The Arabidopsis ZED1-Related Kinase Genomic Cluster Is Specifically Required for Effector-Triggered Immunity

CRISPR/Cas9-mediated deletion of an Arabidopsis gene cluster encoding eight kinases supports their immunity-specific roles in sensing pathogenic effectors.

Dear Editor,ZED1-related kinases (ZRKs) associate with the nucleotide binding, Leu-rich repeat (NLR) protein HOPZ-ACTIVATED RESISTANCE1 (ZAR1) to mediate effector-triggered immunity (ETI) against at least three distinct families of pathogenic effector proteins. However, it is unknown whether ZRKs specifically function in ETI or whether they also have additional roles in immunity and/or development. Eight ZRKs are clustered in the Arabidopsis (Arabidopsis thaliana) genome, including the three members with known roles in ETI. Here, we show that an ∼14-kb CRISPR-mediated deletion of the Arabidopsis ZRK genomic cluster specifically affects ETI, with no apparent defects in pattern-recognition-receptor–triggered immunity (PTI) or development.Phytopathogens deliver effector proteins into plant cells that suppress PTI and promote the infection process (Jones and Dangl, 2006). In turn, plants have evolved NLRs that recognize effectors, leading to an ETI response. This recognition often occurs indirectly, whereby NLRs monitor host “sensor” proteins for effector-induced perturbations (Khan et al., 2016). In the absence of their respective NLRs, some of these sensors are effector virulence targets that modulate immunity and development, while others appear to be decoys that mimic virulence targets, with ETI-specific roles (van der Hoorn and Kamoun, 2008; Khan et al., 2018).The ZAR1 NLR recognizes at least six type-III secreted effector (T3SE) families from bacterial phytopathogens. This remarkable immunodiversity appears to be conveyed through associations with members of the receptor-like cytoplasmic kinase XII-2 (RLCK XII-2) family, which all display characteristics of atypical kinases (Lewis et al., 2013; Roux et al., 2014). The ZAR1-mediated ETI responses against the Pseudomonas syringae T3SEs HopZ1a and HopF1r (formerly HopF2a) require ZED1 and ZRK3, whereas recognition of the Xanthomonas campestris T3SE AvrAC requires ZRK1/RKS1 (Lewis et al., 2013; Wang et al., 2015; Seto et al., 2017). ZRKs currently have no ascribed functions outside of ZAR1-associated ETI responses and are therefore considered decoy sensors or adaptors (Lewis et al., 2013; Wang et al., 2015; Khan et al., 2018). However, functional redundancy may exist among members of the ZRK family, masking phenotypes of individual mutants beyond gene-for-gene–type ETI responses (Lewis et al., 2013). We therefore utilized the CRISPR/Cas9 system to knock out the Arabidopsis genomic region containing eight of the 13 members of the RLCK XII-2, including all ZRK genes known to contribute to ETI, to investigate any non-ETI roles of ZRKs. The 14-kb ZRK gene cluster in Arabidopsis Col-0 plants includes ZRK1, ZRK2, ZRK3, ZRK4, ZED1, ZRK6, ZRK7, and ZRK10. A CRISPR/Cas9-mediated deletion of 13.3 kb was accomplished by designing guide RNAs flanking the ends of the ZRK gene cluster, which would result in a double-stranded break on both sides of the ZRK cluster, leaving only the 5′ end 63 nucleotides (21 amino acids) of ZRK10 and the 3′ end 118 nucleotides (39 amino acids) of ZRK7 (Fig. 1A). We obtained a T1 individual (zrk_1.11) homozygous for the deletion, as well as a T1 individual heterozygous for the mutation (zrk_1.10; Fig. 1B), from which we obtained homozygous T2 (zrk_2.11) and T3 (zrk_3.10) plants, respectively. Sequencing results from zrk_2.11 confirmed that the expected region had been deleted (Supplemental Fig. S1). Plants homozygous for the ZRK gene cluster deletion were morphologically indistinguishable from wild-type Col-0 plants, as well as zar1-1 plants (Fig. 1C). In addition, zrk plant fresh weight did not significantly differ from Col-0 plants (Supplemental Fig. S2), indicating that the ZRK cluster does not play a major role in vegetative plant development.Open in a separate windowFigure 1.Deletion of the ZRK gene cluster results in loss of ZRK-mediated ETI and does not significantly alter vegetative growth. A, Representation of ZRK gene cluster before (top) and after (bottom) CRISPR/Cas9-mediated deletion depicting guide RNAs and primers used for genotyping (see Supplemental Methods S1). ZRK KO primers (magenta) were used to confirm the deletion of the ZRK cluster, while ZRK3 primers (green) were used to check if the ZRK cluster was still present in T1 individuals. B, PCR genotyping for deletion of ZRK gene cluster. Amplification of product by ZRK3 F + R primers indicates lack of deletion; amplification by ZRK KO F + R indicates deletion has occurred. Examples for wild type (WT), heterozygous (HT; zrk_1.10), and homozygous for the deletion (HM KO; zrk_1.11) are shown. T1 lines (zrk_1.10 and zrk_1.11) are compared to wild-type Col-0. C, Uninfected morphology of homozygous zrk KO plants (zrk_3.10 or zrk_2.11) compared to Col-0 and zar1-1 plants. Bar = 1 cm. D, Phenotypes of zrk_2.11 plants 7 d after being sprayed with PtoDC3000(hopZ1a; left) or PtoDC3000(hopF1r; right) relative to wild-type Col-0 and zar1-1 plants. Plant immunity and disease image-based quantification of disease symptoms is presented in Supplemental Figure S3A (Laflamme et al., 2016).Next, we wanted to confirm that