In a screen for suppressors of npr1-5–based salicylic acid (SA) insensitivity, we isolated a semidominant gain-of-function mutation, designated ssi4, that confers constitutive expression of several PR (pathogenesis-related) genes, induces SA accumulation, triggers programmed cell death, and enhances resistance to bacterial and oomycete pathogens. Through map-based cloning, ssi4 was identified and found to encode a putative protein belonging to the TIR-NBS-LRR (Toll Interleukin1 Receptor–Nucleotide Binding Site–Leu-Rich Repeat) class of R (resistance) proteins. Comparison between ssi4 and the corresponding wild-type sequence revealed a single amino acid substitution in the NBS. Epistasis analysis indicated that SA and EDS1 are required for ssi4-induced PR-1 expression and enhanced disease resistance; they also are required for the increased accumulation of SSI4 and EDS1 transcripts detected in the ssi4 mutant. Although high levels of ssi4 transcripts correlate with the appearance of the mutant phenotype, overexpression of the wild-type SSI4 gene failed to induce stunting, spontaneous lesion formation, or increased PR-1 expression associated with the ssi4 mutation. Thus, the ssi4 phenotype does not appear to be caused by overexpression of this R gene; rather, we propose that the NBS substitution generates a constitutively activated R protein. Furthermore, because SA treatment induced the expression of SSI4 and the closely related TIR-NBS-LRR genes RPP1 and RPS4 but had little effect on the expression of the coiled-coil NBS-LRR genes RPM1 and RPS2, we suggest that SA not only functions as a critical signal for downstream resistance events but also upregulates the expression of certain R genes.
For eons, battles have raged between plants and pathogenic microbes intent on using them as a nutritional resource. To ward off infection, plants activate a variety of defense responses. In the infected leaf, these defenses include strengthening of cell walls, activation and/or synthesis of antimicrobial compounds, and expression of many defense-associated proteins, including the pathogenesis-related (PR) proteins (Durner et al., 1997; Dempsey and Klessig, 1999). A hypersensitive response, characterized by the formation of necrotic lesions and the restriction of the pathogen to the cells within and immediately surrounding these lesions, also frequently develops. After these local responses, the uninoculated portions of the plant usually exhibit increased PR gene expression and the appearance of an enduring resistance to a broad spectrum of pathogens known as systemic acquired resistance (SAR). For some plant–pathogen interactions, the signal transduction cascade leading to disease resistance is activated by the direct or indirect interaction between the products of a plant R (resistance) gene and a pathogen Avr (avirulence) gene (Flor, 1971; Keen, 1990). Based on this gene-for-gene model, if the cognate gene from either the plant or the pathogen is missing, plant defense responses either fail to be activated or are induced too weakly and/or too late to prevent pathogen colonization.
A variety of studies have demonstrated that salicylic acid (SA) plays a critical role in the defense signaling pathway. In many plant species, SA levels increase in conjunction with the activation of PR gene expression and disease resistance (Malamy et al., 1990; Métraux et al., 1990; Uknes et al., 1993). Furthermore, plants unable to accumulate SA as a result of the expression of the bacterial nahG gene fail to develop SAR and exhibit increased susceptibility to pathogen infection (Delaney et al., 1994; Vernooij et al., 1994). Analysis of various genetic mutants, including the cpr (Bowling et al., 1994, 1997; Clarke et al., 1998; Silva et al., 1999; Yoshioka et al., 2001), cim (Ryals et al., 1996), acd (Greenberg et al., 1994; Rate et al., 1999), ssi (Shah et al., 1999, 2001; Kachroo et al., 2001), and lsd (Dietrich et al., 1994; Weymann et al., 1995) mutants, also has revealed a correlation between increased SA levels, constitutive PR expression, and SAR. By contrast, mutations in the NPR1 gene cause a SA-insensitive phenotype, which is characterized by heightened disease susceptibility and the inability to develop SAR after SA treatment (Cao et al., 1994; Delaney et al., 1995; Glazebrook et al., 1996; Shah et al., 1997).
During the past few years, many R genes have been cloned. Based on their predicted protein structures, R proteins can be divided into several groups. The largest group contains a nucleotide binding site (NBS) and a Leu-rich repeat (LRR) region (Ellis et al., 2000a). NBS domains have been identified in many prokaryotic and eukaryotic proteins, in which their ability to bind ATP or GTP is essential for their biological activity (Saraste et al., 1990). The ability of plant R proteins to bind nucleotides has yet to be demonstrated; however, they contain several conserved motifs, such as the P loop, which are known to be important for this function. LRR domains are known to mediate protein–protein interactions in diverse proteins (Jones and Jones, 1996). Based on the high degree of divergence between the LRRs of R protein homologs, they are thought to play a role in pathogen recognition (Bent, 1996; Jones, 1996; Ellis et al., 2000b; Nimchuk et al., 2001). The NBS-LRR class of R proteins can be subdivided further into two classes based on the secondary structure of the N terminus. The first class contains a putative coiled-coil region at the N terminus (CC-NBS-LRR). The second class contains an N-terminal TIR region, which shares homology with the Toll protein of Drosophila and the Interleukin1 receptor (IL-1R) of mammals (TIR-NBS-LRR) (Ellis et al., 2000a). Because the Toll protein and IL-1R play roles in activating innate immunity, the TIR domain has been proposed to transduce a signal that activates plant defense responses (Staskawicz et al., 1995; Baker et al., 1997).
Recent evidence indicates that the signals from the TIR- and CC-containing classes of R proteins are transduced through separate pathways (Aarts et al., 1998). The TIR-NBS-LRR proteins appear to induce disease resistance via a pathway that requires the EDS1 gene (Parker et al., 1996). By contrast, the CC-NBS-LRR proteins frequently use a pathway that requires the NDR1 gene (Century et al., 1995). However, a few CC-NBS-LRR proteins have been identified that require neither EDS1 nor NDR1 (McDowell et al., 2000; Bittner-Eddy and Benyon, 2001). Thus, a third defense signaling pathway(s) that is independent of these proteins also appears to exist.
The ability of different R proteins to activate a specific defense pathway suggests that diverse signal transduction strategies are used. Currently, the mechanisms by which a pathogen is perceived and the resistance signal is initiated and transduced are poorly understood. However, several recent studies have demonstrated that overexpression of R genes can result in enhanced disease resistance. For example, overexpression of the Pto gene in tomato induces constitutive PR gene expression, increased levels of SA, spontaneous lesion formation, and enhanced disease resistance (Tang et al., 1999). Broad-spectrum disease resistance also is induced by overexpression of Prf, a CC-NBS-LRR R gene whose product works in conjunction with Pto (Oldroyd and Staskawicz, 1998). In addition, overexpression of the Arabidopsis TIR-NBS-LRR R gene At4g16890 results in enhanced disease resistance (Stokes et al., 2002). It has been hypothesized that R proteins are found normally in a complex with a guard protein; while sequestered in this complex, they are unable to activate defense responses (Dangl and Jones, 2001). After interaction with the appropriate Avr protein, a change in the R protein would allow the complex to dissociate and a signal transduction event would be initiated. Thus, if R proteins are present normally in rate-limiting amounts, their overexpression might be sufficient to activate the defense signaling pathway.
To further investigate the SA signaling pathway leading to plant disease resistance, we used a genetic screen to isolate suppressors of the npr1-5 mutation. One mutant, designated ssi4, was identified based on its ability to constitutively express several PR genes. Analysis of the cloned ssi4 gene revealed that the predicted open reading frame (ORF) encodes a TIR-NBS-LRR protein with a single amino acid substitution in the NBS domain. Consistent with the presence of an altered TIR-NBS-LRR R gene, ssi4 mutants exhibited enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica biotype Emco5. Furthermore, both SA and EDS1 were required for ssi4-induced constitutive PR-1 expression and enhanced disease resistance. All plants exhibiting the ssi4 mutant phenotype contained increased levels of ssi4 transcripts. However, because SSI4-overexpressing plants failed to express PR-1 or to display enhanced disease resistance, the NBS mutation, rather than overexpression of this TIR-NBS-LRR gene, appears to be responsible for signaling defense responses. Interestingly, SA treatment induced the expression of SSI4 and several other TIR-NBS-LRR R genes. This finding suggests that SA is involved in a positive feedback loop that regulates the expression of a variety of defense-associated genes, including certain R genes.
To identify signaling components of the SA defense pathway, 3- to 4-week-old M2 progeny of ethyl methanesulfonate–mutagenized npr1-5 seeds (ecotype Nö) were screened for individuals that exhibited constitutive PR gene expression. One individual, designated ssi4 (suppressor of salicylic acid insensitivity of npr1-5), was found to express the PR-1, PR-2, and PR-5 genes (Figure 1A) . By contrast, the jasmonic acid– and ethylene-inducible PDF1.2 gene was not expressed (data not shown). The ssi4 mutant also exhibited several morphological abnormalities, including stunted growth, severe chlorosis, and the development of spontaneous lesions (Figure 1B). Because these lesions contained large areas of dead cells (Figure 1C) and were associated with the presence of autofluorescent material (data not shown), the cell death induced by ssi4 is similar to that seen during the hypersensitive response.
