Nuclear Dynamics during Plant Innate Immunity

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Plants are sessile organisms living in an environment rich in microbes that are potentially able to cause disease. As a result, plant survival depends on the ability to couple rapid pathogen detection to an efficient defense response. In contrast to the somatic adaptive immune system of mammals involving mobile defender cells, plant immune responses rely on the ability of each cell to recognize and respond to pathogen invasion and on systemic signals originating from infection sites. A first line of plant defense is activated after recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs), which are highly conserved among microbes. PAMP recognition by specific pattern recognition receptors leads to PAMP-triggered immunity (PTI), which induces basal defenses thereby preventing further pathogen ingress (Jones and Dangl, 2006). To counteract PTI, thriving pathogens gained the ability to secrete virulence effectors able to target crucial PTI regulators. In turn, plants evolved to produce resistance (R) proteins that recognize pathogen-produced effectors or their action on host components, leading to effector-triggered immunity (ETI). ETI provides a second layer of plant defense that is frequently associated to the development of a form of programmed cell death, the so-called hypersensitive response (HR; Jones and Dangl, 2006). Plant immunity against microbial infection engages highly dynamic responses that involve multiple organelles during the recognition and signaling mechanisms associated to defense. A particularly important role in plant defense responses has been attributed to nuclear dynamics, since a growing number of reports revealed that nuclear localization of pathogen effectors, R proteins, and key host components, including transcription factors (TFs) and regulators, is essential for plant immunity (Deslandes and Rivas, 2011). Spatial restriction of defense regulators by the nuclear envelope as well as their stimulus-induced nuclear translocation provide an important mechanism for defense regulation, as their level of nuclear accumulation determines the output of the defense response. Interestingly, mutations in cellular factors involved in the transport of macromolecules through the nuclear envelope, compromise plant resistance signaling, underlining the importance of nucleocytoplasmic trafficking during plant innate immunity (Deslandes and Rivas, 2011). Together, these findings situate the nucleus at the forefront of the mutual recognition between plants and pathogens. Here, I provide a summary of our current knowledge about nuclear dynamics during plant immune responses. Communication between the cytoplasm and the nucleus is a fundamental feature conserved among eukaryotic systems. Transport of macromolecules across the nuclear envelope occurs through nuclear pore complexes (NPCs), which are composed of nucleoporins, and depends on import and export receptors, importins and exportins that respectively recognize nuclear localization signals (NLSs) and nuclear export signals on cargo proteins (Meier and Brkljacic, 2009). The Ras-related nuclear (Ran) protein provides the directionality of transport through its binding to GDP (cytoplasmic side) or GTP (nuclear side). For a more detailed account on dynamics at the nuclear envelope, refer to Meier (2007). Several cellular factors involved in the transport of macromolecules through the nuclear envelope, including nucleoporins, importins, and Ran-GTP-related components, are essential to mount an efficient immune response in response to different pathogens (Palma et al., 2005; Zhang and Li, 2005; Tameling and Baulcombe, 2007; Cheng et al., 2009). In addition, several reports strongly suggest that components of the NPC specifically mediate the transport of R proteins, immunity components, as well as TFs and regulators that are necessary for activation of disease resistance (García and Parker, 2009). These and other findings support the notion that specific modulation of the nuclear concentration of a set of defense regulators is crucial for the fine tuning of plant immunity. Following delivery into the plant cell, microbial effectors may be targeted to different cellular compartments where they may manipulate a variety of host cellular functions. Importantly, a significant number of effector proteins from different pathogens, including nematodes, fungi, viruses, bacteria, and oomycetes, are targeted to the nucleus by co-opting the host nuclear import machinery (Deslandes and Rivas, 2011). This observation suggests that effectors may manipulate host transcription or directly target nuclear essential host components for the benefit of the pathogen. It has been additionally proposed that some effectors may affect histone modification and chromatin remodelling. Alternatively, nuclear translocation of effectors may affect subcellular localization of their cognate R proteins in a process that