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Spontaneous symbiotic reprogramming of plant roots triggered by receptor-like kinases
Symbiosis Receptor-like Kinase (SYMRK) is indispensable for the development of phosphate-acquiring arbuscular mycorrhiza (AM) as well as nitrogen-fixing root nodule symbiosis, but the mechanisms that discriminate between the two distinct symbiotic developmental fates have been enigmatic. In this study, we show that upon ectopic expression, the receptor-like kinase genes Nod Factor Receptor 1 (NFR1), NFR5, and SYMRK initiate spontaneous nodule organogenesis and nodulation-related gene expression in the absence of rhizobia. Furthermore, overexpressed NFR1 or NFR5 associated with endogenous SYMRK in roots of the legume Lotus japonicus. Epistasis tests revealed that the dominant active SYMRK allele initiates signalling independently of either the NFR1 or NFR5 gene and upstream of a set of genes required for the generation or decoding of calcium-spiking in both symbioses. Only SYMRK but not NFR overexpression triggered the expression of AM-related genes, indicating that the receptors play a key role in the decision between AM- or root nodule symbiosis-development.https://doi.org/10.7554/eLife.03891.001
eLife digest
Like all plants, crop plants need nutrients such as nitrogen and phosphate to grow. Often these essential elements are in short supply, and so millions of tons of fertiliser are applied to agricultural land each year to maintain crop yields. Another way for plants to gain access to scarce nutrients is to form symbiotic relationships with microorganisms that live in the soil. Plants pass on carbon-containing compounds—such as sugars—to the microbes and, in return, certain fungi provide minerals—such as phosphates—to the plants. Some plants called legumes (such as peas, beans, and clovers) can also form relationships with bacteria that convert nitrogen from the air into ammonia, which the plants then use to make molecules such as DNA and proteins.
To establish these symbiotic relationships with plants, nitrogen-fixing bacteria release chemical signals that are recognized via receptor proteins, called NFR1 and NFR5, found on the surface of the plant root cells. These signals trigger a cascade of events that ultimately lead to the plant forming an organ called 'root nodule' to house and nourish the nitrogen-fixing bacteria. A similar signalling mechanism is thought to take place during the establishment of symbiotic relationships between plants and certain soil fungi.
A plant protein called Symbiosis Receptor-like Kinase (or SYMRK for short) that is also located on the root cell surface is required for both bacteria–plant and fungi–plant associations to occur. However, the exact role of this protein in these processes was unclear. Ried et al. have now investigated this by taking advantage of a property of cell surface receptor proteins: if some of these proteins are made in excessive amounts they activate their signalling cascades even when the initial signal is not present.
Ried et al. engineered plants called Lotus japonicus to produce high levels of SYMRK, NFR1, or NFR5. Each of these changes was sufficient to trigger the plants to develop root nodules in the absence of microbes. Genes associated with the activation of the signalling cascade involved the formation of root nodules were also switched on when each of the three proteins was produced in large amounts. In contrast, only an excess of SYMRK could activate genes related to fungi–plant associations. Ried et al. also found that, while SYMRK can function in the absence of the NFRs, NFR1 and NFR5 need each other to function. These data suggest that the receptor proteins play a key role in the decision between the establishment of an association with a bacterium or a fungus.
As an excess of symbiotic receptors caused plants to form symbiotic structures, Ried et al. propose that this strategy could be used to persuade plants that usually do not form symbioses with nitrogen-fixing bacteria to do so. If this is possible, it might lead us to engineer crop plants to form symbiotic interactions with nitrogen-fixing bacteria; this would help increase crop yields and enable crops to be grown in nitrogen-poor environments without the addition of extra fertiliser.https://doi.org/10.7554/eLife.03891.002
Plants circumvent nutrient deficiencies by establishing mutualistic symbioses with arbuscular mycorrhiza (AM) fungi or nitrogen-fixing rhizobia and Frankia bacteria (Gutjahr and Parniske, 2013; Oldroyd, 2013). One of the first steps in the reciprocal recognition between rhizobia and the legume Lotus japonicus is the perception of bacterial lipo-chitooligosaccharides, so called nodulation factors, by the two lysin motif (LysM) receptor-like kinases (RLKs) Nod Factor Receptor 1 (NFR1) and NFR5 (Madsen et al., 2003; Radutoiu et al., 2003, 2007; Broghammer et al., 2012). Nodulation factor application induces two genetically separable calcium signatures in root hair cells; an early transient influx into the cytoplasm and within minutes calcium-spiking - periodic calcium oscillations in and around plant cell nuclei (Ehrhardt et al., 1996; Miwa et al., 2006; Oldroyd, 2013). (Lipo)-chitooligosaccharides have also been isolated from AM fungi (Maillet et al., 2011; Genre et al., 2013) and a NFR5-related LysM-RLK from Parasponia has been pinpointed as a likely candidate for their perception (Op Den Camp et al., 2011). The common symbiosis genes of legumes are required for AM and root nodule symbiosis. A subset of these genes is essential for either the generation or the decoding of calcium-spiking. In L. japonicus, the former group encodes the RLK Symbiosis Receptor-like Kinase (SYMRK; Stracke et al., 2002; Antolín-Llovera et al., 2014), two cation-permeable ion channels CASTOR and POLLUX (Imaizumi-Anraku et al., 2005; Charpentier et al., 2008; Venkateshwaran et al., 2012) as well as the nucleoporins NUP85, NUP133, and NENA (Kanamori et al., 2006; Saito et al., 2007; Groth et al., 2010). The latter group encodes Calcium Calmodulin-dependent Protein Kinase (CCaMK; Tirichine et al., 2006; Miller et al., 2013) and CYCLOPS (Yano et al., 2008), which form a complex that has been implicated in the deciphering of calcium-spiking (Kosuta et al., 2008). Phosphorylation by CCaMK activates CYCLOPS, a DNA-binding transcriptional activator of the NODULE INCEPTION gene (NIN; Schauser et al., 1999; Singh et al., 2014). NIN itself is a legume-specific and root nodule symbiosis-related transcription factor and regulates the Nuclear Factor-Y subunit genes NF-YA1 and NF-YB1 that control the cell division cycle (Soyano et al., 2013; Yoro et al., 2014). The paradigm of a common signalling pathway for both symbioses bears important open questions about the molecular mechanisms that ensure the appropriate cellular response for AM fungi on the one hand and for rhizobia on the other hand.
SYMRK carries an ectodomain composed of a malectin-like domain (MLD), and a leucine-rich repeat (LRR) region that experienced structural diversification during evolution (Markmann et al., 2008) and is cleaved to release the MLD (Antolín-Llovera et al., 2014). Although SYMRK has been cloned several years ago (Stracke et al., 2002), its precise function in symbiosis is still enigmatic. While nfr mutants lack most cellular and physiological responses to rhizobia (Radutoiu et al., 2003), including nodulation factor-induced calcium influx and calcium-spiking, root hairs of symrk mutants respond with calcium influx to nodulation factor but not with calcium-spiking, and do not develop infection threads with rhizobia (Stracke et al., 2002; Miwa et al., 2006). Based on these phenotypic observations, SYMRK was positioned downstream of the NFRs (Radutoiu et al., 2003; Miwa et al., 2006). Importantly, it has not been conclusively resolved whether SYMRK plays an active signalling role in symbiosis or, alternatively, is involved in mechanical stress desensitisation (Esseling et al., 2004). To approach this issue, we built on the observation that specific mutations in, or over-abundance of mammalian receptor tyrosine kinases on the cell surface is linked with the development of some cancers caused by spontaneous receptor complex formation and inappropriate initiation of signalling (Schlessinger, 2002; Wei et al., 2005; Shan et al., 2012). We hypothesized that similar behaviour could be triggered by overexpression of symbiosis-related plant RLKs, providing a tool to further dissect the specific signalling pathways they address.
Symbiotic RLKs trigger spontaneous formation of root nodules
To achieve overexpression, we generated constructs expressing functional SYMRK (Antolín-Llovera et al., 2014), NFR5, or NFR1 under the control of the strong L. japonicus Ubiquitin promoter and added C-terminal mOrange fluorescent tags for detection purposes (pUB:SYMRK-mOrange, pUB:NFR5-mOrange, pUB:NFR1-mOrange). The functionality of the NFR constructs was confirmed by their ability to restore nodulation in the corresponding, otherwise nodulation deficient, nfr mutant roots to the level of L. japonicus wild-type roots transformed with the empty vector (Figure 1—figure supplement 1). Intriguingly, transgenic expression of any of the three symbiotic RLK versions in L. japonicus roots was sufficient to spontaneously activate the entire nodule organogenesis pathway as evidenced by the formation of nodule-like structures in the absence of rhizobia (Figure 1; Figure 1—figure supplement 2). The presence of peripheral vascular bundles instead of a central root vasculature unambiguously identified these lateral organs as spontaneous nodules (Figure 1C). Spontaneous nodule primordia or nodules were present on 90% (116 out of 129), 23% (30 out of 133), 11% (16 out of 182), and 0% (0 out of 164) of L. japonicus root systems at 60 days post transformation (dpt) with, respectively, pUB:SYMRK-mOrange, pUB:NFR5-mOrange, pUB:NFR1-mOrange, or the empty vector (Figure 1A; Figure 1—figure supplement 2). A total of 810 empty vector roots generated throughout the course of this study did not develop spontaneous nodules in any of the genetic backgrounds and time points tested. Roots expressing functional SYMRK-RFP from its native promoter (pSYMRK:SYMRK-RFP; Kosuta et al., 2011) and grown in the absence of rhizobia did not develop spontaneous nodules, indicating that spontaneous nodulation was triggered by SYMRK expression from the Ubiquitin promoter and not by the addition of a C-terminal tag alone (Figure 1—figure supplement 3). Moreover, the expression of non-tagged SYMRK under the control of the Ubiquitin promoter triggered the formation of spontaneous nodules. In comparison to roots transformed with the tagged SYMRK version, a lower number of roots transformed with non-tagged SYMRK contained spontaneous nodules (Figure 1—figure supplement 4). One explanation for this observation is that the C-terminal mOrange tag might result in alterations in the relative amount of signalling-active SYMRK. Another possibility is that the presence of the tag improves homo- and/or hetero-dimerization, which subsequently leads to downstream signalling. Our results demonstrate that overexpression of NFR1-mOrange, NFR5-mOrange, or SYMRK results in the activation and execution of the nodule organogenesis pathway in the absence of external symbiotic stimulation.