Phenotypic Effects of the ssi4 Mutation.
(A) Expression of the PR-1, PR-2, and PR-5 genes in npr1-5, npr1-5 ssi4/SSI4, or npr1-5 ssi4/ssi4 plants. RNA gel blot analysis was performed on 10 μg of total RNA extracted from 3-week-old soil-grown plants. Ethidium bromide staining of rRNA (bottom gel) was used as a loading control.
(B) Comparison of the morphological phenotypes displayed by plants heterozygous or homozygous for the ssi4 mutant allele in the npr1-5 background. Plants were grown on soil and photographed when 3 weeks old.
(C) Microscopy of trypan blue–stained leaves from npr1-5 and ssi4 npr1-5 plants. Leaves from ssi4 npr1-5 plants contain intensely stained areas of dead cells.
To investigate the nature of the ssi4 mutation, we analyzed the progeny of a backcross between the ssi4 npr1-5 double mutant and the SSI4 npr1-5 parent. All of the F1 progeny exhibited constitutive PR gene expression and the stunted morphology associated with the ssi4 mutation. However, all of the ssi4-associated phenotypes were less pronounced in these heterozygous plants, suggesting that ssi4 is a semidominant mutation (data not shown). Analysis of F2 progeny confirmed this finding (Figures 1A and 1B).
To determine whether the ssi4 mutation suppresses the npr1-5 phenotype by functioning as an intragenic suppressor of the npr1-5 allele, ssi4 npr1-5 and wild-type (ecotype Nö) plants were crossed. ssi4 homozygous F2 progeny that also were homozygous for NPR1 were identified using a codominant cleaved amplified polymorphic sequence marker for npr1-5 (Shah et al., 1999). The ssi4 NPR1 plants showed the same phenotypes as ssi4 npr1-5 plants (data not shown). Thus, the ssi4 mutation is not a simple revertant of npr1-5, and the effects of this mutation are independent of the npr1-5 mutation.
We next determined whether the ssi4 mutation is attributable to a dominant gain of function or to haploinsufficiency by crossing ssi4 homozygous plants with Columbia tetraploid plants (CS3432). RNA gel blot analysis of the resulting triploid F1 progeny revealed that they constitutively expressed PR-1, and these plants also displayed extensive leaf chlorosis and formed lesions spontaneously (data not shown). This result suggests that the ssi4 phenotype is caused by a gain-of-function mutation rather than by haploinsufficiency.
To determine the map position of the ssi4 locus, we crossed ssi4 plants with wild-type plants from ecotype Landsberg. As expected, the F2 progeny segregated in a 3:1 ratio (PR-1+:PR-1−) when scored for constitutive PR-1 gene expression. Because of the semi-dominant nature of the ssi4 mutation, we determined map position by analyzing F2 plants exhibiting the wild-type (PR-1−) phenotype. A total of 1179 wild-type–like F2 plants were analyzed; SSI4 was mapped to a 132-kb region of chromosome V. This region corresponds to 37 kb at the 3′ of the P1 clone MBK23, the entire region covered by overlapping clones MUF8 and K16L22, and 2 kb of MJC20 beyond the region of overlap between K16L22 and MJC20, as defined by the Arabidopsis sequencing project (Figure 2A) .
Isolation and Identification of the ssi4 Mutation.
(A) Genetic and physical map of the ssi4 region of chromosome V. The top line represents chromosome V, with the broken lines showing the points of recombination. The middle line represents a larger scale of the region within the recombination breakpoints, with markers indicated above it, the number of recombination events for each marker designated below it, and the MUF8.2 ORF (ssi4) denoted as a black box. P1 and TAC clones spanning this region are represented as labeled boxes (MBK23, MUF8, K16L22, and MJC20). Directly beneath these boxes are the numbered TAC-based clones representing the contig of this region generated from ssi4 genomic DNA. Clone 94, marked with an asterisk, contains the ssi4 mutation and maps to the MUF8.2 ORF.
(B) PR-1 expression in wild-type (Nö wt), ssi4/SSI4, and T1 (#94-1) plants. Ten micrograms of total RNA was used for RNA gel blot analysis. rRNA stained with ethidium bromide served as a loading control.
(C) Amino acid sequence of SSI4. Basic Local Alignment Search Tool (BLAST) analysis of the predicted SSI4 protein sequence revealed three conserved regions identified by the vertical lines: TIR, NBS, and LRR. The ssi4 mutation (G422R) is indicated in boldface type. The arrowheads indicate the intron positions in the corresponding genomic SSI4 sequence. The aligned spaces in the LRR region indicate the putative β-turn/β-strand motif as aligned with the porcine ribonuclease inhibitor structure.
Because ssi4 is a gain-of-function mutation, complementation analysis could be performed only using the ssi4 gene. Thus, a transformation-competent artificial chromosome (TAC) library containing ssi4 genomic DNA was constructed with an average insert size of 10 to 20 kb. After screening 100,000 colonies, those clones spanning the ssi4-containing region were isolated and organized into a contig (Figure 2A). Each clone was introduced into wild-type (ecotype Nö) plants by vacuum infiltration. Hygromycin-resistant T1 plants then were screened for the ssi4 phenotype. Only plants containing clone 94, which encompasses the MUF8.2 reading frame, exhibited stunted growth and extensive leaf chlorosis (data not shown). These plants also displayed constitutive PR-1 gene expression (Figure 2B), confirming that this clone contains the ssi4 mutation.
Sequence analysis of the MUF8.2 ORF from TAC clone 94, as well as reverse transcriptase (RT)–mediated PCR of the ssi4 mutant and SSI4 wild-type mRNAs, identified the ssi4 mutation as a G-to-A transition at codon 422 of MUF8.2, resulting in a Gly-to-Arg amino acid substitution. Sequencing of PCR-generated fragments using genomic DNA or cDNA from mutant or wild-type plants as the template produced the same result. Because the ssi4 mutation does not alter any restriction enzyme site, a derived cleaved amplified polymorphic sequence marker was created to monitor the segregation of mutant genes in transgenic plants containing clone 94. T2 analysis of these plants further confirmed that the ssi4 mutation is responsible for the mutant phenotype; the ssi4 transgene cosegregated with constitutive PR-1 gene expression and the stunted, chlorotic morphology exhibited by ssi4 plants.
Analysis of TAC clone 94 revealed that it contains a single ORF of 3434 nucleotides. Comparison of RT-PCR–generated SSI4 or ssi4 cDNA with genomic DNA indicated that this gene contains three introns that are spliced out to produce a transcript with a predicted protein of 1055 amino acids. A search of the GenBank database indicated that SSI4 is highly similar to several genes that encode functional R proteins, including N (Whitham et al., 1994), L6 (Lawrence et al., 1995), RPS4 (Gassmann et al., 1999), members of the RPP5 gene family (Parker et al., 1997; van der Biezen et al., 2002), and RPP1-WsA (Botella et al., 1998). Like the R proteins encoded by these genes, SSI4 contains a TIR-NBS-LRR structure (Figure 2C). Downstream of the TIR domain, SSI4 contains seven conserved motifs that are associated with the NBS domains of all other TIR-NBS-LRR R proteins (Meyers et al., 1999). The Gly-to-Arg substitution in ssi4 is located in a nonconserved region of the NBS just upstream of the resistance nucleotide binding site D TIR motif. The C-terminal portion of SSI4 contains an LRR domain consisting of 13 imperfect LRRs that range in length from 20 to 25 amino acids.
The discovery that SSI4 encodes a TIR-NBS-LRR protein raised the possibility that other defense responses, in addition to PR gene expression, might be activated by the NBS mutation. To assess this possibility, we assayed ssi4 mutants for enhanced disease resistance and increased levels of SA. SA levels in ssi4 npr1 homozygous plants were 300-fold higher than those detected in wild-type plants or in the parental npr1-5 mutant, and SA glucoside (SAG) levels were >200-fold greater (Figure 3A) . Plants heterozygous for ssi4 in the npr1-5 background exhibited intermediate levels of SA and SAG. By contrast, ssi4 homozygous and heterozygous mutants carrying the NPR1 allele accumulated substantially lower levels of SA and SAG than the corresponding double mutants.
The ssi4 Protein Stimulates SA Accumulation and Enhanced Disease Resistance.
(A) SA and SAG levels in the leaves of 3-week-old, soil-grown npr1 single mutants, npr1 mutants heterozygous or homozygous for ssi4, wild-type (Nö wt) plants, and ssi4 heterozygous and homozygous single mutants. The values presented are averages of four replicates.
(B) Growth of P. syringae pv maculicola ES4326. Leaves of different ssi4 plant genotypes were infiltrated with P. syringae pv maculicola ES4326 (OD600 = 0.002). Four leaf discs were collected immediately after inoculation (white bars), and six leaf discs were collected at 3 days after inoculation (gray bars). Colony-forming units (cfu) per leaf disc are expressed ±sd and represent averages of four or six samples.
