequence reduced but did not abolish nuclear entry, whereas complete abrogation of nuclear entry was observed upon mutation of the basic cluster overlapping epitope 2. In the case of the PY-NLSs in PHYSO_480605 and PHYSO_251824, an extra basic cluster downstream of the PY-NLS consensus markedly enhanced the nuclear entry, effectively constituting a fourth epitope (Figures 5, 6). These results suggest that extra positive charges may facilitate the binding of PY-NLSs to the presumptive P. sojae Kapβ2. Similar combinatorial organization of a PY-NLS was observed in the Xenopus Kapβ2 substrate ELYS (RRTRRRIIAKPVTRRKMR), in which a variant PY dipeptide (PV) is flanked by a C-terminal basic cluster (Lau et al., 2009). Thus, our results may explain why the four PY-NLS prototypes from human and yeast functioned poorly in P. sojae, as they lack the additional basic residues required for efficient targeting to the P. sojae nucleus.

Like the nuclear entry mediated by the P. sojae cNLS and PY-NLS motifs, entry by highly conserved nuclear proteins also showed distinctive features in P. sojae. Amino acid sequences of many ribosomal proteins and core histones are highly conserved among different eukaryotes, though within an organism different conserved proteins are imported into the nucleus via different NLSs. This suggests that the responsible karyopherins from different organisms should show similar specificity. However, in P. sojae, NLS motifs that could act autonomously in human and yeast were required to act together to deliver conserved proteins into the nucleus. For example, in yeast L28, two sequences (7-KTRKHRG-13 and 24-KHRKHPG-30) could individually serve as an NLS. However, the corresponding sequences in P. sojae L28 were required together for efficient nuclear localization (thus constituting a bipartite NLS). In the case of histone H3, where the amino acid sequences are nearly identical in all eukaryotes, two adjacent sequences that could each serve as an NLS in human (residues 1–32 and 33–75 of H3) were jointly required for NLS activity in P. sojae. Similarly, in H4 three adjacent sequences that could serve separately as NLSs in human were required together in P. sojae H4 for nuclear entry. The results suggest that the P. sojae karyopherins that presumably bind to these NLSs do so more weakly than in other characterized eukaryotes, and perhaps therefore that binding by multiple karyopherins is required to produce nuclear entry in P. sojae.

During the course of this study, we evaluated three software tools for predicting regions of the proteins that might have NLS activity, conferring nuclear localization. PSORT II (PSORT, 1997) was released in 1997, and since then, a variety of experimentally defined NLSs have been found that do not match any of the consensus sequences (Kosugi et al., 2009). Based on those findings, additional NLS prediction tools have been developed to include those newly identified NLS. We evaluated NLStradamus and cNLS Mapper. We also evaluated PredictNLS, but it produced so few predictions as to not be useful, as has been reported elsewhere (Nguyen Ba et al., 2009). Overall we found that NLStradamus and cNLS Mapper identified long regions but with little indication of which amino acid residues might be most responsible for nuclear localization (Tables S3, S4). On the other hand, PSORT II identified large numbers of very specific predicted NLSs; this was useful at the outset of our study when we were searching for sequences important for nuclear localization in P. sojae, though in the end many of these proved to be false positives (in P. sojae). After we had experimentally tested a large number of protein segments for nuclear localization activity in P. sojae, it emerged that NLStradamus was most successful in identifying regions that could confer partial or full nuclear localization in P. sojae, albeit only 60% successful (Tables S3, S4). Of 10 regions predicted in 9 P. sojae proteins, six were wholly or partially correct, while four were incorrect. Of the four, two were false positives and two were false negatives. More accurate prediction tools for oomycetes will need to take into account the combinatorial nature of NLSs we observed in P. sojae, as exemplified most strikingly by PHYSO_251824.

In summary, using P. sojae as a model, we identified distinctive features of nuclear localization in oomycetes, based on characterization of multiple classes of NLS from 10 nuclear proteins. A consistent pattern has emerged in which individual NLS sequences that are sufficient for autonomous nuclear localization in other eukaryotes function weakly or not at all in P. sojae, but can collaborate with each other or with patches rich in basic residues to produce efficient localization. Several studies have shown that karyopherins from different families and organisms exhibit preferences for specific NLSs (Sekimoto et al., 1997; Köhler et al., 1999; Fang et al., 2001; Mason et al., 2002; Kosugi et al., 2009; Chang et al., 2012); further characterization of P. sojae karyopherin/NLS structures may help us to explain the distinctive features that we observed in our studies. Because nuclear localization has not been dissected comprehensively in a wide diversity of eukaryotes or even in other oomycetes, it is currently unclear whether the differences we observe in P. sojae are part of a wider pattern of diversity in eukaryotes, or have a narrower functional significance, related perhaps to the pathogenic lifestyle of this organism.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.