Symbiotic RLKs trigger spontaneous nodulation-related signal transduction
To establish whether the development of nodule-like structures was associated with nodulation-related gene activation, we analysed the expression behaviour of marker genes induced during root nodule symbiosis (NIN and SbtS; Kistner et al., 2005) via quantitative real-time PCR (qRT-PCR; Figure 2A). The SbtS gene is also induced during AM symbiosis (Kistner et al., 2005). In comparison to control roots transformed with the empty vector, the SYMRK construct resulted in a highly significant increase in NIN and SbtS transcript levels (mean fold increase of 137 and 24, respectively). A slighter but statistically significant increase in transcript levels could be observed in roots overexpressing either NFR1-mOrange (NIN, mean fold increase 3; SbtS, mean fold increase 7) or NFR5-mOrange (NIN, mean fold increase 8; SbtS, mean fold increase 15) (Figure 2A).
To monitor the spontaneous activation of NIN and SbtS by an independent and histochemical method, we made use of stable transgenic L. japonicus reporter lines carrying either a NIN promoter:β-glucuronidase (GUS) fusion (pNIN:GUS; Radutoiu et al., 2003) or a SbtS promoter:GUS fusion (pSbtS:GUS; Takeda et al., 2009) (Figure 2B). In addition, we employed the symbiosis-reporter line T90 that was isolated in a screen for symbiosis-specific GUS expression from a promoter-tagging population (Webb et al., 2000) (Figure 2B). The T90 reporter is activated in roots treated with nodulation factor or inoculated with Mesorhizobium loti and—similar to pSbtS:GUS—also shows GUS expression during AM (Radutoiu et al., 2003; Kistner et al., 2005). GUS activity was determined in roots by histochemical staining with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc; Figure 2B). Either of the three symbiotic RLKs but not the empty vector activated the pNIN:GUS, the pSbtS:GUS, as well as the T90 reporter in the absence of M. loti or AM fungi (Figure 2B).
This histochemical analysis of GUS activity, in combination with the qRT-PCR results, provide strong evidence that overexpression of symbiotic RLKs leads to the activation of nodulation-related genes in the absence of external symbiotic stimulation (Figure 2). However, the three RLK genes were not equally effective in inducing the symbiotic program: NFR5 or NFR1 overexpression resulted in a lower percentage of root systems showing promoter activation and formation of spontaneous nodules when compared to SYMRK overexpression (Figure 1; Figure 1—figure supplement 2; Figure 2B). Interestingly, SYMRK- as well as NFR5-mediated T90 or NIN promoter activation was first observed in the root and retracted to nodule primordia and nodules over time, while NFR1-mediated T90 or NIN promoter activation could only be detected in nodule primordia or in nodules (Figure 2B). The Ubiquitin promoter drives expression of the receptors in all cells of the root (Maekawa et al., 2008), which is in marked contrast to the highly specific and developmentally controlled expression patterns of the marker genes observed. These incongruences thus reveal the presence of additional layers of regulation, operating downstream of the receptors, which dictate the precise expression patterns of the reporters.
SYMRK triggers spontaneous AM-related signal transduction
Since SYMRK is not only required for nodulation but also for AM symbiosis, we investigated the potential of dominant active RLK alleles to spontaneously activate AM-related marker genes or a promoter:GUS reporter (Figure 3). Blue copper-binding protein 1 (Bcp1) and the subtilisin-like serine protease gene SbtM1 are induced during AM symbiosis (Liu et al., 2003; Kistner et al., 2005; Takeda et al., 2009), and both genes are predominantly expressed in arbuscule-containing and adjacent cortical cells (Hohnjec et al., 2005; Takeda et al., 2009, 2012). Furthermore, in L. japonicus, SbtM1 expression marks root cells that contain an AM fungi-induced prepenetration apparatus (Takeda et al., 2012)—an intracellular structure that forms prior to invasion by fungal hyphae (Genre et al., 2005). Transcript levels of SbtM1 and Bcp1 were determined via qRT-PCR, and both were significantly increased in roots transformed with pUB:SYMRK-mOrange compared to the empty vector control (Figure 3A). To determine SbtM1 activation by an independent, histochemical approach, we employed a stable transgenic L. japonicus line harbouring a SbtM1 promoter:GUS fusion (pSbtM1:GUS; Takeda et al., 2009). In line with the results from the qRT-PCR experiments, overexpression of SYMRK-mOrange in roots of the pSbtM1:GUS reporter line resulted in activation of the SbtM1 promoter at 40 and 60 dpt (Figure 3B). In contrast, no SbtM1 promoter activation or AM-related gene induction could be detected upon overexpression of either of the NFRs (Figure 3). The absence of AM-related gene expression in NFR5-expressing roots is not a consequence of the overall lower induction power of the NFR5 construct. In SYMRK- vs NFR5-expressing roots, the relative ratio of transcripts was 1.6:1 for SbtS and 17:1 for NIN (Figure 2A). In contrast, SbtM1 was undetectable in NFR5- but more than 1100-fold above detection limit in SYMRK-overexpressing roots (Figure 3A). These data clearly demonstrate a strong difference in the gene repertoire activated by SYMRK vs NFR5. Together with the spontaneous nodulation, these results demonstrate that overexpression of NFR1-mOrange, NFR5-mOrange, or SYMRK-mOrange activates the nodulation pathway as evidenced by spontaneous organogenesis and gene expression results at the level of endogenous transcripts as well as promoter:GUS expression. In contrast, only the SYMRK construct but neither of the NFR constructs induced AM-related gene expression. This suggests that signalling specificity towards the two different symbiotic programs is achieved at the level of the receptors.
SYMRK associates with NFR1 and NFR5 in Lotus japonicus roots
Spontaneous receptor complex formation caused by overexpression offers itself as a likely explanation for the observed activation of symbiosis signalling in the absence of an external trigger or ligand. This is a scenario described in the context of cancer formation, where receptor tyrosine kinase overexpression or specific mutations in the receptor lead to receptor dimerization in the absence of a ligand, which results in ectopic cell proliferation (Schlessinger, 2002; Wei et al., 2005; Shan et al., 2012). Upon expression in Nicotiana benthamiana leaves in the absence of symbiotic stimulation, we observed previously weak association between full-length SYMRK and NFR1 as well as NFR5, but not between SYMRK and the functionally unrelated RLK Brassinosteroid Insensitive 1 (BRI1; Li and Chory, 1997; Antolín-Llovera et al., 2014; Figure 4—figure supplement 1). To test whether overexpression is associated with receptor complex formation in L. japonicus roots, we employed the overexpression constructs of NFR1, NFR5, or the unrelated EF-Tu receptor kinase (EFR; Zipfel et al., 2006) for co-immuno-enrichment experiments. The EFR construct did not interfere with nodulation in wild-type plants (Figure 1—figure supplement 1). Endogenous full-length SYMRK was co-enriched with NFR1 and NFR5, but not with EFR demonstrating association of SYMRK and both NFRs (Figure 4). However, it should be noted that the expression strength of EFR was lower than that of NFR1 and NFR5. SYMRK-NFR association was detected in the absence of nodulation factor. We did not observe an effect of M. loti on this association at 10 days post inoculation (Figure 4).
Epistatic relationships between SYMRK and other common symbiosis genes
The availability of dominant active receptor gene alleles offers an attractive tool for their positioning in the genetic pathway required for nodule organogenesis and symbiosis-related gene expression. We asked whether the pUB:SYMRK-mOrange construct induced spontaneous nodules or the symbiosis-specific T90 reporter in mutants of common symbiosis genes (Figure 5; Figure 5—figure supplement 1 and 4). SYMRK-induced spontaneous nodules were absent from pollux-2, castor-12, nup133-1, or ccamk-13 mutant roots. Likewise T90 reporter (GUS) activation was not detectable in the castor-2 x T90 (Kistner et al., 2005) or ccamk-2 x T90 (Gossmann et al., 2012) lines (Figure 5—figure supplement 4). This epistasis revealed that the ion channel genes CASTOR and POLLUX, the nucleoporin gene NUP133, and the calcium- and calmodulin-dependent protein kinase gene CCaMK, operate downstream of SYMRK in a pathway leading to spontaneous nodulation and activation of T90 (Figure 5; Figure 5—figure supplement 1 and 4). In contrast, SYMRK induced spontaneous nodules on cyclops-3 mutant roots (Figure 5; Figure 5—figure supplement 1). Spontaneous nodule formation on the cyclops-3 mutant (Figure 5; Figure 5—figure supplement 1) corresponds to the formation of bump-like structures upon inoculation with M. loti on cyclops mutants (Yano et al., 2008). While bacterial infection is strongly impaired in L. japonicus cyclops or M. truncatula ipd3 mutants, nodule primordia or nodules, respectively, develop upon rhizobia inoculation (Yano et al., 2008; Horvath et al., 2011; Ovchinnikova et al., 2011). Furthermore, an autoactive version of CCaMK is able to induce the formation of mature spontaneous nodules in cyclops mutants (Yano et al., 2008). The ability of SYMRK to mediate spontaneous nodule organogenesis in the cyclops mutant is consistent with these results and points towards the existence of redundancies in the genetic pathway leading to organogenesis at the level of CYCLOPS (Singh et al., 2014).