(C) Growth of P. parasitica ecotype Emco5. Cotyledons of 7-day-old seedlings from the various plant genotypes listed at left were inoculated by applying a drop of conidiospore suspension (105 spores/mL). There was no difference in the size of cotyledons between wild-type and mutant plants at the time of infection. Pathogen growth was assayed by counting the number of sporangiophores per cotyledon at 7 days after inoculation. The shade of each box indicates the severity of infection, based on the number of sporangiophores per cotyledon (see key at right). Numbers to the right of the sample boxes indicate the number of cotyledons assayed.
To determine whether ssi4 plants exhibit enhanced disease resistance, inoculations were performed using the bacterial pathogen P. syringae pv maculicola ES4326 and the oomycete pathogen P. parasitica biotype Emco5. As a result of the extreme stunting of ssi4 homozygous plants, inoculations with P. syringae pv maculicola ES4326 were performed only on ssi4 heterozygous plants. By 3 days after inoculation with P. syringae pv maculicola ES4326, ssi4/SSI4 npr1 plants contained ∼15- or 20-fold less bacteria than either wild-type or npr1-5 parental plants, respectively (Figure 3B). ssi4-induced resistance was even higher in the NPR1 background; these plants contained 150-fold less bacteria than wild-type plants. Similar results were observed after inoculation with P. parasitica biotype Emco5. At 7 days after inoculation, the level of sporulation on the cotyledons of ssi4/ssi4 npr1 or ssi4/ssi4 NPR1 plants was reduced greatly compared with that detected on wild-type or npr1-5 single-mutant plants (Figure 3C).
After activation, R proteins stimulate many defense responses via an SA-dependent pathway(s). To determine whether SA is required for ssi4-induced PR gene expression and disease resistance, a cross between ssi4 and nahG plants (ecotype Nö) was performed. None of the resulting F1 progeny constitutively expressed the PR-1 gene (Figure 4A) , became chlorotic (Figure 4B), or exhibited enhanced resistance to P. syringae pv maculicola ES4326 (Figure 3B). Thus, SA is required for these ssi4-mediated phenomena. However, nahG-expressing ssi4 heterozygous plants exhibited some stunting, and their leaves were curled (Figure 4B). At the microscopic level, trypan blue staining revealed a significant number of dead cells surrounding the leaf vascular area (data not shown). Thus, ssi4 appears to induce stunting and spontaneous lesion formation via a pathway that is at least partly independent of SA. Although the residual level of SA in ssi4 nahG plants was not determined, it is likely to be similar to that in wild-type plants, because PR-1 gene expression was no longer constitutive and previous studies of ssi1 nahG and ssi2 nahG plants indicated that nahG in the ecotype Nö background was able to suppress SA accumulation to basal levels (Shah et al., 1999, 2001).
Most ssi4 Phenotypes Are Activated via an SA- and EDS1-Dependent Pathway.
(A) Expression of the PR-1, nahG, and ssi4 genes in wild-type (Nö wt), nahG, ssi4/SSI4, and ssi4/SSI4 nahG plants. Ten micrograms of total RNA was used for RNA gel blot analysis, and ethidium bromide staining of rRNA was used as a loading control.
(B) The phenotypes of 3-week-old soil-grown nahG, ssi4/SSI4 nahG, and ssi4/SSI4 plants.
(C) Phenotypes of ndr1 plants and ndr1 plants heterozygous or homozygous for the ssi4 mutation. Plants were grown in soil and photographed when 3 weeks old.
(D) Phenotypes of eds1 and ssi4 eds1 plants. Plants were grown in soil and photographed when 3 weeks old.
Previous studies have demonstrated that several members of the TIR-NBS-LRR class of R genes activate disease resistance via an EDS1-dependent pathway, whereas most members of the CC-NBS-LRR class use NDR1 (Aarts et al., 1998). To determine whether ssi4 induces defense responses via either of these signal transducers, ssi4 plants were crossed with eds1-1 and ndr1-1 plants. Analysis of the resulting ssi4 ndr1 double mutants revealed that they were nearly as stunted and chlorotic as the ssi4 single mutant in the npr1-5 background (Figure 4C). In addition to these abnormalities, ssi4 ndr1 double mutants developed spontaneous lesions and constitutively expressed PR-1 (Figure 5A and data not shown). Furthermore, after inoculation with P. syringae pv maculicola ES4326 or P. parasitica biotype Emco5, ssi4 ndr1 plants exhibited greater resistance than ndr1 single mutants (Figures 3B and 3C). However, they were much less resistant than ssi4 single mutants; this result suggests that NDR1 either plays some role in the enhanced resistance conferred by the ssi4 mutation or has an additive effect on resistance. By contrast, ssi4 eds1 double mutants exhibited wild-type morphology, even in the ssi4 homozygous condition (Figure 4D). Furthermore, these plants did not develop spontaneous lesions, failed to constitutively express PR-1 (Figure 5A and data not shown), and exhibited heightened susceptibility to infection by P. syringae pv maculicola ES4326 and P. parasitica biotype Emco5 (Figures 3B and 3C). Based on these results, ssi4, like other TIR-NBS-LRR genes, requires a functional EDS1 gene to signal defense responses and disease resistance.
ssi4 Expression Requires EDS1 and Is SA Inducible.
(A) Expression of the PR-1, SSI4, EDS1, and PAD4 genes in wild-type (Nö wt) plants, ssi4, npr1, ndr1, and eds1 single mutants, and ssi4 npr1, ssi4 ndr1, and ssi4 eds1 double mutants (all of the mutant genes were present in the homozygous state). RNA gel blot analysis was performed using 10 μg of total RNA, and ethidium bromide staining of rRNA was used as a loading control.
(B) RT-PCR analysis of wild-type plants treated with 500 μM SA or sterile water. Plants were sprayed with SA or water, and leaves were harvested at the times indicated after treatment. RT-PCR was performed using total RNA and gene-specific primers; the products were visualized on an ethidium bromide–stained agarose gel. The level of Actin2 (ACT2) was used as an internal control to normalize the amount of cDNA template. A control with RNA from ssi4 processed without the addition of reverse transcriptase (−RT) is shown at right.
It was demonstrated previously that overexpression of the R genes Pto, Prf, and At4g16890 induces constitutive PR gene expression, spontaneous cell death, increased SA levels, and/or enhanced disease resistance (Oldroyd and Staskawicz, 1998; Tang et al., 1999; Stokes et al., 2002). Because the ssi4 mutant exhibits a similar phenotype, we tried to determine whether the presence of the NBS substitution alters the expression of this gene. RNA gel blot analysis revealed that transcripts for the ssi4 gene accumulate to moderate levels in ssi4 mutant plants (Figure 5A). By contrast, the level of SSI4 transcripts in wild-type plants was extremely low and could be detected only by RT-PCR (Figures 5A and 5B). This increase in ssi4 expression does not appear to be caused by a promoter mutation, because analysis of 981 base pairs 5′ of the translational start site revealed no difference between the ssi4 and wild-type sequences. ssi4 transcripts also were readily detectable in ssi4 npr1 and ssi4 ndr1 mutant plants, but not in ssi4 eds1 (Figure 5A) and ssi4 nahG mutant plants (Figure 4A). These results suggest that increased expression of ssi4 requires a functional EDS1 gene and is SA dependent.
Because ssi4-induced defense responses appear to be activated via an EDS1-dependent pathway, we tested whether ssi4 plants also constitutively express EDS1. Correlating with ssi4 expression, transcripts for EDS1 were detected readily in ssi4 and ssi4 npr1 plants as well as in the ssi4 ndr1 double mutant (Figure 5A). By contrast, few or no transcripts were detected in wild-type plants or in the ssi4 eds1 double mutant. Because eds1-1 contains a point mutation resulting in a nonfunctional protein (Falk et al., 1999), the lack of EDS1 transcripts in ssi4 eds1 plants suggests that a functional EDS1 protein is required for the upregulation of both ssi4 and EDS1 expression. Analysis of PAD4, which encodes a protein that forms a complex with EDS1 (Feys et al., 2001), revealed that it, like ssi4 and EDS1, is expressed at readily detectable levels only in ssi4, ssi4 npr1, and ssi4 ndr1 plants (Figure 5A). Strikingly, transcripts for RPP1, a closely related TIR-NBS-LRR gene, also accumulate to increased levels in ssi4 plants (Figure 5B). Thus, the ssi4 mutation causes increased expression of a wide variety of genes involved in plant defense.
It is possible that the high levels of SA found in the ssi4 mutant induce the expression of ssi4, EDS1, and PAD4. Consistent with this possibility, EDS1 was shown to be induced by SA treatment (Falk et al., 1999). To determine whether SA also induces SSI4 expression, we treated wild-type (ecotype Nö) plants with SA. As expected, SA treatment strongly induced PR-1 expression by 6 h after treatment, and a very weak band corresponding to SSI4 also was detected at this time (data not shown). Thus, SA treatment induces SSI4 expression, although to a much lower level than that detected in ssi4 mutant plants. We then tested whether other TIR-NBS-LRR genes could be induced by SA treatment. Because R genes frequently are expressed at very low levels, this analysis was performed using RT-PCR. Consistent with our RNA gel blot analysis, transcripts for SSI4 increased until 6 h after SA treatment and then decreased gradually during the remainder of the time course (Figure 5B). The same trend was seen with RPP1, another TIR-NBS-LRR gene (Figure 5B). By contrast, the CC-NBS-LRR gene RPM1 exhibited little or no induction by SA treatment. In addition, RPS4, a TIR-NBS-LRR gene, was activated by SA, whereas RPS2, another CC-NBS-LRR gene, was not (data not shown).
