Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae

Abstract

To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast.

Nuclear localization of a putative Phytophthora sojae bZIP1 transcription factor is mediated by multiple targeting motifs

“…Positive control, SV40 (SV40 NLS) fused to 2XGFP; negative control, 2XGFP alone. Histone H2B fused to mCherry was used as a nuclear marker (Fang et al ., ). Bottom, PsbZIP1 was excluded from the nucleolus, as confirmed by the nucleolar marker FIB‐mCherry (Fang et al ., ).…”

“…Histone H2B fused to mCherry was used as a nuclear marker (Fang et al ., ). Bottom, PsbZIP1 was excluded from the nucleolus, as confirmed by the nucleolar marker FIB‐mCherry (Fang et al ., ). Scale bar corresponds to 5 μm in this and all subsequent figures.…”

“…Histone H2B fused to mCherry was used as a nuclear marker (Fang et al, 2017a). Bottom, PsbZIP1 was excluded from the nucleolus, as confirmed by the nucleolar marker FIB-mCherry (Fang et al, 2017a). localized to the nuclei (LNC 5 0.75; slightly but significantly greater than 2XGFP, LNC 5 0.56; false discovery rate adjusted p value, FDR p 0 < 0.05) ( Fig.…”

“…Although there is an additional arginine preceding the N‐terminal basic cluster of the bipartite cNLS within PsbZIP1(9‐DTKVRRREQCRANQARYRDKQRN‐31), it conferred only very weak nuclear localization. Comparison of this bipartite cNLS motif with those functional bipartite cNLSs reported in our previous study (Fang et al ., ) revealed that the C‐terminal basic cluster of the bipartite cNLS within PsbZIP1 lacks the consecutive lysine or arginine residues characteristic of bipartite NLSs that are strongly active in P. sojae .…”

“…PY-NLSs are defined based on three rules: they are structurally disordered in the unbound state; they have overall basic character; and they possess a central hydrophobic or basic motif followed by the motif [R/H/K]-X 2-5 -P[Y/L] (Lee et al, 2006;Suel et al, 2008). In a recent study (Fang et al, 2017a), we found that many cNLSs and PY-NLSs that have been well-characterized in mammalian cells or Saccharomyces cerevisiae functioned poorly in P. sojae. To mediate efficient nuclear localization of proteins, fully functional P. sojae cNLSs and PY-NLSs required additional basic amino acids compared to yeast or human NLSs or else collaboration with other weak NLSs.…”