Epistatic relationships between symbiotic RLK genes
We used the dominant active alleles to determine the hierarchy of the symbiotic RLK genes in the spontaneous nodulation and T90 activation pathways. Control roots of mutant lines transformed with the empty vector (218 root systems) or SYMRK driven by its own promoter (33 root systems) did not carry spontaneous nodules or nodule primordia (Figure 5; Figure 5—figure supplement 1–3). Expression of pUB:SYMRK-mOrange spontaneously activated the nodulation program in nfr1-1, nfr5-2, and symrk-3 mutant roots (Figure 5; Figure 5—figure supplement 1). Spontaneous nodules on nfr1-1 or nfr5-2 roots overexpressing SYMRK-mOrange indicate that the simultaneous presence of both NFRs is not necessary for spontaneous SYMRK-mediated nodulation (Figure 5; Figure 5—figure supplement 1). Consistent with this result, overexpression of SYMRK-mOrange resulted in spontaneous GUS expression in the nfr1-1 x T90 line (Gossmann et al., 2012) (Figure 5—figure supplement 4). NFR-mediated formation of spontaneous nodules could only be observed in the wild-type or the respective nfr mutant (Figure 5; Figure 5—figure supplement 2 and 3). Neither NFR construct spontaneously induced nodule organogenesis in a symrk-3 (null mutant) or symrk-10 (kinase dead mutant) background, indicating that the formation of nodules is depended on the presence of kinase-active SYMRK (Figure 5; Figure 5—figure supplement 2 and 3). Spontaneous NFR5-mediated nodulation was completely abolished in the nfr1-1 mutant, demonstrating that NFR1 is essential for this NFR5 function (Figure 5; Figure 5—figure supplement 2). This dependence of NFR5 on NFR1 is further supported by the observation that overexpression of NFR1-mOrange and SYMRK-mOrange but not of NFR5-mOrange activated the T90 reporter in the nfr1-1 mutant background (Figure 5—figure supplement 5). These results position SYMRK downstream of or at the same hierarchical level as NFRs. Moreover, while SYMRK-mediated spontaneous signalling does not require the simultaneous presence of NFR1 and NFR5, NFR5-mediated spontaneous signalling is dependent on the presence of NFR1.
Spontaneous signalling induced by receptor overexpression
A hallmark of the nitrogen-fixing symbiosis of legumes is the accommodation of rhizobia inside plant root cells in specialised organs—the nodules—that provide a favourable environment for nitrogen fixation. Given that the common symbiosis pathway is operating in AM symbiosis in most land plants, the discovery that expression of either of the three symbiotic RLK constructs from the strong Ubiquitin promoter leads to the spontaneous formation of nodules in transgenic L. japonicus roots (Figure 1, Figure 1—figure supplement 2; Figure 1—figure supplement 4; Figure 2; Figure 5, Figure 5—figure supplement 1–5) could pave the way towards the synthetic transfer of nitrogen-fixing root nodules to important non-leguminous crop species. As the symbiotic RLKs act at the entry level of root nodule symbiosis signalling, auto-active versions provide a valuable tool to study the entire nodulation pathway uncoupled from bacterial infection. Furthermore, dominant active RLK versions could be useful for probing and dissecting the symbiotic signalling pathway, also in those plant lineages that are presently unable to develop nitrogen fixing root nodule symbiosis.
SYMRK has an active and direct role in symbiosis signalling
It has been observed that cytoplasmic streaming in root hairs of a symrk-3 mutant did not resume after mechanical stimulation, which raised the possibility that the absence of calcium-spiking upon injection of calcium-sensitive dyes into mutant root hair cells was a pleiotropic effect of this increased touch sensitivity (Esseling et al., 2004; Miwa et al., 2006). If touch desensitisation was the only function of SYMRK, its overexpression would not lead to spontaneous nodule formation. We therefore unambiguously demonstrated a direct role of SYMRK in symbiosis signalling, while eliminating the possibility that the symbiosis defects of symrk mutants are due to pleiotropic effects only.
SYMRK is positioned upstream of genes involved in calcium-spiking
Mutants defective for either of the common symbiosis genes SYMRK, CASTOR, POLLUX, NENA, NUP85, or NUP133 produce very similar phenotypes in symbiosis, in that they abort infection at the epidermis and are impaired in calcium-spiking (Kistner et al., 2005; Miwa et al., 2006; Groth et al., 2010), which placed them at the same hierarchical level. Consequently, a genetic resolution of the relative position of the common symbiosis genes upstream of calcium-spiking was missing. Epistasis tests revealed that SYMRK initiates signalling upstream of other common symbiosis genes implicated in the generation and interpretation of nuclear calcium signatures (Figure 5, Figure 5—figure supplement 1 and 4). These findings support the conceptual framework in which SYMRK activates the calcium-spiking machinery and consequently the CCaMK/CYCLOPS complex, a central regulator of symbiosis-related gene expression and nodule organogenesis (Gleason et al., 2006; Tirichine et al., 2006; Singh and Parniske, 2012; Singh et al., 2014). This is in line with the observation that dominant active variants of CCaMK were able to restore nodulation and infection in symrk mutant backgrounds, indicating that a main function of SYMRK in symbiosis is the activation of CCaMK (Hayashi et al., 2010; Madsen et al., 2010).
Interaction between SYMRK and the NFRs
We observed association between SYMRK and either NFR1 or NFR5 upon NFR overexpression in L. japonicus roots (Figure 4). Interestingly, under these conditions, the SYMRK-NFR association was detected in the absence of nodulation factor (Figure 4). In mammalian receptor tyrosine kinases as well as plant RLKs, ligand-induced receptor dimerization is the single most critical step in signal initiation (Li et al., 2002; Nam and Li, 2002; Schlessinger, 2002; Chinchilla et al., 2007; Schulze et al., 2010; Liu et al., 2012; Sun et al., 2013a, 2013b). However, ligand-independent dimerization of receptor tyrosine kinases mediated by specific mutations in the kinase domain (Shan et al., 2012) or by overabundance of receptor tyrosine kinases (Wei et al., 2005) results in signalling activation and is a scenario well described in the context of cancer formation (Schlessinger, 2002). Similarly, overexpression of symbiotic RLKs might trigger ligand-independent receptor complex formation and activation of downstream signalling, thus providing an explanation why the interaction was also detected in the absence of external symbiotic stimulation. Unfortunately, we could not address the question whether SYMRK-NFR interaction is ligand-induced at endogenous levels of NFR expression since NFR1 and NFR5 were difficult to detect under these conditions.
The relationship between NFR1, NFR5 and SYMRK
We observed that NFR5 requires NFR1 as well as SYMRK for the spontaneous initiation of symbiosis signalling. This provides support for a model first put forward by Radutoiu et al. (2003), in which NFR1 and NFR5 engage in a nodulation factor perception complex. This model has received additional support through their synergistic effect on promoting cell death in N. benthamiana (Madsen et al., 2011; Pietraszewska-Bogiel et al., 2013). The finding that NFR1 and NFR5 interact with SYMRK upon overexpression suggests that the three RLKs engage in a receptor complex (Antolín-Llovera et al., 2014; Figure 4, Figure 4—figure supplement 1), and that this interaction might activate SYMRK for signal transduction. The observation that SYMRK operates independently of NFR1 or NFR5 brings about a new twist into current models of the signalling pathway (Downie, 2014) (Figure 5; Figure 5—figure supplement 1 and 4). NFR1 and NFR5 are only essential in the epidermis (Madsen et al., 2010; Hayashi et al., 2014), and it is likely that—at least partially—other members of the LysM-RLK gene family of L. japonicus (Lohmann et al., 2010) take over their role in the root cortex. NFR1 or NFR5 dispensability may be explained by other LysM-RLKs that might engage in alternative receptor complexes with SYMRK. Alternatively, spontaneous SYMRK-mediated signalling might be independent of any LysM-RLK, however, given the large number of LysM-RLKs in legumes (17 in L. japonicus; Lohmann et al., 2010), it is difficult to test the latter hypothesis conclusively.