The correlation between increased ssi4 expression and the constitutive activation of defense responses suggested that overexpression of this TIR-NBS-LRR gene may be responsible for these effects. To assess this possibility, we transformed ecotype Nö plants with an SSI4 cDNA fused to the 35S promoter of Cauliflower mosaic virus in a pBI121 binary vector. Kanamycin-resistant T1 plants were isolated, and their level of SSI4 expression was determined by RNA gel blot analysis for eight independent T1 plants (Figure 6) . None of the T1 plants, including those with highly increased levels of SSI4 transcripts, developed the severe ssi4 phenotype or constitutively expressed PR-1 (Figure 6 and data not shown). This was confirmed further by analyzing ∼30 to 40 T2 plants of T1 lines 9, 15, 16, and 21. Interestingly, overexpression of SSI4 resulted in a corresponding increase in EDS1 transcripts, suggesting that the increased levels of EDS1 mRNAs in ssi4 plants are attributable in part to overexpression of the ssi4 gene. However, increased levels of SSI4 and EDS1 transcripts in 35S::SSI4 T2 plants did not confer enhanced resistance to the oomycete pathogen P. parasitica biotype Emco5; ∼200 to 300 T2 plants of T1 lines 9, 15, and 16 were tested and found to be as susceptible as nontransformed control plants (data not shown). These results suggest that the constitutively activated defense responses exhibited by ssi4 mutant plants are not simply the result of overexpression of the ssi4 gene; rather, they are caused by the amino acid substitution in the NBS.
Defense Gene Expression in Transgenic Plants Carrying the SSI4 cDNA Fused to the 35S Promoter of Cauliflower mosaic virus.
Ten micrograms of total RNA extracted from 4-week-old T1 plants was subjected to RNA gel blot analysis. The blot was probed sequentially for the SSI4, EDS1, and PR-1 gene transcripts. The numbers at top correspond to the different T1 plants. Ethidium bromide staining of rRNA served as a loading control.
To elucidate the components involved in defense signaling, a screen for suppressor mutations of npr1-5 was performed. One semidominant gain-of-function mutant, designated ssi4, was identified based on its ability to constitutively express several PR genes. In addition, ssi4 plants exhibited a stunted, chlorotic morphology and spontaneously developed lesions. Map-based cloning of the ssi4 gene revealed that it encodes a TIR-NBS-LRR protein. Thus, ssi4 exhibits structural similarity to a large class of plant R genes; nearly 100 members have been identified in the Arabidopsis genome (Dangl and Jones, 2001). Comparison with the wild-type SSI4 sequence revealed that the ssi4 gene contains a point mutation leading to a single amino acid substitution in the NBS domain. Although the crystal structures of a TIR domain and several LRR domains, all from other organisms, were solved recently, structural information on the NBS domain remains elusive (Xu et al., 2000; Kobe and Kajava, 2001). Recent sequence analysis of many R protein NBS domains has identified several conserved TIR-NBS-LRR–specific motifs that are dispersed between highly divergent nonconserved regions (Meyers et al., 1999). The Gly-to-Arg substitution caused by the ssi4 mutation was detected in a nonconserved region just upstream of the resistance NBS D motif. Thus, it is unclear whether this mutation affects the putative NBS of ssi4 or some other activity performed by this domain.
The combined discoveries that SSI4 encodes a TIR-NBS-LRR–type R protein and that the Gly-to-Arg substitution in its NBS confers constitutive PR gene expression led us to suspect that ssi4 is a constitutively activated R protein. Consistent with this hypothesis, ssi4 plants accumulated high levels of SA and SAG and exhibited enhanced resistance to both P. syringae pv maculicola ES4326 and P. parasitica biotype Emco5. Analysis of ssi4 nahG plants revealed that most of the ssi4-induced phenotypes, including constitutive PR gene expression, enhanced disease resistance, and chlorosis, require increased levels of SA. Because the ssi4 mutation was isolated in the npr1-5 background, all of these SA-dependent defenses must be activated via an NPR1-independent pathway. Analyses of several other Arabidopsis mutants also have revealed an SA-dependent, NPR1-independent pathway(s) that mediates PR gene expression and/or disease resistance (Bowling et al., 1997; Clarke et al., 1998; Li et al., 1999, 2001; Rate et al., 1999; Shah et al., 1999, 2001; Kachroo et al., 2001; Yoshioka et al., 2001). Because SA alone does not induce PR expression or enhanced resistance in npr1 plants, the activation of the SA-dependent, NPR1-independent pathway(s) is thought to require a second signal that works in conjunction with SA. As has been proposed by others, this second signal could be generated by pathogen infection or by various mutations, such as ssi4.
It is interesting that although NPR1 is not required for the ssi4-mediated activation of defense responses, it appears to play an important role in regulating SA accumulation. SA and SAG levels were substantially greater in ssi4 npr1-5 double mutants than in wild-type plants. SA and SAG levels in the ssi4 single mutant also were increased with respect to wild-type plants, although they were lower than those in the double mutant. A similar phenomenon was observed in snc1, cpr5, cpr6, and ssi2 single mutants, which accumulate less SA than snc1 npr1, cpr5 npr1, cpr6 npr1, and ssi2 npr1 double mutants (Clarke et al., 2000; Li et al., 2001; Shah et al., 2001). Together, these results suggest that NPR1 not only transduces the SA signal but also plays a role in downregulating SA levels, as was proposed previously by Clarke et al. (2000).
Most R proteins activate disease resistance via either an EDS1- or an NDR1-dependent pathway, with the structure of the R protein determining which pathway is used (Aarts et al., 1998). Like other TIR-NBS-LRR proteins, ssi4 requires EDS1 to activate resistance to P. syringae pv maculicola ES4326 and P. parasitica biotype Emco5. Indeed, ssi4 eds1 double mutants were indistinguishable from wild-type plants, demonstrating that EDS1 is required for all of the ssi4-induced phenotypes. In addition to a strong requirement for EDS1, NDR1 also appears to play a minimal role in signaling the ssi4 phenotype. ssi4 ndr1 double mutants exhibited less pronounced levels of PR gene expression, stunting, and resistance to P. syringae pv maculicola ES4326 compared with ssi4 single mutants. Similarly, the presence of an ndr1 allele reduced snc1- and RPS4-mediated resistance to strains of P. syringae and decreased RPP-mediated resistance to various P. parasitica isolates (Aarts et al., 1998; Li et al., 2001). Thus, these EDS1-dependent R genes also may incrementally activate defense responses via an NDR1-dependent pathway.
There have been several reports that R gene overexpression results in enhanced disease resistance (Oldroyd and Staskawicz, 1998; Tang et al., 1999; Stokes et al., 2002). Consistent with this possibility, increased levels of ssi4 transcripts correlated with constitutive SA accumulation, PR gene expression, and enhanced disease resistance. However, because overexpression of the wild-type SSI4 gene did not lead to stunting, constitutive PR-1 expression, or enhanced resistance to P. parasitica biotype Emco5, the Gly-to-Arg substitution in the NBS, rather than or in addition to overexpression of this R gene, appears to be required to activate defense responses. There are several possible explanations for how the NBS substitution in ssi4 can lead to constitutive defense response activation. For example, the Gly-to-Arg mutation might generate an aberrant protein that stresses the cell, thereby activating a nonspecific pathway for defense responses. Consistent with this possibility, previous studies have demonstrated that expression of various foreign or endogenous transgenes can induce PR gene expression and SAR (Durner et al., 1997). However, because SSI4 is a TIR-NBS-LRR–type R protein and ssi4 requires EDS1 but not NDR1 for defense response activation, it seems unlikely that this protein activates defenses via a nonspecific pathway. An alternative possibility is that the NBS mutation abolishes ssi4's ability to bind a guard protein that sequesters (and thereby silences) SSI4 in the absence of the appropriate Avr protein. Arguing against this possibility is the finding that overexpression of the SSI4 gene, which should titrate out the guard protein (as may have happened with overexpressed Pto, Prf, and At4g16890), did not induce defense responses. A more likely explanation is that the NBS mutation produces a constitutively activated R protein that constantly stimulates the defense signaling pathway, causing increased levels of SA, constitutive PR gene expression, and enhanced disease resistance. The observation that ssi4 is a semidominant gain-of-function mutation is consistent with this model. Note that the latter two models are not mutually exclusive.