the deletion of the ZRK gene cluster compromised ZRK-mediated ETI responses. We sprayed the zrk_2.11 line with PtoDC3000(hopZ1a) or PtoDC3000(hopF1r), as both T3SEs require a ZRK as well as the NLR ZAR1 for their recognition in Arabidopsis (Lewis et al., 2013; Seto et al., 2017). We observed that the zrk_2.11 line was susceptible to both PtoDC3000(hopZ1a) and PtoDC3000(hopF1r), and this susceptibility was to the same level as zar1-1 plants as quantified by plant immunity and disease image-based quantification (Fig. 1D, Supplemental Fig. S3, A and C; Laflamme et al., 2016). We observed a similar phenotype for the zrk_3.10 line, confirming that the ZRK cluster deletion compromised ZRK-mediated ETI responses (Supplemental Fig. S3, B and C). Furthermore, the ZAR1-mediated ETI responses against the P. syringae T3SEs HopBA1a, HopX1i, and HopO1c were also lost in zrk_2.11, demonstrating the ZRK-dependence of these ETI responses (Supplemental Fig. S4; Laflamme et al., 2020). To ensure that the ZRK gene cluster deletion specifically impacted ZRK-related ETI responses, the zrk_3.10 and zrk_2.11 lines were also sprayed with PtoDC3000(avrRpt2), an ETI elicitor that does not require a ZRK or ZAR1 for its recognition (Mackey et al., 2003). zrk_3.10 and zrk_2.11 plants remained resistant to PtoDC3000(avrRpt2), indicating that the ZRK gene cluster deletion specifically impacts ZRK-mediated ETI responses (Supplemental Fig. S3C). In addition, growth of virulent PtoDC3000 on the zrk_3.10 and zrk_2.11 lines was unchanged compared to wild-type Col-0 plants, indicating that the ZRKs within this cluster likely do not represent virulence targets (Supplemental Fig. S5).We then examined whether knocking out the ZRK gene cluster impacted PTI. We first measured induction of peroxidase (POX) enzyme activity, as POX enzymes are produced in response to PTI (Mott et al., 2018). After treatment with the PTI elicitor flg22, addition of the POX substrate 5-aminosalicylic acid produces a brown end-product in the presence of active POX enzymes, which is quantified by reading at an optical density of 550 nm (OD₅₅₀; Mott et al., 2018). Twenty h after leaf discs were treated with flg22, zrk_3.10 and zrk_2.11 plants showed the same level of PTI-associated POX activity as wild-type Col-0 plants (Fig. 2A). To further examine the role of the ZRK gene cluster in PTI, we quantified the growth of PtoDC3000ΔhrcC, which is defective in T3SE secretion and is sensitive to altered host PTI responses under high humidity conditions such as those used in our growth assays (Guo et al., 2009; Xin et al., 2016). Growth of PtoDC3000ΔhrcC on zrk_3.10 and zrk_2.11 plants was not significantly different compared to wild-type Col-0 plants (Fig. 2B). In addition, we monitored reactive oxygen species (ROS) production and found that zrk_3.10 and zrk_2.11 plants did not show a significant difference in the ROS burst observed in wild-type Col-0 plants (Fig. 2, C and D). Finally, we treated seedlings with flg22, and found that growth of zrk_3.10 and zrk_2.11 seedlings was inhibited by the same amount as in wild-type Col-0 seedlings, indicative of a similar induction of PTI responses (Fig. 2, E and F; Gómez-Gómez et al., 1999). Together, these results indicate that the ZRK gene cluster does not play a significant role in Arabidopsis PTI responses.Open in a separate windowFigure 2.Deletion of the ZRK gene cluster does not alter pattern-recognition-receptor–triggered immune responses. A, Response to the PTI elicitor flg22 measured by POX activity. Activity from leaf discs was quantified 20 h after treatment with 1 μm of flg22 at a measurement of OD₅₅₀ (n = 6; Mott et al., 2018). B, Bacterial growth of the T3SS-compromised PtoDC3000ΔhrcC on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants 3-d postinoculation. Plants were domed for the duration of the experiment (n = 8). C, Response of Col-0, zrk KO plants (zrk_3.10 and zrk_2.11), and fls2 to the PTI elicitor flg22 measured using luminol-based detection of ROS over a time course of 60 min, with relative light units measured every 2 min (n = 12). D, Boxplots of total relative light units over a period of 30 min from treatments in C (n = 12). E, Growth inhibition of seedlings 7 d after treatment with 1 μm of flg22. F, Seedling growth inhibition was quantified by measuring fresh weight of flg22-treated seedlings as a percentage of water-treated controls (n = 4). Error bars in A, B, C, D, and F, represent se. Lowercase letters represent significantly different statistical groups by Tukey’s honest significant difference test (P < 0.05). Experiments were replicated three times with similar results.Overall, our results support an ETI-specific role for ZRKs in Arabidopsis, acting as sensors of the ZAR1 NLR. Structural insights have revealed important residues required for ZAR1-ZRK1 complex formation, and these are conserved across the RLCK XII-2 family, which includes ZRKs outside the genomic cluster (Supplemental Fig. S6; Lewis et al., 2013; Wang et al., 2019). This suggests that the ZRKs outside this genomic cluster may also play a similar role as ZAR1 sensors. As such, the ZRK family would have evolved to mimic and/or interact with the numerous kinase virulence targets of pathogenic effectors, thereby expanding the surveillance potential of ZAR1.Supplemental DataThe following supplemental materials are available.