SYMRK undergoes cleavage of its ectodomain, resulting in a truncated RLK molecule called SYMRK-ΔMLD (Antolín-Llovera et al., 2014). In competition experiments in N. benthamiana leaves, NFR5 binds preferentially to SYMRK-ΔMLD, which experiences rapid turnover in N. benthamiana and in L. japonicus (Antolín-Llovera et al., 2014). As our SYMRK antibody does not recognise endogenous SYMRK-ΔMLD, we were not able to assess whether overexpressed NFR1 or NFR5 also associates with this truncated SYMRK variant in L. japonicus roots. In a hypothetical scenario, the SYMRK-ΔMLD complex with NFR5 forms constitutively to prevent inappropriate signalling, for example in the absence of rhizobia. The recruitment of NFR1, a hypothetical signal initiation event, would be promoted by the presence of nodulation factor. Our observation that upon overexpression in L. japonicus both NFR1 and NFR5 seem to interact with full-length SYMRK (Figure 4) suggests the formation of a ternary complex. This hypothetical complex has dual functionality: it signals through SYMRK on one hand to activate CCaMK and through the NFR1-NFR5 complex on the other hand to trigger the infection-related parallel pathways discovered by Madsen et al. (2010) and Hayashi et al. (2010). It is possible that SYMRK has a dual—positive and negative—regulatory role: on the one hand SYMRK promotes signalling but on the other hand SYMRK-ΔMLD may be involved in preventing inappropriate signalling. A negative regulatory role would explain the exaggerated root hair response of symrk mutants to rhizobia (Stracke et al., 2002), since NFR1–NFR5 interaction is no longer under governance by SYMRK-ΔMLD. It has been demonstrated recently that expression of the intracellular kinase domain of SYMRK (SYMRK-KD) from Medicago truncatula or Arachis hypogaea in M. truncatula roots from the CaMV 35S promoter induces nodule organogenesis in the absence of rhizobia (Saha et al., 2014). However, in the presence of Sinorhizobium meliloti, nodules on plants overexpressing AhSYMRK-KD were poorly colonized and bacteria were rarely released from infection threads (Saha et al., 2014).
Heterocomplexes between SYMRK and alternative LysM-RLKs may govern nodulation- vs mycorrhiza signalling
The origin of AM dates back to the earliest land plants (∼400 mya) and recent angiosperms maintained a conserved genetic program for the intracellular accommodation of AM fungi (Gutjahr and Parniske, 2013). During the evolution of the nitrogen-fixing root nodule symbiosis, this ancient genetic programme has been co-opted, as evidenced by the common symbiosis genes (Kistner et al., 2005). The discovery that the ancient SYMRK might act as a docking site for the recently evolved nodulation factor perception system (Antolín-Llovera et al., 2014; Figure 4, Figure 4—figure supplement 1), highlights the role of this putative interface during the recruitment of the ancestral AM signalling pathway for root nodule symbiosis. Since a LysM-RLK closely related to NFR5 has been implicated in AM signalling (Op Den Camp et al., 2011), this finding also provides a conceptual mechanism for the integration of signals from the rhizobial and fungal microsymbiont through alternative complex formation between SYMRK and NFRs or AM factor receptors.
Specificity originates from the receptors
One question that has puzzled the community since the postulate of a common symbiosis pathway is how the decision between the developmental pathways of AM or root nodule symbiosis is made when the signalling employs identical signalling components. Models proposed involved different calcium-spiking signatures with symbiosis-specific information content (Kosuta et al., 2008) or additional yet unidentified pathways that operate in parallel to the common symbiosis pathway to mediate exclusive and appropriate signalling (Takeda et al., 2011). Our observation of differential gene activation triggered by NFRs and SYMRK provides evidence that an important decision point is directly at the level of the receptors (Figure 2 and 3). Moreover, the observation that the dominant active SYMRK allele activates both pathways, which is not detected by stimulation with AM fungi or rhizobia, implies the existence of negative regulatory mechanisms that prevent the activation of the inappropriate pathway upon contact with either bacterial or fungal microsymbiont. The SYMRK-mediated loss of signalling specificity may be explained by simultaneous complex formation of SYMRK with NFR1 and NFR5, and related LysM-RLKs that mediate recognition of signals from the AM fungus (Maillet et al., 2011; Op Den Camp et al., 2011), which results in the release of both negative regulatory mechanisms, or by an unbalanced stoichiometry of SYMRK and putative specific negative regulators of AM- and root nodule symbiosis signalling. Candidates for such regulators include the identified interactors of the kinase domains of NFR1, NFR5, and SYMRK (Kevei et al., 2007; Zhu et al., 2008; Lefebvre et al., 2010; Mbengue et al., 2010; Chen et al., 2012; Den Herder et al., 2012; Ke et al., 2012; Toth et al., 2012; Yuan et al., 2012). The loss of signalling specificity upon SYMRK overexpression is reminiscent of expression of the deregulated CCaMK314 deletion mutant that also induces spontaneous nodules and AM-related gene activation (Takeda et al., 2012). It is therefore possible that SYMRK overexpression imposes a deregulated state on CCaMK that is otherwise attainable artificially through the deletion of its regulatory domain.
Materials and methods
DNA constructs and primersRequest a detailed protocol
For a detailed description of the constructs and primers used in this study, please see Supplementary file 1.
Agrobacterium tumefaciens-mediated transient transformation of Nicotiana benthamiana leavesRequest a detailed protocol
Transient transformation of N. benthamiana leaves was performed as described previously (Antolín-Llovera et al., 2014).
Plant growth, hairy root transformation and inoculationRequest a detailed protocol
L. japonicus seed germination (Groth et al., 2010) and hairy root transformation (Charpentier et al., 2008) were performed as described previously. Plants with emerging hairy roots systems were transferred to Fahraeus medium (FP) plates containing 0.1 µM of the ethylene biosynthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine at 2.5 weeks after transformation. For spontaneous nodulation experiments, promoter activation assays, or qRT-PCR experiments, plants were transferred to sterile Weck jars containing 300 ml dried sand/vermiculite and 25 ml FP medium at 23 dpt. For co-enrichment experiments, plants were transferred to sterile Weck jars containing 300 ml dried sand/vermiculite at 23 dpt, mock treated with 20 ml FP medium or inoculated with 20 ml of a M. loti MAFF303099 DsRED suspension in FP medium set to an OD600 of 0.05, and incubated for 10 days. Plants for the SYMRK- and CCaMKT265D-mediated T90 activation in the nfr1-1, cyclops-2, and ccamk-2 mutants were transferred to FP plates containing 0.1 µM of the ethylene biosynthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine at 21 dpt and kept on FP plates for 17 days. Transformants of the pSbtM1:GUS line were directly transferred to Weck jars containing 300 ml dried sand/vermiculite and approximately 25 ml ddH2O at 2.5 weeks after transformation. It is important to avoid free water at the bottom of the Weck jar. Plants were grown in Weck jars in a growth chamber (16 hr light/8 hr dark; 24°C) for 1.5–6 weeks. For complementation experiments, plants were transferred from FP plates to open pots containing 300 ml dried sand/vermiculite and 75 ml FP medium at 23 dpt. After 1 week, plants were inoculated with 25 ml per pot of a M. loti MAFF303099 DsRED suspension in FP medium set to an OD600 of 0.05. Roots were phenotyped 15 days after inoculation.
Non-denaturing protein extraction from Nicotiana benthamiana leaves and immunoprecipitation experimentsRequest a detailed protocol
Protein extraction and immunoprecipitation was performed as described previously (Antolín-Llovera et al., 2014).
Non-denaturing protein extraction from Lotus japonicus hairy roots and immuno-enrichment experimentsRequest a detailed protocol
Plant tissue was ground to a fine powder in liquid nitrogen with mortar and pestle. Proteins were extracted by adding 200 µl extraction buffer per 100 mg root tissue (50 mM Hepes, pH 7.5, 10 mM EDTA, 150 mM NaCl, 10% sucrose, 2 mM DTT, 0.5 mg/ml Pefabloc, 1% Triton-X 100, PhosSTOP [Roche, Germany], Plant Protease Inhibitor [P9599; Sigma–Aldrich, Germany], 1% polyvinylpolypyrrolidone). Samples were incubated for 10 min at 4°C with 20 rpm end-over mixing, and subsequently centrifuged for 15 min at 4°C and 16,000 RCF. 30 µl of each protein extract was mixed with 10 µl 4× SDS-PAGE sample buffer (input; 25% (vol/vol) 0.5 M Tris–HCl (pH 6.8), 35% (vol/vol) 20% SDS, 40% (vol/vol) 100% Glycerol, 0.03 g/ml DTT, dash of Bromphenol blue). For immuno-enrichment procedures, 30 µl RFP binder coupled to magnetic particles (rtm-20; Chromotek, Germany) were washed in wash buffer (WB; 50 mM Hepes, pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% Triton-X 100). Between 500 and 1000 µl of the protein extract was added to the beads and immuno-enrichment was performed for 4 hr at 4°C with 20 rpm end-over mixing, followed by 15 min magnetic separation at 4°C. Supernatant was removed and beads were washed twice with WB. 40 µl 2× SDS-PAGE sample buffer was added to the beads and both beads and input were incubated 10 min at 56°C. After heating, beads were magnetically collected at the tube wall for 5 min and 40 µl of the supernatant (eluate) was taken. For SDS-PAGE, 20 µl of the input or eluate were loaded on each gel.
Western blot analysisRequest a detailed protocol
Western blot analysis was performed as described previously (Antolín-Llovera et al., 2014).
T90, NIN, SbtM1, and SbtS promoter analysis in Lotus japonicusRequest a detailed protocol
GUS activity originating from the activation of promoter:GUS reporters was visualized by X-Gluc staining as described previously (Groth et al., 2010).
Expression analysisRequest a detailed protocol
Transgenic root systems of L. japonicus plants were harvested 40 dpt. 80 mg root fresh weight per sample was applied for total RNA extraction using the Spectrum Plant Total RNA kit (Sigma–Aldrich, Germany). For removal of genomic DNA, RNA was treated with DNase I (amplification grade DNase I, Invitrogen, Germany). RNA integrity was verified on an agarose gel and the absence of genomic DNA was confirmed by PCR. First strand cDNA synthesis was performed in 20 µl reactions with 600 ng total RNA using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen, Germany) with oligo(dT) primers. qRT-PCR was performed in 20 µl reactions containing 1× SYBR Green I (Invitrogen, Germany) in a CFX96 Real-time PCR detection system (Bio-Rad, Germany). PCR program: 95°C for 2 min, 45 × (95°C for 30 s; 60°C for 30 s; 72°C for 20 s; plate read), 95°C for 10 s, melt curve 60°C–95°C: increment 0.5°C per 5 s. Expression was normalized to the reference genes EF-1alpha and Ubiquitin, and EF-1alpha was used as a reference to calculate the relative expression of the target genes. The empty vector samples were used as negative control. Three biological replicates were analysed in technical duplicates per treatment. A primer list can be found in the supplementary files (Supplementary file 1B).