In addition to stimulating downstream defense responses, the increased SA levels in the ssi4 mutant appear to enhance the expression of the ssi4 gene. Supporting this conclusion, ssi4 plants that express the nahG gene failed to accumulate increased levels of ssi4 transcripts. Analysis of the wild-type SSI4 gene and the closely related TIR-NBS-LRR R genes RPP1 and RPS4 suggests that their expression also is upregulated by SA. All of these genes were induced by SA treatment. Additionally, transcripts for RPP1 accumulated to higher levels in the ssi4 mutant than in wild-type plants. Another TIR-NBS-LRR R gene, 162J11T7, was shown recently to be highly induced by treatment with the SA analog BTH and to a lesser extent by infection with bacterial or oomycete pathogens (Maleck et al., 2000). Thus, increased levels of SA, such as those generated by pathogen infection, may upregulate the expression of at least some TIR-NBS-LRR genes. By contrast, SA treatment did not induce the CC-NBS-LRR genes RPM1 or RPS2, which suggests that upregulation of their expression either does not occur or involves a different mechanism.
Together, our results suggest that a positive feedback loop links SA accumulation and the expression of several TIR-NBS-LRR R genes, including SSI4. In this model, pathogen infection would lead to activation of the SSI4 protein; the activated protein then would transduce the resistance signal primarily via an EDS1-dependent signaling pathway. Increased SA levels generated during this response would not only activate downstream defenses directly but also would upregulate the expression of some R genes, thereby amplifying the defense response. Because defense signaling would not be initiated until the SSI4 protein was activated (presumably by a direct or indirect interaction with the pathogen Avr protein), plants that overexpress the SSI4 gene would not accumulate SA or display constitutive defense responses. By contrast, plants that express a constitutively activated R protein, such as ssi4, would accumulate increased levels of SA, which would stimulate additional expression of the ssi4 gene and activate the resistance signaling pathway.
Mutagenesis and screening for the ssi4 mutant were conducted as described previously (Shah et al., 1999). Briefly, 5000 seeds from Arabidopsis thaliana plants homozygous for the npr1-5 allele were mutagenized with 0.3% ethyl methanesulfonate (Sigma), and the M2 seeds from these plants were harvested in pools of ∼10 ethyl methanesulfonate–mutagenized M1 plants. Pooled M2 plants were screened for putative PR-1 overexpressors. Backcrosses were performed by pollinating flowers of the npr1-5 parental line (SSI4 npr1-5) with pollen from the ssi4 npr1-5 mutant. To isolate ssi4 mutants homozygous for the NPR1 wild-type allele, ssi4 npr1-5 mutant plants were crossed with wild-type ecotype Nö plants. Cleaved amplified polymorphic sequence (CAPS) marker analysis of 36 F2 plants then was used to identify plants that contained only the wild-type NPR1 allele. The genotype at the ssi4 locus in these F2 plants was determined by monitoring the segregation of PR gene expression in the F3 population using RNA gel blot analysis. To isolate ssi4 ndr1 and ssi4 eds1 double mutants, ndr1-1 (ecotype Columbia) or eds1-1 (ecotype Wassilewskija) plants were crossed with ssi4 homozygous plants. PCR markers for the ndr1-1, eds1-1, and ssi4 (see below) alleles then were used to identify the genotypes of the F2 progeny.
ssi4 homozygous plants were crossed with a wild-type Landsberg plant, and F2 progeny lacking constitutive PR gene expression (homozygous for the SSI4 allele) were identified by RNA gel blot analysis. CAPS and simple sequence length polymorphism marker analysis performed on these F2 plants localized the ssi4 mutation to chromosome V, between PHYC and DFR. Sequence information (www.kazusa.or.jp/kaos/) then was used to generate novel CAPS and simple sequence length polymorphism markers in this region. Of 1179 F2 plants examined using these markers, two recombination events were identified using MBK23.42kb and three recombination events were detected using MJC20.2kb. No recombination events were identified using additional markers within this 132-kb region. Thus, ssi4 is located within a region spanning the latter half of P1 clone MBK23, the entire region of P1 clone MUF8, and the entire region of transformation-competent artificial chromosome (TAC) clone K16L22, as defined previously (www.kazusa.or.jp/kaos/).
An ssi4 TAC library was created as described by Liu et al. (1999). Briefly, genomic DNA from ssi4 homozygous mutant plants was partially digested with HindIII, and 10- to 20-kb fragments were purified by agarose gel electrophoresis and cloned into pYLTAC7 (Liu et al., 1999). The library was screened using the K16L22 TAC clone and PCR fragments from P1 clones MBK23 and MUF8 as probes. A contig was constructed based on the results of end-sequencing positive clones with vector-based primers. Two gaps in the contig were identified and filled by PCR-generated clones (Advantage-HF2; Clontech, Palo Alto, CA). Clones representing the entire contig then were transformed into wild-type ecotype Nö plants using the vacuum infiltration method (Bechtold and Pelletier, 1998). Transformants were selected on Murashige and Skoog (1962) medium containing 15 μg/mL hygromycin, and the presence of the entire T-DNA region was confirmed by PCR analysis using primers for the sacB and hygromycin resistance genes, indicating the left and right borders of the vector, respectively (Liu et al., 1999).
Segregation analysis of the ssi4 mutation among F2 and T2 plants was conducted by creating a derived CAPS marker. An ∼100-bp fragment was amplified using primers MUF8.2BglIIF (5′-GGTTCATCTTTGCGTGGGGAGAGCAAGCAA-3′) and MUF8.2BglIIR (5′-GTGAAGGAATAGAGATTGATTTTTCTTCGACAATCTTTCAGATC-3′) and digested with BglII. This generated either a 140-bp undigested fragment in the amplified product from the wild-type plants or 100- and 40-bp fragments in the amplified product from the ssi4 plants.
Total RNA was extracted from 3- to 4-week-old leaves using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). RNA was separated on a 1.2% denaturing agarose gel, transferred onto a Hybond-NX membrane (Amersham Pharmacia), and hybridized with radiolabeled probes that were generated as described previously (Shah et al., 1997). PCR-amplified products of SSI4 (SSI4F, 5′-CTC-AAGAGAGTATGCTTCTCTTTCCATAACCC-3′; SSI4R, 5′-CTGGTT-TGGTCTTCATGAGACTCCATGAG-3′), EDS1 (EDS3F, 5′-GGATAG-AAGATGAATACAAGCC-3′; EDS1R, 5′-ACCTAAGGTTCAGGTATC-TGT-3′), and PAD4 (PAD4F, 5′-ATGGACGATTGTCGATTCGAG-3′; PAD4R, 5′-CTAAGTCTCCATTGCGTCACT-3′) were used as probes to detect these transcripts by RNA gel blot analysis.
Leaf samples were taken from 2-week-old plants grown on soil. Trypan blue staining was performed as described previously (Bowling et al., 1994).
Salicylic acid (SA) and SA glucoside were extracted and measured from 0.25 g (fresh weight) of leaf tissue as described previously (Bowling et al., 1994).
Infection with Pseudomonas syringae pv maculicola ES4326 was performed on 4-week-old soil-grown plants as described previously (Shah et al., 1997). Leaves were infiltrated with a bacterial suspension (OD600 = 0.002) in 10 mM MgCl2. For each genotype, four leaf discs (4 mm in diameter) were harvested at day 0 and six discs were harvested at day 3; the discs were homogenized in 10 mM MgCl2, and colony-forming units were counted. Infection with Peronospora parasitica biotype Emco5 was performed by spraying or applying a single drop of asexual inoculum suspension (105 conidiosporangia/mL) per cotyledon of 7-day-old seedlings. The seedlings were grown at 19°C and >90% RH with an 8-h photoperiod. P. parasitica biotype Emco5 growth was assayed visually by counting the number of sporangiophores per cotyledon at 7 days after inoculation.
Three-week-old plants were sprayed and irrigated for 10 min with a solution of SA (500 μM) or water. Leaves were harvested at the times indicated (in Figure 5B), and RNA was extracted as described above.
cDNA for reverse transcriptase (RT)–mediated PCR was generated using SuperScript Reverse Transcriptase (Invitrogen Life Technologies) according to the manufacturer's instructions. PCR was performed using Advantage 2 polymerase (Clontech). The gene-specific primers used for RT-PCR analysis were as follows: for SSI4, SSI4F (5′-CTCAAGAGAGTATGCTTCTCTTTCCATAACCC-3′) and SSI4R (5′-CTGGTTTGGTCTTCATGAGACTCCATGAG-3′); for RPP1, RPP1F (5′-GTGGAGCTCCCCGCTATCGAGAATGCGAC-3′) and RPP1R (5′-GCAAGGGAATCTGGAAGTTGGGGGAGTGATACC-3′); for RPM1, RPM1F (5′-GCATACATGGGACCTAGGTTGCGTTTTGCACAAGG-3′) and RPM1R, (5′-GCCTTGGCCGCCTAAGATGAGAGGCTCAC-3′); and for Actin2, ACT2F (5′-CTAAGCTCTCAAGATCAAAGGCTT-AAAAAGCTGGGG-3′) and ACT2R (5′-CTTATACAATACTTATATTAACATTGCAAAGAGTTTCAAGGT-3′).