• Supplemental Figure S1. Sequencing confirmation of the ZRK gene cluster deletion.
• Supplemental Figure S2. Fresh weight of zrk knockout (KO) plants (zrk_3.10 and zrk_2.11) relative to wild-type Columbia-0 (wild-type Col-0) and zar1-1 plants.
• Supplemental Figure S3. ZRK gene cluster deletion specifically compromises ZRK-dependent ETI responses.
• Supplemental Figure S4. ZRK gene cluster compromises the ZAR1-dependent ETI responses against HopBA1a, HopO1c, and HopX1i.
• Supplemental Figure S5. Bacterial growth of the virulent PtoDC3000 strain on zrk KO plants (zrk_3.10 and zrk_2.11) relative to wild-type Col-0 (wild-type Col-0) plants 0- and 3-d post-inoculation via syringe infiltration.
• Supplemental Figure S6. Multiple sequence alignment of RLCK XII-2 family shows high conservation of putative ZAR1-interacting residues.
• Supplemental Methods S1. Generation and characterization of ZRK cluster deletion lines.

     

Mitogen-Activated Protein Kinases 3 and 6 Are Required for Full Priming of Stress Responses in Arabidopsis thaliana

In plants and animals, induced resistance (IR) to biotic and abiotic stress is associated with priming of cells for faster and stronger activation of defense responses. It has been hypothesized that cell priming involves accumulation of latent signaling components that are not used until challenge exposure to stress. However, the identity of such signaling components has remained elusive. Here, we show that during development of chemically induced resistance in Arabidopsis thaliana, priming is associated with accumulation of mRNA and inactive proteins of mitogen-activated protein kinases (MPKs), MPK3 and MPK6. Upon challenge exposure to biotic or abiotic stress, these two enzymes were more strongly activated in primed plants than in nonprimed plants. This elevated activation was linked to enhanced defense gene expression and development of IR. Strong elicitation of stress-induced MPK3 and MPK6 activity is also seen in the constitutive priming mutant edr1, while activity was attenuated in the priming-deficient npr1 mutant. Moreover, priming of defense gene expression and IR were lost or reduced in mpk3 or mpk6 mutants. Our findings argue that prestress deposition of the signaling components MPK3 and MPK6 is a critical step in priming plants for full induction of defense responses during IR.     