is essential for R-protein-mediated plant immunity. Most of the molecular mechanisms by which nuclear localization of microbial effectors contributes to promotion of virulence remain to be elucidated. However, some well-characterized examples, mainly from the bacterial field, have been seminal to illustrate pathogen strategies hidden behind effector nuclear targeting. To promote virulence, some nuclear effectors are able to induce transcriptional reprogramming in host cells. For example, the Xanthomonas campestris pv vesicatoria effector AvrBs3 and likely other members of the transcription activator-like effector family provoke developmental reprogramming of host cells by mimicking eukaryotic TFs (Kay and Bonas, 2009). AvrBs3 dimerizes in the plant cell cytoplasm and is translocated to the nucleus via the specific recognition of its NLSs by importin α. In the nucleus, AvrBs3 is able to bind to a specific DNA sequence, the Up-regulated by AvrBs3 (UPA) box, and induce the expression of the so-called UPA genes, via its acidic transcriptional activation domain. Among the UPA genes, UPA20, which encodes a TF containing a basic helix-loop-helix domain, has been identified as a master regulator of plant cell hypertrophy (Kay et al., 2007; Fig. 1A). Plants have evolved a sophisticated strategy to recognize AvrBs3 using the promoter of the R gene Bs3 as a molecular trap. In resistant pepper (Capsicum annuum) plants activation of Bs3 leads to HR development (Römer et al., 2007; Fig. 1A). Nuclear translocation of bacterial effectors and plant resistance proteins. A, After dimerizing in the plant cell cytoplasm, AvrBs3 is translocated into the nucleus, where it is able to bind to the UPA box and act as a TF. Transcriptional activation of UPA20 induces plant cell hypertrophy, whereas in resistant pepper, activation of the R gene Bs3 leads to HR. B, XopD nuclear interaction with the Arabidopsis TF AtMYB30 is mediated by the HLH domain of XopD, which is necessary and sufficient to repress AtMYB30 transcriptional activation and thereby the plant defense response. C, In resting cells, the N immune receptor presents a dual cytoplasmic and nuclear localization. Upon TMV infection, the viral replicase p50 recruits to the cytoplasm the tobacco NRIP1 protein, which is localized in the chloroplast of uninfected cells. The NRIP1/p50 prerecognition complex interacts with and activates cytoplasmic N, which is then able to cross the nuclear envelope and/or send a signal to activate nuclear N, thereby activating defense signaling. D, The PopP2 effector induces nuclear accumulation of its cognate R protein RRS1-R, which acts as a transcriptional repressor in resting cells, and of the vacuolar protease RD19. The nuclear interaction between the PopP2/RD19 complex and RRS1-R leads to activation of plant defense, perhaps through modification of the transcriptional activity of RRS1-R WRKY domain or by activation of additional TFs. It has been additionally proposed that PopP2 acetyltransferase activity may disrupt higher-order packaging of the chromatin or alter DNA-binding activities and interaction properties of host TFs, leading to modification of their activity. Similar to transcription activator-like effectors, HsvG and HsvB effectors of gall-forming Pantoea agglomerans, which present two functional NLSs required for their nuclear targeting and pathogenicity, are able to bind DNA and activate transcription (Nissan et al., 2006; Weinthal et al., 2011). Although the mode of action of HsvG and HsvB remains unknown, it has been hypothesized that it involves modulation of host phytohormones associated with gall formation. The PopP2 effector from Ralstonia solanacearum presents an NLS that is required for its nuclear targeting (Deslandes et al., 2003). It has been reported that PopP2 is able to display acetyltransferase activity, suggesting that PopP2 may directly manipulate host transcription (Tasset et al., 2010). Indeed, acetylation of Lys residues of histone tails facilitates access of TFs to DNA by disrupting higher-order packaging of the chromatin and also by neutralizing the positive charge of histones, which reduces their affinity for DNA. Acetylation also impairs the ability of the Lys side chain to form hydrogen bonds, thereby enhancing specific or inhibiting nonspecific DNA-binding activities of TFs. In addition, acetylation forms docking sites for recruitment of transcriptional coactivators. Therefore, it has been proposed that PopP2 autoacetylation and/or acetylation of its host targets may affect gene transcription in host cells (Tasset et al., 2010; Fig. 1D). XopD, a modular effector of 760 amino acids from X. campestris pv vesicatoria, displays SUMO protease activity and is targeted to plant cell subnuclear structures named nuclear bodies (or nuclear foci), suggesting that it may target SUMO-conjugated nuclear proteins (Hotson et al., 2003; Canonne et al., 2010). The molecular mechanism allowing XopD nuclear import remains unknown, but a truncated XopD