Statistics and data visualisationRequest a detailed protocol
All statistical analyses and data plots have been performed and generated with R version 3.0.2 (2013-09-25) 'Frisbee Sailing' (R Development Core Team, 2008) and the packages 'Hmisc' (Harrell, 2014), 'agricolae' (Mendiburu de, 2014), 'car' (Fox and Weisberg, 2011), 'multcompView' (Graves et al., 2012) and 'multcomp' (Hothorn et al., 2008). For statistical analysis of the numbers of nodules, nodule primordia, or total organogenesis events, a Kruskal–Wallis test was applied followed by false discovery rate correction. Quantitative real-time PCR data were power transformed with the Box–Cox transformation and a one-way ANOVA followed by a Dunnett's test was performed, in which every treatment was compared to the empty vector samples.
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Article and author information
Author details
Deutsche Forschungsgemeinschaft
- Martin Parniske
European Union Seventh Framework Programme (FP7/2007-2013 no. 340904 (EvolvingNodules))
- Martin Parniske
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Michael Bartoschek for technical support as a student helper and Verena Klingl for technical assistance. This work was supported by the DFG priority program SPP1212 'Microbial Reprogramming of Plant Cell Development'. This project has received funding from the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 340904 (EvolvingNodules).
Reviewing Editor
- Maria J Harrison, Boyce Thompson Institute for Plant Research, United States
Publication history
- Received: July 4, 2014
- Accepted: October 29, 2014
- Version of Record published: November 25, 2014 (version 1)
- Version of Record updated: December 15, 2020 (version 2)
© 2014, Ried et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
- Plant Biology
The plant kingdom contains a stunning array of complex morphologies easily observed above-ground, but more challenging to visualize below-ground. Understanding the magnitude of diversity in root distribution within the soil, termed root system architecture (RSA), is fundamental in determining how this trait contributes to species adaptation in local environments. Roots are the interface between the soil environment and the shoot system and therefore play a key role in anchorage, resource uptake, and stress resilience. Previously, we presented the GLO-Roots (Growth and Luminescence Observatory for Roots) system to study the RSA of soil-grown Arabidopsis thaliana plants from germination to maturity (Rellán-Álvarez et al., 2015). In this study, we present the automation of GLO-Roots using robotics and the development of image analysis pipelines in order to examine the temporal dynamic regulation of RSA and the broader natural variation of RSA in Arabidopsis, over time. These datasets describe the developmental dynamics of two independent panels of accessions and reveal highly complex and polygenic RSA traits that show significant correlation with climate variables of the accessions' respective origins.
- Ecology
- Plant Biology
By capitalising on positive biodiversity-productivity relationships, intercropping provides opportunities to improve agricultural sustainability. Intercropping is generally implemented using commercial seeds that were bred for maximal productivity in monocultures, thereby ignoring the ability of plants to adapt over generations to the surrounding neighbourhood, notably through increased complementarity, i.e. reduced competition or increased facilitation. This is why using monoculture-adapted seeds for intercropping might limit the benefits of crop diversity on yield. However, the adaptation potential of crops and the corresponding changes in complementarity have not been explored in annual crop systems. Here we show that plant-plant interactions among annual crops shifted towards reduced competition and/or increased facilitation when the plants were growing in the same community type as their parents did in the previous two generations. Total yield did not respond to this common coexistence history, but in fertilized conditions, we observed increased overyielding in mixtures with a common coexistence history. Surprisingly, we observed character convergence between species sharing the same coexistence history for two generations, in monocultures but also in mixtures: the six crop species tested converged towards taller phenotypes with lower leaf dry matter content. This study provides the first empirical evidence for the potential of parental diversity affecting plant-plant interactions, species complementarity and therefore potentially ecosystem functioning of the following generations in annual cropping systems. Although further studies are required to assess the context-dependence of these results, our findings may still have important implications for diversified agriculture as they illustrate the potential of targeted cultivars to increase complementarity of species in intercropping, which could be achieved through specific breeding for mixtures.


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Biology Discussion board
After reading the linked article below please discuss the following question. Researchers have identified certain gene mutations that indicate a high likelyhood of developing the inherited form of alzheimer's disease later in life. Screening for these mutations could lead to early treatment of the disease and a prolonging of neuronal health. Of course, once these mutations have been detected, they would become part of a person's health reacord. The question is, who should have access to this type of information and how should it be used? For example, is it acceptible for health insurance or life insurance companies to have access to this informaton and use it to deny coverage or raise premiums? Would you want to know if you had this mutation?

subset: commoncrawl


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Dan Whitesides Gets His Record Breaking Zit Popped
Footage of Dan Whitesides' zit getting popped while at Big Bear 2010 has been posted at The Used's YouTube channel. The Utah band joked that the blemish was "the largest zit on record by both the Guiness Book of Records and by Dr. Michael P. Keaton D.D.S. With Dr. Keaton's help and continued support this video has been published for medical research and instructional films worldwide. In addition to helping billions in the classroom, this film, and afore mentioned zit itself, have been able to raise money for O.R.B.Z. (Orphans with Really Bigass Zits) by being the producer of the main ingredient in what is now known as ProActive solutions." Watch the drummer's puss-filled predicament below.


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For much of the past two weeks, I've been wrestling with my mental health. I could sense a crisis coming, so I scheduled some time away. I didn't want to have to be worrying about blog posts while I was worrying about everything else. Thus, my "summer vacation".
Long-time readers are aware that I've struggled with depression for most of my life.
In sixth grade, I missed five weeks of school with what my father called "parrot fever". (We had parrots, and he attributed my issues to a parrot allergy.) After our family physician could find nothing wrong with me, Dad took me to his therapist. Hushed conversations followed the appointment. The verdict: I was dealing with depression.
In junior high, I was briefly suicidal but made a deliberate decision to turn things around. In high school and college, the depression was always there, looming in the shadows. As a young adult, it mostly went away…but then it came back as I got older.
In 1999, when I was thirty, I experienced something new: anxiety. At one point, I thought I was having a heart attack. Nope. It was a panic attack. When the second panic attack came a few weeks later, I knew it wasn't my heart. It was me stressing about life.
Interesting note: It was after the second panic attack that my doctor strongly encouraged me to start drinking red wine. For real. Before that, I was a teetotaler.
During my divorce in 2011-12, Kris asked me a favor. "Please see a counselor," she said. I did, and it helped. My therapist gave me advice for coping with depression and anxiety, plus she diagnosed me with ADD. For a few years, I was able to manage my symptoms.
Last year, though, things got bad. March and April and May were a struggle. In June, I published an article here about my ongoing battle with depression. During the summer, my mental health improved, however, and I forgot about how hard the spring had been.
A Sneaky Little, Sticky Bitch
In February of this year, my anxiety returned. The depression followed soon after. When my heart-attack scare in mid-March turned up no physical issues (other than high blood pressure), my doctor suggested that the problem was anxiety. She asked me to start seeing a therapist again. So, I did.
Since early May, I've been attending talk therapy once a week. We're exploring why I feel so anxious, and how using alcohol to cope with anxiety is a "maladaptive behavior". We're exploring other ways to make things work.
The trouble? When I don't drink in the afternoon, I get more anxious.
The frustrating thing is that the depression and anxiety lead me to act like a completely different person.
For instance, I love people. I love spending time with people. Social interaction energizes me. Right now, though? I hate it. I don't want to deal with anyone in any capacity. I don't want to spend time with friends. I don't want to be in crowds. (I make an exception for Portland Timbers games.) I don't even want to go to the grocery store.
Here are some ways this manifests itself:
- Today, I had a lunch appointment with a colleague and friend. Karl is a great guy and I enjoy spending time with him. Normally. Today, though, all I could think about were the reasons I might be able to cancel.
- Yesterday, I taped a TV interview with a local station. I wanted to cancel that too. Afterward, I ought to have driven out to the family box factory. But I didn't. I didn't want to spend time with my brother and cousin.
- This Sunday evening, there's another Portland Timbers game. Kim can't go with me, so I need to find somebody else to join me. I have zero desire to do so. I may end up selling the tickets and skipping the game because of my anxiety.
My medical doctor has prescribed propranolol to simultaneously deal with my high blood pressure and my anxiety. While it seems to be helping the former, it's not helping the latter. (According to wikipedia, it's really only useful for performance anxiety.)
Meanwhile, the depression is even worse. If you look at the symptoms of depression, I'm exhibiting every single one. Some of my symptoms are severe.
- Fatigue? Have it.
- Insomnia? You bet.
- Feelings of guilt and worthlessness? Oh boy.
- Irritability? Yes, and it's so not me. I'm not an irritable guy — but I am lately.
- Loss of interest in things once pleasurable? Absolutely, and it's SO FRUSTRATING. Nothing appeals to me. I'm numb.
- Trouble concentrating, remembering details, and making decisions? You have no idea. Everything is a chore.