SNF1-Related Protein Kinases Type 2 Are Involved in Plant Responses to Cadmium Stress

Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd²⁺ showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd²⁺ treatment. Our data show significantly lower Cd²⁺-induced ROS accumulation in the mutants’ roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions.Cadmium is one of the most toxic soil pollutants. Cadmium ions accumulate in plants and affect, via the food chain, animal and human health. In plants, cadmium is taken up by roots and is transported to aerial organs, leading to chromosomal aberrations, growth reduction, and inhibition of photosynthesis, transpiration, nitrogen metabolism, nutrient and water uptake, eventually causing plant death (for review, see DalCorso et al., 2008). Plants are challenged not only by cadmium ions themselves, but also by Cd²⁺-induced harmful effects including oxidative stress (Schützendübel et al., 2001; Olmos et al., 2003; Cho and Seo, 2005; Sharma and Dietz, 2009). The extent of the detrimental effects on plant growth and metabolism depends on the level of cadmium ions present in the surrounding environment and on the plant’s sensitivity to heavy metal stress.Tolerant plants avoid heavy metal uptake and/or induce the expression of genes encoding products involved, directly or indirectly, in heavy metal binding and removal from potentially sensitive sites, by sequestration or efflux (Clemens, 2006). The best-characterized heavy metal binding ligands in plants are thiol-containing compounds metallothioneins and phytochelatins (PCs), whose production is stimulated by Cd²⁺. PCs bind metal ions and transport them to the vacuole, thus reducing the toxicity of the metal in the cytosol (for review, see Cobbett, 2000; Cobbett and Goldsbrough, 2002). PCs are synthesized from reduced glutathione (GSH). Therefore, production of compounds involved in cadmium detoxification and, at the same time, in cadmium tolerance closely depends on sulfur metabolism. So far, our knowledge on the cellular processes induced by cadmium that lead to changes in sulfur metabolism in plants has been rather limited.Protein kinases and phosphatases are considered major signal transduction elements. However, until now only a few of them have been described to be involved in cadmium stress response or sulfur metabolism. For instance, excessive amounts of cadmium or copper activate mitogen-activated protein kinases (MAPKs) in Medicago sativa (Jonak et al., 2004), rice (Oryza sativa; Yeh et al., 2007), and Arabidopsis (Arabidopsis thaliana; Liu et al., 2010). Studies on rice MAPKs involved in heavy metal stress response indicate that the activity of these kinases depends on the oxidative stress induced by Cd²⁺. Moreover, Yeh et al. (2007) suggested that the activation of MAPKs in rice by cadmium or copper required the activity of calcium-dependent protein kinase (CDPK) and PI3 kinase, since the MAPK pathways involved in cadmium and copper stress response could be inhibited by a CDPK antagonist (W7) or a PI3 kinase inhibitor (wortmannin). However, so far the function of the identified kinases in plant adaptation to heavy metal pollution has not been established. There is some information concerning an involvement of CDPK in sulfur metabolism (Liu et al., 2006). Soybean (Glycine max) Ser acetyltransferase (GmSerat2;1), the enzyme that catalyzes the first reaction in the biosynthesis of Cys from Ser, is phosphorylated by CDPK. The phosphorylation has no effect on GmSerat2;1 activity, but it renders the enzyme insensitive to the feedback inhibition by Cys (Liu et al., 2006). There is growing evidence that SnRK2s (for SNF1-related protein kinase2) play a role in the regulation of sulfur metabolism. Most information showing a connection between SnRK2s and sulfur metabolism comes from experiments on the lower plant Chlamydomonas reinhardtii (Davies et al., 1999; Irihimovitch and Stern, 2006; González-Ballester et al., 2008, 2010). SNRK2.1 is considered a general regulator of S-responsive gene expression in C. reinhardtii (González-Ballester et al., 2008).In higher plants the SnRK2 family members are known to be involved in plant response to drought, salinity, and in abscisic acid (ABA)-dependent plant development (Boudsocq and Laurière, 2005; Fujii et al., 2007, 2011; Fujii and Zhu, 2009; Fujita et al., 2009; Nakashima et al., 2009; Kulik et al., 2011). Ten members of the SnRK2 family have been identified in Arabidopsis and in rice (Boudsocq et al., 2004; Kobayashi et al., 2004). All of them, except SnRK2.9 from Arabidopsis, are rapidly activated by treatment with different osmolytes, such as Suc, mannitol, sorbitol, and NaCl, and some of them also by ABA. Results presented by Kimura et al. (2006) suggest that in Arabidopsis, similarly to C. reinhardtii, some SnRK2s are involved in the regulation of S-responsive gene expression and O-acetyl-l-Ser accumulation under limited sulfur supply, indicating that also higher plants’ SnRK2s could be involved in sulfur metabolism.As it was mentioned before, oxidative stress induced by cadmium ions significantly contributes to the metal toxicity. Reactive oxygen species (ROS) can be produced in many different reactions in various compartments of the cell in response to cadmium (Romero-Puertas et al., 2004; Heyno et al., 2008; Tamás et al., 2009). The best-characterized ROS-generating enzymes that take part in the response to cadmium are the plasma-membrane-bound NADPH oxidases (Olmos et al., 2003; Romero-Puertas et al., 2004; Garnier et al., 2006). There are some indications that plant NADPH oxidases are phosphorylated by SnRK2s (Sirichandra et al., 2009), therefore it is highly plausible that SnRK2s play a role in the regulation of ROS accumulation in plants subjected to cadmium stress. Taking into consideration all facts mentioned above we hypothesized that SnRK2s could be involved in the plant response to stress induced by cadmium ions. To verify this conjecture, we analyzed the activity and potential role of selected SnRK2s, in tobacco (Nicotiana tabacum) cells and Arabidopsis plants, in the response to cadmium ions.     