version containing only the helix-loop-helix domain of XopD (amino acids 216–405), and not comprising its putative NLS, is sufficient for XopD nuclear import and subnuclear targeting (Canonne et al., 2011). Expression of XopD appears to induce reorganization of the host nuclear structure that leads to nonspecific relocalization of all tested nuclear proteins into nuclear bodies. In addition, 4′,6-diamino-phenylindole staining showed that DNA accumulation is weaker in nuclear bodies, where XopD is expressed, compared to the nucleoplasm, where DNA distribution remains otherwise unaltered (Canonne et al., 2011). It is thus tempting to speculate that XopD-induced modification of the nuclear structure and protein distribution may be part of a general virulence strategy, which allows Xanthomonas to perturb plant cell responses to bacterial infection. Along these lines, it has been proposed that XopD may affect host transcription by affecting chromatin remodelling (Kay and Bonas, 2009). Significantly, in agreement with the idea that plant TFs and/or regulators might be direct targets of XopD, recent data show that XopD is able to interact with AtMYB30, a previously described Arabidopsis (Arabidopsis thaliana) MYB TF that positively regulates defense and HR responses through the activation of the lipid biosynthesis pathway that leads to the production of very-long-chain fatty acids (VLCFAs; Raffaele et al., 2008). Indeed, XopD-specific interaction with AtMYB30 leads to inhibition of the transcriptional activation of AtMYB30 target genes and suppression of plant defense during Xanthomonas infection (Canonne et al., 2011; Fig. 1B). Importantly, XopD interaction with AtMYB30 appears to be independent of nuclear foci formation. Different R proteins have been reported to dynamically traffic between the cytoplasm and the nucleus where they act as cotranscriptional regulators to activate innate immune responses. For example, the tobacco (Nicotiana tabacum) immune receptor N, which confers resistance to Tobacco mosaic virus (TMV), has been shown to function in the nucleus. In resting cells, N is found in the cytoplasm and the nucleus. During TMV infection, the viral replicase p50 is delivered into the plant cell cytoplasm where it recruits the tobacco rhodanase sulfurtransferase NRIP1, which otherwise localizes to the stroma of chloroplasts. As part of this cytoplasmic prerecognition complex, NRIP1 interacts with and activates cytoplasmic N (Caplan et al., 2008). Shuttling of p50-activated N from the cytoplasm to the nucleus appears to be required for an efficient defense response, although it is also possible that cytoplasmic N is able to send a signal that activates the N nuclear pool (Fig. 2C). Nuclear relocalization of NRIP1 is required to provide full resistance to TMV infection (Caplan et al., 2008). The molecular mechanism behind p50-mediated NRIP1 nuclear relocalization remains unclear. It has been proposed that p50 may disrupt global chloroplast import by an unknown mechanism that would affect NRIP1 translocation. Otherwise, interaction with p50 may mask NRIP1 chloroplast targeting signal and allow its nuclear recruitment. Alternatively, NRIP1 may be released from chloroplasts into the cytoplasm and the nucleus following p50-induced permeabilization of the outer membrane. Finally, since close contact between stromules and nuclei has been observed, it has been proposed that this close association may enhance the nuclear import of chloroplastic factors, such as NRIP1. Nucleo-cytoplasmic trafficking of TFs and regulators during plant defense responses. A, The secreted phospholipase AtsPLA2-α is partially relocalized from Golgi-associated vesicles to the nucleus, where it interacts with the positive defense and HR regulator AtMYB30. This nuclear protein interaction leads to down-regulation of AtMYB30 transcriptional activation of VLCFA-related genes and, therefore, suppression of HR and defense responses. B, Pathogen-induced SA and reactive oxygen species (ROS) production leads to a change in the oligomerization state of the transcriptional coactivator NPR1, which is translocated into the nucleus in its monomeric active form. In the nucleus, active NPR1 interacts with TGA TFs and enhance their DNA-binding properties during the establishment of NPR1 nuclear accumulation is by the of the immune response mediated by the R protein, which confers resistance to the also its nuclear the protein is also found in the cytoplasm et al., Indeed, recognition of cognate effector induces the nuclear association of and and which to the WRKY of plant TFs and act as of In this appears to with the WRKY repressor thereby PAMP-triggered basal defense responses. In activation of would allow rapid of PTI by directly pathogen with of transcriptional reprogramming in the nucleus. As previously the solanacearum effector PopP2 is targeted to the nucleus of plant cells. cognate Arabidopsis R protein RRS1-R is an R protein that confers resistance to solanacearum and presents