An SSI4 cDNA of 3226 bp was amplified from Arabidopsis wild-type ecotype Nö RNA by RT-PCR using primers MUF8.2F (5′-CTT-TCTTTCAAGCATTTGTGATCTCTCATGGCTTCT-3′) and MUF8.2R (5′-CTGGTTTGGTCTTCATGAGACTCCATGAG-3′) and cloned into the pCR2.1-TOPO vector (Invitrogen Life Technologies). The cDNA clone was sequence verified, and the SSI4 coding sequence then was subcloned into the XbaI and SacI sites of pBI121 (Clontech), which placed the SSI4 gene under the control of the 35S promoter of Cauliflower mosaic virus. This construct then was transformed into wild-type ecotype Nö plants by vacuum infiltration (Bechtold and Pelletier, 1998). Transformants were selected on Murashige and Skoog (1962) medium containing 50 μg/mL kanamycin for 2 weeks, transferred to soil, and grown for 2 additional weeks before analysis of SSI4 expression.
The GenBank accession number for the SSI4 genomic sequence is AY179750.
We thank Daisuke Shibata for kindly providing the pYLTAC7 vector used in this study as well as S. Tabata for the P1 and TAC clones. We also thank J.E. Parker for eds1-1 seeds and P. Repetti and B.J. Staskawicz for ndr1-1 seeds and marker information. We are grateful to Shashi Sharma for conducting HPLC analysis for SA and SAG quantitation and to Keiko Yoshioka and Fasong Zhou for helpful advice. We also gratefully acknowledge D'Maris Dempsey for critical reading of the manuscript. This work was supported by Grant MCB-9723952/0110404 from the National Science Foundation to D.F.K.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.005348.
↵1 Current address: Department of Plant Pathology, University of Kentucky, Lexington, KY 40546.
↵2 Current address: Division of Biology, Kansas State University, 303 Acker Hall, Manhattan, KS 66506.
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62 Christmas Trees, 4000 Gumballs, and the White House Winter Wonderland
by: Kenzi Wilbur
To get to the place where you're about to be shown one of the first family's favorite recipes, you're first escorted down a hallway with a ceiling made of snowflakes, past the first dogs—Sunny and Bo—playing fetch. (They get the run of the house.) Casual.
this is the White House and here are Bo and Sunny playing fetch and you're going to have to start planning my funeral because I am literally *ded* (how's my hyperbole, @nytimes?)
A photo posted by Kenzi Wilbur (@kenziwilbur) on Dec 3, 2015 at 3:17pm PST
After your lucky encounter with the real McCoys, you stumble on two larger-than-life replicas of the dogs, husky yarn structures made from seven thousand yarn pom-poms lovingly crafted from fifty-five thousand feet of yarn. Through a grand hallway and up the stairs past the presidential portraits (these rotate per term, like the national, wall-art version of musical chairs); then through the State Dining Room and past the almost 500-pound gingerbread house. (It has electricity. It has East and West wings. You briefly consider asking if you can have a tour of this, too.)
This year's house was built with *over* 250 pounds of gingerbread dough.
The epic gumball tree, left; the epic teddy bear, right.
It's been 20 minutes, and you've passed many glitzed-up trees (at least 7 of the whole house's 62, plus one constructed entirely from gumballs, four thousand of them), probably 100 ornaments (of the whole house's 70,000), and you-don't-even-know how many representations of holly or pointsetta or other such festive, bushy Christmas plants. You've seen a 6-foot teddy bear and a family of penguins in glittering sleighs (they fancy) and enough garland to string the distance from D.C. to New York and back again. This is the White House, and you are in its holiday wonderland.
We weren't kidding when we said they had their own sleighs.
The aptly-named Red Room, left; an 18 1/2-foot Fraser Fir on display in the Blue Room, right.
It's a little like Narnia, with more security: Instead of entering through a wardrobe, you find your way through a few gates—at one of which you dial a number quickly dictated to you into a phone hidden behind its own little door—and down the front drive with an escort. And it's just as disorienting, at times: You are inside for a moment, looking at yarn dogs and Paul Bunyan-sized trees, and then you are outside, walking through a corridor, and then you are back in. You might ask your guide how long it took her to learn her way around the grounds; she'll probably laugh and say she's been here years.
And then you're standing in the kitchen of the White House with executive chef Cris Cumerford and she's making you cauliflower mac and cheese, full of multi-colored florets and an exuberant amount of cheese and a little bit of mustard for good measure. Cris helms the kitchen through the epic marathon of White House parties: 12,000 guests in 20 days, she'll tell you. She'll steam the cauliflower while telling you she's been cooking at the White House since the Clinton administration; while the water boils, she'll let you in on why this recipe is so good: It's all about the breadcrumbs you blanket over its top. The kitchen hums in the background. The 20 days aren't yet up.
The mac and cheese she's making is requested by the first family often; it's also perfect for the quiet moments after the house—White or otherwise—is restored to its pre-holiday state. When the decorations are down and the guests have gone on their merry way. But if that day feels a little too distant, put it on your holiday table—who says cheesy pasta doesn't belong? If your family protests, just tell them it's a direct order, straight from the White House.
Cauliflower Mac and Cheese
1 pound penne pasta
1 cup 2% or skim milk
1 pound shredded cheddar cheese (or a mixture of your favorite melting cheeses)
1/2 head cauliflower, cut into florets
3 tablespoons chopped flat-leaf parsley
1 tablespoon melted butter
daffy
kpeck
Written by: Kenzi Wilbur
I have a thing for most foods topped with a fried egg, a strange disdain for overly soupy tomato sauce, and I can never make it home without ripping off the end of a newly-bought baguette. I like spoons very much.
by: Theresa
by: cindy
by: ETZ
Saffron3 January 18, 2016
I liked the article, copied the recipe. I also know that many of the Christmas decorations, trees, other generic holiday items and all are mostly gifts to the White House. State Councils of the Arts, National Endowment for the Arts, national, state garden associations and clubs, various clubs and schools from states; all these organizations are judged to be part of the White House celebration. I suggest many folks would be very proud to do that. America is a democracy of creativity and tastes. Kudos to everyone represented for such festivities.
daffy December 20, 2015
Yarn 'dogs', 'gum ball' Christmas tree? Who thought THOSE were 'good ideas' & got PAID for them?
GOOD FRIEF! I don't know if it's just me, but was this article a classic example of REALLY BAD writing? Who 'helms' anything?
kpeck December 20, 2015
So thrilled to see where my hard-earned tax money from my three jobs is going...yay Government!

redpajama_set_name: RedPajamaCommonCrawl


text:
import './export_sideeffect';
import {A} from './export_default';
import {A as X} from './export_default';
import {foo} from './export_named';
import * as namespace from './export_namespace';
import {D} from './export_both';
import * as DExports from './export_both';
import {bar} from './export_both';

// Use imports from a namespace.
namespace.foo();
namespace.bar(namespace.x);

// Use destructured import.
foo();

// Use import from a file that has both default and named exports.
const D;
bar(DExports.x);

// Use imports with default and local names.
const aInstance = new A();
const aInstanceAsX = new X();

redpajama_set_name: RedPajamaGithub


Darsheel the successful child artist of Taare Zameen Par, currently is in demand and has signed for a couple of more movies. He is in demand now, Ganesha thus wishes to know and let know how will be his success in the coming years as an actor.
At the outset one finds that neither Mercury nor Venus are in any such strong position as per signs to make such a successful actor.
True, but on studying the planets in Navamsa, D-9, Ganesha observes that Mercury, Jupiter and Venus are in dual signs, signs of higher intelligence and communication than the other 10 signs. Additionally, Mars is in the Navamsa sign of Venus along with Sun. This leads to the acting profession of glamour and success.
Considering Aquarius Sun sign horoscope, when the film was released, transiting Jupiter was over its natal position, eleventh from the sun sign, indicating success.
This was all fine to lay the basis why he was successful, now looking far into the future, Ganesha observes that while transiting Saturn is opposite to his natal Sun and Jupiter is in conjcuntion and then sextile to its natal position, he will continue getting films and success, if the same are released within this period, which is till the end of 2009.
After that, though Darsheel will remain in the news and papers, he may not be a successful actor and may not maintain the same level of recognition.
Ganesha is happy to know that he has kept his first interest as studies and not films.
This article was posted on 30th May 2008 on the world wide web.


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text:
Study: Natural causes, not human activity, behind Plains drought
Michael Martinez, CNN
Updated 9:12 PM EDT, Fri April 12, 2013
Extreme heat, drought ravage Midwest —
The drought had a negative impact on corn in Le Roy, Illinois. Drought occurred in six Plains states between last May and August because moist Gulf of Mexico air "failed to stream northward in late spring," and summer storms were few and stingy with rainfall, said a report by the National Oceanic and Atmospheric Administration.
A calf strains for mother's milk as they forage amid dry wheat husks on the Becker farm August 24 in Logan, Kansas.
Farmer Darren Becker sifts through arid topsoil under a ruined crop on the family farm on August 24 in Logan, Kansas.
Rancher Gary Wollert pauses before heading out for work on August 23 near Eads, Colorado. The nation's severe drought has been especially hard on cattlemen. Much of eastern Colorado and virtually all of Nebraska and Kansas are still in extreme or exceptional drought, according to the University of Nebraska's Drought Monitor.