Two Mitogen-Activated Protein Kinases,MPK3 and MPK6, Are Required for Funicular Guidance of Pollen Tubes in Arabidopsis

Double fertilization in flowering plants requires the delivery of two immotile sperm cells to the female gametes by a pollen tube, which perceives guidance cues, modifies its tip growth direction, and eventually enters the micropyle of the ovule. In spite of the recent progress, so far, little is known about the signaling events in pollen tubes in response to the guidance cues. Here, we show that MPK3 and MPK6, two Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinases, mediate the guidance response in pollen tubes. Genetic analysis revealed that mpk3 mpk6 double mutant pollen has reduced transmission. However, direct observation of mpk3 mpk6 mutant pollen phenotype was hampered by the embryo lethality of double homozygous mpk3^–/– mpk6^–/– plants. Utilizing a fluorescent reporter-tagged complementation method, we showed that the mpk3 mpk6 mutant pollen had normal pollen tube growth but impaired pollen tube guidance. In vivo pollination assays revealed that the mpk3 mpk6 mutant pollen tubes were defective in the funicular guidance phase. By contrast, semi-in vitro guidance assay showed that the micropylar guidance of the double mutant pollen tube was normal. Our results provide direct evidence to support that the funicular guidance phase of the pollen tube requires an in vivo signaling mechanism distinct from the micropyle guidance. Moreover, our finding opened up the possibility that the MPK3/MPK6 signaling pathway may link common signaling networks in plant stress response and pollen-pistil interaction.In flowering plants, successful fertilization is dependent on extensive cell-cell communication between male and female gametophytes. After landing on a compatible stigma surface, a mature pollen grain germinates to form a pollen tube, which penetrates the stigma, perceives guidance cues along the growth path, and modifies its tip growth direction toward the ovule (Hülskamp et al., 1995). In Arabidopsis (Arabidopsis thaliana), the pollen tube guidance can be divided into two phases: funicular guidance, in which the pollen tube emerges from the septum and proceeds to a funiculus, and micropylar guidance, in which the pollen tube grows toward and enters the micropyle of an ovule (Hülskamp et al., 1995).In pollen tube, it is believed that receptors on the tube tip perceive various guidance cues and regulate downstream signaling pathways to modify tip reorientation toward the ovule (Higashiyama, 2010; Takeuchi and Higashiyama, 2011). Two receptor-like kinase genes, Lost In Pollen tube guidance1 (LIP1) and LIP2, are involved in guidance control of pollen tubes. LIP1 and LIP2 were anchored to the membrane in the pollen tube tip region via palmitoylation, which was essential for their guidance control (Liu et al., 2013). Therefore, LIP1 and LIP2 are the essential components of the receptor complex in micropylar guidance. The Glu receptor-like channels facilitate Ca²⁺ influx across the plasma membrane and regulate pollen tube growth and morphogenesis (Michard et al., 2011). This interesting work revealed that there is a signaling mechanism between the male gametophyte and pistil tissue that is similar to the amino acid-mediated communication in animal nervous systems (Michard et al., 2011). Recent findings also highlight the importance of the endoplasmic reticulum (ER), ion homeostasis, and protein processing in pollen tube guidance (Li et al., 2011; Lu et al., 2011; Li and Yang, 2012). Two pollen-expressed cation proton exchangers (CHXs), CHX21 and CHX23, were reported to mediate K⁺ transport in ER and are essential for the pollen tube to respond to directional signals from the ovule in Arabidopsis (Lu et al., 2011). POLLEN DEFECTIVE IN GUIDANCE1 plays an important role in micropylar guidance in pollen tube (Li et al., 2011). It is an ER luminal protein involved in ER protein retention and interacts with a luminal chaperone involved in Ca²⁺ homeostasis and ER quality control (Li et al., 2011). Therefore, the ER quality control is likely an important mechanism in surveillance of signaling factors in pollen tube guidance (Li and Yang, 2012).In spite of the recent progresses, so far, little is known about the cytoplasmic signaling events in pollen tubes in response to the guidance cues. Mitogen-activated protein kinase (MAPK, or MPK) cascades are conserved signaling pathways that respond to extracellular stimuli and regulate various cellular activities. In Arabidopsis, MPK3 and MPK6 are induced by various biotic and abiotic stresses and collaboratively play important roles in defense response and plant development (Zhang, 2008). Here, we show that MPK3 and MPK6 are also critical to pollen tube guidance. Utilizing a fluorescent reporter-tagged complementation method, we demonstrated that mpk3 mpk6 pollen was defective in pollen tube guidance at the funicular guidance phase. Intriguingly, the micropylar guidance of mpk3 mpk6 pollen tube is not affected.     

