Together with MOS6, the Arabidopsis genome contains nine IMP-α paralogs (Wirthmueller et al., 2013). The genome of the single cellular eukaryote Saccharomyces cerevisiae only encodes one IMP-α whereas several paralogs have been identified in higher eukaryotes. The genomes of humans, mice, rice or Drosophila melanogaster, contain seven, six, five or three IMP-αs, respectively (Merkle, 2001; Ouyang et al., 2007;
Ratan et al., 2008; Hu et al., 2010; Kelley et al., 2010; Wirthmueller et al., 2013). The relatively high number of IMP-αs in higher eukaryotes indicates specialization of IMP-α family members in nuclear protein import or might be explained by possibly redundant roles of different IMP-αs. The complexity of higher eukaryotic organisms demands for regulation of nuclear import in order to meet the specific requirements of different tissues, developmental or physiological stages and stimulus-specific nucleocytoplasmic dynamics. Research from the mammalian field provides important insights into regulation of IMP-α activities via tissue-specific expression patterns (Köhler et al., 1997; Tsuji et al., 1997; Yasuhara et al., 2007). Another way to allow for complex regulation is the specialization of NTRs to import a specific group of cargos. In fact, several examples from the mammalian field show the preferential nuclear import of cargo proteins by specific IMP-α adapters (Köhler et al., 1999; Melen et al., 2003; Miyamoto et al., 1997; Nadler et al., 1997; Quensel et al., 2004).
The nuclear import of NF-B TFs (1.3,Huang et al., 2000; Malek et al., 2001) following IB degradation in human cells is a well-studied example for IMP-α specificity.
Here, human IMP-α3 and IMP-α4 are mainly responsible for import of NF-B p50/p65 heterodimers although at least seven different IMP-α isoforms are present in the human genome (Pemberton and Paschal, 2005; Fagerlund et al., 2005). During this process, IMP-α3 binds to NF-B p50 with its major NLS binding pocket while the minor pocket mediates binding to NF-B p65 (Fagerlund et al., 2005). Another example for IMP-α specificity in human cells is the import of the nuclear protein Ran guanine nucleotide exchange factor wich selectively depends on IMP-α3 (RCC1, Quensel et al., 2004).
Like in mammals, Drosophila melanogaster immune responses also depend on the action of NF-B proteins whose activity is controlled at the level of nuclear transport
Introduction _______________________________________________________________
and occurs after activation of the Toll signaling cascade. The nuclear transport receptor specifically involved in transport of NF-B transcription factors is NTF2 (NUCLEAR TRANSPORT FACTOR 2). NTF2 is usually involved in importing Ran-GDP back to the nucleus after a round of nucleocytoplasmic transport (Bhattacharya and Steward, 2002;
Ribbeck et al., 1998). Direct binding of NTF2 to NF-Bs, however, has not been shown and the possibility that NTF2 indirectly influences import of these proteins by regulating the function of IMPs or Ran must be considered as well. Another group of transcription factors whose activity is regulated on a spatial level are the mammalian signal transducers and activators of transcription (STAT). STAT proteins dimerize and cross the nuclear envelope upon activation of the canonical STAT-signaling pathway. Stimulus induced signaling leads to phosphorylation which in turn results in homo- or hetero-dimerization (Lim and Cao, 2006). STAT1 homodimers and STAT1/STAT2 heterodimers specifically interact with IMP-α5 (Melen et al., 2001; Fagerlund et al., 2002) and loss of IMP-α3 via RNAi leads to impaired nuclear translocation of STAT3, but not of STAT1 (Liu et al., 2005).
This finding leads to the conclusion that STAT3 specifically interacts with IMP-α3 and corroborates the notion that some IMP-αs preferentially bind to particular STAT transcription factors. In addition to transcription factors that use the nucleocytoplasmic transport system to enter the nucleus in a biotic stress induced manner, the vertebrate NLRs CIITA and NLRC5 (CLASS II TRANSACTIVATOR and NLR CASPASE RECRUITMENT DOMAIN (CARD) CONTAINING PROTEIN 5) both contain NLS motifs and ultimately regulate gene expression via interaction with DNA-binding proteins inside the nucleus (Meissner et al., 2012b; Meissner et al., 2012a; Cressman et al., 2001; Spilianakis et al., 2000). However, exclusive binding to a specific IMP-α or a subset of IMP-α proteins has not yet been shown.