A billboard, "Jesus in the Wheat," stands alongside Interstate 70 on August 24 in Colby, Kansas. The billboard was erected by local residents Tuffy and Linda Taylor. "We just put it up there to minister," Linda Taylor told the Hays Daily News.
A boat dock is left high and dry at the Morse Reservoir in Noblesville, Indiana, on August 16, where water depths are significantly lower than average because of a widespread drought.
Fish remains bake in the heat in an area that is usually underwater at the Morse Reservoir in Indiana.
President Obama inspects a drought-stricken area of Missouri Valley, Iowa, with corn farmer Roger McIntosh on August 13 as he campaigns in the area. Since mid-June, corn prices have risen about 60% because of declining crop yields.
Heat and drought have destroyed corn yields.
A car kicks up dust as it drives by corn fields on dry dirt road in State Center, Iowa.
Jeremy Tilton adds hay to a feed mixer for cattle he raises on pastureland near Cuba, Illinois.
A tractor cuts down corn in a field designated as zero-yield on a farm in Vigo County, Indiana, on Tuesday, July 31. The U.S. Department of Agriculture has declared more than half the counties in the country natural disaster areas as drought sears millions of acres of pasture and cropland.
bloomberg/getty images
A field of dead corn sits next to the Lincolnland Agri-Energy ethanol plant in Palestine, Illinois, on Wednesday, July 25.
An employee stocks dairy products at a supermarket in New York on Wednesday, July 25. The U.S. Department of Agriculture said prices of dairy products like cheese, milk and eggs are expected to rise 2% to 3% because of the drought.
Corn stalks struggle to survive in a drought-stricken farm field on Thursday, July 19, near Oakton, Indiana. The corn and soybean belt in the middle of the nation is experiencing one of the worst droughts in more than five decades.
A field of corn shows the effects of the drought on a farm near Fritchton, Indiana, on Wednesday, July 17.
Farmer Ed Schoenberg and his son harvest oats early in attempt to salvage their drought damaged crop near Burlington, Wisconsin, on July 17.
Landov
Corn is watered with an irrigation system near Fritchton, Indiana, on July 17.
A single stalk of corn grows in a drought-stricken field near Shawneetown, Illinois, on July 16.
Brown and dry, a field of corn sturggles to survive drought conditions near Uniontown, Kentucky, on July 16.
Illinois Gov. Pat Quinn examines a drought-damaged ear of corn near Waltonville, Illinois, on July 16.
A weed grows thorugh the dried and cracked earth where a pond used to br near Ashley, Illinois, on July 16.
Farmer Marion Kujawa looks over a dried-up pond where his cattle used to water, near Ashley, Illinois, on July 16. Kujawa is digging the pond deeper so that the water will last longer in the future.
Farmer Albert Walsh walks through his drought-damaged corn field in Carmi, Illnois, on July 11.
The drought plaguing the Midwest has taken a harsh toll on America's corn crops, such as this one in Grayville, Illinois.
Swimmers relax in the shallow waters of the Mississippi River at Meeman-Shelby Forest State Park in Tennessee on July 6. Drought conditions have lowered the river's levels considerably from this time last year.
Fish skeletons line the bottom of a drainage ditch in Skelton, Indiana, on July 12.
Soybean seedlings push their way through dry soil in Skelton, Indiana, on July 12.
Cattle graze in a field on July 13 near Paris, Missouri. Many ranchers are rushing to sell off their herds as hay supplies dwindle and feed prices soar.
Cracked, dry ground marks the area where a pond normally stands in Crossville, Illinois, on July 11.
Brown corn stalks stand in a drought-stricken field on July 11 in Carmi, Illinois.
Photos: Heat wave, drought ravage Midwest
NEW: Climate scientist disputes study findings
"Every drought that's occurring isn't a result of climate change," author says
Study found a lack of moist air from Gulf of Mexico and "severe rainfall deficits"
The drought in six Great Plains states was the worst since records began in 1895
Extreme natural events, not man-made climate change, led to last summer's historic drought in the Great Plains, a new federal study said Friday.
Drought occurred in six Plains states between last May and August because moist Gulf of Mexico air "failed to stream northward in late spring," and summer storms were few and stingy with rainfall, said a report by the National Oceanic and Atmospheric Administration.
"Neither ocean states nor human-induced climate change, factors that can provide long-lead predictability, appeared to play significant roles in causing severe rainfall deficits over the major corn-producing regions of central Great Plains," the report summary said.
The drought in Wyoming, Colorado, Kansas, Nebraska, South Dakota and North Dakota was the worst since record keeping began in 1895, even eclipsing the notorious Dust Bowl droughts of 1934 and 1936, said study leader Martin Hoerling, a NOAA meteorologist.
The drought of 2012 had a devastating impact on farmers in the United States. But one industry that actually benefits from dry conditions is wine. This is Breaux Vineyards in Loudon County, Virginia.
Larry Lazo/CNN
Drought hits vineyards
exp point delgado wild weather explainer_00014809.jpg
What's behind wild weather swings?
Candiotti Pray for Rain_00004225
2012: Rancher tries to better bad decision
marketplace africa drought kenyan farmers_00004010
Protecting farmers from drought
"The event was rare, and we estimated maybe a once in a couple of hundred years event," Hoerling said. "But for as extreme as it was, it didn't have any strong indications for early warning.
"It's highly unlikely that this upcoming May to August will be as dry as last year's," he said, referring to the six states.
Although he believes in global warming, Hoerling said greenhouse gas concentrations weren't behind the heat wave in the Midwest region.
"I'm an advocate of global warming because science tells me that greenhouse gases have warmed the planet by about 1 degree Celsius in the last 100 years. So there's no question about that," he said. "But the science also tells that every drought that's occurring isn't a result of climate change."
Last summer's dryness had caused at least $12 billion in economic losses, as of last July, before the drought peaked, the study said. Crop yields and commerce on major rivers fell during the sweltering weather, the report said.
"It remains to be seen if the economic effects of the 2012 drought will approach prior events, including the 1988 drought that inflicted $78 billion in losses and the 1980 event that caused $56 billion in losses (adjusted for inflation to 2012 dollars)," the report said.
The study was prepared by 20 scientists from several universities and NOAA. "It was highly vetted through a review process," Hoerling said of the study.
A climate scientist disputed the study's findings and said it failed to address the role of global warming in an appropriate way.
"The analysis failed to take account of the record low snow pack in the Southwest, the development of the drought there and the record high temperatures, the inadequacies of the models used, or why there was no monsoon," said Kevin Trenberth of the National Center for Atmospheric Research, a federally funded facility.
"We know that drought forms naturally, but the role of global warming, via the extra heat available … is to increase the intensity of the drought, and thus the subsequent heat waves and wildfire risk," Trenberth said. "The study does not answer why there were so any records broken last year throughout the U.S."
Major snow disappoints with minor moisture
Hundreds of U.S. counties labeled disaster areas due to drought
2012 broke U.S. heat records
How severe weather affects food supply


text:
City council awards road project contract
dprice@lahontanvalleynews.com
Steve Ranson / LVN photo
A & K Earth Movers was awarded a contract for the New River Parkway extension project by the Fallon City Council Tuesday night during its regularly scheduled meeting.
The City Council approved the contract in the low-bid amount of $371,000 for an extension required as part of the terms of the city's land sale agreement for the Dairy Farmers of America Fallon Plant. The extension is set to connect Rogers Road and Wildes Road with work that will include roadway construction, curb, gutter, sidewalk and underground utilities.
City Engineer Jim Souba informed council members six bids were received for the project. The vote to approve was 2-0 (councilman James Richardson was absent).
The plant, targeted by DFA officials to open by the end of 2013, will be equipped to process 2 million pounds of raw milk daily and at full capacity will produce 90 million pounds of dried dairy ingredients annually for customers in the U.S. and worldwide.
Council members also discussed the following items:
• Heard a presentation regarding activities and projects of the Western Nevada Development District from Ron Radil, executive director. The WNDD region covers a six-county area that includes Churchill, Lyon, Humboldt, Pershing, Mineral, Storey, Douglas, Carson City and Washoe counties.
• Heard a presentation of the Police Department report for February.
Note: Mayor Ken Tedford Jr. noted the city has scheduled its 13th annual Keep Fallon Green Day for April 8-13. This is a time when city refuse crews collect large items such as appliances and furniture on regular pick-up days as part of a week-long effort that is capped off by a community-wide cleanup by volunteers on Saturday, April 13.


In answer to caravanners' concerns about stability, ATC Trailer Control and shock absorbers are standard. Bailey Pageant Series 7 Monarch 2008.
Rather than playing it safe with pale cabinetwork, the new Pageants sport a more contemporary continental look. The Monarch's level of luxury and finish are evident.
Dark wood 'Bailey Teak' cabinetry and black sockets lend a touch of class, while the large rooflight and abundant spotlights help lift the interior. The upholstery in the photos is the optional 'Savoy' treatment, but the more traditional 'Hilton' fabric is standard.