The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.When cells divide, they must accurately segregate the duplicated genetic material between two daughter cells such that each receives a single complete set of chromosomes. This complex biomechanical feat is achieved through the action of a bipolar microtubule-based scaffold called the mitotic spindle (36). Microtubules are primarily nucleated by centrosomes that sit at the spindle poles (37). However, microtubule nucleation also occurs in the vicinity of the chromosomes and within the spindle itself (12, 13). These activities combine to ensure the efficient capture of sister chromatids as well as the maintenance of a robust structure capable of resisting the considerable forces required for chromosome separation.Spindle assembly is regulated in large part by reversible phosphorylation, and a number of protein kinases are activated during mitosis, localize to specific regions of the spindle, and phosphorylate spindle-associated proteins. These include the master mitotic regulator Cdk1/cyclin B, the polo-like kinase Plk1, and the Aurora family kinases Aurora A and B (25). More recently, members of the NIMA-related kinase family have also been implicated in mitotic spindle regulation (27, 29). NIMA was first identified in Aspergillus nidulans as a kinase required for mitotic entry, possibly through triggering the relocation of Cdk1/cyclin B to the nucleus (6, 38). NIMA can also phosphorylate S10 of histone H3 to promote chromatin condensation (7). The fission yeast NIMA-related kinase Fin1 contributes to multiple steps in mitotic progression, including the timing of mitotic entry, spindle formation, and mitotic exit (14, 15). However, the detailed mechanisms by which these fungal kinases contribute to mitotic regulation remain far from understood.In mammals, there are 11 NIMA-related kinases, named Nek1 to Nek11, and of these, 4 have been directly implicated in mitotic regulation, as follows: Nek2, Nek6, Nek7, and Nek9 (also known as Nercc1) (26, 27, 29). Nek2 is the most closely related mammalian kinase to NIMA and Fin1 by sequence and has been studied in the most detail. It localizes to the centrosome, where it phosphorylates and thereby regulates the association of a number of large coiled-coil proteins implicated in centrosome cohesion and microtubule anchoring (1, 10, 11, 21, 22, 30). These activities facilitate the early stages of spindle assembly at the G₂/M transition. Interestingly, Aspergillus NIMA and fission yeast Fin1 also localize to the fungal equivalent of the centrosome, namely the spindle pole body (15, 20, 38). Here, they may participate in positive feedback loops that promote the activation of Cdk1/cyclin B and mitotic entry.Nek6, Nek7, and Nek9 act together in a mitotic kinase cascade, with Nek9 being upstream of Nek6 and Nek7. Nek9 was identified as an interacting partner of Nek6 and subsequently shown to phosphorylate Nek6 at S206 within its activation loop (2, 33). Both Nek9 and Nek6 have been reported to be activated in mitosis (2, 33, 39), although other studies dispute this (18, 23). NIMA-related kinases are characterized by having a conserved N-terminal catalytic domain, followed by a nonconserved C-terminal regulatory domain that varies in size and structure. Nek6 and Nek7 are significant exceptions to this, in that they are the smallest of the kinases and consist only of a catalytic domain with a very short N-terminal extension. They share significant similarity with each other, being 87% identical within their catalytic domains. Hence, although they exhibit distinct tissue expression patterns (8), it has generally been assumed that they are likely to have very similar properties and functions, with both being downstream substrates of Nek9.Functional studies of Nek9 reveal that it has major roles to play in the organization of the mitotic spindle. Expression of inactive and truncated Nek9 mutants led to the missegregation of chromosomes, while injection of anti-Nek9 antibodies into prophase cells caused aberrant mitotic spindle formation (33). Similarly, depletion of Nek9 from Xenopus egg extracts led to a reduction in the formation of bipolar spindles in vitro (32; J. Blot and A. M. Fry, unpublished results). The basis for these defects remains unclear, but a number of binding partners have been identified that suggest possible functions in microtubule nucleation and anchoring, including components of the γ-tubulin ring complex (γ-TuRC), the Ran GTPase, and BicD2 (18, 32, 33).While Nek9 is proposed to act upstream of Nek6 and Nek7, the proportion of its activities being channeled through these kinases is not known. Limited studies have been performed by looking at the consequences of expressing kinase-inactive Nek6 or Nek7 constructs or depleting the proteins by RNA interference (RNAi). Interference with Nek6 has been reported by one group to lead to metaphase arrest and apoptosis (39), although this is disputed by another study (23). Interference with Nek7 apparently leads to an increase in the mitotic index and apoptosis (19, 40). A decrease in centrosome-associated γ-tubulin and microtubule nucleation was also detected upon RNAi of Nek7, which is interesting in light of the interaction between Nek9 and γ-tubulin. Furthermore, defects in cytokinesis were found upon Nek7 depletion if cells were allowed to progress past the spindle checkpoint by codepletion of Mad2 (19). Importantly, both Nek9 and Nek7 localize to centrosomes, further supporting the model that this is a major site of action for this family of kinases in spindle formation (19, 32, 40).In this study, we set out to clarify the mitotic roles of Nek6 and Nek7 by examining the consequences of expression of mutants with different levels of kinase activity as well as depletion of the proteins by RNAi. Our results demonstrate that Nek6 and Nek7 are both activated in mitosis and that interference with either kinase leads to apoptosis following mitotic arrest. Interestingly, expression of inactive mutants or small interfering RNA (siRNA)-mediated depletion leads to a metaphase delay with fragile mitotic spindles, whereas expression of hypomorphic mutants or depletion in the presence of a spindle assembly checkpoint (SAC) inhibitor leads to an accumulation of cells in cytokinesis. Based on additional localization data, we propose that these kinases regulate microtubule organization not only at spindle poles but also within the mitotic spindle itself and possibly at the central spindle during late mitosis. This study therefore provides important novel insights into how Nek6 and Nek7 contribute to distinct molecular events in mitotic progression.     