a WRKY and a putative NLS (Deslandes et al., 2003). PopP2 nuclear accumulation of RRS1-R, by preventing its PopP2 and RRS1-R interact in the nucleus, but and this protein interaction host transcription remain to be In to the previously putative modulation of host transcription that may be mediated through PopP2 acetyltransferase activity, it has been proposed that PopP2 interaction with RRS1-R may to of gene expression directly via the RRS1-R domain or through the action of additional plant TFs (Deslandes et al., 2003; et al., 2010; Fig. 1D). Nuclear recognition of PopP2 by RRS1-R an additional molecular to pathogen with signaling. the Arabidopsis protein, which confers resistance to pv et al., has been shown to function with RRS1-R in resistance against a variety of pathogens et al., et al., 2009). which a functional NLS, between and nuclear accumulation is necessary for immunity through activation by et al., Finally, recognition of the protein of virus by the immune receptor confers resistance to the virus in a process that is as resistance et al., The identified protein interaction between and suggests the of components of the nuclear pore in the of plant immune responses et al., 2007; Tameling and Baulcombe, This idea additionally by the that of impairs resistance et al., 2007; Tameling and Baulcombe, data show that the of an NLS its sequence, localizes to the cytoplasm and the nucleus in cells and that this dual localization is required for full et al., 2010; Tameling et al., 2010). The interaction between and cytoplasmic in of in the cytoplasm, of activity. This leads to immune signaling, which with HR and resistance to In nuclear HR and resistance responses to This is in agreement with the that is activated in the cytoplasm, where recognition of the and be activated in the nucleus et al., 2010). Together, these data as a cytoplasmic that regulates nucleocytoplasmic of in a process that is required for of defense signaling. have been for nuclear and cytoplasmic during ETI Nuclear may a role in transcriptional leading to ETI whereas cytoplasmic may directly activate an mechanism in the cytoplasm, where and detection by et al., 2010; Tameling et al., 2010). In to R and effector proteins, essential regulators of the plant immune response between the cytoplasm and the nucleus, a possible for defense signal trafficking between the two of the of this dynamic traffic of immunity components is by the plant immune complex by and activities and are essential for responses to multiple et al., Together, and a family of proteins with to eukaryotic although activity has been reported for these proteins and their mode of action remains in the cytoplasm and in the nucleus with its are are found in cytoplasm and nucleus and complexes are nuclear et al., recognition an in the nuclear which transcriptional reprogramming of and genes (García et al., 2010). Importantly, such of nuclear to and with the cytoplasmic which is also required for full nuclear may affect transcription is but interaction with a number of TFs in suggests that nuclear complexes may by binding TFs and/or in the to activities and with the DNA (García et al., 2010). between the cytoplasmic and nuclear via the NPC trafficking machinery is to full plant For example, resistance by the of which a in an R is associated with in of cytoplasmic and nuclear (García et al., 2010). In addition, the nuclear pool during the immune response and associated transcriptional a cytoplasmic pool is during defense. to the cytoplasm using a nuclear export signal resistance and basal defense responses. through nuclear and cytoplasmic activities to mediate the and of genes, thereby allowing the plant to mount an immune response (García et al., 2010). A recent that not between and in a complex et al., 2011). resistance signaling is more likely to be by complexes of with or which are to have in plant defense. A has been proposed in which molecular between complexes different of signal After pathogen the of and but not their is sufficient to the resistance response by activating localized host cell death, which leads to pathogen at infection sites. In in cells the complexes a different function necessary for basal resistance to pathogen infection, which with transcriptional of This the of resistance to systemic and of defenses during systemic resistance an form of plant defense immunity to infection. In this complexes would the HR at infection sites with the of resistance at the of the HR. complexes would thus a state between the of cell and and the of resistance in et al., 2011). Transcriptional reprogramming is a key of plant defense as to of Arabidopsis genes respond to pathogen infection by their et al., et al., 2003). This dynamic of gene expression on an of TFs and regulators and adaptive of plants in highly of several TF such as and MYB factors, have been shown to bind to promoter of genes and to their expression and As by the following examples, cytoplasmic of TFs or transcriptional regulators, by