The kitchen, like the washroom, is high-spec, with an oven, grill, four-burner hob, 115-litre Dometic 8-series fridge and a microwave oven. There's plenty of work surface beside the sink and a well-placed 240V socket.
The main feature of this van is its end washroom. And what a washroom it is. On the offside there's a huge walk-in shower cubicle, which has anti-slip matting and plenty of space for a scrub.
The large plastic door is easier to keep clean than a shower curtain and is nice and solid. Alongside is the new Thetford C-250 toilet, while a sink plus toothbrush and towel holders complete the specification.
Fitted drop-in carpets keep your feet warm on a winter's morning, and the chunky washroom door adds to the homely feel.
In the daytime, the two facing sofas are perfect for lounging or listening to the stereo system that's fitted as standard. Come bedtime, they make up into a big double that's over six feet long.
Storage for two people's clothes is ample, too, and the wardrobe has enough shelves to section your socks from your shirts.


Helmed by James Lees via La Pac; The film, #ShakeParkinson'sOff launched on World Parkinson's Day on April 11, 2018 is part of a campaign to raise awareness about its early onset and an appeal for donations.
France is home to 1,50,000 Parkinson's patients with 8000 new patients each year. This powerful campaign by France's ICM (Institut du Cerveau et de la Moelle épinière / the Brain and Spinal Cord Institute) uses haptic technology to let mobile viewers experience the devastating tremors of Parkinson's by triggering mobile vibrations.
The film shows us the life of Tom, right from his childhood to old age. It highlights the key emotional experiences such as the birth of his child or him going through an accident that would surely leave him trembling. But as the ad progresses, we are bound to question if this is because of the intense emotions he experiences or is it a sign of a dangerous disease.


text:
Ek Var Piyu Ne Malva Aavje
Unix filesystem
Variety (botany)
Malva (disambiguation)
Ek Var Piyu Ne Malva Aavje (Gujarati: એકવાર પીયુને મળવા આવજે ) is a 2006 Gujarati romantic action film produced by Gunvant Thakor, Ramesh Patel, directed by Harsukh Patel, starring Hiten Kumar, Vikram Thakor, Firoz Irani, Minaxi, Mamata Soni, Jayendra Maheta, Jaimini Trivedi and Mayur Vankani in the lead roles, released in 2006.
Vikram (Vikram Thakor), a stage performer, loved Radha (Mamta Soni), a daughter of rich Vajesang Thakor. Vajesang disapproved their marriage due to poor financial condition of Vikram. Suraj (Hiten Kumar), brother of Vikram, gives in and help Vikram to marry Radha.
Hiten Kumar as Suraj
Vikram Thakor as Vikram
Firoz Irani as Roopsinh
Minaxi as Tejal
Mamata Soni as Radha
Jayendra Maheta as Vajesang Thakor
Jaimini Trivedi as Kadavi Foi
Bhaskar Nayak as Mangalsinh
Mayur Vakani as Popat
The film was debut of Vikram Thakor which was commercially successful. The film completed 25 weeks.
The music of the film is by Appu and all the songs were composed and written by Gunvant Thakor. The music album was released by Jay Shree Mahaveer Audio.
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Ek_Var_Piyu_Ne_Malva_Aavje
Var, VAR, VAr, VaR, or var may refer to:
Varna Airport IATA airport code
Vacuum arc remelting, a process for production of steel and special alloys
Value-added reseller, a company that adds some feature(s) to an existing product(s), then resells it, common in the electronics industry
Vector autoregression, an econometric method of analysis
Visual Aural Radio Range, VAR, a radio navigation aid for aircraft, predecessor to VHF omnidirectional range
Free Anti Revolutionary Party (Vrije Anti Revolutionaire partij), a Dutch political party
Value at risk in economics and finance
Var (department), a French department
Vár, a goddess in Norse mythology
Var (river) in France
Var, Iran, a village in West Azerbaijan Province, Iran
Var, Iran (disambiguation), other places in Iran
Uar, the name of a proto-Mongolian tribe placed in charge of the Uyghurs by the Rouran
Var, (Hungarian: Vár), a village in Obreja Commune, Caraş-Severin County, Romania
Var, a village in Jibou town, Sălaj County, Romania
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Var
In Unix and operating systems inspired by it, the file system is considered a central component of the operating system. It was also one of the first parts of the system to be designed and implemented by Ken Thompson in the first experimental version of Unix, dated 1969.
Like in other operating systems, the filesystem provides information storage and retrieval, as well as interprocess communication, in the sense that the many small programs that traditionally comprise a Unix system can store information in files so that other programs can read these, although pipes complemented it in this role starting with the Third Edition. Additionally, the filesystem provides access to other resources through so-called device files that are entry points to terminals, printers, and mice.
The rest of this article uses "Unix" as a generic name to refer to both the original Unix operating system as well as its many workalikes.
The filesystem appears as a single rooted tree of directories. Instead of addressing separate volumes such as disk partitions, removable media, and network shares as separate trees (as done in MS-DOS and Windows: each "drive" has a drive letter that denotes the root of its file system tree), such volumes can be "mounted" on a directory, causing the volume's file system tree to appear as that directory in the larger tree. The root of the entire tree is denoted /.
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Unix_filesystem
In botanical nomenclature, variety (abbreviated var.; in Latin: varietas) is a taxonomic rank below that of species and subspecies but above that of form. As such, it gets a three-part infraspecific name.
The pincushion cactus, Escobaria vivipara (Nutt.) Buxb., is a wide-ranging variable species occurring from Canada to Mexico, and found throughout New Mexico below about 2,600 metres (8,500 ft). Nine varieties have been described. Where the varieties of the pincushion cactus meet, they intergrade. The variety Escobaria vivipara var. arizonica is from Arizona, while Escobaria vivipara var. neo-mexicana is from New Mexico.
The term is defined in different ways by different authors. A variety will have an appearance distinct from other varieties, but will hybridize freely with those other varieties (if brought into contact). Usually varieties will be geographically separate from each other.
Other nomenclature uses
In plant breeding nomenclature, at least in countries that are signatory to the UPOV Convention, "variety" or "plant variety" is a legal term.
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Variety_(botany)
Malva is a genus of about 25–30 species of herbaceous annual, biennial, and perennial plants in the family Malvaceae (of which it is the type genus), one of several closely related genera in the family to bear the common English name mallow. The genus is widespread throughout the temperate, subtropical and tropical regions of Africa, Asia and Europe. The word "mallow" is derived from Old English "malwe", which was imported from Latin "malva", cognate with Ancient Greek μαλάχη (malakhē) meaning "mallow", both perhaps reflecting a Mediterranean term. A number of species, previously considered to belong to Lavatera, have been moved to Malva.
The leaves are alternate, palmately lobed. The flowers are from 0.5–5 cm diameter, with five pink or white petals.
The colour mauve was in 1859 named after the French name for this plant.
Cultivation and uses
Several species are widely grown as garden flowers, while some are invasive weeds, particularly in the Americas where they are not native.
Many species are edible as leaf vegetables and commonly foraged in the West. Known as ebegümeci in Turkish, it is used as vegetable in Turkey in various forms such as stuffing the leaves with bulgur or rice or using the boiled leaves as side dish. Malva verticillata (Chinese: 冬寒菜; pinyin: dōngháncài, Korean: 아욱 auk) is grown on a limited commercial scale in China; when made as a herbal infusion, it is used for its colon cleansing properties and as a weight loss supplement.
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Malva
Malwa is a natural region in west-central India occupying a plateau of volcanic origin. Geologically, the Malwa Plateau generally refers to the volcanic upland north of the Vindhya Range. Politically and administratively, the historical Malwa region includes districts of western Madhya Pradesh and parts of south-eastern Rajasthan. The definition of Malwa is sometimes extended to include the Nimar region north of the Vindhyas.
The Malwa region had been a separate political unit from the time of the ancient Malava Kingdom. It has been ruled by several kingdoms and dynasties, including the Avanti Kingdom, the Mauryans, the Malavas, the Guptas, the Parmaras, the Malwa sultans, the Mughals and the Marathas. Malwa continued to be an administrative division until 1947, when the Malwa Agency of British India was merged into Madhya Bharat (also known as Malwa Union) state of independent India.
Although its political borders have fluctuated throughout history, the region has developed its own distinct culture, influenced by the Rajasthani, Marathi and Gujarati cultures. Several prominent people in the history of India have hailed from Malwa, including the poet and dramatist Kalidasa, the author Bhartrihari, the mathematicians and astronomers Varahamihira and Brahmagupta, and the polymath king Bhoja. Ujjain had been the political, economic, and cultural capital of the region in ancient times, and Indore is now the largest city and commercial centre.
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Malwa
Malva was a Dacian settlement in present day Romania, where the Roman city of Romula was built. It may also refer to:
Malva, a genus of plants
Malva, Zamora, a Spanish municipality of Castile and León
Malwa, an alternative spelling for the Indian region and state
the pseudonym of Syrian artist Omar Hamdi
Malva pudding, a South African sweet pudding
Malva, a fictional character from Pokémon X and Y
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Malva_(disambiguation)
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