Only few examples for IMP-α cargo selectivity exist in plants. In the following, examples are summarized where pathogen effector proteins were found to preferentially bind to certain IMP-α proteins. The Agrobacterium tumefaciens Vir proteins are a prominent example for pathogen derived proteins that take advantage of the plant nucleocytoplasmic transport machinery to promote infection (Durrenberger et al., 1989;
Shurvinton et al., 1992; Howard et al., 1992; Ballas and Citovsky, 1997; Bhattacharjee et al., 2008). For transformation, the Agrobacterium derived transfer DNA (T-DNA) needs to
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be transported into the host nucleus. For this, a T-DNA/protein complex (T-complex) is formed in the cytoplasm of infected host cells. The effectors VirD2 and VirE2 form a covalently linked complex with the T-DNA (Durrenberger et al., 1989). Both VirD2 and VirE2 harbor bipartite NLS (Gelvin, 2010; Pitzschke and Hirt, 2010). Several Arabidopsis IMP-αs interact with these NLS motifs and subsequently mediate transfer of the T-complex to the nucleus (Ballas and Citovsky, 1997; Bhattacharjee et al., 2008).
Knock-out of IMP-α4 alone, however, has been shown to be sufficient to reduce A. tumefaciens transformation rates in Arabidopsis root tissue. Interestingly, this phenotype can be complemented by ectopic overexpression of other IMP-α paralogs (Bhattacharjee et al., 2008). IMP-α4 is the predominantly expressed IMP-α in Arabidopsis root tissue. This indicates that the specialized function of IMP-α4 in the transport of the T-complex in roots may be explained by its specific expression. Hence, tissue-specific expression rates of IMP-αs add an additional level of regulation for cargo selectivity in nucleocytoplasmic transport. It could be shown that the rate of NLS-cargo/NTR complex formation is an important factor for efficiency of nuclear import.
This implies that nuclear import rates can be elevated by either increasing protein levels of the cargo or IMP-α, or by increasing the affinity of the NLS for the NTR (Riddick and Macara, 2005; Hodel et al., 2006; Timney et al., 2006; Wirthmueller et al., 2015).
Additional examples for preferential binding to IMP-αs by effector proteins were found in a directed yeast two hybrid screen aimed to search for interactions between 83 effectors from H. a. and Pseudomonas syringae pv. tomato (Pst) with numerous Arabidopsis proteins (Mukhtar et al., 2011). In this screen two interactions between plant IMP-αs and effectors were detected (Mukhtar et al., 2011). The H. a. effector HaRxLL445 was shown to interact with MOS6 while the effector HaRxL106 interacted with MOS6, IMP-α1, IMP-α2 and IMP-α4 (Mukhtar et al., 2011). Specific interaction of MOS6, IMP-α1, IMP-α2 and IMP-α4 with HaRxL106 could be verified in CoIP experiments (Wirthmueller et al., 2015). Selective interaction with IMP-α proteins was also reported for effectors from the oomycete pathogen Phytophthora infestans and the Phytoplasma asteris effector SAP11 (SECRETED AY-WB protein). Nuclear import of these effectors could be attenuated by silencing of NbIMP-α1 or NbIMP-α2 in N. benthamiana (Kanneganti et al., 2007a; Bai et al., 2009). SAP11 contains an eukaryotic bipartite NLS which probably is
Introduction _______________________________________________________________
involved in IMP-α binding (Bai et al., 2009). Nuclear import of the bipartite NLS-containing P. infestans effectors Nuk6 and Nuk7 is also specifically inhibited by silencing of NbIMP-α1 and NbIMP-α2 while nuclear localization of another Nuk effector (Nuk12) was not affected (Kanneganti et al., 2007a).
The examples above illustrate that selective binding of cargo proteins to a specific IMP-α or a subset of IMP-α proteins occurs in animals and could be demonstrated for some effector proteins in plant-pathogen interactions. However, besides these reports little is known about nuclear transport mechanisms that mediate exchange of proteins between the cytoplasm into the nucleoplasm in plant cellular immune responses. To date no plant host defense regulator has been reported as cargo substrates of any IMP-α.
Therefore, it is feasible to postulate that the identification and analysis of defense-related cargo proteins could provide important insights in plant immune responses.