D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.     

AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1''s defense function, which is linked to the activation of ETI.     

Sphingosine Kinases Are Not Required for Inflammatory Responses in Macrophages

Sphingosine kinases (Sphks), which catalyze the formation of sphingosine 1-phosphate (S1P) from sphingosine, have been implicated as essential intracellular messengers in inflammatory responses. Specifically, intracellular Sphk1-derived S1P was reported to be required for NFκB induction during inflammatory cytokine action. To examine the role of intracellular S1P in the inflammatory response of innate immune cells, we derived murine macrophages that lack both Sphk1 and Sphk2 (MΦ Sphk dKO). Compared with WT counterparts, MΦ Sphk dKO cells showed marked suppression of intracellular S1P levels whereas sphingosine and ceramide levels were strongly up-regulated. Cellular proliferation and apoptosis were similar in MΦ Sphk dKO cells compared with WT counterparts. Treatment of WT and MΦ Sphk dKO with inflammatory mediators TNFα or Escherichia coli LPS resulted in similar NFκB activation and cytokine expression. Furthermore, LPS-induced inflammatory responses, mortality, and thioglycolate-induced macrophage recruitment to the peritoneum were indistinguishable between MΦ Sphk dKO and littermate control mice. Interestingly, autophagic markers were constitutively induced in bone marrow-derived macrophages from Sphk dKO mice. Treatment with exogenous sphingosine further enhanced intracellular sphingolipid levels and autophagosomes. Inhibition of autophagy resulted in caspase-dependent cell death. Together, these data suggest that attenuation of Sphk activity, particularly Sphk2, leads to increased intracellular sphingolipids and autophagy in macrophages.     