The latter is especially difficult to deal with. When Karl asked where to meet for lunch today, I couldn't decide. Why not? That's so simple! Last night, Kim wanted me to make dinner. But I didn't because I couldn't decide what to fix. That's ridiculous!
A Horrible, Terrible, No Good, Very Bad Day
In fact, yesterday was miserable. It might have been the worst day of my entire life.
My head was a mess of negative thoughts and emotions, all of them swirling and swirling and swirling in a never-ending dark cloud of despair. I couldn't focus on anything. I did tape the TV interview (the first segment went very well, but the second bordered on incoherent) but that's the only productive thing I did all day.
On the drive home, I bought — and then consumed — a big bowl of clam chowder, a big bag of potato chips, and an entire package of chocolate chip cookies. Then I sat in the hot tub and played a videogame for five hours. (At least I didn't drink alcohol!)
When Kim came home, she asked, "What's for dinner?" I admitted that I hadn't made dinner — but I didn't tell her how messed up my head had been all day. (She knows I'm struggling but she doesn't know how badly.) While she changed out of her scrubs, I fried some frozen potstickers.
Naturally, all of this makes me feel even more guilty and worthless and depressed. It's a vicious cycle.
I'm sure you can see how this would translate in an inability to get work done, both here at Get Rich Slowly and in my real life.
It's a problem.
What's the solution to the problem? I'm not sure. There must be one. But I don't know what it is. Drink every afternoon? That's what I've been doing, and it works. But, as my therapist says, it's a maladaptive behavior. I think we all know where that road leads.
My therapist is patient. She keeps giving me homework assignments…and I keep avoiding them. Exercise! Meditate! Set goals! These all sound awesome. They're all things I know I like to do. But they also sound like tremendous effort, so I don't do them.
Bringing Gratitude
Instead of canceling my lunch appointment with Karl today, I went. I'm glad I did.
I've known Karl for almost a decade. He's one of the most uplifting, supportive people I've ever met. I love that his work is centered on positivity. He runs a site called Bring Gratitude and he published a book by the same name. (Six months ago, he shared a guest article here at Get Rich Slowly about practicing gratitude with a daily journal.)
As we sat down for lunch, I told Karl point blank about the issues I'm going through.
"I can totally relate," he said, and he shared some of his own past struggles.
"You know," I said, "my therapist has been urging me to try meditation. But I don't know how to start."
Karl nodded. "I meditate. I meditated just this morning. But it can be tough to get going. You have so many thoughts racing through your head. Here's one thing that might work, though. Give yourself one minute. Only a minute. For that minute, meditate on all of the things that you're thankful for."
"I like that idea," I said. "I like it a lot. Normally, I'm a grateful guy. I'm a lucky man, and I know it. Usually. Lately, though, I've forgotten how awesome life is. Meditating on the things I'm grateful for would be a great way to remind me of what I've got."
Thank You
On my drive home, I put Karl's idea into practice. I took back roads. As I drove slowly through the countryside, I thought about all of the things that I'm thankful for.
- I'm thankful for Kim. She's a not just a wonderful partner in life, but she's a wonderful person. She's a good soul.
- I'm thankful for my dog. Tahlequah is a handful (a pawful?), and I do get frustrated with her. But I'm also grateful to have such an enthusiastic hound dog in my life.
- I'm thankful for my health. I haven't taken care of myself much lately, but that's on me. Generally speaking, my body is in fine shape. And with a little work, it could be in great shape once again.
- I'm grateful for music. I don't mention it much, but music brings great joy to my life. I love music of all sorts. Taylor Swift, yes, but also U2 and Mozart and Styx and ABBA and Public Enemy.
- I'm thankful for Portland. I love the green of it. I love its quirky die-hard (sometimes absurd) liberalism. I love the food scene and the Timbers and the passion for books. Speaking of which…
- I'm grateful for words. Books bring me joy. So does writing. I've managed to make a living from my words, and I hope to continue doing so in the future.
- I'm grateful for life.
Here at home, I had a call with my business partner, Tom. We spent two hours talking about behind-the-scenes details here at Get Rich Slowly. We made plans for the future. But we also took a lot of time to talk about nothing.
It was awesome. It was just what I needed.
When I got off the call, the dog wanted to play. She looked up with puppy-dog eyes and made her little whine that means, "Dad, throw the ball for me." We went outside into the sunshine and I threw the ball for her. Then, I got down on my knees and wrestled with her. She loves when I wrestle with her.
"I really do have a good life," I thought after the dog and I were done chomping on each other. I went into the kitchen to put away the clean dishes. "I'm thankful for all of it."
You know what? I'm thankful for Get Rich Slowly too. And for you, the readers. This site has been a huge blessing in my life — and I'm not one to talk much about blessings. I've put a lot into GRS, it's true, but I've gotten so much more out of it. I've gotten so much from you folks.
So, thank you. I mean it. Thank you for reading. Thank you for contributing. Thank you for everything.
Few and Far Between
As Karl and I chatted at lunch today, I caught a Natalie Merchant song playing on the restaurant's radio. At first I thought it was "Wonder", but then I recognized it as "Few and Far Between".
"How fitting," I thought. Some of the lyrics:
"'Til you make your peace with yesterday, you'll never build a future. I swear by what I say: Whatever penance you do, decide what it's worth to you, and then respect it. However long it will take to weather your mistakes? Why not accept it?"
So, that's what has been going on in my life lately. It's been a struggle. But I can see a light at the end of the tunnel. And I can see some money articles at the end of the keyboard. (Thank goodness, right?)
What's been going on with you?


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(Photo by Mark Wilson/Getty Images)
WASHINGTON — A budget agreement between key Republicans and Democrats. Even President Barack Obama was on board. All without anyone threatening to repeal this or shut down that.
Gridlock, however briefly, took an early holiday in the bitterly polarized, Republican-run House.
But across the Capitol, the high-minded Senate remained in the grip of some of the worst partisan warfare in its history after majority Democrats curbed the Republicans' power. A round-the-clock talkathon is the result, putting no one in the mood for cooperation. Majority Leader Harry Reid threatened to shorten the Senate's cherished Christmas vacation if need be.
A Republican called his bluff. "What's new about that? What's even threatening about that?" challenged Sen. Mike Johanns, R-Neb.
Traditionally effective prods to action are often less so in the divided, crisis-managed Congress. Lawmakers have lurched from sequester to shutdown over spending, national health care and more in the three years since Republicans won control of the House with a sizable group of newcomers reluctant to compromise. Their approach proved costly — to the nation's credit rating, to Congress' standing among voters and to the GOP, which took the brunt of public blame for the partial government shutdown in October.
The scene has been no better in the Senate. What remained of that chamber's deliberative nature blew apart last month when majority Democrats, citing GOP obstructionism, curtailed the Republicans' power to block some presidential nominees. Republicans have tried this week to do what they can to protest, but Reid's slate of 11 nominations didn't appear in peril. Early Thursday morning, the Senate approved the first of those, voting 51-44 to confirm Cornelia "Nina" Pillard to the influential U.S. Court of Appeals for the District of Columbia Circuit.
President Barack Obama praised the confirmation of Pillard, the second judge seated on the D.C. Circuit this week, noting that Pillard would give the court five active female judges for the first time.
"Throughout her career, Ms. Pillard has displayed an unwavering commitment to justice and integrity," Obama said.
Democrats continued their promised march of confirmation votes on Thursday morning, approving Chai Rachel Feldblum to be a Member of the Equal Employment Opportunity Commission by a vote of 54-41. Two Republicans, Sens. Susan Collins of Maine and Lisa Murkowski of Alaska voted for Feldblum.
The string of roll calls continued with a 70-29 vote confirming Elizabeth A. Wolford to be a U.S. district judge for the Western District of New York. Several other procedural votes and confirmation votes on district judges and other government officials were planned throughout the day.
Still, with the 2014 midterm election year fast approaching, there was something unexpected this week: Instead of the standoffs, demands and disrespect that have become routine, key Republicans and Democrats announced a budget deal. Authored by Rep. Paul Ryan, R-Wis., and Sen. Patty Murray, D-Wash., and quickly endorsed by Obama, the agreement would avert another government shutdown in January and reverse $63 billion in across-the-board spending cuts. It would offset the higher spending with $85 billion over a decade from higher fees and modest curbs on government benefit programs.
What's more, House conservatives didn't kill it outright, although some grumbled about the spending levels. Liberals, meanwhile, complained that the deal would not include an extension of unemployment benefits set to expire Dec. 28 for 1.3 million Americans.
But the deal survived the day.
The negotiators spoke Wednesday of finding common ground, however narrow, in pursuit of a larger goal.
"On balance, my view is this is a step forward," said Rep. Chris Van Hollen, D-Md. "A small one, but a step forward."
Ryan told the House Rules Committee, "We have shown that we can work together."
In the Senate, it wasn't immediately clear whether Republican conservatives would follow their House counterparts and grudgingly accept the Ryan-Murray budget, or rebel against it.
Sen. Marco Rubio, R-Fla., who like Ryan is counted among his party's presidential contenders, criticized the deal. "'I think to walk away from the already agreed-upon reductions in spending that were so difficult to achieve, I think opens the floodgates that really threaten to put us right back in these spending habits, and really, we're going to continue to have a government that spends more money than it takes in," he said.
In any case, GOP senators were focused Wednesday and early Thursday on doing what they could to protest the Democrats' change in Senate rules. On Nov. 21, Democrats pared the threshold for stopping filibusters from 60 votes to a simple majority. The lower threshold applies to nearly all presidential nominations but does not affect nominated Supreme Court justices or legislation.