their activation and translocation into the nucleus, allows plants to signal at the cell and cytoplasmic signal to defense gene activation in a Transcriptional activation of VLCFA-related genes by the Arabidopsis TF AtMYB30 is required to mount an efficient defense response during bacterial infection et al., 2008). Several data that of AtMYB30 transcriptional activity is during plant defense. In to the inhibition of AtMYB30 activity by the bacterial effector XopD that leads to AtMYB30 transcriptional activation is also by the plant via the secreted phospholipase Indeed, AtsPLA2-α identified in a as an protein et al., 2010). As for a secreted protein, AtsPLA2-α localized in Golgi-associated vesicles and secreted to the However, AtsPLA2-α and AtMYB30 are in targeting of AtsPLA2-α is partially from cytoplasmic vesicles to the plant cell nucleus, where the interaction between proteins has been This protein interaction leads to of transcriptional activity and of plant HR and defense responses et al., 2010; Fig. These data identified AtsPLA2-α as a regulator of defense signaling and the importance of dynamic protein trafficking to the nucleus for the of As previously the solanacearum effector PopP2 interacts with the Arabidopsis R protein RRS1-R in the nucleus, although the of this protein interaction on host transcription is Interestingly, PopP2 induces nuclear targeting of the Arabidopsis protease otherwise localized to mobile vesicles and to the et al., 2008). expression is by Ralstonia infection, interacts with but not RRS1-R, in the plant cell nucleus. is required for Arabidopsis resistance to it proposed that with PopP2 to form a nuclear complex that is required for activation of the plant resistance response (Fig. 1D). to the vacuolar protease is relocalized to the nucleus in response to a et al., In the nucleus, acts as a TF to activate the expression of the although the mechanism involved is Therefore, it is possible that nuclear as a transcriptional and/or with RRS1-R for or in the of genes et al., 2008). or AtsPLA2-α not a NLS and the molecular mechanism that allows their nuclear recruitment is However, different have been proposed to the nuclear targeting of these proteins. In the of and it proposed that an permeabilization process would a of the vacuolar membrane. This would to the of and from compartments into the cytoplasm, where they may for et al., 2005; et al., 2008). AtMYB30 might AtsPLA2-α from compartments to the nucleus through an unknown mechanism that may involve AtsPLA2-α et al., 2010). Indeed, Lys residues with of are present in and and interacts with SUMO in Therefore, it has been that of these proteins may the signal required for their nuclear translocation. Alternatively, AtMYB30 may AtsPLA2-α on its to the through transport from the which has some with the nuclear A mechanism previously proposed for nuclear translocation of RD19. Finally, binding of AtMYB30 to AtsPLA2-α may mask AtsPLA2-α targeting signal or AtMYB30 may disrupt global affecting the translocation of AtsPLA2-α et al., 2010). SA is the plant during is a of and activation of its expression is on SA production and by members of the TGA family of basic TFs, which bind to two in the acts as a transcriptional coactivator of expression In uninfected cells, NPR1 forms its functional NLS and the protein in the cytoplasm et al., 2003). Upon pathogen SA induce a change in the of the cell that NPR1 are partially to a monomeric state and of NPR1 NLS allows its nuclear translocation. in the nucleus, NPR1 interacts with TGA TFs and their DNA-binding activity to positive and in of genes, such as and WRKY TFs et al., Fig. Therefore, the of the fine tuning of responses. Interestingly, of nuclear NPR1 of transcription in cells and allows full transcriptional activation during et al., Fig. Finally, a mechanism of defense signaling is by the repressor NPR1, that expression by histone et al., 2006). The plant cell nucleus a key role in signals and in gene expression to counteract pathogen ingress the associated to the defense regulators such as TFs of the MYB or TGA and immunity components such as are nuclear in their localization. However, during the from our have revealed an number of proteins that are dynamically translocated across the nuclear For example, immune regulators such as NPR1, and as well as several R proteins and their cognate effectors, show and in subcellular localization that are key to the modulation of their activity during defense signaling. R proteins N, and a nuclear localization for whereas is activated in the Here, I have recent in nuclear dynamics associated to plant immunity. However, recent the signaling mechanisms that direct in protein subcellular localization and activity are to be In this strategies a of cell and to are Finally, to a about the mechanisms by which plants defense in response to our knowledge about nuclear trafficking of immunity components to be in the of cellular dynamics, by by additional including or the I to all not be of