Tyrosine Kinases Are Required for Catecholamine Secretion and Mitogen-Activated Protein Kinase Activation in Bovine Adrenal Chromaffin Cells

Abstract: Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by >90% in cells stimulated by nicotine, 55 m M KCI, or the Ca²⁺ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie down-stream of Ca²⁺ influx and upstream of MEK activation. This Ca²⁺-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca²⁺-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.     

Variable Interactions between Sucrose Non-fermented 1-Related Protein Kinases and Regulatory Proteins in Higher Plants

WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.     

Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [(3)H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, and odc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.     

The Histidine Kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 Regulate Vascular Tissue Development in Arabidopsis Shoots

The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CKI1 forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 overexpression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1^H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis.     

Ethylene Signaling Regulates Accumulation of the FLS2 Receptor and Is Required for the Oxidative Burst Contributing to Plant Immunity

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response.A rapid and transient increase in reactive oxygen species (ROS), termed an “oxidative burst,” is often associated with responses to abiotic and biotic stresses and could trigger changes in stomatal aperture or programmed cell death in defense against pathogens (Kwak et al., 2003; Torres and Dangl, 2005). ROS production can occur extracellularly through activities of plasma membrane-resident NADPH oxidases (Kangasjärvi et al., 2005; Torres and Dangl, 2005). In plants, Rboh proteins, which are homologs of mammalian NADPH oxidase 2, were shown to be the predominant mediators of apoplastic ROS production (Torres et al., 1998; Galletti et al., 2008). Respiratory burst oxidase homologs D and F (RbohD and RbohF) were identified by mutation to be the responsible oxidases in Arabidopsis (Arabidopsis thaliana) defense responses (Torres et al., 2002). While most ROS generated in response to avirulent Pseudomonas syringae bacteria and Hyaloperonospora oomycete pathogens depend on RbohD function, the induced cell death response by these pathogens appears to be mostly regulated by RbohF. Cell death provoked upon infection with the necrotizing fungus Alternaria, however, is under the control of RbohD (Pogány et al., 2009). The contribution of NADPH oxidases to plant immunity was also described in barley (Hordeum vulgare) and tobacco (Nicotiana benthamiana), where resistance to powdery mildew fungi and the oomycete Phytophthora infestans, respectively, was dependent on Rboh functions (Yoshioka et al., 2003; Trujillo et al., 2006).An early layer of active plant defense is mediated by pattern recognition receptors, which sense microbes according to conserved constituents, so-called pathogen-associated molecular patterns (PAMPs). These initiate a plethora of defense responses referred to as PAMP-triggered immunity (Boller and Felix, 2009). The Arabidopsis receptor kinase Flagellin Sensitive2 (FLS2) recognizes and physically interacts with flg22, the elicitor-active epitope of bacterial flagellin (Felix et al., 1999; Gomez-Gomez and Boller, 2000; Chinchilla et al., 2006). FLS2 is plasma membrane localized and expressed throughout the plant (Robatzek et al., 2006). FLS2 requires the receptor kinase BRI1-Associated Kinase1 (BAK1), which forms a heteromeric complex upon flg22 binding (Chinchilla et al., 2007). Subsequently, a rapid and transient flg22-stimulated oxidative burst occurs that is dependent on RbohD (Zhang et al., 2007). In addition, flg22 triggers early responses, such as ethylene biosynthesis, activation of mitogen-activated protein (MAP) kinase cascades, and changes in gene expression (Felix et al., 1999; Asai et al., 2002; Zipfel et al., 2004). Late flg22 responses include the accumulation of salicylic acid (SA), callose deposition, and an arrest of seedling growth (Gomez-Gomez et al., 1999; Mischina and Zeier, 2007). This collectively contributes to plant immunity (Zipfel et al., 2004; Melotto et al., 2006).Little is known about the regulatory components of FLS2-activated early flg22 responses and their relevance in plant resistance to pathogens. Here, we investigated flg22-triggered ROS production in Arabidopsis seedlings and have identified ethylene signaling as a critical component of the oxidative burst in response to flg22, partly through promoting the accumulation of FLS2. We further provide evidence that the flg22-triggered oxidative burst is required for resistance to bacterial infection at the point of pathogen entry through stomata.     

Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

The Ras–extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice.     

The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 Are Required for Autophagic Degradation in Arabidopsis

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit VACUOLAR PROTEIN SORTING2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.