On Monday, Sen. Lamar Alexander, R-Tenn., objected to Reid's request for unanimous approval of 76 nominees, including Janet Yellen, Obama's choice to head the Federal Reserve. Also on the list are Jeh Johnson, Obama's choice for Homeland Security secretary, and Deborah Lee James, for secretary of the Air Force. Most of the nominations, though, are for middle- or lower-level posts like ambassadors and federal judges.
On Tuesday, Republicans invoked a little-known rule to postpone a committee vote on the nomination of John Koskinen, Obama's choice to head the Internal Revenue Service.
Reid had set in motion the votes for 11 of the nominees, most of them non-controversial. Late Wednesday, he asked for unanimous consent to waive some of the required debate time and begin voting at 9 a.m. EST Thursday morning. Republicans objected, and an all-night talkathon began. If Republicans refuse to give up their allotted debate time, the Senate could be in session continuously into Saturday — or longer.
"If we have to work through Christmas, we're going to do that," Reid warned from the Senate floor.
"The Republicans are wanting to waste more of this body's time, this country's time," he added. Senate aides carted in Listerine, fruit, chocolate and mints for what appeared to be a long night ahead. "We are here … looking at each other, doing basically nothing, as we have done for vast amounts of time, because of the Republicans' obstructionism."
"This isn't about obstructionism," fired back Sen. Tom Coburn, R-Okla. "You limited our rights."
Associated Press writer Henry C. Jackson contributed to this report.
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In March, 2007, the virus that causes tomato yellow leaf curl popped up in greenhouse tomatoes being grown by a high school science class in Imperial County, Calif.
Because this disease is new to California and potentially devastating for tomato production, agencies have produced an informational brochure to help curtail its spread.
Tomato yellow leaf curl virus is a member of a family of viruses that are spread by whiteflies or leafhoppers. It infects peppers, some tobacco species, and a range of weed species, but tomatoes are by far the most important host.
With input from University of California, Davis Plant Pathologist Robert Gilbertson, the University of California statewide integrated pest management program has developed a brochure to help growers and pest control advisors learn about the disease, how to identify it, and what to do if they suspect that plants are infected. Several color photos illustrate disease symptoms and the whiteflies that spread the virus.
A PDF file of the brochure can be downloaded from the UC IPM Web site at www.ipm.ucdavis.edu/PMG/r783103311.html.
A pest management guideline on tomato yellow leaf curl will be posted on the site in the near future.
Anyone finding tomatoes with symptoms that look like tomato yellow leaf curl should contact a UC Cooperative Extension office; UC Davis Plant Pathologist Robert Gilbertson, (530) 752-3163; or California Department of Food and Agriculture Scientist Tonyan Tian, (916) 262-1127.
The California Tomato Research Institute, Inc., and California Tomato Growers Association, Inc., are supporting the project.


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This post is related to Eye Makeup for Valentine's Day 2014. So, girls if you are planning out a romantic Valentine's Day date, then spice up your look with these eye makeup ideas. We have some suggestion and tips for the eye makeup related to this Valentine's Day. Eye make is as essential part of makeup. It's a prominent part of your face and if you have a little experience of applying make up on your eyes, you can change your complete look. It will definitely help you in enhancing your eyes.
Its Valentine's Day and no doubt you will be trying to get a look which will make you look just like a princess on this special day. To start your eye makeup the first step is to have a good skin. There are simple steps that will be discussed in this article.
- First brush on neutral beige eye shadow and blend shimmery mocha eye shadow on top.
- Apply it from the lash line to the socket wrinkle.
- For a closer look, blend plum eye shadow into the hollow of the socket focusing on the outer corners.
- Applying a fine eye liner brush, apply a thin line of shimmery mocha shadow just under the roots of the lower lashes and blend to touch in the inner corners to open up your eyes.
For enhancing the Eyes, draw along the upper lash line with a black eye liner pencil and solidify the line gradually from the mid of the eyelid towards the outer corner of the eye, covering it slightly beyond the eye and make a curving path it upward to create a delicate glance. Draw along the inner rim of the lower lid to extend the eye. Curl lashes and apply mascara at the end. And it is finished here so, girls get a good and bright look by following these steps. We hope you will like these makeup ideas.


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I love spring! In honor of this time of year, I thought I would have a sale to help any of you who are on the fence about purchasing anything I sell. A majority of what I offer on my site is FREE, but there are a few things I do sell.
My "store" webpage outlines all of the different items and has a shopping cart feature, so you can make one purchase, instead of one for each item. You can see all bundles I offer in one place and read more about each one.
From now through April 6, 2013, with the code spring50 you will receive
50% off of your entire shopping cart!
Click here to shop and enter code: spring50
I am also hosting the same discount in my Teacher's Notebook Shop, where I have a few smaller items for sale. The discount is already applied over there, you'll see the sale prices for each item. Below is just a few of the items on sale and their sale price shown!
Thanks to all of you who support our family and the work I do online through your purchases of my items!
Please remember, if you have any trouble downloading or printing anything from my site {or others} I have a PDF troubleshooting post!


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|Posted On
When Fear Looks Like Control
posted on May 14, 2014
Some of my favorite works of art are Julian Beever's sidewalk illusions. If you haven't seen them they are truly amazing! He employs a technique called trompe l'oeil which is French for deceive the eye. He creates images that, when…… Read More
Band-Aids Heal More Than You Can See
posted on April 25, 2014
Giving and receiving love may seem like an easy thing, but for many adopted and foster kids it is hard. They have given love to people only to have those people disappear from their lives or not return their love.…… Read More
Can I Give My Child a Joyful "YES" Instead?
posted on April 22, 2014
One of the things I've learned in my journey as a mom is the need for me to raise the level of nurture I bring to parenting in order to help my children build trust. My children need to trust…… Read More


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CBS Films and Lionsgate's period supernatural thriller Winchester, starring Helen Mirren, won the Friday box-office race with $3.6 million for a projected $9 million debut.
However, holdover Jumanji: Welcome to the Jungle (Sony) is widely expected to rumble past Winchester on Saturday and top Super Bowl weekend — one of the quietest frames of the year in terms of moviegoing — with $10 million-$11 million.
Winchester could also be beat by Maze Runner: The Death Cure (Fox), which looks to earn $9.6 million in its second outing, a 64 percent drop.
Directed by Michael Spierig and Peter Spierig, Winchester is inspired by the real-life tale of Sarah Winchester, the eccentric 19th-century heiress to the Winchester Repeating Arms Co. fortune. Winchester spent her life constructing an enormous mansion in San Jose, California, complete with secret passages and trap doors, in order to keep at bay what she thought were the angry spirits of the people killed by her family's firearms.
In the film, Winchester (Mirren) is visited by a skeptical San Francisco psychiatrist (Jason Clarke) who discovers that her obsession may not be so insane after all.
CBS Films acquired rights to the movie for a modest $3.5 million.
Between Maze Runner, The Greatest Showman and The Post, Fox could have three films in the top five. Greatest Showman, boasting another great hold, looks to place No. 4 for the weekend with $7.8 million, followed by The Post with a projected $5.2 million.
Scott Cooper's indie Western Hostiles, starring Christian Bale, hopes to beat The Post, but it will be close.
Elsewhere, several films nominated for top Oscars are hoping to score major points, led by Guillermo del Toro's The Shape of Water (Fox Searchlight), which is upping its theater count from 1,854 locations to 2,341. The film is projected to pace No. 9 with $4.1 million.
Searchlight also has Three Billboards Outside Ebbing, Missouri, which is increasing its theater count from 1,457 locations to 1,726. It's the first time that Fox Searchlight has had films playing in 4,000 or more theaters on any given weekend.
Both Shape of Water and Three Billboards are up for the best picture Oscar, the gold standard when it comes to enjoying a box-office bump. Other movies scoring top noms can also benefit this weekend, with I, Tonya hoping that's the case as it moves into 1,400 cinemas. The dramedy, from Neon and 30West, stars Oscar-nominated Margot Robbie as disgraced ice skater Tonya Harding.
Three Billboards is projected to gross $2.9 million for the weekend; T, Tonya, $2.4 million.
Feb. 3, 8:20 a.m. Updated with Friday grosses and revised weekend estimates.
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Thursday, January 22, 2015
I started with a phrase - Winning Hearts and Minds. From a conversation about Apocalypse Now, half listening to the men in my life dissecting the film, I wandered through history to the underlying thesis of the war - to win the hearts and minds of the populace. If they could be convinced that Ho Chi Minh had nothing on Uncle Sam, the battle would be won.
Using napalm and supporting corrupt officials didn't help our cause, of course, but I was going elsewhere. Does changing someone's beliefs count as a win? By whose lights are beliefs right or wrong? Because you are the other, with skin tinted differently, are you always in the wrong? Was it necessary for the people to look at the world through an American lens in order for us to win?
And why did we need to win? The Domino Theory (if Vietnam falls to Communism, the rest of Asia will not be not far behind), perhaps? Because we'd never lost a war? Because our soldiers had fallen and their sacrifices shouldn't be in vain? The backstory to our involvement in the conflict may never be resolved; the notion of winning lies crumpled on the rooftop of the American Embassy in Saigon, pictured in the faces of the Vietnamese we left behind.
They had hitched their wagons to those they perceived as winners. Being left behind was their reward.
I remember the vindication I felt when the war was finally over. We'd done it. The passion of America's youth had changed the course of history. We'd stopped a war. I thought we had won - the we being those who agreed with me. Our country had certainly abandoned, if not lost, the war. The Vietnamese were left with a damaged countryside and a communist regime; not what America considered a winning scenario.
But I thought that I had won.
Perspective, it seems, makes a difference.
Before being perforated, I never would have tolerated finishing last in a race. ... or in anything else, for that matter. Showing up was not enough. I had to try to win. I compared myself to the first across the finish line, and I measured my competence accordingly. In the first 10K I ran, back in 1979, I decided that winning would be completing the course in under an hour. I was two minutes over that time. Though proud of the accomplishment, I felt that I had failed myself.
Failed myself. Let myself down. Not measured up to my own expectations. It was a hard pill to swallow, even as I accepted congratulations for finishing the wet and muddy course. I never ran another race for time again.
When Brenda Starr and I crossed the finish line at the Pecan Run last Fall, many minutes after the everyone else had packed up and gone home, my reaction was at the other extreme. We had done it! We hadn't sat down or given up or taken a lengthy break. We'd put one gimpy foot in front of the other and, with the encouragement of friends and family, we were done. It was a modest goal, but an achievable one. We were ecstatic.
Some piece of me still longed for the exhilaration of passing a slower participant. Some piece of me wanted to fly by, smiling inside, knowing that someone was less than I was.
That's where I get caught up in winning. Someone has to lose. In my youth, this was less of a problem. I was more self-absorbed, looking for ways to propel myself forward. Now, as age and bullets and grandchild have combined to create a more mellow version of me, I'm less concerned with coming out on top. I'm looking to win my own heart and mind, to make my own contribution the best it can be, even if it did take us an hour to walk 2.3 miles last Saturday.
We finished. We didn't stop. We were smiling. And no one had to lose or sacrifice for us to feel as pleased with ourselves as we were. I had adjusted my expectations. I had satisfied myself. We certainly didn't win the race in a conventional sense, but we won the race we were running.
I've converted my heart and mind.
Thanks to Rain Truax for the post (here) which prompted this.
3 comments:
Thanks for the mention. I think winning and figuring out what it is is a big deal and I certainly hadn't thought of it before that dream. I don't like to participate in win/lose situations. I played a lot of games in my life and finally realized the stress of that set up wasn't worth it to me. I don't want to lose or win. I guess I am not a competitive person by nature. But winning now seems to me about a lot more than games or wars. It's about us figuring it out for ourselves. To me you won when you entered the race. The ones who lost are those, like me, who aren't entering at all. It's definitely something to think about.ReplyDeleteReplies
There's no judgment implied, Rain. You are winning if you are comfortable with the path you have chosen. I'm trying to learn to take the perception of others out of my equations. It's not easy, but it seems to be worthwhile.Delete
No, it's no judgment but it is something for me to think about as to how often do i not enter into something because I don't like to lose. That is the real loss if I am doing that ;)Delete


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1689 London Baptist Confession -Ch. 22 para.7
Westminster Confession –Ch. 21 para.7
"As it is the law of nature, that in general a proportion of time, by God's appointment, be set apart for the worship of God, so by his Word, in a positive moral, and perpetual commandment, binding all men, in all ages, he hath particularly appointed one day in seven for a sabbath to be kept holy unto him, which from the beginning of the world to the resurrection of Christ was the last day of the week, and from the resurrection of Christ was changed into the first day of the week, which is called the Lord's day: and is to be continued to the end of the world as the Christian Sabbath, the observation of the last day of the week being abolished."
Monday, January 23, 2012
The Lord's Day
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Map dota v6.79 test 8.w3x download and update by Icefrog
A public test version of 6.79 has been released to help increase bug testing coverage before the official version is published. Comment here to report any bugs you find. Certain bugs can be discovered in single player games while others will require full 5v5 games.
Link download: Dota v6.79.w3x
Mirror 2: Game2e.com DotA v6.79.zip
Mirror 3: https://www.mediafire.com/?xdjemyl8mj4d7ti


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Monroe Shocks & Struts Coupon: I believe it was rapper, Ice Cube, who boasted about having "more bounce to the ounce."Well, Monroe would probably be happy to promise you the exact opposite. And, with these coupons, Advance Auto Parts would be happy to promise you more bang for the buck! (which all of us can appreciate)
With over 9,000 shocks and struts in stock, (including top name, Monroe) Advance Auto Parts has you covered, with parts for every model car or truck. The best part is that when you use a coupon, you can save a big on your entire order. Don't forget that any order can be picked up at your local store, and orders over $75 ship free.
Monroe Shocks Coupons:
Advance Auto Parts: (recommended)
- 30% off Monroe shocks (click, then use promo code)
- View all coupons for Advance Auto – Updated daily!
AutoZone.com: AutoZone no longer carries Monroe parts, but instead have Gabriel, Sachs & Bilstein brand shocks
- View all shocks & struts
- View all coupons (some printable)
What do shock Monroe absorbers do, and where can I get a coupon? Monroe shock absorbers are one of the most popular brands sold by Advance Auto Parts. Let's take a look at a few specific models, but before doing this let's also review what a shock absorber actually does.These mechanical parts are designed to smooth out and dampen the kinetic energy of small impacts of a moving car along a differential terrain.
Commonly called "shocks," these parts can be pneumatic or hydraulic, and are used with cushions and springs. The main components include a bar, piston and oil seal, cylinder, oil reservoir, floating piston, and air chamber.
If you apply one of the coupons mentioned above on your purchase of specific Monroe shocks, let's see what you are getting in addition to a serious discount and promo.
One of the most popular and well-rated is the Monroe Reflex Monotube, (Monroe Reflex Shocks) engineered to improve handling, control and car performance for light trucks and SUV's. The high-pressure gas charge keeps the internal oil and nitrogen separate, further enhancing functioning.
Monroe Shocks & Struts Cheap? Best Price Online With This Coupon!
Another popular product from Advance Auto it the Monroe Gas-Matic LT. No, this is not gas-matic in the sense of what happens to your bowels after eating three bratwursts and a plate of sauerkraut but is rather the formalized name of a part that is so easy to install its automatic. These are heavy-duty soldiers for your car's suspension.
*More coupons: Snapfish | Winc Wine | WW
So please don't hesitate to check out related products in this category, including mounts, coil springs, shock and strut hardware, and other Monroe products that can be bought through AdvanceAutoParts.com using a fantastic promotional code, often saving you 15-40%! Also, don't forget to check out their oil change deals and rebates to save even more.


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Allie Thompson
Associate Attorney
Allie was born and raised in Muscle Shoals, Ala., and graduated from the University of Alabama in 2012. After college, Allie earned her J.D. in 2015 from the Cumberland School of Law at Samford University. While in law school, Allie developed a strong passion for domestic relations and family law which has only grown during her time with the Isaak Law Firm.
Since joining the firm in 2016 as an associate in the Montgomery office, Allie has handled a wide variety of cases. She has represented men in divorce, modification, contempt, child support, adoption, and domestic violence related matters.
When she's not practicing law, Allie enjoys spending time with family and friends, seeing live music, listening to podcasts, being outdoors, and spending time with her dog, Tallulah.


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Amaya, D., S.-P. Xie, A. J. Miller and M. J. McPhaden, 2015:
Seasonality of tropical Pacific decadal trends associated with the 21st century global warming hiatus
Journal of Geophysical Research-Oceans
, 6782-6798.
Equatorial Pacific changes during the transition from a non-hiatus period (pre-1999) to the present global warming hiatus period (post-1999) are identified using a combination of reanalysis and observed data sets. Results show increased surface wind forcing has excited significant changes in wind-driven circulation. Over the last two decades, the core of the Equatorial Undercurrent intensified at a rate of 6.9 cm s-1 decade-1. Similarly, equatorial upwelling associated with the shallow meridional overturning circulation increased at a rate of 2.0 x 10-4 cm s-1 decade-1 in the central Pacific. Further, a seasonal dependence is identified in the sea surface temperature trends and in subsurface dynamics. Seasonal variations are evident in reversals of equatorial surface flow trends, changes in subsurface circulation, and seasonal deepening/shoaling of the thermocline. Anomalous westward surface flow drives cold-water zonal advection from November to February, leading to surface cooling from December through May. Conversely, eastward surface current anomalies in June-July drive warm-water zonal advection producing surface warming from July to November. An improved dynamical understanding of how the tropical Pacific Ocean responds during transitions into hiatus events, including its seasonal structure, may help to improve future predictability of decadal climate variations.


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They promised you a projector and a screen but when you arrived the meeting room had changed and all you have is your trusty laptop - still think you can win the deal or train their staff on your marketing plans?
Business people can't leave things to chance so why not stack the cards in your favour - take the astonishing PK301 Pico Pocket Projector with you and take control - whatever the circumstances. This pocket size device has an amazing WVGA resolution, VGA/HDMI input and a Micro SD slot, ideal for storing and sharing all kinds of media. With images up to 120" diagonal, the PK301 turns your PC or laptop into a thing of real power. The PK301 goes with you from room to room, town to town or country to country. When it's not winning business it's there to entertain your family - the PK301 works for up to 1.5 hours on its internal battery or an amazing 5 hours with the extended battery pack.
- A 120" Image in your Pocket
- Built-in Media Player with Micro SD Slot for up to 32GB
- Play Films, show images direct from the Projector
- Connects to most equipment with AV out capability
- Up to 1.5 hours use on the supplied rechargeable battery
- Up to 5 hours use on the optional High Capacity rechargeable battery
- Power adaptor supplied for direct power and charging
- WVGA (854 x 480) Resolution
The following resources are available for the Optoma PK301 Pico Projector